Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

12(2) [Special Section]: 1-27 (e165).

Perspective

Integrating current methods for the preservation of

amphibian genetic resources and living tissues to

achieve best practices for species conservation

^Breda M. Zimkus, ^Craig L. Hassapakis, and ^Marlys L. Houck

'Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, Massachusetts 02138, USA Mmphibian Conservation

Research Center and Laboratory (ACRCL), 12180 South 300 East, Draper, Utah 84020-1433, USA ^San Diego Zoo Institute for Conservation

Research, 15600 San Pasqual Valley Road, Escondido, California 92027, USA

Abstract —Global amphibian declines associated with anthropogenic causes, climate change, and amphibian-

specific infectious diseases (e.g., chytridiomycosis) have highlighted the importance of biobanking amphibian

genetic material. Genetic resource collections were the first to centralize the long-term storage of samples for

use in basic science, including disciplines such as molecular evolution, molecular genetics, phylogenetics,

and systematics. Biobanks associated with conservation breeding programs put a special emphasis on

the cryopreservation of living cells. These cell lines have a broader application, including the potential for

genetic rescue and use in species propagation for population enhancement, such as captive breeding and

reintroduction programs. We provide an overview of the most commonly used methods for the preservation of

genetic resources, identify ways to standardize collection processes across biobanks, and provide decision

trees to assist researchers in maximizing the potential use of their samples for both scientific research and the

practice of species conservation. We hope that the collection and deposition of tissues preserved using methods

that enable eventual cell line establishment will become routine practice among researchers, particularly

herpetologists working in the field. While many major museums do not yet cryopreserve reproductive cells or

cell lines, they contain the infrastructure and staff to maintain these collections if protocols and procedures

are adapted. Collaboration between organizations can play an important future role in the conservation of

amphibians, especially biobanks associated with research institutions and those pioneering techniques used

in breeding programs.

Keywords. ARTs, biobanks, cryopreservation, cell lines, tissue sampling, tissue culture, in vitro fertilization

Citation: Zimkus BM, Hassapakis CL, Houck ML. 2018. Integrating current methods for the preservation of amphibian genetic resources and living

tissues to achieve best practices for species conservation. Amphibian & Reptile Conservation 12(2) [Special Section]: 1-27 (e165).

NoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided

the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication

credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation-, official journal website <amphibian-

reptile-conservation.org>.

Received: 30 November 2018; Accepted: 18 December 2018; Published: 31 December 2018

Global Amphibian Declines

With approximately one-third of all known amphibian

species worldwide considered threatened, amphibians

are currently the most threatened vertebrate group (Stu¬

art et al. 2004; Wake and Vrendenberg 2008; Ceballos et

al. 2015). Distressingly, these estimates do not take into

account a large percentage of amphibians that are consid¬

ered “data deficient” by the standards of the International

Union for Conservation of Nature (lUCN 2018). The risk

of underestimation is that even more species are threat¬

ened, especially in regions of the world that are known

to be understudied (e.g., Madagascar, Southeast Asia;

Corr6SpondenC6. * bzimkus@oeb.harvard.edu

Rowley et al. 2010; Vieites et al. 2009). The hypoth¬

esized drivers of global amphibian decline include an¬

thropogenic factors, such as habitat degradation or loss,

overexploitation, pollution, and introduction of invasive

species (Sodhi et al. 2008; Hof et. al 2011; Ficetola et

al. 2014). Disease and climate change first emerged as

the most commonly cited causes because almost 50% of

amphibian species were characterized as having rapid

and unexplained decline in areas where suitable habitat

remained (Stuart et al. 2004).

Chytridiomycosis, an infectious disease in amphib¬

ians caused by the chytrid fungus Batrachochytrium den-

drobatidis (Bd), is now known to be one of the proximate

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 65

Zimkus et al.

GENETIC ^

TISSUE COLLECTION

CELL

ASSISTED REPRODUCTIVE

TECHNOLOGIES (ARTs)

in vitro ferttlizatian (IVF)

Embryo transfer*

Somatic cell nuclear transfer

REINTRODUCTION - ^IVE OFFSPRING

INTO WILD

CAPTIVE

BREEDING

Fig. 1. Role of genetic resource collections in the research and

conservation of amphibians. Green indicates the storage of tissues

in biobanks. Purple indicates procedures associated with ARTs

that lead to achieving multiple goals in amphibian research and

conservation. Asterisk (*) denotes tissue or methodologies that are

not currently used in ARTs but may be possible in the future. NOTE:

For a more complete list of ARTs reference Clulow et al. (2014).

2001; Carey and Alexander 2003). In addition, global

warming has led some amphibians in temperate regions

to breed earlier, making them vulnerable to early season

freezes and floods induced by snowmelt (Beebee 1995;

Blaustein et al. 2001; Gibbs and Breisch 2001). This

trend was found to vary regionally for a single species,

leading researchers to believe that climate change may

be affecting amphibian populations in more subtle and

complex ways. Hayes et al. (2010) suggested that inter¬

actions between multiple factors, including atmospheric

change, environmental pollutants, habitat modification,

invasive species, and pathogens, are the cause of amphib¬

ian declines. More recent work has found that although

some amphibian communities are sensitive to changes in

climate, observed declines can not be explained by the

impact of climate change (Miller et al. 2018).

Regardless of the specific causes of global amphibian

declines, there is an increased need to help prevent am¬

phibian species extinction. One key approach to conser¬

vation is biobanking genetic resources of all types (e.g.,

somatic tissues, cell lines, gametes) before these resourc¬

es are no longer available. We believe that integrating

current methods used to preserve genetic resources and

living tissues will facilitate stakeholder efforts and pro¬

mote more effective cooperation to conserve amphibians

(Hassapakis 2014).

History of Biobanking Amphibians

drivers of amphibian decline (Berger et al. 1998; Johnson

2006). The rapid decline of amphibians was linked with

the emergence of Bd; the geographic ranges of declining

species overlapped with areas most suitable for the fungus

(Letters et al. 2009). Zoospores produced by the fungus

are dispersed in water and infect the keratin-containing

epidermis in adults and the mouthparts in tadpoles (Berger

et al. 2005). Bd has been documented to infect all extant

orders of Amphibia and was detected in 41% of amphib¬

ian species across 63% of the countries in which sampling

has been reported (Gower et al. 2013; Olson et al. 2013).

A second species, Batrachochytrium salamandrivorans

(Bsal), is known to cause the disease only in salamanders

(Martel et al. 2013). Mitigating the effects of chytridio-

mycosis remains a major challenge, but data suggests that

temperature range and precipitation may be of particular

importance as the odds of Bd detection decrease with in¬

creasing temperature (Olson et al. 2013). In addition, re¬

cent work has found that increasing the salinity in aquatic

habitats can block transmission and reduce the severity

and mortality associated with Bd, hence, this tactic may be

a promising focus for future management of this disease

(Klop-Toker et al. 2017; Clulow et al. 2018).

Climate change has also been identified as a proxi¬

mate cause of population declines because amphib¬

ians are sensitive to small changes in temperature and

moisture given their permeable skin, biphasic lifestyle,

and unshelled eggs (Pounds et al. 1999; Kiesecker et al.

Biobanking, the practice of storing and curating genetic

resources and their associated data, including cryopre-

served living tissue, is one of numerous complementary

methods that should be used to counteract global amphib¬

ian extinction and was included in the 2005 lUCN Global

Amphibian Summit as one of 11 priorities relevant for

amphibian conservation (Wren et al. 2015). Genetic re¬

source collections form a critical basis for advances in

scientific understanding of species (and species limits),

evolutionary histories, and phylogenies. Many institu¬

tional biobanks that include amphibian genetic resources

are associated with natural history museums (e.g.. Har¬

vard’s Museum of Comparative Zoology, Smithsonian’s

National Museum of Natural History), while laboratories

or departments within colleges/universities (e.g., Kan¬

sas University Biodiversity Institute, The University of

Texas at El Paso) have also become de facto biobanks be¬

cause individual researchers have amassed large and/or

important sample collections (Zimkus and Ford 2014b).

Amphibian genetic resources are also essential for con¬

servation breeding programs (CBPs; species propagation

for population enhancement), which store primary cell

cultures for current and future use in habitat restoration,

reintroduction from captivity to the wild, and captive

management (Fig. 1).

Tissue samples traditionally collected and used in

phylogenetic and systematic studies have long aided in

understanding global amphibian diversity, resolving phy-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Preservation of amphibian genetic resources and living tissues

logenetic relationships for closely-related species and

revealing cryptic species that were morphologically in¬

distinguishable (Fouquet et al. 2007; Vieites et al. 2009).

Species delimitation is needed for population assess¬

ments and assists in the understanding of species ranges,

and accurate taxonomic assignments allow the identifi¬

cation of characteristics (e.g., endemic species/lineage,

population declines, threatened species) informative

for assisting in conservation assessments and priorities

(Mace 2004). In addition, modern molecular approaches

have allowed a greater understanding of amphibian de¬

clines, characterizing the prevalence of infectious dis¬

eases and assessing the effects of habitat alteration on

population connectivity (Storfer et al. 2009). Therefore,

genetic vouchers from newly described species, especial¬

ly if there are limited voucher specimens or the species

has a highly restricted distribution, should be deposited

in established biobanks for future conservation options

(Garcia-Castillo et al. 2018).

Those collecting amphibian specimens for molecular

analysis have known for decades that the traditional prep¬

arations used primarily for morphological studies (e.g.,

fixation in formalin) are not ideal for DNA sequencing

protocols because these methods induce DNA interstrand

crosslinks, cause base modifications, and induce frag¬

mentation (Campos and Gilbert 2012; Do and Dobrovic

2012; Quach et al. 2004; Wong et al. 2014). Procedures,

therefore, evolved to include sub-sampling of tissues

before specimens were exposed to formalin and low-

concentration ethanol, thereby avoiding extensive DNA

damage. Many institutions found the value in the genetic

resources collected for project-based purposes and began

to curate these collections for long-term preservation, in¬

cluding the formation of centralized biobanks (Zimkus

and Ford 2014b). Although these genetic resources were

likely collected for specific purposes associated with the

original research, it was clear that samples could be used

in future studies, making it possible for others to avoid

costly and time-consuming fieldwork required to collect

new samples (Astrin et al. 2013). In addition, institutions

realized that the utility and value of samples may actu¬

ally increase as rapidly-changing technology and newly-

developed methods allow samples to be used in ways that

are not currently possible.

The primary goals of amphibian conservation breed¬

ing programs include building genetically representative

captive populations, and maintaining the health, reli¬

able reproduction, and perpetuation of genetic variation.

Storage of genetic material in the case of amphibians is

important insurance against possible extinction and can

be used to reduce the loss of genetic diversity in cap¬

tive colonies and in declining wild populations (Fig. 1).

Some biobanks house samples for purposes of species

propagation using Assisted Reproductive Technologies

(ARTs), including gamete cryopreservation and in vitro

fertilization (IVF), where resulting offspring may be

used in captive breeding or re introduction programs. A

Amphib. Reptile Conserv. 3

number of these types of biobanks exist, including the

Memphis Zoo (Department of Research/Conservation)

and San Diego Zoo Global in the United States (Frozen

Zoo®, San Diego Zoo Institute for Conservation Re¬

search), the Zoological Society of London, the Institute

of Cell Biophysics at the Russian Academy of Sciences

in Moscow, and the University of Newcastle in Australia

(Kouba and Vance 2009). The rapid loss of amphibian

species has more recently resulted in the World Asso¬

ciations of Zoos and Aquariums (WAZA) promoting the

formation of conservation breeding programs supported

by research as a key element of their conservation plans

(WAZA 2005), which has likely led to an increase in the

number of biobanks in recent years. Kouba et al. (2013)

report that biobanks are being initiated or planned at the

Smithsonian Conservation Biology Institute in the U.S.,

the Toronto Zoo in Canada, the New Zealand Centre for

Conservation Medicine at Auckland Zoo, and the Uni¬

versity of Wollongong in Australia.

Considerations for Preservation

Aims and Goals

Numerous methods are currently being used to preserve

amphibian tissue and likely depend on the specific aim of

a scientific study or goals of an institutional or collabora¬

tive program (e.g., biobank, multi-institution initiative).

Samples may be collected for individual research proj¬

ects with explicit and relatively short-term goals (e.g.,

molecular ecology, molecular phylogeny, population ge¬

netics). Studies may also be taxonomically or regionally-

focused, such as rapid biodiversity assessments that use

DNA barcoding techniques to identify species surveyed

in a specific region. In contrast, biobanking initiatives or

collaborative programs involving multiple institutions

may have targeted specific species for long-term conser¬

vation and/or use in ARTs. The ultimate aim of a research

study or conservation initiative may dictate the specific

tissue types or biomolecules needed to fulfill the proj¬

ect goals. Molecular studies traditionally used DNA as

it could be preserved more easily in the field with many

methods. Unfortunately, the various methods used for

DNA preservation are not equally effective, and DNA

may be fragmented or otherwise compromised. Some

preparations may allow high-quality Sanger sequencing

reads but prevent high-quality gDNA needed to sequence

genomes or the high-molecular-weight DNA needed for

long-read sequencing and other technologies (e.g., BAG

library preparation, optical mapping, lOX Chromium

libraries; Mayjonade et al. 2016). RNA is increasingly

being used in gene expression studies but is preserved

using fewer methods and degrades rapidly if not frozen

immediately. Researchers should, therefore, consider all

preservation options as some may allow them to both ful¬

fill their study goals and aid in current or future research

or conservation initiatives.

December 2018 | Volume 12 | Number 2 | e165

Zimkus et al.

Table 1. Tissue types commonly used for genetic study, growth of cell lines, and ARTs in amphibians. Preferred tissues for the

production of cell lines are included in parentheses, although other tissue types that have been successful are listed. NOTE; Asterisk

(*) denotes unsuccesful efforts to cryopreserve to date (Clulow and Clulow 2016).

Preserve for

Genetic Study

Make Cell

Lines

Make Cell Lines

in Future

Collect and Use

Immediately in

ARTS

Preserve for

Future Use in

ARTS

Testes

(X)

Ovaries

(X)

Limb/foot

(X)

Skin (Biopsy)

(X)

Tongue

(X)

Eye

Kidney

Tadpole

Tail clip

Lung

Toe clip

X (if large)

Embryos

Spermic urine

Sperm

Blood

Feces

Glands

Heart

Fiver

Muscle

Oocytes

=1=

Pancreas

Spleen

Swab (e.g., skin, mouth)

Tissue Types

Many different tissue types can be preserved for use in

basic genetic studies as many soft tissues yield high-mo¬

lecular-weight-genomic DNA (Table 1). Liver and skel¬

etal muscle are perhaps the most commonly sampled tis¬

sues for herpetological research (Gamble 2014). A small

incision can allow researchers to push the liver out, caus¬

ing minimal damage to specimens being used for mor¬

phological study. Camacho-Sanchez (2013) also found

that rat (Rattus rattus) liver yielded the best RNA and

DNA quality when compared to blood, brain, ear clips,

muscle, and tail tips. Although liver is widely used by

those collecting amphibian genetic samples, bile salt can

contaminate this organ and affect tissue stability, so tis¬

sue should be preserved as soon as possible and the gall¬

bladder avoided (Dessauer et al. 1990). Muscle can be

dissected from the thigh on one side, leaving the remain¬

ing side intact for morphology, but it has been reported

that yields are small due to tough fibers (Gamble 2014;

Wong et al. 2012). According to Wong et al. (2012), tes¬

tes provide high yields and are the preferred tissue in spe¬

cies with heterogametic or temperature-dependent sex

determination, while liver is recommended for immature

specimens and homogametic individuals (females in XY,

males in ZW systems). Others recommend sequencing

genomes from the heterogametic sex or both sexes for

amphibians as this may provide important information

about sex determination in different species, which is rel¬

evant for managing captive populations and reproduction

(Tony Gamble, pers. comm.). Wong et al. (2012) notes

that soft tissues (e.g., spleen, pancreas, lung, glands) are

prone to faster degradation, so harder tissues (e.g., mus¬

cle, kidney, heart) may be preferable. Lastly, Wong et al.

(2012) suggest that red blood cells are a good source of

high-molecular-weight DNA and are the preferred tissue

for constructing large-insert libraries and for use in long-

read sequencing. Blood collection may be difficult for

small species, but techniques using doppler ultrasound

and fiber-optic lights may make it more feasible (Gamble

2014).

The collection of samples from different tissue types

(stored in separate vials) is desirable for RNA studies,

achieving the highest possible coverage of the diverse

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Preservation of amphibian genetic resources and living tissues

transcriptome, as well as optimizing the chance of es¬

tablishing a successful cell line. Contractile proteins,

connective tissue, and collagen in skeletal muscle, heart,

and skin tissue may result in low RNA yield (Wong et al.

2012). For cell lines, the recommended tissues include

(in order from most to least successful): whole limb (i.e.,

foot), tongue, skin, and gonads (Table 1). Viable cell lines

provide the highest quality material for DNA and RNA,

and additionally can be used for chromosome analysis

and potentially reprogrammed into induced pluripotent

stem cells (Takahashi et al. 2007; Yu et al. 2007; Ben-

Nun et al. 2011) that can differentiate into any type of

cell, including gametes. Species propagation in amphib¬

ians historically requires reproductive cells (e.g., sperm,

oocytes) for ARTs and has proven successful with the use

of testes and ovaries (Table 1).

Destructive vs. Non-Destructive Sampling

Tissue samples traditionally collected and used in phylo¬

genetic and systematic studies are often associated with

whole-animal voucher specimens deposited in natural

history collections. Recently-deceased animals are also a

source of both tissues that can be used for genetic study

and material used in species propagation. A number of

factors may result in the choice of less destructive pro¬

tocols associated with sample collection. For example,

projects may require links between genetic samples and

live animal ‘vouchers’ in zoos, aquaria, universities, and

other institutions (e.g., CryoArks project; U.K. Research

and Innovation 2018). Collection of samples from live

animals require less invasive methods that do not affect

the animal’s fitness and precludes the collection of vital

organs, such as the liver, that require animal euthaniza-

tion. Non-lethal sampling methods may include biop¬

sies, blood draws, feces collection, skin swabs, sperm

or spawn collection (hormonally-induced), toe clips,

and tail clips (Ezaz et al. 2009; Gamble 2014; Mollard

2018; Mollard et al. 2018). The feasibility of obtaining

sperm and eggs in the field and laboratory has been dem¬

onstrated both in frogs and salamanders (Shishova et al.

2011; Figiel Jr 2013; Uteshev et al. 2013; Uteshev et al.

2015). Non-invasive sampling methods for use in genetic

analyses, including the detection of chytrid fungus using

skin swabs, is becoming increasingly common with am¬

phibians (Pichlmuller et al. 2013; Soto-Azat et al. 2009).

Certain specimens, such as those designated as type ma¬

terial, may require similar sample collection methods

that minimize external damage to retain all parts needed

for taxonomic diagnosis. Small animals or early develop¬

mental stages may have low amounts of tissue available,

and hence eggs, tadpoles, metamorphs or juveniles may

need to be collected whole.

Collection of Gametes

Timing of tissue collection should be synchronized with

Amphib. Reptile Conserv.

breeding season if the goal is the collection of gametes

for ARTs. Whenever possible, reproductively active ani¬

mals should be collected during or close to breeding sea¬

son, which makes obtaining gametes (primarily sperm)

much less laborious (Childress 2017). Collecting animals

out-of-season or not of reproductive age requires that an¬

imals be maintained in captivity until gametes can be col¬

lected naturally or breeding is induced through hormone

usage. Housing and monitoring the reproductive status

of animals requires veterinary permits, substantial time,

skill in captive husbandry methods, and species-specific

nutrition, as well as social and behavioral specifications.

Monitoring live animals can become time-consuming

and complex when multiple species have different diets

and housing requirements. In addition, many anurans do

not reproduce easily in captivity because of confinement

stress or lack of critical environmental cues needed to

induce reproduction (Kouba et al. 2009). Given that the

most time-consuming aspect of collecting gametes for

cryopreservation is the timing of natural reproduction,

amphibians can be injected with hormones to induce

spawning and reduce required time in captivity (Fig. 2;

Rugh 1934; Miller 1985; Browne et al. 2006; Trudeau et

al. 2010; Trudeau et al. 2013).

Logistics

Researchers collecting samples need to consider the lo¬

cation of initial preservation and if possible carry out a

feasibility study to ensure that the selected preservation

method(s) will work given any logistical constraints.

Those transporting live animals to permanent laborato¬

ries for sample collection or using mobile labs have the

most choices in regards to preservation methods. Work¬

ing within a short distance from the laboratory or bio¬

bank requires the development of field protocols that

adapt laboratory techniques given local conditions at the

collection site but offers numerous options for sample

preservation. In contrast, fewer methods allow samples

to be collected and transported from remote locations for

a number of reasons: 1) the preservatives or equipment

needed to maintain the samples may not be available in

the specific country or collection site, 2) the duration of

the field trip or time required to transport the samples

may eliminate specific methods, and 3) the ambient tem¬

perature at the collection site or temperatures that the

samples are exposed to during transit may preclude use

of specific methods.

Shipping biological materials requires attention to

the type of material transported, adherence to regulatory

requirements, packaging materials and proper assembly,

labeling, and engaging reputable carriers (Simione and

Sharp 2017). International shipments that include dan¬

gerous goods must follow International Air Transport

Association (lATA) Dangerous Goods Regulations to

meet commercial standards, while domestic shipments

must follow national guidelines. Legal requirements as-

5 December 2018 | Volume 12 | Number 2 | e165

Zimkus et al.

sociated with the transportation of dangerous goods or

hazardous materials may preclude the shipping of some

preservatives either using a courier or in personal airline

baggage; therefore, shipping options should be deter¬

mined given the materials (e.g., infectious agents), pre¬

servatives (e.g., hazardous chemicals), and cold-chain

methods (e.g., dry ice, liquid nitrogen [LN 2 ]) employed.

Courier services that maintain samples at required tem¬

peratures can be considered for viable material but are

expensive.

Ethical and Legal Requirements

Scientific procedures carried out on animals should mini¬

mize adverse effects while maximizing the scientific

benefit gained. These legal and ethical requirement are

included under the laws and regulations of numerous

countries worldwide, including the U.S. Animal Welfare

Act (United States Code, Title 7, Chapter 54, Sections

2131-2159), the U.K. Animals (Scientific Procedures)

Act 1986, the U.K. Animal Welfare Act 2006, the Animal

Health and Welfare Strategy for Great Britain, Animal

Welfare Strategy, Canadian Council on Animal Care in

Science, among others. Researchers should be aware of

laws and regulations associated with their home coun¬

try and possibly the country of origin of the specimens

collected. Within the U.S., an Institutional Animal Care

and Use Committee (lACUC) ensures that all projects

involving the use of live vertebrate animals comply with

federal regulations and guidelines (OLAW/ARENA

2002). An lACUC is required by federal regulations for

most institutions that use animals in research, teaching,

and testing and has a key oversight role, including the

review and approval of animal use activities. lACUC re¬

view of such studies would focus on, but not necessarily

be restricted to, such issues as: number of animals to be

used in a study; stability of the population from which

the animals are to be taken; appropriateness of the meth¬

ods used for capturing, immobilizing, and/or euthanizing

animals; and training and supervision of the personnel

involved with the study. To this end, both collection pro¬

cedures and animal husbandry practices must be planned

in advance and approved to meet the intended goals and

objectives of the research project.

Proper planning for collection of specimens/samples

includes researching the permits needed to conduct re¬

search, collect, and export scientific specimens from a

specific country. The Nagoya Protocol on Access and

Benefit-Sharing (ABS) is (for its contracting parties) a

legally binding supplementary agreement to the Con¬

vention on Biological Diversity (CBD) that affirms that

countries hold sovereign rights over their biological re¬

sources. Those collecting genetic samples should, there¬

fore, determine country-specific permitting requirements

using the ABS Clearing-House (Secretariat of the Con¬

vention on Biological Diversity 2018), including obtain¬

ing Prior Informed Consent (e.g., collecting permit) from

the providing country and establishing Mutually Agreed

Terms (e.g., benefit-sharing agreement) if needed. In

some countries separate permits may be required for col¬

lecting wildlife and taking genetic resources. In addition

to national permits, other permissions and documenta¬

tion may be needed to research and/or collect particular

species or in specific regions (i.e., protected lands), as

well as import specimens into the destination country.

Lastly, indigenous communities may have legal authority

over wildlife and may have requirements associated with

collecting materials (e.g.. New Zealand). Given that the

process of applying for and receiving permission to con¬

duct research and collect specimens may take substan¬

tial time, permits and any other required documentation

should be secured as far in advance as possible to allevi¬

ate complications that might slow or jeopardize research

projects. For those working internationally, collaboration

with in-country partners (e.g., local scientists, wildlife

managers) should be considered as it may facilitate the

permit process and fulfill benefit-sharing obligations.

Best Practices in Tissue Preservation for

Genetic Analyses

Tissue preservation (fixation) methods used for amphib¬

ian samples generally prevent or reduce enzymatic and

thermodynamic degradation of nucleic acids (Yagi et

al. 1996; Prendini et al. 2002). A review of tissue pres¬

ervation methods for use in molecular studies was first

presented by Prendini et al. (2002) and later updated by

Nagy (2010). In addition. Gamble (2014) provided infor¬

mation specific to collecting and preserving genetic ma¬

terial from herpetological specimens. These reviews pro¬

vided thorough overviews of tissue preservation methods

for molecular genetic analyses, outlining the advantages

and disadvantages of each method. A global survey of 45

independent genetic resource collections within 39 dif¬

ferent institutions found that the vast majority (80%) of

genetic resource collections store samples that were ini¬

tially preserved in multiple (2-5) different ways, which

is expected given that the majority of these collections

stored samples collected for individual research projects

(Zimkus and Ford 2014). This survey included numerous

types of institutions and was not taxonomically-focused,

but over two-thirds (64%) of the respondents reported

that their collections stored amphibian samples. Data

from the 29 collections reporting amphibian genetic re¬

sources was analyzed for this study to determine the most

commonly used procedures associated with initial pres¬

ervation in an attempt to provide more accurate statistics;

however, it should be noted that most of these collections

house diverse taxonomic collections, so responses may

also be applicable to non-amphibian collections.

All of the genetic resource collections that included

amphibian samples indicated that they housed samples

preserved with 95-99% ethanol with two noting that

most or all samples were preserved in 99% ethanol. Over

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Preservation of amphibian genetic resources and living tissues

three-fourths of collections (77%) reported that samples

were initially flash-frozen at a temperature of -80 °C or

below; however, the survey question did not ask respon¬

dents to clarify the technique used: frozen on dry ice (at

-78.5 °C), mechanical freezers (-80 °C to -150 °C), im¬

mersion in LN 2 (-196 °C), or storage in the vapor-phase

of nitrogen (<-150 °C). Respondents also reported their

collections included samples initially preserved in di-

methylsulfoxide (DMSO; 45%), which is a commonly

used cryoprotectant, but the survey did not ask respon¬

dents to specify the temperature at which the preservative

was used. Fewer collections used commercial products,

such as RNAtoer® (Ambion; 52%) or Allprotect® Tis¬

sue Reagent (Qiagen; 10%) as an initial preservative;

however, non-hazardous proprietary agents that allow

the preservation of multiple biomolecules at ambient

temperature are being increasingly used (Muller et al.

2016). Herein we will discuss these most commonly used

methods of sample preservation identifled for collections

biobanking amphibian genetic resources, addressing the

advantages and disadvantages for both researchers mak¬

ing the initial collections and biobankers concerned with

long-term storage and downstream use.

Freezing

One of most effective techniques for the long-term sta¬

bilization of genetic samples is cryopreservation, an

approach that is based on the principle that cryogenic

temperatures suspend biological, chemical, and physical

processes (Karlsson and Toner 1996). Depending on the

temperature, freezing samples reduces or inhibits the en¬

zymatic and chemical activity that leads to nucleic acid

damage and degradation (Engstrom et al. 1990; Karls¬

son and Toner 1996). Degradation is virtually absent if

samples are continuously kept in LN 2 storage (generally

between -150 °C and -196 °C, depending on whether

vapor-phase or liquid is used) because of lack of thermal

energy needed for chemical reactions; however, freez¬

ing damage, including cell death, can be caused by ice

formation and crystallization if cryoprotectants are not

used (Karlsson and Toner 1996). Therefore, the initial

preservation method, any subsequent methods used in

long-term storage, and all downstream handling (e.g.,

freeze-thaw events, changes to preservative) will affect

the quality of the sample (Benson et al. 2016).

Many methods use freezing or flash-freezing as one

component to preserve genetic samples. Freezing sam¬

ples without the use of a preservation agent either by

placing them into a laboratory freezer (~-20 °C) or by

using ice within an insulated container is generally not

recommended because temperatures are not low enough

to prevent enzymatic activity nor the formation of intra¬

cellular ice crystals (Stoycheva et al. 2007; Nagy 2010).

Those collecting samples may have access to mechanical

freezers that can maintain samples at ultracold or cryo¬

genic temperatures (-80 °C to -150 °C), but this gener¬

ally excludes samples that are collected in the held. Dry

ice (frozen carbon dioxide; -78.5 °C) stored within insu¬

lated containers may allow the collection or transport of

samples, but a sublimation rate of approximately 10%

or 2-5 kg every 24 hours limits this method to relatively

short trips. In addition, since temperatures near the upper

end of the ultra-low temperature range, some DNA deg¬

radation may occur as a result of weak enzymatic activity

(Nagy 2010).

Cryogenic storage dewars, a specialized type of vac¬

uum flask used to store cryogenic fluids, can be used to

flash-freeze samples in LN 2 . Access to LN 2 increases

held sample collection options, including the preserva¬

tion of tissues useful for cell culture, RNA, and gametes

for multiple weeks. If a LN 2 source is available, samples

can be kept viable in the held until they can be transport¬

ed back to a laboratory. The use of LN 2 requires addition¬

al precautions as it can cause frostbite, cold burns, and

asphyxiation by displacing the oxygen of the surround¬

ing area. In addition, sample vials can shatter when re¬

moved from storage because LN 2 can enter the vials and

rapidly expand upon warming, creating a hazard from

both flying debris and exposure to the contents. Second¬

ary containment (e.g., polyethylene tubing, tin foil) and

protective eyewear is, therefore, recommended. Cross¬

contamination has been reported for samples immersed

directly in LN 2 , so researchers should consider vial type,

secondary containment options, and use of vapor-phase

storage when considering flash-freezing methods (Clark

1999).

Freezing without the inclusion of a preservative was

once thought to maximize future research potential, but

data now suggests that buffered samples or those stored

in a cryoprotectant, such as DMSO or glycerol, may per¬

form better after thawing and refreezing (Nagy 2010).

Cryoprotectants partly protect against degradation occur¬

ring during temperature changes, such as freeze-thaw cy¬

cles. Mulcahy et al. (2016) found that fish tissues stored

in a solution with 25% DMSO or DNAzol yielded higher

quality DNA after thawing than putting tissue directly

into LN 2 or -20 °C storage without buffer, while crab tis¬

sues in DMSO and ethanol did equally well in preserving

DNA quality. If viable cells are desired (e.g., cell culture,

gametes), slow freezing and using a cryoprotectant is re¬

quired to prevent the formation of ice crystals that lead

to fatal cell lysis.

A dry shipper is an insulated cryogenic flask/con¬

tainer that contains LN 2 absorbed into a porous lining.

Dry shippers are not considered a dangerous product and

hence can be used to ship samples by plane if the liq¬

uid is fully absorbed and excess poured off There are

wide variations in dry shippers with regard to size and

temperature (static) hold times. Size ranges include those

with space for a dozen vials to others that can accom¬

modate thousands of vials. Temperature hold times can

vary from a few days to multiple weeks, and variations

also exist in how well they hold temperature under dif-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Zimkus et al.

ferent environmental and handling conditions. Although

dry shippers are expensive to purchase, they can be used

for many years. Performance degradation can be attrib¬

uted to catastrophic or gradual vacuum loss of the dewar,

accumulation of moisture in the LN 2 absorbent material,

or damage and loss of portions of the absorbent material

(Simione and Sharp 2017). It is recommended that the

performance of dry-shipping dewars be checked at regu¬

lar intervals and ideally before each use using a simple

24-hour evaporation test to identify whether there has

been deterioration in any components.

An increasing number of biobanks associated with

natural history collections are using LN 2 cryovats for

long-term storage of samples. Zimkus and Ford (2014b)

surveyed genetic resource collections associated with

natural history museums and found that vapor-phase ni¬

trogen storage that include a standing level of liquid be¬

low a rotating carousel was the most commonly used type

of cryovat. Those managing collections should be aware

of the previously-discussed safety issues associated with

both cryogenic liquids and vials previously immersed in

LN 2 ; for additional information about proper ventilation

and use of oxygen monitors in genetic resource collec¬

tions with LN 2 storage, see Zimkus and Ford (2014a).

Ethyl Alcohol (Ethanol)

Alcohols, and specifically ethanol, are the most fre¬

quently used chemical to preserve amphibian tissues.

Ethanol denatures proteins that may degrade DNA and

is able to preserve samples for long periods of time at

ambient temperature; RNA cannot be preserved us¬

ing ethanol at room temperature. Survey data indicated

that all participating genetic resource collections stored

samples preserved in 95-99% ethanol (Zimkus and Ford

2014b). Ethanol concentration can greatly affect the re¬

sulting quality of the samples with 95-96% (190 proof)

recommended as optimal. In most countries 190 proof

ethanol is widely available for purchase at pharmacies.

Concentrations above 96% (including absolute ethanol)

are not recommended as they likely contain traces of dry¬

ing agents (e.g., benzene) that can affect DNA preserva¬

tion (Ito 1992). Concentrations of 65-75% (commonly

used to preserve whole animals for morphology) are also

not recommended; Seutin et al. (1991) were unable to

recover DNA from bird brain and muscle samples kept

in 70% ethanol for six weeks at room temperature, while

liver samples yielded significantly degraded DNA. Re¬

searchers should avoid using distilled alcoholic beverag¬

es because they may have alcohol concentrations as low

as 35%. Undiluted rectified spirits or neutral spirits (e.g.,

Everclear, Crystal Clear, Primasprit, Spirytus) is highly

concentrated (95-96%) but should be avoided because it

includes denaturing chemicals. Denatured alcohol (i.e.,

methylated spirits), widely used for industrial purposes,

is made of 70-99% ethanol but contains additives that

make it non-consumable for humans (e.g., methyl ethyl

ketone, also known as MEK) and thus should also be

avoided (Post et al. 1993; Dillon et al. 1996).

Using ethanol as a preservative has numerous advan¬

tages for researchers whose primary goal is to preserve

DNA. Ethanol is easy to use and able to preserve DNA

even in areas with elevated ambient temperatures for

long periods of time, although the liquid is fiammable

and considered hazardous. Nagy (2010) suggested that

tissues be cut into small pieces to increase the surface

area, using at least 5:1 volumes of ethanol, while others

suggest higher ratios (Martin 1977). Although the initial

concentration and ratio of ethanol to sample is important,

changing the alcohol during the first one to two days of

storage is also necessary because samples release wa¬

ter and progressively dilute the preservative (Kilpatrick

2002; Nagy 2010). Researchers should be aware that

most inks are soluble in ethanol, so pens used for label¬

ing vials containing ethanol should be tested before use.

A secondary labeling method should also be considered

for redundancy, such as labeling with graphite pencil, en¬

graving or barcoding. Placing paper tags inside vials has

been used as a method of labeling, but it is currently un¬

known whether this may lead to contamination (Zimkus

and Ford 2014b).

The transport of non-infectious ethanol-preserved

specimens has been allowed since 2011 via International

Air Transport Association (lATA) Special Provision

Also, making it possible to transport specimens pre¬

served in ethanol. The following packing and marking

requirements must be met, including: 1) specimens are

placed in vials or other rigid containers with no more than

30 ml of alcohol or an alcohol solution; 2) the specimens

are then placed in a plastic bag that is then heat-sealed;

3) the bagged specimens are then placed inside another

plastic bag with absorbent material then heat-sealed; 4)

the finished bag is then placed in a strong outer packaging

with suitable cushioning material; 5) the total quantity of

flammable liquid per outer packaging must not exceed

one E; and 6) the words “scientific research specimens,

not restricted. Special Provision Al 80 applies” must be

written on both the outside of the package and on the air

waybill in the description of the substance.

Those maintaining archival collections, including

biobanks, can combine initial preservation in ethanol

with long-term cold storage at cryogenic temperatures

to preserve DNA indefinitely. Since the melting/freezing

point of pure ethanol is approximately -114 °C (-173 °F),

high-concentration ethanol thaws almost immediately

after removing from EN 2 storage. Procedures associ¬

ated with sub-sampling can be completed significantly

faster when the storage medium does not require thaw¬

ing. Nucleic acids are sequentially degraded by cycles

of freezing and thawing, but anecdotal evidence suggests

that samples can be thawed and refrozen several times

(Shao et al. 2012). Collections storing samples in vials

with a silicone 0-ring should be aware that, according

to the manufacturers (e.g., Nalgene, NUNC), these types

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Preservation of amphibian genetic resources and living tissues

of vials were designed for tissue culture and are vapor

permeable. After observing that rapid evaporation was

occurring in their ethanol-preserved tissue collections on

a scale of weeks, the North Carolina Museum of Natural

Sciences conducted tests and found that 95-100% etha¬

nol evaporated fastest at room temperature and slowest at

-80 °C (Bryan Stuart, pers. comm.).

Dimethyl Sulfoxide (DMSO)

DMSO is commonly used in aqueous solutions to pre¬

serve DNA as it readily permeates tissues and enhanc¬

es absorption of materials that inhibit nucleases (e.g.,

EDTA, NaCl). In addition, like glycerol, DMSO prevents

cellular damage from formation of ice crystals, making it

an effective cryoprotectant. A number of solutions with

20-25% DMSO, 0.25 M disodium-EDTA, and salt to

saturation have been shown to be effective (Dawson et

al. 1998; Kilpatrick 2002; Seutin et al. 1991). Kilpatrick

(2002) found that a 3:1 DMSO-salt solution provided the

best protection from DNA degradation of mammalian

liver tissues stored for up to two years when compared to

95% ethanol and lysis buffer. Nagy (2010) recommended

that the ratio between DMSO and sample exceed 5:1 but

at the very least be 3:1 for effective preservation. These

cost-efficient solutions can be easily made in the labo¬

ratory, are associated with only minor health concerns

(e.g., skin irritation), and can be shipped without restric¬

tions (Kilpatrick 2002; Nagy 2010).

Although DMSO-salt solutions are an effective pres¬

ervation method, there are a number of drawbacks. One

major issue is that these solutions preserve DNA and not

RNA at room temperature. Although these solutions have

been well-tested with marine invertebrates and mam¬

mals, only anecdotal evidence seems to exist regarding

its effectiveness in the preservation of amphibian tissues.

In addition, there have been no long-term studies to test

effects on tissue and DNA quality over periods of time

relevant to museum collections. For those working with

archival samples, tissue can become encrusted with salt,

making it more difiicult to sub-sample. In addition, it

may be toxic at high levels to living cells.

Commercial Products for Ambient Storage

A number of proprietary products are available for

ambient temperature stabilization with increasingly

more products available every year (Muller et al. 2016).

RNAtoer® is commonly used by researchers depositing

their samples in genetic resource collections associated

with natural history museums (Zimkus and Ford 2014).

PAAAlater is an aqueous, nontoxic tissue storage reagent

marketed to preserve RNAupto one day at37 °C, up to one

week at 25 °C, up to one month at 4 °C, and indefinitely

at temperatures of -20 °C or below. According to both the

manufacturer and published studies, PAAAlater has been

tested and found to be successful in preserving many

animal tissues (e.g., brain, heart, kidney, spleen, liver,

testis, skeletal muscle, fat, lung, and thymus; Nagy 2010;

Camacho-Sanchez 2013). According to the manufacturer,

this product is not recommended for bone because of the

lack of sufficient penetration into the tissue. In addition,

use of PAAAlater to preserve RNA in blood and plasma

have more involved procedures.

PAAAlater should only be used with fresh tissue

and requires that samples be cut into small pieces (i.e.,

less than 0.5 cm in one dimension) and placed in 5-10

volumes of the solution. Previously frozen tissues thaw

too slowly in PA^Alater, preventing the reagent from

diffusing into the tissues quickly enough to prevent

nucleic acid degradation. It is recommended that tissues

are incubated overnight at 4 °C to allow thorough

penetration; however, if ambient temperature is above 25

°C, it is suggested that samples are placed on ice for a

few hours after being placed in PAAAlater before storing

at ambient temperature.

For researchers making initial collections, there are

a number of strengths associated with this product, in¬

cluding this single solution is able to stabilize and protect

both RNA and DNA at ambient temperature. In addition,

PAAAlater is not considered hazardous for shipping, mak¬

ing it easy to transport to and from field collection sites.

This product does have a number of limitations that may

make it difficult to use for researchers at remote sites,

in areas where the ambient temperature is above 25 °C

(unless refrigeration is available and cold-chain can be

maintained during transport), and long duration trips.

This product is considered expensive (e.g., $348 US/250

ml), but researchers have devised homemade versions

that may be more cost-effective, although their efiicacy

is yet untested (Nagy 2010).

If the product is used according to the manufacturer’s

recommendations, samples initially preserved in

PASAlater and stored in biobanks or for archival purposes

can be used to extract both RNA and DNA. In addition,

samples can be thawed at room temperature and refrozen

without significantly affecting the amount or integrity

of recoverable RNA or DNA. It is recommended that

samples be removed from the solution before long¬

term storage because the liquid requires substantial time

to thaw and expands upon freezing, so overfilled vials

may crack or explode. There have yet been no long-term

studies to test effects on tissue and sample quality over

periods of time relevant to museum collections (i.e.,

decades).

AllProtect® has also been used to preserve samples

deposited in genetic resource collections, although less

frequently compared to PAAAlater (Zimkus and Ford

2014b). This gel-like tissue storage reagent preserves

DNA, RNA, and proteins for up to one day at 37 °C, at

room temperature (15-25 °C) for seven days, 2-8 °C for

up to six months, or indefinitely below -20 °C. The reagent

is provided with a pump that dispenses approximately

0.5 ml at a time; other methods of aliquoting may be

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Zimkus et al.

difficult because of the viscosity of the reagent. Fewer

studies have been completed compared to RNAtocr,

but AllProtect is recommended by the manufacturer

for most animal tissues, exeluding bone because of

laek of penetration, and it is not suitable for stabilizing

cultured cells, whole blood, plasma, or serum. Similar to

RNAtoer, fresh tissues (not previously frozen) must be

eut into small pieces (less than 0.5 em in one dimension),

and samples must be plaeed in at least 10 volumes of

solution.

The strengths of AllProtect are associated with the

fact that it allows preservation of multiple biomoleeules

at ambient temperature and is not eonsidered hazardous

when shipping. The reagent is viscous and maybe more

difficult to use when compared to Mater. AllProtect

is eonsidered very expensive (e.g., $645 US/100 ml), al¬

most five times more expensive when eompared by vol¬

ume to PAAAlater, which is already cost-prohibitive for

some researchers. In addition, the reagent is only stable

for six months after the produet is open, so it would need

to be purchased eaeh year for annual fieldwork. The

manufacturer reports that RNA remains intact up to 15

freeze-thaw cycles, while proteins remain intact for five

freeze-thaw cyeles, which is beneficial for biobankers

who may be sub-sampling a specimen numerous times

for different requests. The manufacturer does recom¬

mend that excess product is removed from the sample

surface (e.g., dabbing, rolling over paper towel) before

long-term storage, which may require substantial time

for biobankers. In addition, no long-term studies have

tested the effects on tissue and DNA quality over periods

of time relevant to museum collections.

Best Practices for Gamete Preservation for

Use in ARTs

Gonadotropic Hormones

ARTs for amphibians are based on the use of gonado-

tropie hormones (e.g., hCG, synthetie analogs of lutein¬

izing hormone releasing hormone [LHRH]) to trigger

spawning and the maturation of gametes for collection,

cryopreservation, and potential future use in artificial

fertilization (Ananjeva et al. 2017; Norris and Lopez

2011). Gonadotropic hormones are injected to stimulate

natural reproductive and spawning behavior in amphib¬

ians and are most often used outside of the natural breed¬

ing cyele (Goncharov et al. 1989). Experimentally it has

been shown in frogs and salamanders that these general

protocols induce reproductive behaviors (e.g., amplexus,

deposition of eggs) in amphibians (Kouba et al. 2009;

Kouba and Vanee 2013; Vu and Trudeau 2016). A current

review of Australian frogs reports that usage of gonado¬

tropic hormones (single dose of gonadotropic-releasing

hormone [GnRH] or hCG) is effeetive in Myobatraehi-

dae and Limnodynastidae for induetion of spermiation

and ovulation (Clulow et al. 2018a) but remains much

more problematic for the Pelodryadidae group of speeies.

Hormones are used to obtain mature gametes for imme¬

diate, postponed (i.e., days), or suspended (i.e., months,

years) artifieial fertilization (Ananjeva et al. 2017).

Frog Sperm Collection and Preservation

Anuran sperm from many speeies has been shown to re¬

main viable (defined by motility or membrane integrity)

when refrigerated from days to weeks. Ultimately, the

production of offspring helps to maintain conservation

breeding populations in captivity, bolster natural popula¬

tions, and reintroduce populations into areas where it has

been extirpated. If there are no immediate plans to breed

frogs, cryopreserving sperm allows for future attempts at

species propagation using ARTs, eolleeting these valu¬

able resources while they still exist in nature and are rela¬

tively easy to collect, archiving in biobanks and prioritiz¬

ing endangered and threatened speeies and speeies from

highly threatened and endangered habitats.

Sperm can be sampled from deceased frogs by mae-

erating excised testes and using physiological solution to

prevent activation. Sperm motility depends on solution

osmolarity with initial aetivation occurring in hypotonic

solutions below 250 mOsmol/kg with respeet to blood

plasma; most sperm are activated between 100-50 mOs-

mol/kg, and total activation occurs in solutions with os-

molarities of approximately 50 mOsmol/kg (Ananjeva

et al. 2017). Testieular sperm ean be eryopreserved and

used at a later date using sperm collected by maceration

of testes or by the more recent and non-lethal method

of hormonally induced sperm (HIS) eollected in urine

(Browne et al. 1998; Shishova et al. 2011; Uteshev et al.

2013). Physiological modifications to be considered for

amphibian sperm cryopreservation are related to extracel¬

lular osmolarity variation, effeet of egg jelly eomponents

on sperm physiology, extraeellular environment, role of

calcium and bicarbonate in sperm physiology, and physi¬

ological changes after spermiation (Krapf et al. 2011). A

recent study has reported on the effeet of extraeellular

conditions (i.e., exposure to water, differing tempera¬

tures) on sperm motility and structural properties (i.e.,

morphology, DNA integrity) collected from hormonally

stimvXdXQd Atelopus zeteki (Della Togna et al. 2018). The

study found that sperm longevity and its DNA integrity

depends on the hypo-osmolality of the environment but

not the temperature or hormonal stimulation method.

Spermatozoa can be preserved via the refrigeration of

whole carcasses at 4 °C for later (up to seven days) post¬

mortem collection of testicular sperm (Shishova et al.

2013). Viability has been demonstrated via the produc¬

tion of embryos, although the length of sperm viability

appears to be speeies-dependent. Testicular sperm gath¬

ered from carcasses of Rana temporaria refrigerated at 4

°C could produce viable offspring via in vitro fertiliza¬

tion (IVF) for up to 6 days, while approximately 90% of

sperm from carcasses of Bufo baxteri lost motility after

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Preservation of amphibian genetic resources and living tissues

Table 2. Recovery of viable previously frozen sperm from specific anuran and urodelan families and successful IVF using previously

frozen sperm. NOTE; Asterisk (*) denotes short-term refrigeration storage of spermatozoa at +4 °C, rather than cryopreservation.

Order Family

Species

Recovery of viable

frozeu-thawed sperm

Successful IVF using

frozeu-thawed sperm

ANURA Bufonidae

Anaxyrus (Bufo)

americanus

Beesleyetal. 1998

Bufo (Rhinella) marinus

Browne et al. 1998; Proano

and Perez 2017

Browne et al. 1998

B. bufo

Kaurova et al. 2008

Hylidae

Litoria peronii

Browne et al. 2002a

L. brevipalmata

Browne et al. 2002a

L. fallax

Browne et al. 2002a; Upton

et al. 2018

Upton et al. 2018

L. nasuta

Browne et al. 2002a

L. latopalmata

Browne et al. 2002a

L. dentate

Browne et al. 2002a

L. phyllochroa

Browne et al. 2002a

L. lesueuri

Browne et al. 2002a

L. subglandulosa

Browne et al. 2002a

Feptodactylidae

Eleutherodactylus coqui

Michael and Jones 2004

Myobatrachidae

Limnodynastis peronii

Browne et al. 2002a

Crinia signifera

Browne et al. 2002a

Philoria sp.

Browne et al. 2002a

Pipidae

Xenopus laevis

Sargent and Mohun 2005;

Mansour et al. 2009;

Pearl et al. 2017

Sargent and Mohun

2005; Mansour et al.

2009; Pearl et al. 2017

X. (Silurana)

tropicalis

Sargent and Mohun 2005;

Pearl et al. 2017

Sargent and Mohun

2005; Pearl et al. 2017

Ranidae

Rana temporaria

Kaurova et al. 1996;

Kaurova etal. 1997;

Mansour et al. 2010;

Shishova et al. 2011

Kaurova etal. 1996;

Kaurova etal. 1997;

Mansour et al. 2010;

Shishova et al. 2011

R. sylvatica

Mugnano et al. 1998;

Beesleyetal. 1998

R. pipiens

Beesleyetal. 1998

Pelophylax lessonae

Uteshev et al. 2013

URODEFA Cryptobranchidae

Andrias davidianus

Peng et al. 2011

Cryptobranchus

alJeganiensis bishopi

Unger et al. 2013

C. alJeganiensis

alleganiensis

Nashville Zoo Hellbender

Conservation 2018

Nashville Zoo

Hellbender

Conservation 2018

Ambystomatidae

Ambystoma tigrinum

Marcec et al. 2014;

Marcec 2016

A. mexicanum

Figel 2013

Salamandridae

Pleurodeles waltl

Uteshev et al. 2015*

36 hours, and no motility was present in sperm ofAndrias

japonicas after only two days (Roth and Obringer 2003).

Healthy and reproductive adults that have produced off¬

spring has been achieved via cryopreservation of sperm

(Pearl et al. 2017; Upton et al. 2018; Table 2). Three

primary factors affect the success of amphibian sperm

cryopreservation, including: 1) cryoprotectants used, 2)

Amphib. Reptile Conserv.

sampling and acclimation of sperm to cryoprotectants,

and 3) freezing rates (Browne and Figiel 2011). Specific

published protocols and references for cryopreservation

of sperm and IVF of 28 species and subspecies are listed

in Table 2.

Spermic urine or urinal sperm, the cloacal urine

containing a suspension of seminal plasma and mature

11 December 2018 | Volume 12 | Number 2 | el65

Zimkus et al.

Fig. 2. Procedures used to obtain amphibian eggs or sperm for use in ARTs. A) Gravid female Leopard Frog (Lithobates sp.)

after gonadotropic hormone injection. B) Expressing eggs into container by pressing on abdomen and pushing thumb toward cloaca;

eggs can be fertilized (i.e., IVF) by fresh or cryopreserved sperm. Sperm can similarly be released from males by pushing towards

the cloaca and releasing sperm naturally (in season) or after injection of gonadotropic hormones (e.g., HIS).

sperm, can be a source of gametes that eliminates the

need to sacrifice live frogs. Spermic urine can be induced

from males through the intraperitoneal administration

of 50 micrograms of Luteinizing Hormone-Releasing

Hormone analog (LHRHa) and manual massage of the

area between the bladder and cloaca to induce urination

(Fig. 2 demonstrates similar technique for manual release

of eggs; Ananjeva et al. 2017; Kouba et al. 2012, 2013;

Shishova et al. 2011). Cryopreservation of hormonally-

induced sperm have been successful; concentrations of

200 X 106/mL are mixed in a 1:1 ratio with cryodiluents

(e.g., glycerol, DMSO, sucrose) to form cryosuspensions

with concentrations of 15 x 106/mL of HIS to achieve the

highest fertilization percentage (Shishova et al. 2011).

More recently, sperm has been collected from hormon¬

ally stimulated Atelopus zeteki by Della Togna et al.

(2018) following intraperitoneal injection of gonadotro¬

pin-releasing hormone (GnRH) agonist (4 mg/g of body

weight), hCG (10 lU/gbw), or Amphiplex™ (10 mg/gbw

metoclopramide hydrochloride; 0.4 mg/gbw).

Salamander Sperm Collection and Preservation

The majority of salamander species have internal fertil¬

ization with males laying spermatophores. Sperm has

been successfully collected from salamanders after sac¬

rificing males and isolating the ducti deferens of Pleuro-

deles waltlii, P poireti, and Cynops pyrrhogaster (Jaylet

and Ferrier 1978; Watanabe et al. 2003). Spermatophores

have also been collected from an internally fertilizing

salamander, Ambystoma mexicanum, using two cryodi¬

luents: 10% sucrose solution and Simplified Amphibian

Ringers (SAR); SAR proved better at recovering more

active sperm (Figiel Jr 2013). Urinal sperm has also been

isolated as a suspension of spermatozoa for use in ARTs

(Mansour et al. 2011; Uteshev et al. 2015). In vivo meth¬

ods of obtaining sperm have been developed for Ambys¬

toma mexicanum, Andrias davidianus, Cryptobranchus

a. alleganiensis, and Pleurodeles waltlii (Browne and

Figiel 2011; Mansour et al. 2011; Uteshev et al. 2015).

Cryopreservation of the sperm of Ambystoma tigrinum

has also been successful with subsequent fertilization

achieved via IVF, although no embryos survived passed

the neurula stage (Table 2; Marcec et al. 2014; Marcec

2016).

Caecilian Sperm Collection and Preservation

Caecilians are the only order of amphibians that use

internal insemination. Sperm has been obtained from

Uraeotyphlus narayani by removing lobes of the testis,

washing them thoroughly in amphibian physiological sa¬

line solution (pH 7.4), and macerating them (George et

al. 2005). This study demonstrated that sperm are motile

when released from the testis, not requiring post-testic-

ular physiological maturation. In addition, the secretory

material of the Mullerian gland contributes to enhancing

the speed and duration of motility of the spermatozoa.

Although no known cryopreservation studies exist for

this group, future development of protocols is encour¬

aged, especially for threatened species.

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 65

Preservation of amphibian genetic resources and living tissues

Oocyte/Embryo Collection and Preservation

The collection of oocytes from live frogs can be achieved

by natural collection via spawning or via artificial ma¬

nipulation by stripping, excision from the ovaries, or

through the use of hormones to stimulate release (Kouba

et al. 2012). Natural collection through spawning can be

successful but may require that animals are collected in

the field during specific periods of the reproductive cycle

or housed for periods of time until natural spawning oc¬

curs (Figiel Jr 2013; Fitch 1970). Manually extruding oo¬

cytes by pushing on the abdomen towards the cloaca with

the fingers can greatly decrease experimental procedural

time (Fig. 2). Amplexing males may fertilize oocytes that

have spawned spontaneously, or IVF can be completed.

The size of the spawning enclosure (e.g., plastic box) and

depth of the simplified amphibian Ringer’s (SAR) de¬

pends on the general size of the species and if artificially

or naturally fertilized oocytes will require more solution

to enable IVF (Browne and Zippel 2007). Newer meth¬

ods for obtaining oocytes in vivo include induction of

females using gonadotropic hormones and use of a glass

rod to extended the cloacal sphincter, allowing release of

oocytes (Kouba et al. 2012, 2013). Eighteen species of

frogs, including thirteen genera, and two salamander spe¬

cies of two different genera, have successfully oviposited

after hormone induction to date (Calatayud et al. 2017).

For a review of attempts to induce oviposition using hGH

in anuran (n = 21, including Mixophyes fasciolatus) and

urodela (n = 6) species see Clulow et al. (2012). Manually

stripping ooctyes through palpation may be possible for

some species if females ovulate but do not spawn (Fig.

2; Whitaker 2001). Although effective in some groups

(i.e., ranids), manually stripping oocytes is generally not

suitable for those species with egg masses produced as

paired strings (e.g., toads; Browne and Figiel 2011).

Stimulated release of anuran and urodelan ooctyes

is commonly achieved using hCG, LHRHa, or GnRH

(Ananjeva et al. 2017; Clulow et al. 2018; Uteshev et

al. 2015). For those groups for which current protocols

have not yet been successful, an improved suite of tools,

including access to pure or recombinant endogenous go¬

nadotropins, are likely needed (Clulow et al. 2018). Al¬

though pituitary gland suspension is used commercially

with common species to stimulate oogensis, it is no lon¬

ger recommended for use with threatened species since

pituitary tissue may transmit pathogens (Ananjeva et al.

2017).

The collection of oocytes post-mortem for use in IVF

was first reported by Dabagyan and Sleptsova (1975) for

anurans and by Bordzilovskaya and Dettlaf (1975) for

salamanders with mature oocytes being excised from the

lower part of the oviduct after ovulation (Ananjeva et al.

2017). See Dettlaff and Vassetzky (1991), Dabagyan and

Sleptsova (1991), and Bordzilovskaya and Dettlaf (1991)

for English translations. A recent study using Rana tem-

poraria has shown that oviductal oocytes can be stored

for up to five days in carcasses refrigerated at 4 °C or

when isolated from the oviduct, leading to 70% normal

development (Ananjeva et al. 2017; Uteshev et al. 2018).

The refrigeration of oocytes can be a simple but critical

technique that allows gamete transport when collected

from field populations or between institutions involved

in ARTS. In addition, the period that oocytes remain fer¬

tile can be increased by lowering temperature to reduce

metabolism or increasing osmolarity to slow oocyte gel

coat hydration (Browne et al. 2001).

The cryopreservation of neither mature oocytes nor

embryos has yet been achieved. Oocytes are likely diffi¬

cult to cryopreserve due to their high cellular fat content,

size, shape, and low permeability characteristics lead¬

ing to cell damage during freezing (Eawson et al. 2013).

Although oocytes have not yet been cryopreserved for

future use, they can be collected for use in ARTs, and

can remain viable and fertilized up to as long as 30 days

(Uteshev et al. 2018). A number of techniques appear

promising in lieu of developing a method for cryopre¬

serving oocytes or embryos, including cryopreservation

of blastomeres used in conjunction with somatic cell

nuclear transfer (SCNT) and androgenesis with frozen

spermatozoa (Clulow and Clulow 2016). A recent dis¬

covery that coral larvae can be cryopreserved through

vitrification and thawed to resume swimming after laser

warming provides great promise that breakthroughs in

other taxonomic groups may lead to the successful cryo¬

preservation of amphibian oocytes and embryos (Daly et

al. 2018).

In Vitro Fertilization (IVF)

The terms artificial fertilization (AF) and IVF are both

used in the literature and by amphibian reproductive bi¬

ologists to denote the artificial insemination of eggs in a

Petri dish. Kouba and Vance (2009) suggested that IVF

is more appropriate for salamanders and caecilians given

they exhibit internal fertilization, and AF is a more ap¬

propriate term for anurans where external fertilization

is more common. For clarity, we have chosen to use

the term IVF to denote when fertilization is performed

manually by a researcher. IVF of frog oocytes have been

performed for decades in experimental embryology

(Rugh 1962; Dabagyan and Sleptsova 1975). The general

procedure, which can be used for all anuran species, in¬

volves placing 20-50 oocytes with 200-500 pi of sperm

(urinal or testicular) in a Petri dish. The fertilized eggs

are washed with fresh water and left to develop; success¬

ful fertilization is identified via cleavage approximately

3-8 hours later, although species may vary in develop¬

mental timing and rates (Ananjeva et al. 2017).

Although most salamanders have internal fertiliza¬

tion, IVF has been successful for some species. Gametes

obtained post mortem from Ambystoma mexicanum were

first used in IVF of salamander oocytes (Brunst 1955).

More recently, Mansour et al. (2011) fertilized oocytes

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Zimkus et al.

using gametes sampled from the same species in vivo

with spermatozoa from semen obtained through hor¬

monal induction and abdominal massage. In 2012, the

Nashville Zoo in the U.S. announced the first successful

captive breeding of Eastern Hellbenders {Cryptobran-

chus a. alleganiensis) from eggs produced and artificially

fertilized from captive zoo animals, and later in 2015, the

zoo was successful in hatching a salamander from an egg

that was artificially fertilized with cryopreserved sperm

(Nashville Zoo Hellbender Conservation 2018).

Tissue Preservation for Cell Lines

Viable fibroblast cell lines are one of the most versatile

genetic resources and can play an important role in ex

situ conservation. Cell lines banked in LN 2 can be main¬

tained indefinitely and provide a continual source of ge¬

netic material for a wide variety of purposes (Ryder and

Onuma 2018). These cells can be utilized to obtain chro¬

mosomes, expanded to generate large quantities of DNA/

RNA, and used for SCNT because they are living and

dividing (Houck et al. 2017). Induced pluripotent stem

cells (iPSCs) capable of differentiation into multiple cell

types have been generated from skin cells of mammals,

including humans, mice, and rhesus monkeys, by direct

molecular reprogramming (see Houck et al. 2017 for re¬

view). More recently these methods were successfully

applied to cryopreserved adult fibroblasts of endangered

mammal species, including a primate {Mandrillus leu-

cophaeus) and the northern white rhinoceros (Ceratoth-

erium simum cottoni; Ben-Nun et al. 2011; Korody et al.

2017). Preserving amphibian genetic material as fibro¬

blast cells while populations are still available will allow

the greatest number of options for future genetic rescue

when methods have been adapted for non-mammalian

species. Once a population is reduced to a critically small

number of individuals the feasibility of successfully es¬

tablishing a significant number of cell lines diminishes.

Post-mortem tissue and organ samples should be col¬

lected while they are still viable (not necrotic or frozen),

and, at a minimum, cryopreserved using DMSO so that

fibroblast cell lines can be established and cryopreserved

in genetic resource collections at a later date. Freezing

tissue biopsy samples for later initiation of cell culture

(i.e., “tissue piecing,” Fig. 3) is described in Houck et al.

(1995), Gamble (2014), and Houck et al. (2017, protocol

24.11); herein we summarize this method. Tissue is col¬

lected in vials containing cell culture media with antibi¬

otics and held at 4 °C (or room temperature if refrigera¬

tion is not available); ideally tissues are stored in media

for less than three days but can potentially be stored up to

10 days if no contamination occurs. Under aseptic condi¬

tions tissue is then minced into one mm^ fragments and

placed in cell culture medium containing 10% DMSO

and either transferred to a primed LN 2 dry shipper for

short-term storage during transport or placed directly

into a long-term LN 2 storage. Tissue prepared this way

Fig. 3. The “tissue piecing” protocol used to preserve viable

cells for establishment of cell lines in the future. A) Tissue is cut

into long, thin strips. B) Tissue is diced into 1 mm^ fragments before

adding medium containing 10% DMSO as a cryoprotectant. C)

Prepared tissue is stored in LN 2 until future cell culture is possible;

those without cell culture capability can transport samples using a

dry shipper to maintain cold-chain.

and kept in LN 2 can be stored indefinitely and later trans¬

ported to a lab with experience in tissue culture to estab-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 65

Preservation of amphibian genetic resources and living tissues

Freeze Time

CS"

Ll_

0 56 112 168 224 280 336 392 448 504 560 616

Days

Fig. 4. Length of time from cell culture initiation to freezing for amphibian cell lines in San Diego Zoo’s Frozen Zoo®.

Low = 19 days; high = 596 days; average = 154 days.

lish cell lines. The preferred amphibian tissues to collect

for this method (in order) include: whole limb (i.e., foot),

tongue, skin, and gonads. Other tissues that have been suc¬

cessfully used to establish amphibian cell lines include:

eye, tail (juveniles), whole tadpoles, and kidney.

Challenges Associated with Amphibian Cell Culture

A number of challenges are associated with the establish¬

ment of amphibian cell lines, demonstrated by the fact

that few biobanks contain cell lines, and most of these

hold mammalian, avian, and reptilian cells. Mammalian

cell culture is generally successful following the many

well-described methods, including Freshney (2005) and

Masters (2003), but establishing cells from amphibian

tissue has proven to be more challenging. Although there

are many reports for methods of amphibian cell culture

(see Okumoto 2001 for summary), there are very few

known cryopreserved viable amphibian cell lines in col¬

lections. A literature search yields only a few papers that

describe methods used to establish cell lines. The Ameri¬

can Type Culture Collection (ATCC), a biological ma¬

terials resource and standards organization, has limited

amphibian cell lines (American Type Culture Collection

2017). The largest known collection of amphibian cell

lines is curated at the San Diego Zoo’s Frozen Zoo®

(Chemnick et al. 2009; San Diego Zoo Institute for Con¬

servation Research: Frozen Zoo® 2018), currently con¬

taining 95 cell lines from 83 individuals and 21 species.

Some of the important parameters that were identified for

the success of cell lines established in the Frozen Zoo®

collection include: media similar to that used for mam¬

malian cells, a low oxygen environment, incubation tem¬

peratures of 20-23 °C (for taxa from cool climates) or

27-30 °C (for tropical species), and use of the explant

method instead of enzymatic digestion (Houck et al.

2017; Houck, unpubl. data).

Contamination is a greater challenge in amphibians

than in other groups, such as mammals, in part because

many amphibians dwell in moist environments. Using

antibiotics and antimycotics, such as penicillin, strepto¬

mycin, gentamicin, normocin, and fungizone (ampho¬

tericin B), is a crucial part of sample collection and tis¬

sue culture in this group. Even with use of these widely

effective antibiotics, one of the most common causes

of failure in amphibian cell culture is contamination.

Keeping wild-caught individuals in captivity for several

weeks may also reduce contamination (Tony Gamble,

pers. comm). The other common failure associated with

amphibian cell culture is absence of cell growth. This can

be attributed to poor sample quality (i.e., few viable cells

to begin with), and sub-optimal growth conditions, such

as temperature and media. Optimal conditions vary by

species (as was noted for sperm collection/preservation

previously), and for most species these conditions are not

yet known. One of us (MH) has found that methods and

conditions that are successful for some species grown in

the tissue culture lab can fail to work on other species,

sometimes even those in the same genus.

Methods for amphibian cell culture have not yet been

fully optimized, and the process takes an average of more

than 150 days in culture before a sufficient number of

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 65

Zimkus et al.

cells can be frozen (Fig. 4). This period of time is sig¬

nificantly longer when compared to an average of 21-28

days for mammalian cells (data from San Diego Zoo tis¬

sue culture lab). Cells that are in culture for long periods

of time are prone to chromosomal changes, and this has

been noted in long-term culture of amphibian cells where

the modal diploid number is observed to differ from ex¬

pected diploid numbers based on chromosome numbers

derived from short-term cultures, such as blood or bone

marrow (Okumoto 2001; Houck, unpubl. data). Although

there is a need for further improvements in amphibian

cell culture methods, recent advances have led to the

addition of over 80 cell lines to the Frozen Zoo® and

suggest that widespread success across the community is

possible. Researchers working with these methods will

likely be able to enhance the protocols further, leading to

shorter culture times and broader application to other am¬

phibian taxa. Until these issues are resolved, freezing tis¬

sues with DMSO using the “tissue piecing” method pre¬

viously described is recommended for those who have

access to post-mortem amphibians (Fig. 3; Houck et al.

1995; Gamble 2014; Houck 2017, protocol 24.11). This

procedure allows researchers to preserve samples for

future initiation of cell culture, which provides time for

the improvement of methods, safeguards valuable or rare

samples until methods are more successful for specific

species, and allows those with experience in cell culture

(possibly at a collaborating institution) to propagate the

cell lines.

Captive Breeding Programs

Captive breeding is an important aspect of amphibian

conservation as it ensures the survival of species that

cannot be safeguarded in their natural habitat. It is also

often the only way to collect oocytes of specific species

since they cannot currently be cryopreserved with current

methods. The Amphibian Ark (AArk) focuses on ex situ

programs for species that cannot currently be safeguard¬

ed in the wild, and their survival is dependent on conser¬

vation breeding programs (Amphibian Ark: Establishing

Ex Amphibian Programs 2018). AArk is convinced

that two steps are vital to executing a successful ex situ

conservation program, in particular if release back into

the wild is required: 1) the program must be completed

entirely within the range country, and 2) the population

must be maintained, housed, and confined separate from

populations outside its range. Facilities located within

the species natural range that exclude non-native spe¬

cies are examples of best practice, thereby requiring the

smallest budget and least amount of effort to be success¬

ful (Pessier and Mendelson 2017). Conservation breed¬

ing programs or survival assurance colonies intending to

reestablish amphibians into their natural habitat should

manage animals in perpetual isolation (e.g., committed

buildings or rooms) separated from amphibians origi¬

nating outside of the species native range (Amphibian

Amphib. Reptile Conserv.

Ark: Establishing Ex Situ Amphibian Programs 2018).

Eong-term quarantine of amphibians is also required for

animals outside the natural range of species that will be

subsequently translocated or reintroduced (Pessier and

Mendelson 2017). Mixed collections or those “cosmo¬

politan” in nature (e.g., facilities housing animals from

multiple geographical) pose an increased risk of intro¬

ducing infectious diseases to natural populations in rein¬

troduction and translocation programs. As a result, these

housed animals may be exposed to diseases not already

exhibited in the controlled population and potentially

spread them to wild populations. The ideal situation and

lowest risk position for introducing pathogenic diseases

to native amphibian populations within reintroduction

programs is, therefore, when conservation assurance

colonies are positioned safely within the native country

of the amphibian species or species group, and the con¬

servation breeding facility maintains only species from

within the species native territory or country (Pessier and

Mendelson 2017). Eastly, the use of dedicated equipment

and tools, committed or single purpose footwear, per¬

sonal protective equipment (e.g., lab coat), and workfiow

methods that diminish risk of introducing non-native

pathogens into amphibian aggregations should be en¬

forced as a top priority.

Ex situ conservation efforts focused on amphibian

species can be hampered by inadequate or incomplete

knowledge of presumptive animal species. It is, there¬

fore, recommended that a phylogenetic analysis of the

wild species from its natural habitat be completed before

conservation breeding efforts begin to ensure that cryp¬

tic species do not remain unidentified (Yan et al. 2018).

Crawford et al. (2013) used DNA barcoding of the COI

and 16S genes to review mitochondrial diversity in cap¬

tive communities of ten species of amphibians from the

Neotropics managed as an ex situ assurance plan. Sub¬

stantial cryptic genetic variation was identified within

three of ten ex situ populations, and three other species

exhibited cryptic diversity in natural indigenous popula¬

tions but not in captive populations. DNA barcoding can

provide the first method to identify cryptic diversity, but

an intergrative taxonomic approach that uses data from

multiple sources, including molecular data, morphology,

ecology, and advertisement calls, should ultimately be

used for species delimitation (Vieites et al. 2009; Evans

et al. 2015).

The Future of Species Conservation

Integrating the current practices for the preservation of

amphibian genetic resources and living tissues will ul¬

timately aid in both basic research and the practice of

species conservation. We, therefore, have devised two

decision trees to allow researchers to determine which

type(s) of samples that they can preserve both for re¬

search and amphibian conservation efforts (Fig. 5, 6).

These two workflows are distinct, depending on whether

December 2018 | Volume 12 | Number 2 | e165

Preservation of amphibian genetic resources and living tissues

Cell Culture

Capability;

Trip Duration

3-7 days

Long-term storage

in LNj available;

specimens

euthanized in lab

Trip Duration

> 7 days; ambient

temperature may

exceed 25 T

Trip Duration

< 7 days; ambient

temp, not

exceeding 25 *C

YES

Collect tissue (e.g..,

limbj, tongue, gonad)

in biopsy transport

vial (keep cool,

do not freeze)

YES

Samples collected

remotely

YES

Dry shipper (LN^)

available

Cryo preserve

some tissue for

genetic study

Freeze some tissue

in mechanical

freezer

for genetic study

YES

Cryo preserve

some tissue for

genetic study

YES

Preserve some tissue

with

9S% EtOH/DMSO

solution until use or

frozen storage

YES

Preserve some tissue

with

RNAlater/All Protect

until use or

frozen storage

Trip Duration

YES

Maintain some

< 3 days (optimal

- >

tissue on dry ice

for time sensitive

(CO 2 ) until use or

cell culture)

frozen storage

Process some tissue

for cell culture;

freeze cells with

DMSO and maintain

in LN.,

Process some tissue

with DMSO using

'"tissue piecing"

protocol; maintain

until future cell

culture or ship to

lab with cell culture

capability

Refrigerate [4 *C)

male carcass (”'2-6

days); excise testes

for use in ART

Excise testes,

macerate and place

in cryoprotectant

(see Table 1) for

future use in ART

Process some fresh,

unpreserved tissue

with DMSO using

"tissue piecing"

protocol

(keep CO oh do not

freeze while in field);

maintain in LNj for

future cell culture or

ship to lab with cell

culture capability

Fig. 5. Decision tree used for specimens euthanized to obtain tissue. Blue indicates steps in the decision tree. Green indicates

procedures that will lead to preservation of tissues for genetic study. Purple indicates procedures that lead to achieving multiple

goals, including cell culture and obtaining gametes for current or future ART. NOTE; Breeding and artificial fertilization can result

in offspring that can be used for genetic purposes, thereby achieving multiple goals.

animals will be euthanized (e.g., museum specimens) or

tissues are being collected from live animals. Research¬

ers can thereby determine whether they may be able to

additionally obtain and preserve gametes and/or cell

culture tissues for immediate use in ARTs or long-term

cryopreservation given the laboratory or field conditions

and available equipment. We believe that these decision

trees will allow researchers to more easily integrate cur¬

rent practices for the preservation of amphibian genetic

resources and living tissues. We also hope that these re¬

sources will aid in the development of best practices for

species conservation in assisted reproductive technolo¬

gies.

Researchers can maximize the downstream research

potential of the sample via their selection of preservation

method(s), ultimately allowing the broadest range of fu¬

ture uses in both basic research and species conservation.

Regardless of the study goals, researchers should care-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Zimkus et al.

Fig. 6. Decision tree used to obtain tissue from live animals. Blue indicates steps in the decision tree. Green indicates procedures

that will lead to preservation of tissues for genetic study. Purple indicates procedures that lead to achieving multiple goals, including

obtaining gametes for current or future ART. NOTE; Breeding and IVF can result in offspring that can be used for genetic purposes,

thereby achieving multiple goals.

fully weigh their choices and consider the conditions as¬

sociated with the collection of their genetic resources as

poor preservation can hinder genetic analyses and render

samples useless. Tissue sampling methods and standards

for vertebrate genomics have been proposed that include

four categories for classifying the utility of tissues and

DNA being prepared for Genome lOK (GlOK) and other

similar projects (Wong et al. 2012). The authors suggest

that researchers attempt to collect more high-quality

samples that include: sufficient fiash-frozen tissue or im¬

mediate extraction of DNA for a minimum of one mg

of DNA, multiple tissues for RNA sequencing and tran-

scriptome analysis, and viably frozen tissue pieces suit¬

able for establishing cell lines. If the standard collection

of genetic resources for molecular analyses includes the

collection of more high-quality samples, future use may

include species propagation.

The standardization of pre-analytical variables by

biobanks is also critical for understanding downstream

sample quality. The Standard PREanalytical Code

(SPREC) was developed to provide a comprehensive

and practical tool to document preanalytical (e.g., col¬

lection, processing, storage) biospecimen data (Eehmann

et al. 2012). Biospecimen Reporting for Improved Study

Quality (BRISQ) are additional standards that have been

proposed for information that should be reported about

biospecimens in scientific publications and regulatory

submissions (Moore et al. 2011). Application of qual¬

ity management systems (e.g., SPREC, BRISQ) should

be applied to biobanks working with animal genetic re¬

sources, but they must first be reviewed and adapted to

ensure that they capture the broad range and diversity of

non-human samples (Benson et al. 2016).

WAZA recently approved a resolution calling for ac-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Preservation of amphibian genetic resources and living tissues

celerated efforts to protect biodiversity and aid species

conservation by establishing and biobanking viable cell

lines from tissue (Oliver Ryder, pers. comm.). If the col¬

lection and deposition of tissues preserved for eventual

cell culture can become routine practice among research¬

ers already preserving genetic material, particularly her¬

petologists working in the held, more cell lines may be

biobanked in the future to aid in this effort. Collecting

different tissue types or using specihc methods may not

be possible for those without the necessary equipment

(e.g., dry shippers) or associations with genetic resource

collections that can store the samples long-term; how¬

ever, it may require minimal change for others. Estab¬

lishing cell lines requires a high skill level and fairly

specialized labs, but freezing tissues with DMSO using

the “tissue piecing” method to allow the establishment of

cell lines in the future is feasible when amphibians are

already being euthanized for museum specimens or ge¬

netic analysis (Fig. 3; Houck et al. 1995; Gamble 2014;

Houck 2017, protocol 24.11). An increasing number of

biobanks associated with natural history collections are

already cryopreserving DNA and RNA samples, so the

addition of tissues processed using methods suitable for

future cell culture may not be arduous. The deposition

of amphibian tissue and cell lines as a standard practice

in research projects worldwide (e.g., academic, govern¬

ment, non-governmental agencies, industry) may also

facilitate future inter-institutional research and collabo¬

ration regarding cryopreservation and ARTs. Cell lines

could potentially be reprogrammed into stem cells in

the future, and their pluripotent nature could allow them

to differentiate into gametes, such as sperm and ova.

There is expanded potential for SCNT to create clones

beyond what has been achieved (Gurdon 1962; Branco

2015) and potentially further conservation efforts. Thus

far, fertile adults may be generated if donor nuclei are

obtained from early embryos (Gurdon 1962). Neither of

these methods (SCNT and developing cells lines as po¬

tential generation of gametes) are completely developed

and achievable for potential use in species conservation,

but they may only be attempted for genetic rescue when

it becomes possible if species are preserved either in the

wild or with biobanked cell lines.

Collaboration and Integration of Biobanks

Globally

A number of programs have been initiated to collect the

world’s amphibian species that are threatened with ex¬

tinction. The goal of a project spearheaded by the Am¬

phibian Survival Alliance and Amphibian Specialist

Group is to create a historically permanent record and

resource (publicly accessible in sustainable repositories)

of bioinformatics and tissue for amphibian species con¬

servation and research. To that end, two target areas have

been identihed: bioinformatics of amphibian genomes

and biodiversity preservation of tissues representing

all amphibian species. The latter aims to cryopreserve

tissues, develop cell lines, and promote ARTs for am¬

phibian species, particularly those in immediate danger

of going extinct and those found in highly endangered

habitats. Amphibia Bank is a collaborative effort that has

been proposed as a means to bring together cell culture

and tissue repositories and promote the collection of cell

samples (e.g., blood, cell cultures, tissues, and sperma¬

tozoa with the potential to include eggs and embryos in

the future; Lawson et al. 2013). This project aims to rep¬

resent every amphibian species on earth in participating

biobanks but will hrst focus on collecting threatened and

endangered species and representatives of every genus.

An initial pilot project for this effort involves the col¬

lection of all North American salamander species, which

was identihed as critical because of the spread of the

pathogenic chytrid fungus Bsal (Gray et al. 2015; Has-

sapakis and Clark 2017).

Regional and international research collaborations

between zoos/aquariums, natural history museums, and

other academic institutions (e.g., universities, colleges)

offer a unique opportunity to move amphibian research

and conservation forward. Although research collabora¬

tions may have existed between these institutions, in¬

cluding the accession of specimens originating from zoos

into natural history museums, modernization of these

relationships may include new partnerships between

biobanks. Natural history museum collections may hold

specimens that rehect historic distributions and former

variation in fragment populations, which may be useful

information for conservation programs at zoos/aquari-

ums. Zoos and aquariums are often focused on public

education, but they are unique in their capacity to de¬

velop and sustain long-term projects through fundraising

efforts and dedicated staff In addition, zoos may have

material that is poorly represented in museum collec¬

tions, including endangered species, which can be used

by natural history museums in research regarding aging,

anatomy, functional morphology, pathology, reproduc¬

tive biology, and taxonomy (Kitchener 1997). Formaliz¬

ing partnerships between zoos/aquariums, natural history

museums, universities, and other institutions, including

signing Memoranda of Understanding, would increase

and improve collaboration in areas of common interest.

Collaboration among academic institutions should

make efforts to include input and participation from ad¬

ditional stakeholders that make up the biobanking com¬

munity. One such proposal has been put forth to establish

a genome resource bank (GRB) for threatened Austra¬

lian amphibians (Mahony and Clulow 2005). The major

objectives of this GRB include: 1) captive husbandry to

prevent species extinction, 2) maintenance of genetic di¬

versity, 3) reduction of the number of individuals held

in captivity, thus extending resources for a more diverse

collection of species, and 4) selection for resistance to

Bd. Progress has also recently been made in the United

Kingdom (U.K.) with the CryoArks project, which was

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Zimkus et al.

funded by the Bioteehnology and Biologieal Sciences

Research Council (BBSRC) to increase access, organiza¬

tion, and species coverage in U.K. animal biobanks by

providing infrastructure and expertise (U.K. Research and

Innovation 2018). This biobanking project joins together

various stakeholders, including Cardiff University, the

Natural History Museum, National Museums Scotland,

Royal Zoological Society of Scotland’s Edinburgh Zoo

and Highland Wildlife Park, University of Nottingham,

and University of Edinburgh. CryoArks will also partner

with the Frozen Ark Project and the European Associa¬

tion of Zoos and Aquaria (EAZA) whose biobanks focus

on endangered species and zoo/aquarium animals, re¬

spectively. The first phase of the project will concentrate

on aggregating genetic resources from the collaborating

institutions to make the material discoverable and acces¬

sible, but future goals include the cryopreservation of

living cells (Jacqueline Mackenzie-Dodds, pers. comm.).

Worldwide efforts focused on amphibian biobanking

should target the most biodiverse countries (e.g., Brazil,

China, Colombia, Democratic Republic of the Congo,

Ecuador, India, Indonesia, Madagascar, Malaysia, Mex¬

ico, Papua New Guinea, Peru, Philippines, South Africa,

United States, and Venezuela). High priority should also

be given to threatened habitats (e.g., Madagascar, Bor¬

neo, Micronesia, Polynesia, Mediterranean Basin, Tropi¬

cal Andes) and animal groups with large numbers of

critically endangered species and unique species. Some

examples of specific groups to be targeted include the

genera Atelopus, Pseudophilautus, Craugastor, Litoria,

Mixophyes, Pristimantis, Plectrohyla, Rhinoderma, An-

drias, and Cryptobranchus, the Madagascan frog family

Mantellidae, and monotypic families such as Nasikaba-

trachus sahyadrensis (Biju and Bossuyt 2003). ARTs re¬

quire viable cells, thus the collection of “living tissue”

(e.g., gametes, cell lines, and other tissues) is needed for

future use. These collection efforts and technologies can

no longer be applied as small-scale, final attempts (Clu-

low et al. 2014). The collection and cryopreservation of

these living materials should be prioritized to include

threatened and endangered species, as well as those from

highly endangered habitats before they are no longer

available. In conjuction with these efforts, the technolo¬

gies needed for successful reproduction and the produc¬

tion of pleuripotent stem cells should be optimized.

Increasing capacity building and training, as well as

sharing existing knowledge and technologies with insti¬

tutions and scientists is essential to moving amphibian

conservation efforts forward (Kouba et al. 2013). The

continual improvement of best practices for bioresposi-

tories that incorporate practices and procedures specific

to non-human biological samples also will aid in the im¬

provement to the operation of biobanks associated with

institutions such as natural history museums and zoos

(Campbell et al. 2018; ISBER 2018). Symposia, work¬

shops, online resources, and increased funding can all

be used in education efforts, and the formation and in¬

teractions of research consortia aimed specifically at tis¬

sue collection, cryopreservation, and cataloging, allow

additional opportunities for discussion and collabora¬

tion. Engaging researchers and staff in continents that do

not yet have an active amphibian biobanking programs

(e.g., Africa, South America, Central America, Asia) by

establishing training opportunities will allow the trans¬

fer of knowledge and expertise needed to build stronger

in-country networks. These in-country networks can ulti¬

mately aid in coordinating field biologists that are already

documenting population declines and categorizing diver¬

sity to bank samples from animals in wild populations,

which will greatly assist in these amphibian conserva¬

tion efforts. The Global Genome Biodiversity Network

(GGBN) was created to fill the need for a network of bio¬

banks associated with natural history museums, herbaria,

botanical gardens and other stakeholders, establishing a

portal for locating samples that meet quality standards

for genome-scale applications (Seberg et al. 2016).

GGBN has formed task forces for important topics, such

as data standards, policies, and biobank procedures, and

brought together those working in both biobanks associ¬

ated with natural history collections and zoos at annual

meetings to compare and contrast methodologies. In ad¬

dition, a tissue preservation study has been initiated that

includes numerous GGBN member institutions to estab¬

lish a clearer perspective on some of the most commonly

used to preservative samples deposited in zoological and

veterinary biobanks. The goal of the study is to increase

standardization among animal biobanks and to enhance

suitability of samples for current and future downstream

analysis.

Conclusions

Biomaterials banked from amphibians are a vital re¬

source that can only be acquired and developed while

these resources exist, thus underpinning the importance

of their collection now for both present and future uses.

We expect that scientific advancements associated with

the preservation of tissues and ARTs will be made as

laboratory protocols and methodologies are more widely

used for amphibians. It is critical that researchers stay

up-to-date with new findings and apply best practices to

maximize the potential of the valuable genetic samples

that they collect. Technological developments associated

with the long-term storage of tissue samples may also

allow more institutions to build internal biobanks. These

new biobanks should integrate into larger networks to

aid in regional or global conservation efforts. Eastly, we

hope that cooperation and research partnerships among

stakeholders, as well as education and promotion within

the scientific community, will lead to scientific progress

in these areas to aid in amphibian conservation.

Acknowledgements. —^We thank Julie Strand for man¬

uscript feedback, as well as Julie Fronczek, Catherine Avila,

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Preservation of amphibian genetic resources and living tissues

Ann Misuraca, and Marisa Korody for information regard¬

ing cell culture and karyotyping. Many thanks to Katy

Thomson for assistance with Fig. 4. Thanks goes to Jaclyn

Zelko, Chester Figiel, Jr., William Wayman, and the U.S.

Fish and Wildlife Service Warm Springs Fish Technology

Center for their friendship and collaborative research oppor¬

tunities. A special thanks is extended to Terrence “Terry” R.

Tiersch and for his invaluable friendship, kindness, exper¬

tise, and expanding his world view to include amphibians.

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Breda M. Zimkus is the Cryogenics Collections Manager for Genetic Resources at the

Museum of Comparative Zoology, Harvard University, in Cambridge, Massachusetts, USA.

She received her B.A. from Boston University and Ph.D. from Harvard University. She is

interested in the biodiversity, biogeography, phylogenetics, and conservation of African

amphibians, and her research integrates a broad range of techniques to interpret patterns of

speciation and diversity, including fieldwork, taxonomy, and molecular systematics. She is a

member of a number of organizations working towards developing best practices associated

with genetic resources, including the Society of the Preservation of Natural History Collections

(SPNHC), Global Genome Biodiversity Network (GGBN), and International Society for

Biological and Environmental Repositories (ISBER).

Craig L. Hassapakis is the Founder, Editor, and Publisher of i\\Q']o\xmdX Amphibian & Reptile

Conservation (official journal website: amphibian-reptile-conservation.org), which was

founded in 1996, and a former editor of FrogLog {http://www.amphibians.org/froglog/). He

has been an instructor (first grade through college), non-profit and governmental volunteer

(Public Library of Science [PLoS]), Co-group Facilitator, Genome Resources Working Group,

lUCN/SSC Amphibian Specialist Group (ASG), and is a member of the lUCN/SSC Amphibian

Specialist Group. His interests include biodiversity, evolution, systematics, phylogenetics,

taxonomy, conservation, and behavior of amphibians and reptiles. He is instmmental in

developing and educating people to establish 'Amphibia Bank: A genome resource cryobank

and network for amphibian species worldwide.'’’’ His professional memberships include:

Society for the Study of Amphibian and Reptiles (SSAR), Herpetologists ’ League (HL), and

International Society for Biological and Environmental Repositories (ISBER).

Marlys L. Houck is the Curator of the Frozen Zoo® cell line collection at San Diego Zoo

Global’s Institute for Conservation Research, San Diego, California, USA. She received her B.A.

from California State University Fresno followed by certification as a Clinical Lab Specialist

in Cytogenetics. Professional memberships include: Association of Genetic Technologists

(AGT), Global Genome Biodiversity Network (GGBN), and International Society for Biological

and Environmental Repositories (ISBER). She is a specialist in cell culture and comparative

cytogenetics of critically endangered species including mammals, birds, reptiles, and amphibians.

She currently manages the Frozen Zoo® cell culture and karyotyping team; together they have

compiled one of the largest exotic species karyotype and cell line collections. As one of the few

research teams with this specialty, they share their methods and expertise with scientists around

the world.

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e165

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

12(2) [Special Section]: 28-36 (el66).

The critically endangered species Litoria spenceri

demonstrates subpopulation karyotype diversity

^’^’^Richard Mollard, ^Michael Mahony, "^Gerry Marantelli, and ^’^Matt West

'Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, 3052, Victoria, AUSTRALIA ^Amphicell Pty Ltd, Cairns,

Queensland, AUSTRALIA ^School of Environmental and Life Sciences, The University of Newcastle, 2308 New South Wales, AUSTRALIA "'The

Amphibian Research Centre, PO Box 1365, Pearcedale, Victoria, 3912, AUSTRALIA ^School of BioSciences, University of Melbourne, 3010

Victoria, A USTRALIA ^National Environmental Science Program, Threatened Species Recovery Hub, The University of Queensland, St Lucia, QLD

4072, AUSTRALIA

Abstract—Litoria spenceri is a critically endangered frog species found in several population clusters within

Victoria and New South Wales, Australia. Biobanking of cell cultures obtained from toe clippings of adults

originating from Southern, Northern and Central Site locations, as well as Northern x Central Site hybrid tadpole

crosses was performed. Analysis of biobanked cells demonstrates a 2n = 26 karyotype and chromosomal

morphology characteristic of the Litoria genus. A potential nucleolar organiser region (NOR) on chromosome 9

demonstrates similar designation to L. pearsoniana and L. phyllochroa of the same phylogenetic subgroup. A

second potential novel NOR was also located on the long arm of chromosome 11, and only within the Central

Site population. This Central Site apparent NOR is inheritable to Northern x Central Site tadpole hybrids in

the heterozygous state and appears to be associated with a metacentric to submetacentric morphological

transformation of the Northern Site inherited matched chromosome of that pair. This potential NOR represents

an important genetic marker for distinguishing subpopulations. These data demonstrate the importance of

prospectively establishing biobanks containing genetically characterized cells so that effective markers of

specific subpopulations can be identified and used to help increase the effectiveness of animal husbandry

programs.

Keywords. Frog, biobanking, cryopreservation, cell culture, toe clips, subpopulations, conservation management

Citation: Mollard R, Mahony M, Marantelli G, West M. 2018. The critically endangered species Litoria spenceri demonstrates subpopulation karyotype

diversity. Amphibian & Reptile Conservation 12(2) [Special Section]: 28-36 (el66).

NoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided

the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication

credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation; official journal website <amphibian-

reptile-conservation.org>.

Received: 24 October 2018; Accepted: 21 December 2018; Published: 28 December 2018

Introduction

The Spotted Tree Frog, Litoria spenceri (Spencer 1901),

is an lUCN Red List critically endangered amphibian

endemic to in Victoria and New South Wales, Australia

(Gillespie and Hollis 1996; Skerratt et al. 2016; Fig. 1).

Litoria spenceri is an obligate stream breeder and his¬

torically known to occur at sites between 300-1,100 m

elevation (Gillespie and Hollis 1996). The species has

been intensively studied, particularly since the early-mid

1990’s to: (i) evaluate possible changes in site occupancy

and population dynamics across the species range, and

(ii) identify factors linked to subpopulation declines.

Population decline studies indicate that L. spenceri has

disappeared from 50% of known historic sites, and is now

rare at all other sites (West 2015). Historically, mining

activities and other habitat disturbances have influenced

the viability of some subpopulations (Gillespie and Hol¬

lis 1996; Watson et al. 1991). Today, ongoing population

declines are documented to be driven by non-native fish

predation of tadpoles and infection by the fungus Ba-

trachochytrium dendrobatidis, that is impacting the sur¬

vival of terrestrial life stages (Gillespie et al. 2015; West

2015). Due to such factors, L. spenceri populations are

currently restricted to around 25 sites across nine streams

(West 2015).

Conservationists at the Amphibian Research Centre in

Australia have captured wild L. spenceri frogs from sev¬

eral sites to establish captive insurance breeding colonies

(e.g., Brannelly et al. 2017). Safeguarding populations

from all sites would be ideal because limited genetic

analyses suggest that L. spenceri populations may form

several distinct genetic clusters or evolutionary signifi¬

cant units (Gillespie and Robertson 1998). Distinct ge¬

netic traits may relate to a noted regional but not well

described phenotypic diversity within this species and.

Corr6Spond6nC6. ^ rmollard@unimelb.edu.au; mollard@amphicell.com

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 66

Mollard et al.

Fig. 1. Phenotypes of L. spenceri frogs and site location. (A) Adult frog from the South Site (1). (B) Adult frog from the North

Site (2). (C) A juvenile frog from the Central Site (3). (D) Site identification within the L. spenceri population range. N = north.

Phenotypes are only examples and not necessarily representative.

therefore, perhaps contribute to regional survival advan¬

tages. Establishing a biobank containing viable somatic

cells of all diverse subpopulations would permit genet¬

ic mapping of these traits, provide markers facilitating

conservation of distinct geographic subpopulations and

potentially provide material for assisted reproductive

technologies if specific populations were lost (ART; see

Clulow and Clulow 2016; Kouba et al. 2013; Mollard

2018 a,b; Zimkus et al. 2018).

Small distal toe tissue samples collected from sev¬

eral L. spenceri representative of three diverse popula¬

tion sites within the known population range: South¬

ern, Central and Northern Sites were processed for cell

culture and deposited under liquid nitrogen (see West

2015; Mollard 2018a, b). To demonstrate suitability of

these cells for future research, samples were thawed and

karyotyped. As L. spenceri karyotypes were previously

not described, inter-site as well as male and female in¬

tra-site population comparisons were made. These data

suggest strong conservation of karyotypic morphology

and chromosome 9 nucleolar organiser region (NOR)

between the closely related species L. pearsoniana and

Amphib. Reptile Conserv.

L. phyllochroa (see King et al. 1980). However, DAPI

(4’,6-diamidino-2-phenylindole) staining demonstrates

what appears to be a novel chromosome 11 NOR pres¬

ent only in the Central Site animals. Significantly, when

captive Northern Site and Central Site animals were in¬

terbred, the novel chromosome 11 NOR is inherited by

tadpole offspring in the heterozygous state. Further, the

matched chromosome from the Northern Site parent ap¬

pears to have undergone a metacentric to submetacen-

tric conversion. The study described here provides an

example of the essential nature of establishing biobanks

of cells, prospectively validated at least at the level of

karyotype, for effective species subpopulation manage¬

ment and safeguarding associated diversity.

Methods

Study Design

All tissue from adult and Juvenile frogs used within this

study was obtained from cultures of toe clippings depos¬

ited in the Amphicell Biobank (for previous examples,

December 2018 | Volume 12 | Number 2 | el 66

Litoria spenceri karyotypic diversity

see Mollard 2018a, b). Tadpole tissue was obtained

from cultures of macerates deposited in the Amphicell

Biobank. Clippings from Southern Site female and male

frogs were collected on-location at a designated mark-re¬

capture transect (Fig. 1). A toe clipping from a Northern

Site non-sexed adult was collected from an adult animal

taken from the wild and maintained in captivity for over

15 years (for reference to housing, see Brannelly et al.

2017). Juvenile Central Site derivative animals were bred

in captivity as above. The one tadpole was bred in captiv¬

ity from a Northern Site x Central Site mixed mating as

above. All tissues were collected in compliance with rel¬

evant State governmental and ethical licensing require¬

ments (for a summary, please see https://frogs.org.au/

arc/legal.html). The Code of Ethics of the World Medical

Association of The Declaration of Helsinki and the EU

Directive 2010/63/EU for animal experiments.

Karyotyping

Karyotyping was performed according to previously de¬

scribed methods (Mollard 2018a,b). Briefly, cryotubes

were removed from liquid nitrogen and quickly thawed

by rubbing between thumb and forefinger. Cryopreser-

vation media was diluted 10 fold in diluted and supple¬

mented DMEM (Dulbecco’s Modified Eagle’s Medium;

GIBCO) at room temperature up to a total volume of one

ml in single wells of a 24 well plate as previously de¬

scribed (Falcon Multiwell™, GIBCO; Ferris et al. 2010;

Mollard 2018a). Cells were passaged at approximately

70% confluence into two wells. Cells in both wells were

once again grown until approximately 70% confluence.

Cells from one well were returned to liquid nitrogen and

cells from the other well were processed for karyotyp¬

ing. Cells for karyotyping were treated with 0.1 pg/ml

KaryoMAX® colcemid (GIBCO) for approximately

eight hours. Cells thus arrested in metaphase were lifted

with 0.25% trypsin/0.02% EDTA, centrifuged, washed

with Amphibian Ringer’s solution (Coldspring Harbor),

centrifuged and suspended in one ml of 0.027M Na 3 Ci-

trate for 10 minutes. Cells were centrifuged at 125 g for

five minutes and the pellet was resuspended in methanol:

acetic acid (3:1), centrifuged, and washed a further two

times in methanol: acetic acid (3:1) prior to storage over¬

night at 4 °C. The next day, conventional drop-splash

technique was performed and cells were cover-slipped

with Gelvatol mounting medium (Cold Spring Harbor

Protocols) containing 1 pg/ml DAPI. Homologous chro¬

mosomes were paired and arranged according to size,

with the longest pair being designated as Chromosome 1

= 2n. Image J software with the Eevan plugin was used to

measure chromosome arm length and long arm to short

arm ratios of 1-1.69, 1.7-2.99 and 3-6.99, respectively,

were used to designate metacentric, submetacentric and

subtelocentric configurations (Eevan et al. 1964; Saka¬

moto and Zacaro 2009).

An Olympus BX60 microscope, colour CCD Eeica

DFC425C camera, and EE-6000 Eeica light source were

used for imaging at 1000 x magnification under oil im¬

mersion. Eeica EAS-AF software was used to capture

images.

Table 1. Measurements of L. spenceri chromosomal arm ratios and corresponding chromosomal morphology designations.

Chromosomes where both homologous chromosomes display a DAPI negative region are indicated with an *. Chromosomes where

only one matched chromosome of that pair displays a DAPI negative region are indicated with a #. Metacentric, submetacentric, and

subtelocentric chromosomal designation are defined as a long arm to short arm ratios of 1-1.69, 1.7-2.99, and 3-6.99, respectively.

Chromosome Number

Southern Site Female

1.69 ±0.1

metacentric

2.24 ±0,3

submetacentric

3,27 ±0,6

subtelocentric

1.52 ±0.2

metacentric

3,76 ±0.54

subtelocentric

2.40 ±0.8

submetacentric

2,16 ±0.3

submetacentric

Southern Site Male

1.50 ±0.2

metacentric

2.39 ±0.5

submetacentric

3.61 ±0.9

subtelocentric

1.58 ±0.3

metacentric

4.05 ± 1.1

subtelocentric

2.05 ±0.3

submetacentric

1.89 ± 0.3

submetacentric

Northern Site Adult

1.62 ±0.1

metacentric

2.11±0.2

submetacentric

3.79 ±0.5

subtelocentric

1.63 ±0.2

metacentric

3.60 ±0.3

subtelocentric

2.10 ±0.3

submetacentric

2.01 ±0.3

submetacentric

Central Site, juvenile 1

1.60 ±0.1

metacentric

1.98 ±0.4

submetacentric

3.17 ±0.9

subtelocentric

1.51 ±0.1

metacentric

3.05 ±0.4

subtelocentric

2.13 ±0.5 1

submetacentric

2.09 ±0.4

submetacentric

Central Site, juvenile 2

1.62 ±0.2

metacentric

l.S7±0.3

submetacentric

3.17 ±0.5

subtelocentric

1.35 ±0.2

metacentric

3.57 ±0.4

subtelocentric

2,02 ±0.3 1

submetacentric i

2.00 ±0.1

submetacentric

Northern Site x Central

Site cross

1.64 ±0.2

metacentric

1.79 ±0.1

submetacentric

3.26 ±0.5

subtelocentric

1.42 ±0.2

metacentric

3.44 ±0.4

subtelocentric

2,41 ±0.3 1

submetacentric

1.81 ±0.4

submetacentric

Chromosome Number

11 i

Southern Site Female

2.53 ±0.7

submetacentric

1.73 ±0.5

submetacentric *

1.5 ±0.1

metacentric

1.49 ±0.3 i

metacentric

1.28 ±0.6

metacentric

1.44 ±0.2

metacentric

Southern Site Male

2.92 ±0.6

xos + b.s

1.30 ± 0.2

1.41 ± 0.2

1.50 ±0.4

1.18 ±0.1

submetacentric

submetacentric *

metacentric

Northern Site Adult

2.65 ±0.7

submetacentric

2.22 ±0.3

submetacentric *

1.23 ±0.1

metacentric

1.24 ± b.i

metacentric

1.20 ±0.1

metacentric

1,57 ± 0.4

metacentric

Central Site, juvenile 1

2.43 ±0.6

ill ±0.4

1.65 ± 0.5

i56±b.5

1.60 ±0.3

1.57 ± 0.4

submetacentric

submetacentric *

metacentric

submetacentric * 1

metacentric

Central Site, juvenile 2

2.03 ±0.2

2.01 ±0.2

1.44 ± 0.3

2.12 ±0.4 1

1.66 ± 0.4

1.59 ±0.2

submetacentric

submetacentric *

metacentric

submetacentric * !

metacentric

Northern Site x Central

1.98 ±0.5

1.81 ±0.4

1.34 ±0.2

2.35 ±0.5 1

1.41 ± 0.2

1.34 ±0.3

Site cross

submetacentric

submetacentric *

metacentric

submetacentric # i

metacentric

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 66

Mollard et al.

Fig. 2. Karyotype from a Southern Site L. spenceri adult female. A representative karyotype demonstrates the L. spenceri 2n = 26

karyotype and a DAPI negative area in the long arm of chromosome 9.

T *

4r \ ^

\ 't

Fig. 3. Karyotype from a Southern Site L. spenceri adult male. A representative karyotype demonstrates the L. spenceri 2n = 26

karyotype and a DAPI negative area in the long arm of chromosome 9.

Results

Litoria spenceri^ Southern Site: field study adult spec¬

imens

One female and one male frog were studied from the

Southern Site. Minimal numbers of animals were used in

this study due to the critically endangered nature of this

species, the lack of readily available tissue samples, and

the unwillingness to harm such critically endangered ani¬

mals explicitly for use in experimentation. Twenty of 23

spreads counted were 2n = 26 for the female and nine of

11 spreads in the male. Eight prepared karyotypes (chro¬

mosomes lined up in order) from the female and seven

prepared karyotypes from the male confirmed the 2n =

26 karyotype, and arranged in descending order of size,

revealed four larger, three medium, and six smaller chro¬

mosome pairs (Figs. 2, 3, and data not shown). DAPI

negative regions were evident on the long arms of chro¬

mosome 9 for each sex in all karyotypes (Figs. 2, 3, 4,

and data not shown). Chromosomes 1,4, 10, 11, 12, and

13 appear metacentric, chromosomes 2, 6, 7, 8, and 9

appear submetacentric, and chromosomes 3 and 5 appear

subtelocentric (Table 1).

Litoria spenceri, Northern Site: captive bred adult

specimen

One unsexed frog was studied from the Northern Site.

Eight of 12 spreads counted were 2n = 26. The same

chromosomal metacentric, metacentric, and subtelocen¬

tric configuration was observed as for the Southern Site

animal karyotypes, with DAPI negative regions again

evident on the long arms of chromosome 9 in four karyo¬

types that were prepared (Figs. 4, 5; Table 1 and data not

shown).

Litoria spenceri, Central Site: captive bred juvenile

specimens

Two unsexed juvenile frogs were studied from the Central

Site. For one frog, 30 of 35 spreads counted were 2n = 26

and 17 of 24 in the other. For one frog, five karyotypes were

prepared and for the other, four karyotypes were prepared

(Figs. 6 and 7; data not shown). Chromosomal configurations

differed slightly compared to those from animals originating

from both the Southern and Northern sites, with DAPI nega¬

tive regions being evident on the long arms of chromosomes

9 and 11 for both animals in all karyotypes (Figs. 4,6, 7, and

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el66

Litoria spenceri karyotypic diversity

Chromosome 9

Chromosome 11

Southern Site

(female)

Southern Site

(male)

Northern Site

Central Site

(juvenile 1)

Central Site

(juvenile 2)

Northern Site x

Central Site

(tadpole)

Fig. 4. Chromosomes 9 and 11 from different L. spenceri populations. Three representative chromosomes from each animal

demonstrate a highly conserved DAPI negative region in the long arms of chromosome 9. A DAPI negative region is observed in

the long arm of chromosome 11, but only in the Central Site juveniles and in only one matched chromosome of the Northern Site x

Central Site tadpole hybrid. Arrows indicate the chromosome 11 DAPI negative region. Asterisks indicate the paired submetacentric

chromosome 11 matched pair of the Northern Site x Central Site tadpole hybrid.

data not shown; Table 1). Chromosome 11 here also differed

and was submetacentric and not metacentric.

Litoria spenceri Northern Site x Central Site: captive

bred tadpole

One unsexed mixed Northern and Central Site tadpole

was studied. Thirty-one of 33 spreads counted were 2n =

26. Nine karyotypes were prepared, confirming the 2n =

26 karyotype (Fig. 8; data not shown). When arranged in

descending order of size, the same chromosomal meta¬

centric, submetacentric, and subtelocentric configura¬

tion was observed as for the Central Site animals (Figs.

4, 8; Table 1; data not shown). DAPI negative regions

were evident on the short arms of both homologous chro¬

mosomes of chromosome 9, and one chromosome 11

matched chromosome in all nine karyotypes (Figs. 4, 8;

data not shown). The one matched chromosome of the

chromosome 11 pair that did not display this DAPI nega¬

tive region was submetacentric in all nine karyotypes

(Figs. 4, 8; data not shown).

Discussion

These data demonstrate a 2n = 26 karyotype for L. spen¬

ceri. This karyotype is characteristic of all species of the

genus Litoria described to date, with the exception of L.

infrafrenata which has a 2n = 24 karyotype (see King

1980; Mollard 2018a). The L. spenceri karyotypes de¬

scribed here, display the highly conserved Litoria genus

centromere positions and corresponding arms ratios, with

the characteristic: pairs 1 and 4 metacentric, pairs 2 and

6 submetacentric and pairs 3 and 5 acrocentric chromo¬

somal morphologies (see King 1980). These data further

demonstrate a consistent DAPI negative region approxi¬

mately midway on the long arms of chromosome 9 of all

animals studied. A DAPI negative region approximately

midway on chromosome 11 was also observed only in

representatives of the Central Site population and appar¬

ently inherited in a heterozygous state in a Northern Site

X Central Site hybrid cross tadpole.

The described DAPI negative regions are likely

NORs, where undercondensation or despiralization of

rDNA is known to typically result in this type of rela¬

tive DAPI understaining (see Haaf et al. 1984; McStay

2016). NORs are regions of chromosomes that contain

ribosomal DNA genes, usually present as tandem re¬

peats, that code for the rRNA of interphase nucleoli.

Amphibian NORs have been traditionally detected using

a specific silver staining method and proximal chromo-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 66

Mollard et al.

Fig. 5. Karyotype from a Northern Site L. spenceri unsexed adult. A representative karyotype demonstrates the L. spenceri 2n = 26

karyotype and a DAPI negative area in the long arm of chromosome 9.

1 2 3 4 5 6 7

Fig. 6. Karyotype from a Central Site L. spenceri unsexed juvenile, animal 1. A representative karyotype demonstrates the L.

spenceri 2n = 26 karyotype and DAPI negative areas in the long arms of chromosomes 9 and 11.

1 2 3 4 5 6 7

O -

Fig. 7. Karyotype from a Central Site L. spenceri unsexed juvenile, animal 2. A representative karyotype demonstrates the L.

spenceri 2n = 26 karyotype and DAPI negative areas in the long arms of chromosomes 9 and 11.

somal arms visualized with orcein or Giemsa counter¬

stains (see Bloom and Goodpasteur 1976; Mahony and

Robinson 1986). Visually apparent DAPI-induced under¬

condensations in chromosomal regions strongly overlap

with silver stained NORs detected using these methods,

meaning that DAPI staining represents a good proxy for

their identification (Haaf et al. 1984; McStay 2016). In

studies that have investigated the location of the NOR

in anurans, and specifically the Australian tree frogs of

the genus Litoria, the NOR location is extremely well

conserved and almost always located in the same region

of the same chromosome pair in closely related species

complexes (King 1980; Schmid 1983; King et al. 1990).

Phylogenetic studies have placed L. spenceri within a

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e166

Litoria spenceri karyotypic diversity

1 2 3 4 5 6 7

Fig. 8. Karyotype from a Northern Site x Central Site L. spenceri unsexed tadpole hybrid. A representative karyotype demonstrates

the L. spenceri 2n = 26 karyotype and DAPI negative areas in the long arm of chromosome 9, as well as one matched chromosome

of chromosome 11. The chromosome 11 matched chromosome that does not contain the DAPI negative area is submetacentric.

closely related speeies group with L. pearsoniana {L.

pearsoni) and L. phyllochroa and both these latter two

animals display an NOR on the long arms of chromo¬

some 9 (Donnellan et al. 1999; MeGuigan et al. 1998).

The notion that the DAPI negative region of L. spenceri

chromosome 9 described here is an NOR, therefore, is

strongly supported.

Although species of the genus Limnodynastes may

eontain up to four NORs, most Litoria species contain

one, with exceptions being L. phyllocroa, allocated to

a closely related species group with L. spenceri, and L.

raniformis, which both contain two (King 1980, 1990;

Mahony and Robinson 1986). With the previous demon¬

stration of a second NOR in L. phyllochroa, it is sug¬

gested that the chromosome 11 DAPI negative region

is an NOR representing a distinguishing marker of the

Central Site L. spenceri subpopulation. This suggestion

is supported by the findings that: (i) NORs are heredi-

table in the heterozygous state, and (ii) the one hybrid

tadpole chromosome 11 homologous ehromosomal pair

contained such a DAPI negative region (see King 1980;

Stults et al. 2008). The presence of a life-stage specific

chromosomal restriction is unprecedented. Further, this

ehromosome 11 DAPI negative region was not present

in either the female or male Southern Site wild or North¬

ern Site captive specimens. Together, these observations

suggest it is neither life stage-, sex- nor captivity-linked.

Therefore, representatives of all three speeies of this

closely related group for which karyotypes have been

presented can apparently display different NOR configu¬

rations: L. pearsoniana and L. spenceri, apparently mid¬

way on the long arm of chromosome 9; L. phyllochroa

apparently distal on the long arm of ehromosome 9; L.

pearsoniana with an additional NOR apparently distal on

the short arm of chromosome 8, and the Central Site L.

spenceri subpopulation, apparently mid-way on the long

arm of chromosome 11 (Mahony and Robinson 1986;

Schmid et al. 2002).

Conclusion

Further studies to unequivocally identify: (i) these DAPI

negative regions as NORs and (ii) an apparent metacen-

Amphib. Reptile Conserv.

trie to submetacentric chromosome 11 eonversion in the

Northern Site x Central Site tadpole hybrid are warranted

(see Mahony and Robinson 1986; Zalesna et al. 2017).

Regardless, these DAPI negative regions represent dis¬

tinguishing markers of L. spenceri subpopulations that

may assist with animal husbandry techniques. Using the

techniques described here ean help identify subpopula¬

tions of endangered amphibian species. Therefore, add¬

ing karyotyping with DAPI staining for NORs as part of

biobanking conservation management programs is an

important step in ensuring that specific populations and

their unique traits are preserved.

Acknowledgements. —The authors thank Associate

Professor Jean-Pierre Seheerlinck for aceess to eulture

equipment and Dr. Charlie Pagel for access to micro¬

scopic equipment, both at the University of Melbourne’s

Department of Veterinary and Agricultural Sciences. The

authors also thank FNQ Cpap and Medieal Gas Pty Ltd

and Cairns Regional Council both in Cairns, Australia for

generous donation of liquid nitrogen and MilliQ H20,

respectively. The experimental work was funded and per¬

formed by Riehard Mollard. Prof. Michael Mahony re¬

ceives funds through Earthwatch, Australia. Tissues used

in these studies were supplied from programs supported

by the National Environmental Science Program Threat¬

ened Species Recovery Hub (Dr. Matt West; Southern

samples) and the Amphibian Research Centre (Gerry

Marantelli; Northern, Central, and Hybrid samples).

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Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 66

Litoria spenceri karyotypic diversity

Dr. Richard Mollard is an Honorary Fellow at the Faculty of Veterinary and Agricultural

Sciences at the University of Melbourne. He is owner of Amphicell Pty Ltd (www.

amphicell.com), an Australian native frog conservation advocacy that is building a

biobank to assist in safeguarding the future of amphibian biodiversity within Australia.

Prof. Michael Mahony is based at the University of Newcastle’s Discipline of

Environmental Science and Management. He is a conservation biologist with specific

research in restoration ecology and mitigation of impacts on threatened fauna. He is

a past Head of Discipline Biology, Head of Discipline of Environmental Science and

Management, and Assistant Dean Research Training. Michael is currently teaching

courses that are managed by the discipline of Environmental Science and Management.

Gerry Marantelli is a conservation scientist at the Amphibian Research Centre (ARC)

part of the not-for-profit organisation frogs.org.au. Founded by Gerry in 1994 the

Amphibian Research Centre’s research has focused on establishing husbandry programs

for numerous endangered frogs and facilitating experimental reintroduction programs to

better understand risks, as well as the development of practical adaptive management

strategies for mitigation of threats to amphibians. As Central Site L. spenceri are now

extinct in the wild materials for this study would not have been available had ARC not

rescued and bred the last few specimens discovered at this population in 2006.

Dr. Matt West is a Research Fellow at the School ofBioSciences, University ofMelbourne.

His research is focused on understanding amphibian decline and the management of

threatened species. He is currently working in the Australian Government’s National

Environmental Science Program Threatened Species Recovery Hub.

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 66

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptiie Conservation

12(2) [Special Section]: 37-64 (el67).

urn:lsid:zoobank.org:pub:440CB3D6-450A-463B-B3D3-1CCBCBD8670E

Two new species of Chiropterothton (Caudata:

Plethodontidae) from central Veracruz, Mexico

^Mirna G. Garcia-Castillo, ^Angel F. Soto-Pozos, ^J. Luis Aguilar-Lopez, "^Eduardo Pineda,

and ^Gabriela Parra-Olea

^’^’^Departamento de Zoologia, Instituto de Biologia, UniversidadNacional Autonoma de Mexico, AP 70-153, Tercer Circuito Exterior s/n, Ciudad

Universitaria, Mexico, Distrito Federal, MEXICO ^ ‘^Red de Biologiay Conservacion de Vertebrados, Instituto de Ecologia, A.C., Carretera Antigua

a Coatepec No. 351, El Haya, CP. 91070, Xalapa, Veracruz, MEXICO

Abstract —The lungless salamanders of the tribe Bolitoglossini show notable diversification in the Neotropics,

and through the use of molecular tools and/or new discoveries, the total number of species continues to

increase. Mexico is home to a great number of bolitoglossines primarily distributed along the eastern, central,

and southern mountain ranges where the genus Chiropterothton occurs. This group is relatively small, with 16

described species, but there remains a considerable number of undescribed species, suggested by the use of

molecular tools in the lab more than a decade ago. Most of these undescribed species are found in the state

of Veracruz, an area characterized by diverse topography and high salamander richness. Described herein

are two new species of Chiropterothton based on molecular and morphological data. Both new species were

found in bromeliads in cloud forests of central Veracruz and do not correspond to any previously proposed

species. Phylogenetic reconstructions included two mitochondrial fragments (L2 and COI) and identified are

two primary assemblages corresponding to northern and southern species distributions, concordant with

previous studies. The two new species are closely related but morphologically and molecularly differentiated

from other species of the C. chiropterus group.

Keywords. Salamanders, bolitoglossines, bromeliads, phylogenetics, cryopreservation, living tissue, biobanking

Citation: Garcia-Castillo MG, Soto-Pozos AF, Aguilar-Lopez JL, Pineda E, Parra-Olea G. 2018. Two new species of Chiropterotriton (Caudata;

Plethodontidae) from central Veracruz, Mexico. Amphibian & Reptiie Conservation 12(2) [Special Section]: 37-54 (el 67).

cialNoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided

the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication

credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptiie Conservation- official journal website <amphibian-

reptiie-conservation.org>.

Received: 30 October 2018; Accepted: 01 December 2018; Published: 31 December 2018

Introduction

Due to their unique topography and geological history, the

Mexican highlands have played an important role in the

evolution of plethodontid salamanders (Wake and Lynch

1976; Darda 1994). Particularly, the tribe Bolitoglossini

(Wake 2012) underwent an adaptive radiation and

diversification in the mountainous regions of Mexico

(Wake and Lynch 1976; Wake 1987), resulting in 40% of

the representative biodiversity of the group (AmphibiaWeb

2018). With the aid of molecular tools and recent expedition

activity, the number of described species has increased in

recent years (Parra-Olea et al. 2016; Garcia-Castillo et al.

2017; Sandoval-Comte et al. 2017).

In Mexico, plethodontid richness is concentrated in

regions with rugged topography and a corresponding

great diversity of habitats and microhabitats (Wake et al.

1992; Rovito et al. 2009). These characteristics are found

in the central region of Veracruz, where two important

mountain systems converge: the Trans Mexican Volcanic

Belt (TMVB) and the Sierra Madre Oriental (SMO). The

state of Veracruz has the second highest salamander di¬

versity in Mexico with 36 species, after Oaxaca with 42

species (Parra-Olea et al. 2014).

The genus Chiropterotriton includes 16 described

species with seven populations suggested as candidate

species in previous phylogenetic analyses: C. sp. C, C.

sp. F, C. sp. G, C. sp. H, C. sp. I, C. sp. J, and C. sp.

K (Darda 1994; Parra-Olea 2003). Two of the described

species, C. lavae (Taylor) and C. chiropterus (Cope), and

two candidate species (C. sp. C and C. sp. H) occur in

Veracruz (Fig. 1). Describe herein are two new species

CorrGSpondonCG. * biol.mirnagarcia@gmail.com ^angelfsotop90@gmail.com^jlal.herp@gmail.com eduardo.pineda@inecol.mx

^gparra@ib.unam.mx (Corresponding author)

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 67

Garcia-Castillo et al.

Fig. 1. Map of sampled localities for phylogenetic analyses of the genus Chiropterotriton. White circles correspond to: 1) C. priscus,

2) C. miquihuanus, 3) C. infernalis, 4) C. cieJoensis, 5) C. cracens, 6) C. multidentatus (Cd. Maiz), 7) C. multidentatus (Rancho

Borboton), 8) C. multidentatus (Sierra de Alvarez), 9) C. magnipes, 10) C. mosaueri, 11) C. chondrostega, 12) C. terrestris, 13) C.

arboreus, 14) C. dimidiatus, 15) C. chico, 16) C. sp. G, 17) C. orculus, 18) C. sp. I, and 19) C. sp. K.

of Chiropterotriton based on analysis of two mitochon¬

drial fragments (L2 and COI) and differing morphologi¬

cal characteristics. Specimens were discovered during

recent expeditions in the mountainous regions of central

Veracruz but could not be assigned to any current species

due to their unique morphological and genetic differen¬

tiation. Furthermore, these proposed new salamanders do

not belong to any candidate species postulated by Darda

(1994) and Parra-Olea (2003).

Methods

Molecular Analyses

Genomic DNA was extracted from liver, intestine, and

tail tissue samples from 38 Chiropterotriton individuals

and Aquiloeurycea cephalica and Parvimolge townsendi

using a DNeasy tissue kit (Qiagen, Valencia, California,

USA). Amplified two mitochondrial fragments using

primers LX12SN1 and LX16S1R for L2 (partial 12S ri-

bosomal subunit, the tRNA, and large subunitl6S; Zhang

et al. 2008) and dgLCO and dgHCO for COI (Meyer

2003). PCR conditions were as follows: L2, 35 cycles of

96 °C (120 s), 55 °C (60 s), and 72 °C (300 s), and COI,

35 cycles of 94 "C (30 s), 50 °C (30 s), and 72 "C (45 s).

PCR products were cleaned with ExoSap-IT (USB Cor¬

poration, Cleveland, Ohio, USA) and sequenced with a

BigDye Terminator v3.1 cycle sequencing kit (Applied

Biosystems, Foster City, California, USA). Products

were purified using Sephadex G-50 (GE Healthcare) and

an ABI3730 capillary sequencer to run sequences. Ad¬

ditionally, 13 Chiropterotriton sequences were obtained

from previous studies (Parra-Olea 2003; Rovito et al.

2015) to complete the study. Voucher information for all

sequences are shown in Table 1.

Sequencher 5.0.1 (Gene Codes Corporation) was used

to edit and assemble sequences and Muscle 3.8 (Edgar

2004) to align fasta files. Mesquite v3.40 (Maddison and

Maddison 2018) was applied to review and concatenate

data matrices and calculate Kimura 2-parameter (K2P)

corrected genetic distances (Table 2). DNA substitution

models were calculated using PartitionFinder vl .0 (Ean-

fear et al. 2012) under the Bayesian information crite¬

rion (BIC), and estimated a maximum likelihood (ME)

tree from RAxME v8.2 (Stamatakis 2014) with 1,000

bootstrap replicates and a GTR+G substitution model.

Additionally, MrBayes v3.2 (Huelsenbeck and Ronquist

2001) was applied for Bayesian analysis with 20 million

generations, sampling every 1,000 generations, and four

chains used to construct a majority consensus tree. Tracer

v.1.7 (Rambaut et al. 2018) was administered to check

stationarity and convergence of chains. Eastly, both phy-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el67

Two new Chiropterotriton from central Veracruz, Mexico

Table 1. Voucher information, localities, GenBank accessions, coordinates and elevation data from specimens used for phylogenetic

analyses. Collection abbreviations; CARJE, Coleccion de Referencia de Anfibios y Reptiles del Institute de Ecologia, A.C.; IBH,

Coleccion Nacional de Anfibios y Reptiles, Instituto de Biologia, UNAM; MVZ, Museum of Vertebrate Zoology, University of

California, Berkeley, California, USA. NOTE: Asterisks indicate data inferred indirectly from the available information.

Species

Voucher

Number

Locality

16S GenBank

COI

GenBank

Latitude

Longitude

Elevation

m asl

Hidalgo: 6.8 km SW (by rd)

C. arboreiis

1BH28191

of Zacualtipan on road to

Tianguistengo

MK335386

MK335232

20.702

-98.667

2029

Veracruz: 6,5 km N from Atzalan,

C. aureus

1BH31040

ejido de desarrollo urbano

Quetzalcoatl

MK335395

MK335241

19.843

-97.231

1249

Veracruz: 6.5 kmN from Atzalan,

C. aureus

1BH31041

ejido de desarrollo urbano

Quetzalcoatl

MK335398

MK335244

19.843

-97.231

1249

Veracruz: 6.5 kmN from Atzalan,

C. aureus

1BH31042

ejido de desarrollo urbano

Quetzalcoatl

MK335396

MK335242

19.843

-97.231

1249

Veracruz: 6.5 kmN from Atzalan,

C. aureus

1BH31043

ejido de desarrollo urbano

Quetzalcoatl

MK335394

MK335240

19.843

-97.231

1249

Veracruz: 6.5 kmN from Atzalan,

C. aureus

1BH31044

ejido de desarrollo urbano

Quetzalcoatl

MK335397

MK335243

19.843

-97.231

1249

C. chico

MVZ200679

Hidalgo: 3,8 km S Mineral del

Chico

AY522471

20.180

-98.731

2630

C. chiropterus

CARIE0777

Veracruz: Huatusco

MK335407

MK335253

19.185

-96.959

1280

C. chiropterus

CARIE0719

Veracruz: Huatusco

MK335408

19.185

-96.959

1280

Hidalgo: 1.0 km S (by rd) of Ea

C. chondrostega

1BH30098

Encamacion on road to MX85,

Parque Nacional los Marmoles

MK335383

MK335229

20.866

-99.219

2471

Tamaulipas: 0.2 km E (by air) of

C. cieloensis

1BH28181

Rancho El Cielo, 6,9 km NNW (by

air) of the center of Gomez Farias,

Reserva de la Biosfera El Cielo

MK335385

MK335231

23.100

-99.190

1174

Tamaulipas: Road from Alta Cima

C. cracens

1BH28192

to San Jose, 1,3 km NE (by air) of

San Jose, Reserva de la Biosfera

El Cielo

MK335384

MK335230

23.059

-99.226

1320

Hidalgo: 4.1 km S (by rd) of

C. dimidiatus

1BH28196

Mineral del Chico on the road

to Pachuca, Parque Nacional El

Chico

MK335390

MK335236

20.198

-98.727

2768

Tamps: Cueva del Brinco, Conrado

C. infernalis

MVZ269665

Castillo, ca. 43.5 km SW (by rd) of

Ejido Guay abas

MK335382

MK335228

23.959

-99.474

1920

C. lavae

1BH22369

Veracruz: 200 m N hwy 140 at

La Joya

MK335393

MK335239

19.614

-97.030

2060

Hidalgo: “El Coni,” 900 m SSE of

C. magnipes

1BH28176

the center of Durango, Municipio

Zimapan, Parque Nacional los

Marmoles

MK335387

MK335233

20.888

-99.226

2234

Nuevo Leon: 1.8 km S (by rd) of

C. miquihuanus

1BH30329

La Encantada on road from La

MK335381

MK335227

23.893

-99.803

2803

Bolsa to Zaragoza

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e167

Garcia-Castillo et al.

Table 1 (continued). Voucher information, localities, GenBank accessions, coordinates and elevation data from specimens used for

phylogenetic analyses. Collection abbreviations: CARIE, Coleccion de Referencia de Anfibios y Reptiles del Instituto de Ecologia,

A.C.; IBH, Coleccion Nacional de Anfibios y Reptiles, Instituto de Biologia, UNAM; MVZ, Museum of Vertebrate Zoology, University

of California, Berkeley, California, USA. NOTE; Asterisks indicate data inferred indirectly from the available information.

Species

Voucher

Number

Locality

16S GenBank

COI

GenBank

Latitnde

Longitnde

Elevation

m asl

Hidalgo: “El Coni,” 900 m SSE

C. mosaueri

1BH28179

of center of Durango, Municipio

Zimapan, Parque Nacional los

Marmoles

MK335388

MK335234

20.888

-99.226

2234

San Euis Potosi: Cueva el

Madrono, 900 m NW (by air)

C. multidentatus

1BH28177

of the entrance to Valle de los

Fantasmas on MX70, Sierra de

Alvarez

MK335416

22.071

-100.614

2297

San Luis Potosi: Cueva el

C. multidentatus

1BH30102

Madrono, 900 m NW (by air) of

entrance to Valle de los Fantasmas

on MX70, Sierra de Alvarez

MK335417

22,071

-100,614

2297

San Luis Potosi: 26.2 km E (by rd)

C. multidentatus

1BH28193

of the center of Ciudad del Maiz

on MX80, at turnoff to RMO Las

Antenas San Luis Potosi

MK335412

22.487

-99,473

1223

San Luis Potosi: 26.2 km E (by rd)

C. multidentatus

1BH30104

of the center of Ciudad del Maiz

on MX80, at turnoff to RMO Las

Antenas San Luis Potosi

MK335414

22.487

-99.473

1223

San Luis Potosi: 26.2 km E (by

C. multidentatus

1BH28194

rd) of center of Ciudad del Maiz

on MX80, at turnoff to RMO Las

Antenas San Luis Potosi

MK335413

22.487

-99.473

1223

C. multidentatus

1BH23111

San Luis Potosi: Rancho

Borborton

MK335415

22.116

-100.601

2098

C. nubilus

1BH31045

Veracruz: 8,2 km W ofXico,

Coxmatla

MK335405

MK335251

19.433

-97.080

2023

C. nubilus

IBH31046

Veracruz: 8,2 km W ofXico,

Coxmatla

MK335399

MK335245

19,433

-97.080

2023

C. nubilus

1BH31048

Veracruz: 8,2 km W ofXico,

Coxmatla

MK335402

MK335248

19.433

-97.080

2023

C. nubilus

IBH31049

Veracruz: 8,2 kmW ofXico,

Coxmatla

MK335403

MK335249

19.433

-97,080

2023

C. nubilus

1BH31050

Veracruz: 8,2 kmW ofXico,

Coxmatla

MK335400

MK335246

19.433

-97.080

2023

C. nubilus

IBH31052

Veracruz: 8,2 kmW ofXico,

Coxmatla

MK335401

MK335247

19.433

-97.080

2023

C. nubilus

1BH31053

Veracruz: 4 km W ofXico, road to

Xico Viejo

MK335404

MK335250

19.439

-97,043

1583

C. nubilus

CARIE0739

Veracruz: Bosque Banderilla,

Banderilla

MK335411

19.586

-96.946

1580

C. nubilus

CAEtIE0740

Veracruz: Bosque Rancho Viejo,

Tlalnehuayocan

MK335406

MK335252

19.521

-96.984

1520

C. nubilus

CARIE 1162

Veracruz: Rancho la Mesa,

Banderilla

KP886894

19,582

-96.945

1577

C. orculus

1BH30765

Estado de Mexico: Amecameca,

road to Popocatepetl volcano

MK335391

MK335237

19.072

-98,711

2800*

C. orculus

IBH30746

Estado de Mexico: Amecameca,

road to Popocatepetl volcano

MK335392

MK335238

19.072

-98.711

2800*

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e167

Two new Chiropterotriton from central Veracruz, Mexico

Table 1 (continued). Voucher information, localities, GenBank accessions, coordinates and elevation data from specimens used for

phylogenetic analyses. Collection abbreviations: CARIE, Coleccion de Referencia de Anfibios y Reptiles del Institute de Ecologia,

A.C.; IBH, Coleccion Nacional de Anfibios y Reptiles, Institute de Biologia, UNAM; MVZ, Museum of Vertebrate Zoology, University

of California, Berkeley, California, USA. NOTE; Asterisks indicate data inferred indirectly from the available information.

Species

Voucher

Number

Locality

16S GenBank

COI

GenBank

Latitude

Longitude

Elevation

m asl

C. priscus

1BH22367

Nuevo Leon: 19,4 Km W 18 de

Marzo, Cerro Potosi

MK335380

MK335226

24.891

-100.208

2600

C. terrestris

GP215

Hidalgo: 5,3 km N hwy 105 at

Zacualtipan.

MK335389

MK335235

20,674

-98.696

1860

C. sp, C

MVZ 163635

Veracruz: 3,2 km S Puerto del Aire

AY522453

18.670

-97,338

2406*

C. sp, C

IBH 14317

Veracruz: 3,0 km S Puerto del Aire

AY522454

18.670

-97.338

2400

Puebla: 7.1 km N (by rd) of the

C. sp, F

1BH30112

center of Cuetzalan on road to

Yohualichan

MK335410

MK335255

20,050

-97.500

965

C. sp, F

MVZ 17 8706

Puebla: 3.9 km S Xicotepec de

Juarez

AY522477

20,246

-97.854

1135

Puebla: Xicotepec de Juarez, 3,3

C. sp, F

MVZ200723

km S of Hotel M. Ranchito on

Mexico Hwy. 130, 21 km E on

road to La Union

AY522478

20.246

-97.854

1152

C. sp. F

MVZ 17 8707

Puebla: 3.9 km S Xicotepec de

Juarez

AY522479

20,246

-97.854

1135

C. sp. G

MVZ 17 8700

Puebla: 4 km S Chignahuapan

AY522480

19.801

-98.030

2750

C. sp, G

MVZ 17 8703

Puebla: 4 km S Chignahuapan

AY522481

19.801

-98.030

2750

C. sp, H

1BH22568

Veracruz: Microondas las Lajas

KP886893

19.593

-97,095

3127

C. sp. I

MVZ201387

Puebla: Santa Cruz de

Texmalaquilla

AY522488

18.942

-97.287

3100*

C. sp. I

MVZ201389

Puebla: Santa Cruz de

Texmalaquilla

AY522487

18,942

-97,287

3100*

C. sp. J

1BH30099

Oaxaca: San Bernardo, 4.8 km SW

(by rd) of La Esperanza on MX 177

MK335409

MK335254

18.015

-96.660

1672

C. sp. K

MVZ 173231

Oaxaca: Cerro San Felipe

AY522493

17.160

-96.661

3010*

Aquiloeurycea

cephalica

1BH30253

Hidalgo: 1.0 km S (by rd) of Ea

Encamacion on road to MX85,

Parque Nacional los Marmoles

MK335378

20.866

-99.219

2407

Parvimolge

townsendi

1BH31063

Veracruz: 4 km W Xico, road to

Xico Viejo

MK335379

MK335225

19.439

-97,043

1583

Table 2. Sequence divergence with Kimura two-parameter distances for 16S (left) and COI (right).

C. aureus

C. chiropterus

C. lavae

C. nubilus

C. sp. C

C. sp. F

C. sp. G

C. sp. H

C. sp. I

C. sp. J

C. sp. K

C. aureus

5%/10%

10%/13%

4%/7%

9%/-

6%/ll%

6%/-

9%/-

5%/8%

5%/-

C. chiropterus

5%/10%

Tmevo

3%/10%

7%/-

6%/13%

7%/-

l%/5%

6%/-

C. lavae

10%/13%

7%/16%

8%/15%

1%/-

9%/13%

7%/-

1%/-

8%/15%

7%/-

C. nubilus

4%/7%

3% 10%

8%/15%

7%/-

5%/12%

7%/-

8%/-

7%/-

3%/8%

6%/-

C. sp. C

9%/-

7%/-

1%/-

7%/-

9%/-

7%/-

2%/-

1%/-

7%/-

C. sp. F

6%/ll%

6%/13%

9%/13%

5%/12%

9%/-

8%/-

9%/-

8%/-

7%/12%

7%/-

C. sp. G

6%/-

7%/-

8%/-

7%/-

4%/-

C. sp. H

9%/-

7%/-

1%/-

8%/-

2%/-

9%/-

7%/-

1%/-

8%/-

7%/-

C. sp. I

9%/-

7%/-

1%/-

7%/-

1%/-

8%/-

7%/-

1%/-

7%/-

6%/-

C. sp. J

5%/8%

l%/5%

8%/15%

3%/8%

7%/-

7%/12%

7%/-

8%/-

7%/-

C. sp. K

5%/-

6%/-

7%/-

6%/-

7%/-

4%/-

7%/-

6%/-

7%/-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e167

Garcia-Castillo et al.

logenetic methods were ran through the CIPRES data

portal (Miller et al. 2010).

Morphological Analyses

Analysis compared new taxa morphology with phyloge-

netically and geographically related species (see Results).

Further comparisons included measurements taken from

seven adult specimens of the two new species, twelve C.

chiropterus, nineteen C. lavae, twenty C. orculus (Cope),

and published measurements of C. dimidiatus (Taylor)

from Garcia-Castillo et al. (2017) [Table 3; Appendix 1].

Male and female comparisons were completed separately

due to sexual dimorphic differences.

Basic characters and measurements follow the for¬

mat used by Lynch and Wake (1989): snout-vent length

(SVL), tail length (TL), axilla-groin distance (AX), fore¬

limb length (FLL), hind limb length (HLL), snout to gular

fold distance (head length, HL), head width at the angle

of the jaw (HW), head depth (HD), shoulder width (SW),

intemarial distance (IN), and right foot width (FW). In ad¬

dition, twelve measurements were taken from holotypes:

anterior rim of orbit to snout, eyelid length, eyelid width,

horizontal orbital diameter, interorbital distance, length of

third (longest) toe, length of fifth toe, projection of snout

beyond mandible, snout to anterior angle of the vent,

snout to forelimb, tail depth at the base, and tail width at

the base (All measurements are given in mm, except tooth

counts and adpressed limbs). Maxillary plus premaxillary

(MT+PMT) and vomerine teeth (VT) were recorded for

all specimens. Finally, measurements were documented

for the limb interval (LI) as the number of costal folds

between adpressed limbs (positive values as grooves and

negative values as the overlap between limbs). Descrip¬

tions are based on the color catalogue from Kohler (2012).

Results

Mitochondrial DNA (mtDNA) dataset included the 16

described species of Chiropterotriton plus seven previ¬

ously proposed candidate species (Darda 1994; Parra-Olea

2003). Obtained were a 1,477-bp matrix for ribosomal

12S, tRNA, and 16S genes (including gaps) and 658 bp

for COI gene. The estimated substitution models were as

follows: GTR+G for 12S, tRNA, 16S, the 3rd codon posi¬

tion of COI, K80+G for the 1 st codon position of COI, and

HKY+1 for the 2"^ codon position of COI. Our concat¬

enated phylogeny has a similar topology as shown in pre¬

vious studies (Darda 1994; Parra-Olea 2003; Rovito and

Parra-Olea 2015; Garcia-Castillo et al. 2017), which show

two main groups, a northern and southern species groups.

(Fig. 2). The northern assemblages have a distribution

from central Mexico in Hidalgo to Nuevo Leon, the most

northern limit for the genus, and include the following

species: C. terrestris (Taylor), C. chico Garcia-Castillo

et al., C. infernalis Rovito and Parra-Olea, C. chondro-

stega (Taylor), C. mosaueri (Woodall), C. priscus Rabb,

C. miquihuanus Campbell et al., C. magnipes Rabb, C.

cracens Rabb, C. cieloensis Rovito and Parra-Olea, C.

arboreus (Taylor), and C. multidentatus (Taylor). Where¬

as, the southern assemblages (PP = 1.0, BS = 100) occur

from central Mexico in Hidalgo to the south in Oaxaca

and only have four described species: C. dimidiatus,

C. orculus, C. lavae, and C. chiropterus. However, this

clade includes seven previously proposed candidate spe¬

cies: C. sp. G, C. sp. K, C. sp. H, C. sp. I, C. sp. C, C. sp.

F, and C. sp. J (Fig. 2). Results support the distinctive¬

ness of two additional taxa genetically divergent from all

others and correspond to specimens collected in central

Veracruz. One occurs in only one locality (Atzalan) on

the western side of Sierra de Chiconquiaco, but the sec¬

ond was found in six localities (Coxmatla, Xico, Ban-

derilla, Cinco Palos, La Cortadura, and Tlalnehuayocan)

on the eastern slope of Cofre de Perote (Fig. 2). There

is no molecular data for the Cinco Palos and La Corta¬

dura populations, but these specimens were assigned to

the new taxa according to morphological characters and

concordant geographical distributions (Fig. 1).

The new taxa are phylogenetically related to C. chi¬

ropterus, C. sp. J, and C. sp. F. The genetic distance

(K2P) between specimens from Atzalan and their closely

related taxa are as follows: C. chiropterus 5% (16S) and

10% (COI), C. sp. J 5% (16S) and 8% (COI), C sp. F 6%

(16S) and 11% (COI), and the Cofre de Perote specimens

(average for all 4 populations) 4% (16S) and 7% (COI).

The genetic distance between specimens from Cofre de

Perote (all 4 populations) and their closely related taxa

are as follows: C. chiropterus 3% (16S) and 10% (COI),

C. sp. J 3% (16S) and 8% (COI), C. sp. F 5% (16S) and

12% (COI), and Atzalan specimens 4% (16S) and 7%

(COI) [Table 2]. According to the phylogenetic analysis,

C. chiropterus and C. sp. J are sister taxa (PP = 1.0, BS

= 100) with 1% (16S) and 5% (COI) genetic divergence

between them, and a sister clade to specimens from Co¬

fre de Perote, although with little support (PP = 0.61, BS

= 40). These three taxa are the sister group to specimens

from Atzalan (PP = 1.0, BS = 100). Chiropterotriton sp.

F is the sister taxon of all the aforementioned taxa (PP =

1.0, BS = 100) [Fig. 2]. Given the molecular evidence

and morphological comparisons, proposed herein are the

Atzalan and Cofre de Perote populations as new species.

Systematics

Chiropterotriton aureus sp. nov.

urn: Isid : zoobank.org: act: A288BF9 A-589E-42D5-8675-2AA9E6E55865

Atzalan Golden Salamander

Salamandra Dorada de Atzalan

(Figs. 3A, 4A, and 4B)

Holotype. IBH 31042, an adult male from Atzalan, Vera¬

cruz, 6.5 km N from Atzalan, ejido de desarrollo urbano

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 67

Two new Chiropterotriton from central Veracruz, Mexico

Aquiloeurycea cephalica

icw^^^C. terrestrisGP2^5

Parvimolge townsendi

1.0

0.4S

0 . 89 /

0.42

0.55

C. Chico MVZ200679

C. infernalis MVZ269665

■ C. chondrostega IBH30098

C. /770saL/e/7 IBH28179

C. priscus IBH22367

C. miquihuanus IBH30329

C. magnipes IBH28176

C. cracens IBH28192

C. c/e/oens/s IBH28181

C. arboreus\BH28^9^

IT!

C. multidentatus IBH28193

C. multidentatus IBH28194

C. multidentatus iBH30104

C. multidentatus IBH23111

C. multidentatus IBH28177

C. multidentatus IBH30102

C. dimidiatus IBH28196

C. orculus IBH30765

C. orcty/ws IBH30746

C.sp. G MVZ178703

C.sp. G MVZ17870

C.sp. K MVZ173231

C. /ai/aelBH22369

C.sp. H IBH225568

C.sp. I MVZ201389

C.sp. I MVZ201387

C.sp. C MVZ163635

C.sp. C IBH14317

c. sp. F MVZ178706

C.sp. F MVZ178707

C.sp. F MVZ200723

C.sp. F IBH30112

C. aureus IB FI 31043

C. aureus IBFI31040

C. aureus IBFI31042

C. at/rei/s IBFI31044

C. at/reus IBFI31041

C./7w6f7uslBH31046

C. nt/ftiVus IBH31050

C. nuJbf/ws IBH31052

C. ni/Jb#7i/s IBH31048

C. ni/ibi/i/s IBH31049

C. nt/ib#7i/s IBH31045

C. nubilus \BH3^053

C. nubilus CARIE0740

C. nubilus CAR\E^^62

C. nubilus CARIE0739

* IC. c/7/ropfert/s CARIE0777

C. chiropterus CARIE0719

C.sp. J IBH30099

— —^

= 3

= Ol

= in

I 3

= ^

= QJ

= lO

= (D

= in

0.06

Fig. 2. Bayesian analysis tree for mitochondrial loci. Numbers above branches correspond to posterior probability, and numbers

below branches are bootstrap values from maximum likelihood analysis. Asterisks indicate significant support (posterior probability,

PP > 0.95 and bootstrap, BS > 70) in both analyses. The topology is grouped into northern and southern assemblages according to

species distributions.

Quetzalcoatl, Mexico, 1,249 meters (m) above sea level

(asl), 19.843138N, 97.231194W. Collected on 11 July

2016 by Angel F. Soto-Pozos, M. Delia Basanta, Omar

Becerra-Soria, and Mima G. Garcia-Castillo.

Paratypes. Three specimens from Atzalan, Veracruz,

Mexico. All females: IBH 31041, 31043^4, 6.5 km N

from Atzalan, ejido de desarrollo urbano Quetzalcoatl,

Atzalan, Veracruz, Mexico.

Referred specimens. IBH 31040, 6.5 km N from At¬

zalan, ejido de desarrollo urbano Quetzalcoatl, Atzalan,

Veracmz, Mexico.

Diagnosis. A plethodontid salamander assigned to the

genus Chiropterotriton due to its small size, slender

body, shape of hand and feet digits (relatively long outer

digit), relatively long tail, presence of sublingual fold,

and based on mtDNA sequence data. Phylogenetically

related to C. nubilus, C. chiropterus, C. sp. F, and C. sp. J

(Fig. 2). Chiropterotriton aureus differs from C. nubilus

in being shorter (SVL 28.5 in one male, mean 26.8 in fe¬

males of C. aureus vs. 29.4 in one male, 30.5 in females

of C. nubilus) with a shorter head (HL 6.4 in one male,

mean 6.0 in females of C. aureus vs. 6.6 in one male,

7.4 in females of C. nubilus), narrower head in females

(mean HW 3.6 in females of C. aureus vs. 4.4 in females

of C. nubilus), relatively shorter limbs in females (mean

LI 2.3 in females of C. aureus vs. 1.5 in females of C. nu¬

bilus), and smaller feet (FW 2.4 in one male, mean 1.8 in

females of C. aureus vs. 2.6 in one male, 2.3 in females

of C. nubilus). Digits are narrower at the tip and with

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e167

Garcia-Castillo et al.

Table 3. Mean ± standard deviation (above) and range (below) of morphometric variables from males and females of C. aureus,

C. chiropterus, C. dimidiatus, C. lavae, C. nubilus, and C. orculus. Measurements are given in millimeters (mm), except TL/SLV

(proportional value), LI (limb interval), and tooth counts. NOTE; Data taken from Garcia-Castillo et al. 2017.

Males

C. aureus

C. chiropterus

C. dimidiatus*

C. lavae

C. nubilus

C. orculus

N=\

N=8

N=15

N=\{)

A^=l

A^=10

SVL

28.5

37.5±0.98

24.7±0.97

32.4±0.92

29.4

35.9±1.36

(36.1-38.8)

(23.3-26.7)

(31.0-33.8)

(33.6-38.9)

47.3±3.24

22.0±1.72

(18.5-24.1)

38.5±2.11

(36.2-42.3)

36.6±2.87

36.5

(42.6-52.3)

N=7

40.2

(33.3-41.0)

V=9

1.25±0.08

0.89±0.08

(0.7-1.0)

1.2±0.06

(1.11-1.27)

1.02±0.08

TL/SLV

1.28

(1.13-1.38)

N=7

1.37

(0.86-1.15)

N=9

15.5

19.6±0.59

13.1±0.75

16.2±0.87

15.9

18.6±1.04

(18.7-20.8)

(11.7-14.0)

(14.7-17.4)

(17.1-20.5)

LLL

5.9

9.1±0.44

4.5±0.34

9.3±0.59

6.4

8.9±0.65

(8.2-9.5)

(3.8-5.0)

(8.4-10.2)

(7.4-9.6)

HLL

7.5

10.3±0.47

5.2±0.34

9.9±0.72

7.1

9.3±0.64

(9.5-10.8)

(4.9-5.9)

(8.5-11.0)

(8.2-10.4)

6.4

8.1±0.41

5.3±0.32

7.5±0.33

6.6

7.4±0.47

(7.7-8.9)

(4.8-5.8)

(7.2-8.1)

(6.7-8.1)

4.0

5.6±0.22

3.5±0.21

4.9±0.31

4.0

5.0±0.35

(5.4-6.0)

(3.0-3.7)

(4.5-5.6)

(4.5-5.5)

1.8

2.7±0.07

1.8±0.09

2.5±0.19

2.0

2.4±0.13

(2.6-2.8)

(1.7-2.0)

(2.3-2.9)

(2.2-2.7)

3.4

4.0±0.35

2.9±0.29

3.1±0.30

3.4

3.4±0.30

(3.2-4.4)

(2.3-3.6)

(2.6-3.5)

(3.1-4.0)

1.0

1.9±0.13

1.2±0.08

2.3±0.20

1.2

2.2±0.19

(1.7-2.1)

(1.0-1.3)

(1.9-2.5)

2.4

3.7±0.33

1.7±0.20

3.7±0.39

2.6

3.2±0.22

(3.3-4.4)

(1.4-2.1)

(3.1-4.2)

(2.8-3.5)

2.0

0.3±0.53

3.9±0.35

-0.6±0.52

2.0

1.9±0.88

(-0.5-1.0)

(3.0-4.0)

(-1.0-0.0)

(0.0-3.0)

PMT+MT

14.0

16.3±3.69

9.4±2.59

10.3±3.62

20.0

10.9±2.47

(11.0-21.0)

(5.0-14.0)

(3.0-15.0)

(7.0-14.0)

15.0

10.6±1.06

5.7±1.35

8.9±1.10

10.0

8.6±1.90

(9.0-12.0)

(4.0-8.0)

(7.0-10.0)

(5.0-11.0)

Females

C. aureus

C. chiropterus

C dimidiatus*

C. lavae

C. nubilus

C. orculus

N=3

N=4

N=\5

N=9

N=2

/V=10

SVL

26.8±0.86

33.5±2.55

25.8±1.56

31.6±2.46

30.5±3.89

39.0±2.70

(26.0-27.7)

(30.7-36.7)

(23.1-29.1)

(27.9-34.9)

(27.7-33.2)

(34.9-43.0)

31.1±1.41

39.5±2.35

22.4±1.85

32.5±4.89

34.3±5.16

39.2±3.64

(34.7-44.7)

N=9

(30.1-32.1)

(37.0-42.6)

(19.9-25.2)

(25.7-40.1)

(30.6-37.9)

TL/SLV

1.16±0.00

1.19±0.12

0.87±0.06

l.OiO.lO

1.12±0.03

1.02±0.08

(0.87-1.12)

N=9

(1.16-1.16)

(1.01-1.26)

(0.7-1.0)

(0.85-1.15)

(1.10-1.14)

15.0±0.49

18.5±2.27

14.8±1.24

16.3±1.68

16.4±2.69

21.2±1.58

(14.7-15.6)

(15.4-20.7)

(12.6-17.3)

(13.9-18.5)

(14.5-18.3)

(18.6-23.2)

LLL

5.3±0.42

7.8±0.48

4.3±0.43

8.2±0.72

6.5±0.28

8.9±0.63

(4.8-5.6)

(7.1-8.2)

(3.8-5.1)

(7.1-9.5)

(6.3-6.7)

(7.6-10.0)

HLL

6.7±0.35

8.9±0.31

5.0±0.47

8.8±0.73

7.2±0.14

9.5±0.57

(6.4-7.1)

(8.4-9.1)

(4.4-6.1)

(7.5-9.8)

(7.1-7.3)

(8.6-10.4)

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e167

Two new Chiropterotriton from central Veracruz, Mexico

Table 3 (continued). Mean ± standard deviation (above) and range (below) of morphometric variables from males and females of

C. aureus, C. chiropterus, C. dimidiatus, C. lavae, C. nubilus, and C. orculus. Measurements are given in millimeters (mm), except

TL/SLV (proportional value), LI (limb interval), and tooth counts. NOTE; Data taken from Garcia-Castillo et al. 2017.

Females

C. aureus

N=3

C. chiropterus

N=4

C. dimidiatus*

N=15

C. lavae

N=9

C. nubilus

N=2

C. orculus

/V=10

6.0+0.31

7.3+0.56

5.1+0.34

7.0+0.42

7.4+0.99

8.0+0.52

(5.7-6.3)

(6.5-7.8)

(4.5-5.6)

(6.3-7.6)

(6.7-8.1)

(7.4-8.9)

3.6+0.10

4.8+0.21

3.5+0.25

4.7+0.30

4.4+0.14

5.2+0.29

(3.5-3.7)

(4.5-5.0)

(3.2-4.0)

(4.1-5.0)

(4.3-4.5)

(4.7-5.6)

1.8+0.02

2.5+0.14

2.0+0.20

2.3+0.18

2.0+0.07

2.6+0.32

(1.8-1.8)

(2.3-2.6)

(1.7-2.2)

(2.1-2.7)

(1.9-2.0)

(2.3-3.4)

3.1+0.17

3.6+0.38

3.1+0.26

3.3+0.33

3.3+0.28

3.9+0.46

(3.0-3.3)

(3.3-4.1)

(2.8-3.5)

(2.8-3.8)

(3.1-3.5)

(3.4-4.8)

1.1+0.06

1.7+0.38

1.3+0.15

1.8+0.13

1.2+0.02

2.1+0.25

(1.0-1.1)

(1.4-2.1)

(1.1-1.7)

(1.6-2.0)

(1.2-1.2)

(1.7-2.5)

1.8+0.21

3.1+0.37

1.8+0.26

3.3+0.27

2.3+0.57

3.4+0.37

(1.6-2.0)

(2.6-3.5)

(L3-2.2)

(3.0-3.7)

(1.9-2.7)

(2.6-3.9)

2.3+0.58

2.0+0.41

4.9+0.26

0.6+0.73

1.5+0.71

2.9+0.32

(2.0-3.0)

(1.5-2.5)

(4.0-5.0)

(0.0-2.0)

(LO-2.0)

(2.0-3.0)

PMT+MT

44.7+2.08

54.3+8.08

34.4+4.12

28.0+8.19

48.0+2.83

35.9+4.46

(43.0-47.0)

(47.0-63.0)

(27.0-41.0)

(17.0-45.0)

(46.0-50.0)

(29.0-43.0)

12.3+1.53

12.5+2.38

8.3+1.35

11.4+2.30

13.5+0.71

12.0+1.94

(11.0-14.0)

(10.0-15.0)

(6.0-11.0)

(8.0-15.0)

(13.0-14.0)

(9.0-15.0)

less webbing (just onto the penultimate phalanx) than C.

nubilus (Fig. 3).

Chiropterotriton aureus differs from C. chiropterus

in being shorter (SVL 28.5 in one male, mean 26.8 in

females of C. aureus vs. 37.5 in males, 33.5 in females

of C. chiropterus), relatively shorter limbs in males (LI

2.0 in one male of C. aureus vs. 0.3 in males of C. chi¬

ropterus), shorter head (HL 6.4 in one male, mean 6.0 in

females of C. aureus vs. 8.1 in males, 7.3 in females of

C. chiropterus), narrower head (HW 4.0 in one male, 3.6

in females of C. aureus vs. 5.6 in males, 4.8 in females

of C. chiropterus), and smaller feet (FW 2.4 in one male,

mean 1.8 in females of C. aureus vs. 3.7 in males, 3.1 in

females of C. chiropterus). Chiropterotriton aureus has

narrower digits at the tip and smaller feet and hands than

C. chiropterus (Fig. 3).

Chiropterotriton aureus differs from its geographi¬

cally close species C. lavae by being shorter (SVL 28.5

in one male, mean 26.8 in females of C. aureus vs. 32.4

in males, 31.6 in females of C. lavae), shorter head (HL

6.4 in one male, mean 6.0 in females of C. aureus vs. 7.5

in males, 7.0 in females of C. lavae), narrower head (HW

4.0 in one male, 3.6 in females of C. aureus vs. 4.9 in

males, 4.7 in females of C. lavae), shorter limbs (LI 2.0

in one male, mean 2.3 in females of C. aureus vs. -0.6 in

males, 0.6 in females of C. lavae), and smaller feet (FW

2.4 in one male, mean 1.8 in females of C. aureus vs. 3.7

in males, 3.3 in females of C. lavae) with less webbing in

C. aureus than in C. lavae (Fig. 3).

Chiropterotriton aureus differs from C. orculus by be¬

ing shorter (SVL 28.5 in one male, mean 26.8 in females

of C. aureus vs. 35.9 in males, 39.0 in females of C. orcu¬

lus), longer tail (TL/SVL 1.28 in one male, mean 1.16 in

females of C. aureus vs. 1.02 in both males and females

of C. orculus), relatively larger limbs in females (mean

LI 2.3 in females of C. aureus vs. 2.9 in females of C.

orculus), shorter head (HL 6.4 in one male, mean 6.0 in

females of C. aureus vs. 7.4 in males, 8.0 in females of

C. orculus), narrower head (HW 4.0 in one male, 3.6 in

females of C. aureus vs. 5.0 in males, 5.2 in females of C.

orculus), and smaller feet (FW 2.4 in one male, mean 1.8

in females of C. aureus vs. 3.2 in males, 3.4 in females

of C. orculus).

Chiropterotriton aureus differs from C. dimidiatus in

being longer (SVL 28.5 in one male, mean 26.8 in fe¬

males of C. aureus vs. 24.7 in males, 25.8 in females

of C. dimidiatus), longer tail (TL/SVL 1.28 in one male,

mean 1.16 in females of C. aureus vs. 0.89 in males, 0.87

in females of C. dimidiatus), longer head (HL 6.4 in one

male, mean 6.0 in females of C. aureus vs. 5.3 in males,

5.1 in females of C. dimidiatus), longer limbs (LI 2.0 in

one male, mean 2.3 in females of C. aureus vs. 3.9 in

males, 4.9 in females of C. dimidiatus), and more max¬

illary teeth (PMT+MT 14.0 in one male, mean 44.7 in

females of C. aureus vs. 9.4 in males, 34.4 in females of

C. dimidiatus).

Chiropterotriton aureus is phylogenetically related

to members of the southern assemblages (Fig. 2), which

includes seven undescribed taxa previously suggested

by allozyme data (Darda 1994) and mtDNA (Parra-Olea

2003). Chiropterotriton aureus differs genetically from

its close relatives as follows: 6% (16S) and 11% (COI) to

C. sp. F; 5% (16S) and 8% (COI) to C. sp. J; 9% (16S) to

C. sp. H, C. sp. I, and C. sp. C; 6% (16S) to C. sp. G; and

5% (16S) to C. sp. K (Table 2).

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 67

Garcia-Castillo et al.

1 mm

Fig. 3. Head, hand, and foot morphology of preserved

specimens of Chiropterotriton species from central Veracruz.

Ventral view from right hand and foot. A) C. aureus holotype

IBH 31042, B) C. nubilus holotype IBH 31048, C) C. lavae

MVZ 106436, and D) C. chiropterus MVZ 85590.

Chiropterotriton aureus differs from other members

of Chiropterotriton by its smaller body size (SVL 28.5

in one male, mean 26.8 in females), while C. arboreus

(mean SVL 33.4 in males, 32.2 in females; Garcia-

Castillo et al. 2017), C. cieloensis (mean SVL 32.6 in

males, 31.1 in females; Rovito and Parra-Olea 2015), C.

chico (mean SVL 38.4 in males, 39.3 in females; Gar¬

cia-Castillo et al. 2017), C. infernalis (mean SVL 36.4

in males, 29.7 in one female; Rovito and Parra-Olea

2015), C. magnipes (mean SVL 46.8 in males, 57.5 in

females; Rabb 1965), C. miquihuanus (mean SVL 33.3 in

males, 36.5 in females; Rovito and Parra-Olea 2015), C.

mosaueri (mean SVL 42.8 in males; Woodall 1941), C.

multidentatus (mean SVL 33.6 in males, 34.0 in females;

Rovito and Parra-Olea 2015), and C. priscus (mean SVL

38.5 in males, 41.8 in females; Rovito and Parra-Olea

2015). However, this species is longer than C. chondro-

stega (mean SVL 23.1 in males, 25.4 in females; Gar¬

cia-Castillo et al. 2017), C. cracens (mean SVL 25.7 in

males, 27.4 in females; Rovito and Parra-Olea 2015), and

C. terrestris (mean SVL 24.2 in males, 23.0 in females;

Garcia-Castillo et al. 2017). Chiropterotriton aureus has

smaller feet (FW 2.4 in one male, mean 1.8 in females)

than C. arboreus (mean FW 3.4 in males, 3.5 in females;

Garcia-Castillo et al. 2017), C. cieloensis (mean FW 3.2

in males, 3.1 in females; Rovito and Parra-Olea 2015),

C. chico (mean FW 4.1 in males, 4.2 in females; Garcia-

Castillo et al. 2017), C. infernalis (mean FW 4.2 in males,

2.8 in one female; Rovito and Parra-Olea 2015), C. mul¬

tidentatus (mean FW 3.6 in males, 3.5 in females; Ro¬

vito and Parra-Olea 2015), and C. priscus (mean FW 3.2

in males, 3.5 in females; Rovito and Parra-Olea 2015).

Chiropterotriton aureus has shorter limbs (LI 2.0 in one

male, mean 2.3 in females) than C. arboreus (mean LI

0.2 in males, 1.0 in females; Garcia-Castillo et al. 2017),

C. cieloensis (mean LI -0.2 in males, 0.1 in females; Ro¬

vito and Parra-Olea 2015), C. infernalis (mean LI -0.7 in

males, -0.5 in one female; Rovito and Parra-Olea 2015),

C. multidentatus (mean LI 0.1 in males, 1.0 in females;

Rovito and Parra-Olea 2015), but it has longer limbs than

C. dimidiatus (mean LI 3.8 in males, 4.9 in females; Gar¬

cia-Castillo et al. 2017), C. miquihuanus (mean LI 4.2 in

males, 4.3 in females; Rovito and Parra-Olea 2015), and

C. priscus (mean LI 3.2 in males, 3.7 in females; Rovito

and Parra-Olea 2015).

Description. A small species of Chiropterotriton, mean

SVL 28.5 in one adult male (with mental gland) and 26.8

in three adult females (range 26.0-27.7). Head narrow

and moderately long (HW 4.0 in one male, mean 3.6 in

females; HL 6.4 in one male, mean 6.0 in females), HW/

SVL=14% in one male a mean of 13% in females (range

13-14), and is wider than the shoulders (SW 3.4 in one

male, mean 3.1 in females). Nostril oval shaped. Men¬

tal gland in one male small and almost circular shaped.

Snout narrow and squared shaped. Eyes slightly protu¬

berant. Jaw muscles are visible as grooves in the “V” be¬

hind the eyes. Few maxillary teeth in one male (mean MX

10.0) but a moderately large number in females (mean

MX 38.3, range 37^0). Premaxillary teeth in one male

are not enlarged and not piercing the lip. Few vomerine

teeth in one male (VX 15.0) and females (mean VX 12.3,

range 11-14), and arranged in a well-defined line nearly

to outer margin of the choanae. Xail is longer than SVL,

XL/SVL 1.28 in one male and 1.16 in females. Limbs are

short and slender, FLL+HLL 47% of SVL in one male

and 45% in females (range 43-46). Adpressed limbs

separated by 2.0 costal folds in one male (LI 2.0) and 2.3

in females (mean LI 2.3, range 2.0-3.0). Digits slender

and narrower at the tip with moderate webbing just onto

the penultimate phalanx. Subterminal pads present. Pha¬

langeal formulae: hand 1-2-3-2, foot 1-2-3-3-2. Digits in

order of increasing length: hand I-IV~II-III, foot I-V-II-

IV-III.

Coloration in life (from photos). Upper side of head

Buff (5) or Yellow Ocher (14) on Dark Carmine (61) sur¬

face, Cream Yellow (82) on the tip of head and part of the

eyelids, and lateral and gular region Pale Buff (1). Dor¬

sum Buff (5), Yellow Ocher (14) or Olive Horn (16) on

Pale Buff (1) surface, venter and costal sides Pale Buff

(1). Upper side of tail with progressively darker Dark

Carmine (61) with Buff (5) and Light Pratt’s Rufous (71)

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 67

Two new Chiropterotriton from central Veracruz, Mexico

speckles, or uniform Yellow Ocher (14), or Olive Horn

(16) with Peach Red (70) speckles. Underside of tail

Pale Buff (1). Forelimbs Chamois (84), and hands nearly

translucent. Hindlimbs Buff (5), feet nearly translucent.

Underside of limbs Pale Buff (1). Iris Orange-Rufous

(56).

Coloration in alcohol. Upper side of head and dorsum

Drab (19) and underside of head Pale Horn Color (11).

Venter Pale Pinkish Buff (3) and costal region Cream

Color (12) or Cinnamon-Drab (50). Upper side of tail

Dark Drab (45), Cinnamon (225) or Hair Brown (277),

and underside of tail Buff (5) or Drab (19). Upper side

of limbs Drab (19) and underside of limbs Cream Color

( 12 ).

Measurements of the holotype, tooth counts, and limb

intervals. SVL 28.5, TL 36.5, AX 15.5, SW 3.4, HL 6.4,

HW 4.0, HD 1.8, projection of snout beyond mandible

0.7, anterior rim of orbit to snout 1.8, interorbital dis¬

tance 1.9, eyelid length 1.7, eyelid width 1.3, horizontal

orbit diameter 0.8, distance between corners of eyes 3.6,

FLL 5.9, HLL 7.5, snout to forelimb 9.2, snout to anterior

angle of vent 26.7, tail width at base 2.0, tail depth at

base 1.9, FW 2.4, length of fifth toe 0.5, length of third

(longest) toe 0.9, mental gland length 1.2, and mental

gland width 1.0. Premaxillary teeth four, maxillary 4-6

(right-left sides) and vomerine 8-7 (right-left sides). Ad-

pressed limbs separated by two costal folds.

Habitat and distribution. Western side of Sierra de Chi-

conquiaco, part of the Sierra Madre Oriental in central

Veracruz. Specimens found in a cloud forest with exten¬

sive deforestation (near crops and paddocks), exclusively

in arboreal bromeliads over oaks at 1,249 m asl (Figs. 5A

and 5B).

Natural History. Chiropterotriton aureus was found

exclusively in bromeliads in cloud forest around 1,200

m asl. Examined were approximately 40 bromeliads and

found only five specimens, including four adults (one

male and three females). Sampled bromeliads were at

1.5-3.0 m from the ground and small (approximately 20-

40 cm in diameter). Sampling site was disturbed and de¬

forested, but adjacent zones with similar environmental

conditions could be explored to delimit the distributional

range of this species. Species possibly sympatric with C.

aureus may be Aquiloeurycea cafetalera, Bolitoglossa

platydactyla, Isthmura gigantea, Pseudoeurycea lynchi,

and Thorius minydemus.

Etymology. Latin epithet aureus (feminine aurea, neuter

aureus) is derived from ''aurunC gold + derivational suf¬

fix meaning made of gold or gold in color, which

is the featured characteristic color of the species.

Fig. 4. Photos in life of two new species from central Veracruz.

A) C. aureus (male) holotype IBH 31042, B) C. aureus (female)

paratype IBH 31044, C) C. nubilus (male) paratype CARIE

0739, and D) C. nubilus (female) holotype IBH 31048. Photo

credit: Maria Delia Basanta (A, B, D) and J. Luis Aguilar-

Lopez (C).

Chiropterotriton nubilus sp. nov.

urn:lsid:zoobank.org:act:F785D7FD-301F-4D53-BD7F-AC680A0BB875

Cloud Forest Salamander from Cofre de Perote

Salamandra del Bosque de Niebla del Cofre de Perote

(Figs. 3B, 4C, and 4D)

Chiropterotriton sp.: Rovito et al. 2015

Holotype. IBH 31048, an adult female from Coxmatla,

Veracruz, 8.2 km W of Xico, Veracruz, Mexico, 2,023 m

asl, 19.433264N, 97.080639W. Collected 25 June 2017

by Angel F. Soto-Pozos, Fabiola A. Herrera-Balcazar,

M. Delia Basanta, Omar Becerra-Soria, and Mima G.

Garcia-Castillo.

Paratypes. One male: CARIE 0739, Banderilla,

19.586667N, 96.946111W. One Female: IBH 31049,

Coxmatla, 8.2 km W of Xico.

Referred specimens. IBH 31045-46, IBH 31050-52,

Coxmatla, 8.2 km W of Xico; IBH 31047, IBH 31053 4

km W of Xico, road to Xico Viejo; CARIE 0718, La Cor-

tadura, Coatepec, 19.491389N, 97.027778W; CARIE

0740, CARIE 1269, Bosque Rancho Viejo, Tlalnehuayo-

can; CARIE 1162, Rancho La Mesa, Banderilla; CARIE

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 67

Garcia-Castillo et al.

Fig. 5. Microhabitat and landscape photographs for new species from central Veracruz. A) Landscape from type locality of C. aureus

(Atzalan, Veracruz), B) bromeliad from type locality of C. aureus, C) view of type locality of C. nubilus (Coxmatla, Veracruz), and

D) bromeliad from locality of C. nubilus (Xico, Veracruz). Photo credit: Mima G. Garcia-Castillo and Angel F Soto-Pozos.

1267, Banderilla; CARIE 1272, Cinco Palos, Coatepec,

19.5N, 97.002778W.

Diagnosis. A plethodontid salamander assigned to the

genus Chiropterotriton due to its slender body with a rel¬

atively long tail, shape of hand and feet digits, presence

of sublingual fold, and based on mtDNA sequence data.

Phylogenetically related to C. aureus, C. chiropterus, C.

sp. F, and C. sp. J (Fig. 2). Chiropterotriton nubilus dif¬

fers from C. aureus in females being longer (mean SVF

30.5 in females of C. nubilus vs. 26.8 in females of C.

aureus), longer tail in males (TF/SVF 1.37 in one male

of C. nubilus vs. 1.28 in one male of C. aureus), rela¬

tively longer limbs in females (mean FI 1.5 in females

of C. nubilus vs. 2.3 in females of C. aureus), a longer

head (mean HF 7.4 in females of C. nubilus vs. 6.0 in

females of C. aureus), and broader head (mean HW 4.4

in females of C. nubilus vs. 3.6 in females of C. aureus).

Chiropterotriton nubilus has longer feet (mean FW 2.3

in females of C. nubilus vs. 1.8 in females of C. aureus)

with more rounded digits and slightly more webbing

(just above penultimate phalanx) than C. aureus (Fig. 3).

Chiropterotriton nubilus differs from C. chiropterus

by being shorter (SVF 29.4 in one male, mean 30.5 in

females of C. nubilus vs. 37.5 in males, 33.5 in females

of C. chiropterus), with relatively shorter limbs in males

(FI 2.0 in one male of C. nubilus vs. mean 0.3 in males

of C. chiropterus), shorter head in males (HF 6.6 in one

male of C. nubilus vs. mean 8.1 in males of C. chiropter¬

us), narrower head (HW 4.0 in one male, mean 4.4 in fe¬

males of C. nubilus vs. 5.6 in males, 4.8 in females of C.

chiropterus), jaw muscles less pronounced and eyes less

protuberant than C. chiropterus (Pig. 3). Chiropterotriton

nubilus has smaller feet (FW 2.6 in one male, mean 2.3

in females of C. nubilus vs. 3.7 in males, 3.1 in females

of C. chiropterus), with rounded digits, and fourth finger

of hand and fifth toe of foot longer than C. chiropterus.

Fikewise, C. nubilus has more webbing that covers just

above the penultimate phalanx while C. chiropterus has

webbing under the penultimate phalanx (Fig. 3).

Chiropterotriton nubilus differs from geographically

proximate species C. lavae in males being shorter (SVF

29.4 in one male of C. nubilus vs. mean 32.4 in males of

C. lavae), a longer tail (TF/SVF 1.37 in one male, mean

1.12 in females of C. nubilus vs. 1.2 in males, 1.0 in fe¬

males of C. lavae), narrower head (HW 4.0 in one male,

mean 4.4 in females of C. nubilus vs. 4.9 in males, 4.7 in

females of C. lavae), relatively shorter limbs (FI 2.0 in

one male, mean 1.5 in females of C. nubilus vs. -0.6 in

males, 0.6 in females of lavae), and more maxillary teeth

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el67

Two new Chiropterotriton from central Veracruz, Mexico

(PMT+MT 20.0 in one male, mean 48.0 in females of C.

nubilus vs. 10.3 in males, 28.0 in females of C. lavae). In

general, C. nubilus is morphologically similar to C. lavae

in body size and proportions (Table 3), but C. nubilus has

smaller feet (FW 2.6 in one male, mean 2.3 in females of

C. nubilus vs. 3.7 in males, 3.3 females of C. lavae) and

less webbing (Fig. 3).

Chiropterotriton nubilus differs from C. orculus in

being shorter (SVL 29.4 in one male, mean 30.5 in fe¬

males of C. nubilus vs. 35.9 in males, 39.0 in females of

C. orculus), longer tail (TL/SVL 1.37 in one male, mean

1.12 in females of C. nubilus vs. 1.02 in both males and

females of C. orculus), relatively longer limbs in females

(mean LI 1.5 in females of C. nubilus vs. 2.9 in females

of C. orculus), shorter head (HL 6.6 in one male, mean

7.4 in females of C. nubilus vs. 7.4 in males, 8.0 in fe¬

males of C. orculus), narrower head (HW 4.0 in one male,

mean 4.4 in females of C. nubilus vs. 5.0 in males, 5.2 in

females of C. orculus), more maxillary teeth (PMT+MT

20.0 in one male, mean 48.0 in females of C. nubilus vs.

10.9 in males, 35.9 in females of C. orculus), and smaller

feet (FW 2.6 in one male, mean 2.3 in females of C. nubi¬

lus ys. 3.2 in males, 3.4 in females of C. orculus).

Chiropterotriton nubilus differs from C. dimidiatus

in being shorter (SVL 29.4 in one male, mean 30.5 in

females of C. nubilus vs. 24.7 in males, 25.8 in females

of C. dimidiatus), longer tail (TL/SVL 1.37 in one male,

mean 1.12 in females of C. nubilus vs. 0.89 in males, 0.87

in females of C. dimidiatus), longer head (HL 6.6 in one

male, mean 7.4 in females of C. nubilus vs. 5.3 in males,

5.1 in females of C. dimidiatus), broader head (HW 4.0

in one male, mean 4.4 in females of C. nubilus vs. 3.5 in

both males and females of C. dimidiatus), relatively lon¬

ger limbs (LI 2.0 in one male, mean 1.5 in females of C

nubilus vs. 3.9 in males, 4.9 in females of C. dimidiatus),

more maxillary teeth (PMT+MT 20.0 in one male, mean

48.0 in females of C. nubilus vs. 9.4 in males, 34.4 in

females of C. dimidiatus), more vomerine teeth (VT 10.0

in one male, mean 13.5 in females of C. nubilus vs. 5.7 in

males, 8.3 in females of C. dimidiatus), and longer feet

(FW 2.6 in one male, mean 2.3 in females of C. nubilus

vs. 1.7 in males, 1.8 in females of C. dimidiatus).

Chiropterotriton nubilus is related to an undescribed

taxon of the southern assemblages with genetic diver¬

gences as follows: 5% (16S) and 12% (COI) to C. sp. F;

3% (16S) and 8% (COI) to C. sp. J; 8% (16S) to C. sp. H;

7% (16S) to C. sp. I, C. sp. C, and C. sp. G; and 6% (16S)

to C. sp. K (Table 2).

Chiropterotriton nubilus differs from other species of

Chiropterotriton by being shorter (SVL 29.4 in one male,

mean 30.5 in females) other than C. arboreus (mean

SVL 33.4 in males, 32.2 in females; Garcia-Castillo et

al. 2017), C. chico (mean SVL 38.4 in males, 39.3 in fe¬

males; Garcia-Castillo et al. 2017), C. magnipes (mean

SVL 46.8 in males, 57.5 in females; Rabb 1965), C.

miquihuanus (mean SVL 33.3 in males, 36.5 in females;

Rovito and Parra-Olea 2015), C. mosaueri (mean SVL

42.8 in males; Woodall 1941), C. multidentatus (mean

SVL 33.6 in males, 34.0 in females; Rovito and Parra-

Olea 2015), and C. priscus (mean SVL 38.5 in males,

41.8 in females; Rovito and Parra-Olea 2015). Chirop¬

terotriton nubilus has a longer body size than C. chon-

drostega (mean SVL 23.1 in males, 25.4 in females;

Garcia-Castillo et al. 2017), C. cracens (mean SVL 25.7

in males, 27.4 in females; Rovito and Parra-Olea 2015),

C. dimidiatus (mean SVL 24.6 in males, 25.8 in females;

Garcia-Castillo et al. 2017), and C. terrestris (mean SVL

24.2 in males, 23.0 in females; Garcia-Castillo et al.

2017). Chiropterotriton nubilus has smaller feet (FW 2.6

in one male, mean 2.3 in females) other than C. arboreus

(mean FW 3.4 in males, 3.5 in females; Garcia-Castillo et

al. 2017), C. cieloensis (mean FW 3.2 in males, 3.1 in fe¬

males; Rovito and Parra-Olea, 2015), C. chico (mean FW

4.1 in males, 4.2 in females; Garcia-Castillo et al. 2017),

C. infernalis (4.2 in males, 2.8 in one female; Rovito

and Parra-Olea, 2015), and C. priscus (mean FW 3.2 in

males, 3.5 in females; Rovito and Parra-Olea 2015). Chi¬

ropterotriton nubilus has relatively shorter limbs (LI 2.0

in one male, mean 1.5 in females) other than C. arboreus

(mean LI 0.2 in males, 1.0 in females; Garcia-Castillo et

al. 2017), C. cieloensis (mean LI -0.2 in males, 0.1 in fe¬

males; Rovito and Parra-Olea 2015), C. infernalis (mean

LI -0.7 in males, -0.5 in one female; Rovito and Parra-

Olea 2015), C. multidentatus (mean LI 0.1 in males, 1.0

in females; Rovito and Parra-Olea 2015), but relatively

longer limbs than C. dimidiatus (mean LI 3.8 in males,

4.9 in females; Garcia-Castillo et al. 2017), C. miquihua¬

nus (mean LI 4.2 in males, 4.3 in females; Rovito and

Parra-Olea 2015), and C. priscus (mean LI 3.2 in males,

3.7 in females; Rovito and Parra-Olea 2015).

Description. Moderate-sized species of Chiropterotri¬

ton, SVL 29.4 in one adult male and mean 30.5 in two

adult females (range 27.7-33.2). Head relatively narrow

and moderately long (HW 4.0 in one male, mean 4.4 in

females; HL 6.6 in one male, mean 7.4 in females), 14%

of HW/SVL in one male and 15% in females (range 14-

16), and wider shoulders (SW 3.4 in one male, mean 3.3

in females). Nostrils moderately sized and oval shaped.

Snout narrow and truncated. Eyes slightly protuberant.

Jaw muscles appear as a bulging mass behind the eyes

and beyond the margin of the jaw, when viewed from

above. Premaxillary teeth in one male not enlarged and

not piercing lip. Few maxillary teeth in males (MT 13.0)

but many in females (mean MT 41.5, range 40-43). Few

vomerine teeth in males (VT 10.0) and females (mean VT

13.5, range 13-14), arranged in a well-defined line nearly

to outer margin of the choanae. Tail large, mean TL/SVL

1.37, in one male and moderate, 1.12, in females (range

1.10-1.14). Limbs short and slender, FLL+HLL 46% of

SVL in one male and 45% in females (range 42^8). Ad-

pressed limbs separated by 2.0 costal folds in one male

(LI 2.0) and 1.5 in females (mean LI 1.5, range 1.0-2.0).

Digits slender with distinct terminal pads and moderate

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e167

Garcia-Castillo et al.

webbing just above the penultimate phalanx. Phalangeal

formulae: hand 1-2-3-2, foot 1-2-3-3-2. Digits in order

of increasing length: hand I-IV-II-III, foot I-V-II-IV-III.

Coloration in life (from photos). Predominating colors

on the upper side of the head and dorsum are Flesh Ocher

(57) or Salmon (58) on Sepia (286) background. Lateral

side of the head is Cream White (52), and underside of

head and venter are Cream White (52) background with

Glaucous (291) marks. Dorsum flanks Glaucous (291)

on Cream White (52) surface with Smoky White (261)

stipples. Tail Flesh Ocher (57) with Sepia (286) marks

on flanks and underside Perl Gray (262) with Glaucous

(291) marks. Upper side of limbs Maroon (39) with toe

tips Magenta (236) and underside of limbs Cream White

(52) surface with Glaucous (291) marks. Iris Gem Roby

(65) [Fig. 4D].

Variation of coloration in life (from photos). CARIE

0739 adult male. Upper side of head Pale Horn Color

(11) on Dark Brownish Olive (127) surface, lateral head

Cream White (52) and underside of head Pale Buff (1).

Dorsum with two stripes Pale Horn Color (11) on Sepia

(286) surface, lateral dorsum Light Lavender (201) and

underside of dorsum Pinkish White (216) with Medium

Bluish Purple (212) small dots. Upper side of tail Pale

Horn Color (11) on Sepia (286) surface and underside

of tail Pinkish White (216) with Medium Bluish Purple

(212) small dots and some Pale Horn Color (11) speck¬

les. Forelimbs Cream Color (12) and hindlimbs Fawn

Color (258) with toe tips Magenta (236). Iris Light Yel¬

low Ocher (13) [Fig. 4C].

Coloration in alcohol. Upper side of head Drab (19), lat¬

eral Dusky Brown (285) line and underside Smoke Gray

(266) with Smoky White (261) marks. Upper side of

dorsum and tail Dark Yellow Buff (54) on Dusky Brown

(285) surface, dorsum flanks Olive-Gray (265) and un¬

derside of dorsum Smoke Gray (266). Underside of tail

Grayish Horn Color (268). Upper side of limbs Olive-

Brown (278) and upper side of limbs Smoke Gray (266).

Variation in alcohol preserved coloration. Three speci¬

mens: one adult male (CARIE 0739) and two juvenile

(CARIE 0740, CARIE 1267). Upper side of head Cream

White (52) on Raw Umber (23) surface and underside

of head Smoky White (261). Dorsum with two stripes

Cream White (52) on Raw Umber (23) surface, flanks

and underside of dorsum Smoky White (261). Upper side

of tail Cream White (52) on Raw Umber (23) surface and

underside Smoky White (261). Upper side of forelimbs

Olive Horn Color (16), hindlimbs Fawn Color (258) and

underside of limbs Smoky White (261).

Measurements of holotype, tooth counts, and limb in¬

tervals. SVE 33.2, TE 37.9, AX 18.3, SW 3.5, HE 8.1,

HW 4.5, HD 2.0, projection of snout beyond mandible

0.8, anterior rim of orbit to snout 2.0, interorbital dis¬

tance 3.9, eyelid length 1.9, eyelid width 1.5, horizontal

orbit diameter 0.7, distance between corners of eyes 2.5,

FEE 6.7, HEE 7.3, snout to forelimb 10.0, snout to ante¬

rior angle of vent 31.4, tail width at base 2.2, tail depth

at base 2.3, FW 2.7, length of fifth toe 0.5, and length

of third (longest) toe 0.8. Premaxillary teeth 23, maxil¬

lary 7-20 (right-left sides) and vomerine 7-6 (right-left

sides). Adpressed limbs are separated by two costal folds.

Habitat and distribution. Eastern slopes of Cofre de Pe-

rote in central Veracruz among cloud forest between 1,520

and 2,023 m asl. Specimens found in arboreal bromeli-

ads of cloud forest fragments with low or moderate dis¬

turbance of habitat. The majority of the specimens found

were juveniles so the possibility of Ending them in ter¬

restrial environments (under cover objects) is not rejected

(Figs. 5C and 5D). Two localities where C. nubilus occurs

are within protected areas: municipal (Ea Cortadura) and

the other under private ownership (Rancho Viejo).

Natural History. Chiropterotriton nubilus was exclu¬

sively found in bromeliads and six localities on the east¬

ern slope of Cofre de Perote. Distribution could include

a fragmented band along cloud forests from Coxmatla to

Banderilla at 1,500-2,000 m asl. Samples included three

collections in three study locations (Banderilla, Ea Cor¬

tadura, and Rancho Viejo) for a total of nine sampling

events. Each sampling event applied 16 person-hours for

a total sampling effort of 144 person-hours. In four of the

nine sampling events collected were C. nubilus, varying

between one to three specimens per sampling event. Bro¬

meliads where C. nubilus were found measured 1.5-5.0

m from the ground and were medium in size (approxi¬

mately 40-60 cm in diameter). Species found in sym-

patry with C. nubilus were Aquiloeurycea cafetalera,

Parvimolge townsendi, Pseudoeurycea lynchi, and Tho-

rius pennatulus. It is conceivable that C. nubilus could

be found in sympatry with C. lavae because distributions

converge at the W slope of Cofre de Perote at 2,000 m

asl. However, C. lavae (Ea Joya) is found eight km away

from the nearest location (Banderilla) C. nubilus occurs.

Etymology. Eatin epithet nubilus (adjective: feminine

nubile, neuter nubilum) means cloudy or rain clouds, re¬

ferring to the cloud forest of Cofre de Perote where it

occurs.

Discussion

Due to recent systematic reviews, expeditions to poorly

explored areas, and recurrent held samplings in relatively

well-studied regions, the number of described species of

bolitoglossine salamanders has increased at a slow but

steady pace in recent years (e.g., Rovito et al. 2015;

Kubicki and Arias 2016; Parra-Olea et al. 2016; Garcia-

Castillo et al. 2017; Arias and Kubicki 2018). The Cofre

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 67

Two new Chiropterotriton from central Veracruz, Mexico

de Perote area has been well studied and is notable for

its salamander richness, which now includes 20 species

representing 16% of Mexican bolitoglossines (Wake et

al. 1992; Parra-Olea et al. 2001). The description of these

two new species increases the salamander diversity in the

state of Veracruz from 37 to 39 (Parra-Olea et al. 2014),

including the recently described Isthmura corrugata

(Sandoval-Comte et al. 2017).

The number of species in the genus Chiropterotriton

has increased by approximately 50% in the last four years

(Campbell et al. 2014; Rovito and Parra-Olea 2015; Gar-

cia-Castillo et al. 2017), but phylogenetic relationships

are not fully resolved. Although the phylogeny exhibited

in this study includes a greater number of well-supported

clades (PP > 0.95, BS > 70), some relationships still lack

strong support. However, previous studies (Parra-Olea

2003; Rovito and Parra-Olea 2015; Garcia-Castillo et

al. 2017) and results here show a well-supported clade

with species from central and southern Mexico, in which

C. dimidiatus is sister to the group. This group also in¬

cludes three more described species (C. chiropterus, C.

lavae, and C. orculus) plus seven previously proposed

candidate species (Figs. 1 and 2). Within the southern as¬

semblages, Chiropterotriton species form two subclades

with three sister taxa groups. The first group includes two

terrestrial forms, sister taxa C. orculus + C. sp. G and C

sp. K, for which only juveniles are known. The second

group includes C. lavae + C. sp. H and C. sp. I + C. sp.

C. The first sister pair occur in geographical proximity to

Cofre de Perote but in different elevation ranges and dif¬

ferent environmental conditions (one terrestrial and one

arboreal): Chiropterotriton lavae at 2,000 m asl in cloud

forest and C. sp. H at 3,000 m asl in pine forest. The

second sister pair (C. sp. C + C. sp. I) occur near Pico de

Orizaba, with C. sp. C at 2,400 m asl in cloud forest and

C. sp. I at 3,000 m asl in pine forest, again one species

being arboreal and the other terrestrial. In contrast to the

previous two groups, the third group is formed by five

arboreal species (C. sp. F, C. aureus, C. nubilus, C. chi-

ropterus, and C. sp. J), all distributed in similar elevation

ranges (1,200 to 2,000 m asl), and similar environmental

conditions along the cloud forest from Sierra Madre Ori¬

ental to Sierra de Juarez, Oaxaca. This continuous cloud

forest belt may have promoted a progressive coloniza¬

tion process enabling species formation through time and

isolation and could very well explain the phylogenetic

link between the species of Veracruz and Oaxaca, a pat¬

tern also seen in other bolitoglossine groups like Thorius

(Rovito et al. 2013) and Isthmura (Sandoval-Comte et

al. 2017).

The two new species of Chiropterotriton have not

been previously reported, although a sequence of C. nu¬

bilus (GenBank number KP886894) was used as a rep¬

resentative of Chiropterotriton in a bolitoglossine study

(Rovito et al. 2015). The discovery of these specimens

in a relatively well-studied area is reason to continue ex¬

plorations, especially if localities are progressively being

deforested (Williams-Linera 2007). Likewise, salaman¬

der diversity numbers are likely underestimated for cen¬

tral Veracruz (C. sp. C and C. sp. H), Puebla (C. sp. F, C.

sp. G and C. sp. I.) and Oaxaca (C. sp. J and C. sp. K)

and investigations should therefore continue as species

knowledge is more completely appreciated.

Tropical salamanders are at high risk of extinction

(Rovito et al. 2009), including the genus Chiropterotri¬

ton. It is imperative, now more than ever, to make the

best use of available bioresources by biobanking ge¬

netic material and living tissue for current and future

uses (Hassapakis and Clark 2017; Zimkus et al. 2018).

Cryobanked genetic material has been essential for sys¬

tematic and evolutionary studies of tropical salamanders,

and allowed the description of taxa thought to be extinct

(i.e., Isthmura naucampatepetl), make taxonomic rear¬

rangements (Wake et al. 2012), discover cryptic taxa

(Parra-Olea et al. 2016), and propose large genus level

phylogenies (Parra-Olea et al 2004; Rovito et al. 2013)

but may now benefit and contribute to best practices in

species conservation (Zimkus et al. 2018). Biobanking

for amphibian conservation may enable us to mitigate or

prevent the complete loss of species already at high risk

(e.g., Bolitoglossa jacksoni. Cryptotriton alvarezdelto-

roi) and archive these bioresources (e.g., cryopreserved

sperm, cell cultures, somatic tissue) and make them

available for present and future conservation technolo¬

gies (i.e.. Assisted Reproductive Technologies [ART];

Kouba et al. 2012; Kouba and Vance 2013) and method¬

ologies. Those with access to specimens (e.g., field biolo¬

gists, zoo and aquarium personnel, et al.) should consider

in their research activities and grant proposals to allow

resources and time for biobanking and preserving am¬

phibian genetic resources and living tissues to enhance

species conservation efforts (Zimkus et al. 2018). Final¬

ly, the existence of the Genome Resource Banks (GRBs)

can alleviate other issues not related to the biology of

species but rather to pressing political (difficulties of ob¬

taining field sampling due to safety) issues and economic

troubles faced by many countries worldwide.

Acknowledgements. —MGGC thanks the Posgrado

en Ciencias Biologicas program, Universidad Nacional

Autonoma de Mexico, and CONACyT for a scholarship

grant (CVU/Becario: 413761/262662). A supporting

grant for our research was kindly provided by Programa

de Apoyo a Proyectos de Investigacion e Innnovacion

Tecnologica (PAPIIT-UNAM) IN203617. We also would

like to thank Laura Marquez-Valdemar and Andrea

Jimenez for assisting us with laboratory work. Special

thanks to Aldo Lopez-Velazquez for the excellent pho¬

tography instruction and the use of his equipment. We

thank Jean H. Raflfaelli, David Darda, and an anonymous

reviewer who all made valuable comments to improve

our manuscript. Finally, we thank Omar Becerra-Soria,

Fabiola A. Herrera-Balcazar, Adriana Sandoval-Comte,

and Flor Vazquez for field assistance and the Soto-Po-

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | e167

Garcia-Castillo et al.

zos family for providing lodging in Teocelo, Veracruz.

Collection permits were provided by the Secretaria del

Medio Ambiente y Recursos Naturales (SEMARNAT):

SGPA/DGVS/00947/16, SGPA/DGVS/03038/17 and

FAUT-0303.

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(el65).

Appendix 1. Specimens examined for morphological comparisons.

Chiropterotriton aureus: Mexico, Veracruz: IBH 31041^4, 6.5 km N of Atzalan, ejido de desarrollo urbano Quetzal-

coatl.

Chiropterotriton chiropterus: Mexico, Veracruz: MVZ 85588-92, 85594, 85597-99, 85605, 85613, 85632, 1.4 miles

(mi) SW (by road), SW edge of Huatusco de Chicuellar.

Chiropterotriton lavae: Mexico, Veracruz: MVZ 106537,106548, W edge of Ea Joya along Highway (Hwy). 140; MVZ

163912-13, 163915, 171873-74, 171876, 171881, 171885, 171901, 173394-95, 173398, 192788-89, 197788, Ea

Joya; 178685, Ea Joya, Mexico Hwy. 140; MVZ 200638, forest W of Ea Joya.

Chiropterotriton nubilus: Mexico, Veracruz: IBH 31048^9, Coxmatla, 8.2 km W of Xico. CARIE 0739, Banderilla.

Chiropterotriton orculus: Mexico, Estado de Mexico: MVZ 76161, 138686, 138688, 138694, 138696-97, 138700,

138776-79,138781,138783-84,138793,138796-97,138804,200629-30, Ridge between Volcanoes Popocatepetl

and Iztaccihuatl along Mexico Hwy. 196, 16.2 km E (by road) of hwy. 115.

Amphib. Reptile Conserv.

December 2018 | Volume 12 | Number 2 | el 67

Garcia-Castillo et al.

Amphib. Reptile Conserv.

Mima Grisel Garcia-Castillo is a Mexican Ph.D. student from Universidad Nacional Autonoma de

Mexico in Mexico City. She began her study of salamanders in Dr. Parra's Lab in 2011 where she

received a Master’s degree focused on molecular phylogeny of the genus Chiropterotriton. Since that

time, she’s worked on molecular systematics, taxonomy, morphology, biogeography, and evolution of

this group as part of her doctoral thesis she is currently finishing.

Angel Fernando Soto-Pozos received a Master’s degree in Biological Sciences in 2018 from the

Institute de Biologia, Universidad Nacional Autonoma de Mexico. His research interests include edge

effect in communities of amphibians and reptiles, diversity, conservation of endangered species of

salamanders in fragmented forest from Veracruz State, and the historic role of diseases in salamander

declines.

Jose Lnis Agnilar Lopez received his Master's degree in 2010 from the Institute de Ecologia A.C.

and is currently a doctoral student at the same institution. His research interests include diversity

patterns, taxonomy, and conservation of amphibians and reptiles in tropical forests.

Eduardo Pineda is a titular researcher at the Institute de Ecologia, A.C. in Xalapa, Mexico. His

research is focused on understanding the relationship between the transformation of tropical forest

and biodiversity at different spatial scales, recognizing the importance of conserved areas and

modified habitats to maintain amphibian diversity, and assess the current situation, through fieldwork,

of amphibian species in imminent danger of extinction. Currently he has several graduate students

addressing topics in ecology and/or conservation of amphibians in Mexico and Eatin America.

Gabriela Parra Olea is a titular researcher at the Institute de Biologia, UNAM, Mexico. Her research

is focused on molecular systematics and conservation of Mexican amphibians. Her laboratory is formed

by students and postdocs from different countries as Mexico, Guatemala, Costa Rica, Colombia, and

Argentina, all working on research projects in systematics and taxonomy, conservation genetics, and

the impact of infectious diseases, specifically chytridiomycosis, on the conservation of amphibians.

54 December 2018 | Volume 12 | Number 2 | el67

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptiie Conservation

12(2) [General Section]: 1-29 (el67).

urn:lsid:zoobank.org:pub:98D842A8-470C-46Dl-A60F-6CF474D8B902

Four new species of the genus Cnemaspis Strauch, 1887

(Sauria: Gekkonidae) from the northern Western Ghats, India

^Amit Sayyed, ^Robert Alexander Pyron, and ^R. Dileepkumar

'Wildlife Protection and Research Society, INDIA ^Department of Biological Sciences, The George Washington University, 2029 G St. NW,

Washington, D.C. 20052, USA ^YIPB Department of Biotechnology, University of Kerala, INDIA

Abstract —^We report four new species of geckos of the genus Cnemaspis Strauch, 1887 from the northern

Western Ghats, India. Cnemaspis iimayei sp. nov. is diagnosable by the following combination of characters:

dorsal scales heterogeneous; spine-like tubercles absent on flank; pre-anal scales larger than ventral; 26-27

scale rows across the belly, between lowest rows of dorsal scales; supralabial I narrowly contacting nasal;

mental posteriorly pointed; two pairs of postmentals; males with 4-5 femoral pores on each side. C. ajijae

sp. nov. is diagnosable by: dorsal scales heterogeneous; granular keeled scales intermixed with large keeled

depressed scales; conical and spine-like tubercles absent on flank; 29-30 scale rows across the belly; three

pairs of postmentals; males with 3-4 femoral pores on each side. C. amboiiensis sp. nov. is diagnosable by:

dorsal scales heterogeneous; granular, keeled small scales intermixed with some large keeled scales; conical

and spine-like tubercles on flank; scales on snout feebly keeled; dorsal scales on forelimb and hindlimb

tricarinate; males with 3-4 pre-anal pores and 3-4 femoral pores on each side of the thigh. C. mahabaii sp.

nov. is diagnosable by: dorsal scales on body heterogeneous; conical and spine-like tubercles absent on

flank; 26-27 scale rows across the belly; scales on ventral part of neck feebly carinate; dorsal scales on

forelimb and hindlimb are strongly keeled; three femoral pores on each side. These four new species are

distinguished by morphological comparison, morphometric, and genetic analysis, leading to a re-appraisal of

the genus Cnemaspis in India. The description of these new species from the Western Ghats suggests that our

understanding of species richness within this genus is still incomplete. Understanding the diversity of species

in Cnemaspis will help in determining the conservation status of these threatened taxa.

Keywords. Species conservation, morphology, Cnemaspis Iimayei sp. nov., Cnemaspis ajijae sp. nov., Cnemaspis

amboiiensis sp. nov., Cnemaspis mahabaii sp. nov.

Citation: Sayyed A, Pyron RA, Dileepkumar R. 2018. Four new species of the genus Cnemaspis Strauch, (Sauria: Gekkonidae) from the northern

Western Ghats, India. Amphibian & Reptile Conservation 12(2) [General Section]: 1-29 (el57).

NoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided

the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication

credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation-, official journal website <amphibian-

reptile-conservation. org>.

Received: 18 December 2017; Accepted: 29 May 2018; Published: 24 August 2018

Introduction

The genus Cnemaspis Strauch, 1887, is one of the most

species-rich genera of the family Gekkonidae and is

distributed from Africa to Southeast Asia. Despite their

superficial morphological similarity, molecular phylog-

eny has shown that the genus Cnemaspis is not a mono-

phyletic group (Gamble et al. 2012; Pyron et al. 2013).

Because the type species C. boulengerii Strauch, 1887 is

from Southeast Asia (Strauch 1887), South Asian and Af¬

rican members of the genus will probably require differ¬

ent generic names in future revisions. While a full-scale

revision of the genus remains out of reach due to a lack

of sampling, ongoing fieldwork in areas of high gecko di¬

versity continues to yield apparently new species, which

must therefore be described under the name Cnemaspis.

In addition to molecular phylogenetics, these descrip¬

tions are facilitated by a rich literature on morphological

variation in the group.

The genus is characterized by slender digits, clawed,

rarely dilated; two distal phalanges compressed, forming

an angle with the basal portion of the digits, the lower

surface with rows of plates; body more or less depressed,

granular, or tubercular above; tail more or less cylin¬

drical; pupils round; eyelid distinct all around the eye;

males with or without pre-anal or femoral pores; a well-

developed hypo-ischium, post-anal bones and sacs, and

a reduced hyoid apparatus, with only one pair of basi-

branchials; presence of three or four sternal ribs; inter¬

clavicles well developed and cruciform (in the Oriental

CorrespondGnCG. * amitsayyedsatara@gmail.com, ^ rpyron@colubroid.org, ^ dileepkamukumpuzha@gmail.com (Corresponding author)

Amphib. Reptile Conserv. 1 August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

species) or much reduced and with only very small trans¬

verse arms (in the African species); adhesive toe pads

absent (in the Oriental species) or present (in the Afri¬

can species); leaf toes and paraphalanges absent (Smith

1933, 1935; Gamble et al. 2012).

The genus currently includes 135 recognized species

(Uetz and Hosek 2017), 29 of which are known from

India. However, the true diversity of the group is likely

much higher throughout its range, and in India in particu¬

lar. During our fieldwork in India, we identified several

populations that varied from the 29 known species with

respect to several of the characters described above. Sub¬

sequent molecular phylogenetic analyses revealed them

to be distinct species, and also clarified species limits in

related taxa. In this study we describe four new species of

Cnemaspis from northern Western Ghats of Maharashtra

State, India. We show that the proposed species are mor¬

phologically distinct from their Indian congeners. Fur¬

ther, we also provide a molecular phlyogenetic analysis

of Cnemaspis species from India based on mitochondrial

16S rRNA sequences.

Materials and Methods

Specimen collection and museum details: Specimens of

the four new species were collected by hand from differ¬

ent localities in Maharashtra State, India from February

2015 to December 2016. See Appendix and data sources

for the details of specimens of the known species col¬

lected from parts of the Western Ghats for genetic analy¬

sis. The specimens were photographed in life, then eutha¬

nized (George 1973), fixed in formalin, and preserved in

70% ethyl alcohol. For the genetic analysis, a few of each

species were preserved in absolute alcohol. All speci¬

mens used in this work were collected by Amit Sayyed

and Abhiiit Nale. The materials referred to are deposited

in the collection of the Bombay Natural History Society

(BNHS), Mumbai, and in the collection of Zoological

Survey of India (ZSI) Akurdi, Pune, Maharashtra, India.

During the study period we have collected 43 addi¬

tional specimens for examination from several localities

in Goa, Karnataka, and Maharashtra which are morpho¬

logically similar to C. goaensis; out of these, 12 speci¬

mens were used for the molecular work, including topo-

types of C. indraneildasii, BNHS 2460 and BNHS 2461,

collected from Gund, Uttara Kannada, Karnataka, and

specimen no. BNHS 2462 and BNHS 2463, collected

from Dandeli, Karnataka. Specimen no. CnKh 33, ChKh

34, CnKo 48, and CnKo 49, were collected from the hu¬

man habitation at Kolhapur, district Maharashtra. Speci¬

men no. CnInAr 1 and CnInAr 2 were collected from

Agumbe road. Specimen no. CnInA 1 and CnInA 2 were

collected near Agumbe, Shimoga district, Karnataka. We

have examined 18 males and 13 females including BNHS

2460 and BNHS 2461, collected near the type locality

of C. indraneildasii. Other than C. indraneildasii we en¬

countered only C. heteropholis in the ranges of Gund and

Agumbe, Karnataka. We also examined live specimens

(not collected) of C. goaensis at Danoli, Sawantwadi

Sindhudurg district, Maharashtra.

Morphological study: The following measurements

were taken with a Yamayo digimatic caliper (to the near¬

est 0.1 mm): snout vent length (SVL; from tip of snout

to vent), trunk length (TRL; distance from axilla to groin

measured from posterior edge of forelimb insertion to

anterior edge of hind limb insertion), trunk width (TW;

maximum width of body), tail length (TL; from vent to

tip of tail), tail width (TW; measured at widest point of

tail), head length (HL; distance between retroarticular

process of jaw and snout-tip), head width (HW; maxi¬

mum width of head), head depth (HD; maximum depth of

head, from occiput to underside of jaws), forearm length

(FL; from base of palm to elbow), tibia length (TBL;

knee to tarsal), eye to nares distance (E-N; distance be¬

tween anterior most point of eye and nostril), eye snout

to distance (E-S; distance between anterior most point of

eye and tip of snout), eye to ear distance (E-E; distance

from anterior edge of ear opening to posterior corner of

eye), ear length (EE; maximum distance end to end of ear

opening), distance between nares (IN; right to left nare),

orbital diameter (OD; greatest diameter of orbit), and in¬

ter orbital snout distance (10; narrowest distance greatest

diameter between orbits on frontal bone). Meristic data

recorded for all specimens were: the number of supral-

abial scales (SupraE), infralabial scales (InfraE), femoral

pores (FPores), pre-anal pores (PaPores), and lamellae

under digits of manus (MEam) and pes (PEam) for both

left (E) and right (R) sides (lamellae counts taken from

the scale just behind claw to first interphalangial joint ex¬

cluding large scansors), as well as scales across the belly

between the lowest rows of dorsal scales (MVS), spine

like tubercles (Sptub), and lamellae under IVth digit of

pes (Eamp IVth). Scale counts and external observations

of morphology were made using a Eeica stereo micro¬

scope. For the geographical coordinates, altitude, and for

temperature readings, we used a Kestrel 4500 receiver.

Genetic analysis: Muscle tissue was collected from 33

fresh specimens. DNA extraction, PCR amplification,

and sequencing protocols follow Sayyed et al. (2016).

Sequences were edited in Geneious (Biomatters Etd.) and

analyzed with the BEAST tool (Altschul et al. 1990) for

similar sequences in the NCBI (www.ncbi.gov) database.

These sequences have been deposited in GenBank (ac¬

cession numbers provided in Table 7). Sequences were

included in the genetic data from Sayyed et al. (2016).

We included Eublepharis (Family Eublepharidae), Hemi-

dactylus, and SE Asian Cnemaspis as outgroups.

Gene sequences were aligned using MUSCEE (Edgar

2004) under the default settings. The best-fit nucleotide

substitution model was selected from 56 models avail¬

able in PhyME (Guindon et al. 2010) using TOPAEi v2

(Milne et al. 2008) based on minimum Bayesian Infor-

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

0.07

100

loor

■

loor

100

—

100

[;

loor

100

IOC

if;;;;;;:

c;;;;;;

1001

loor

901

Cnemaspis goaensis ZSI R 1044

Cnemaspis goaensis ZSI R 1045

Cnemaspis 'indraneiidasii' BNHS 2462

Cnemaspis 'indraneiidasii' BNHS 2463

Cnemaspis 'indraneiidasii' BNHS 2460

Cnemaspis 'indraneiidasii' BNHS 2461

Cnemaspis amboliensis CnGo35

Cnemaspis amboliensis CnGo36

Cnemaspis amboliensis CnAmSl

Cnemaspis amboliensis CnAmSO

Cnemaspis adii BNHS 2464

Cnemaspis adii BNHS 2465

Cnemaspis yercaudensis ASPC144

Cnemaspis yercaudensis ASPC145

Cnemaspis otai ASPC146

Cnemaspis otai ASPC147

Cnemaspis gracilis A131

Cnemaspis gracilis A132

Cnemaspis indica ASPC116

Cnemaspis indica ASPC117

Cnemaspis girii BNHS 2445

Cnemaspis girii BNHS 2446

Cnemaspis limayei ZSI R 1052

Cnemaspis limayei ZSI R 1053

Cnemaspis mahabali A136

Cnemaspis mahabali A137

Cnemaspis mahabali A138

Cnemaspis ajijae ZSI R 1059

Cnemaspis ajijae ZSI R 1060

Cnemaspis ajijae ZSI R 1057

Cnemaspis ajijae ZSI R 1058

Cnemaspis ajijae ZS I R 1055

Cnemaspis ajijae ZSI R 1056

Cnemaspis flaviventralis ZSI-WRC 1042

Cnemaspis flaviventralis ZSI-WRC 1043

Cnemaspis littoralis ASPC118

Cnemaspis littoralis ASPC119

Cnemaspis koihapurensis BNHS 2447

Cnemaspis koihapurensis BNHS 2448

Cnemaspis heteropholis BNHS 2466

Cnemaspis kottiyoorensis ASPC148

Cnemaspis wynadensis ASPC149

Hemidactylus brookii AY517570

Hemidactylus mercatorius AY517583

Hemidactylus flaviviridis AY496699

Cnemaspis limi HQ896026

Eublepharis macularius AB028762

Fig. 1. Maximum likelihood tree of mitochondrial 16S rRNA gene. Eublepharis macularius is used as an out-group. Values along

the node are percent bootstraps for 1,000 bootstrap iterations.

mation Criterion (BIC) value (Schwarz 1978; Nei and

Kumar 2000). The best model was used to perform maxi¬

mum likelihood (ML) analysis using PhyML (Guindon

et al. 2010). Reliability of the phylogenetic tree was es¬

timated using bootstrap values run for 1,000 iterations.

Phylogenetic tree was edited in FigTree vl .4.2 (Rambaut

2009). The ML tree is provided with bootstrap values

converted to percentages. We also calculated K2P-cor-

rected pairwise distances between sequences in MEGA 7

(Kumar et al. 2016).

Results

In addition to the four apparently new species, topotypic

C. goaensis and C. indraneiidasii formed a monophyletic

group (Fig. 1) with minimal genetic distances (Table 5). It

is therefore likely that C. indraneiidasii is a synonym of

C. goaensis. However, pending further investigation, the

taxonomic status of C. indraneiidasii will be discussed

elsewhere. According to our study, C. goaensis is widely

distributed in the ranges of southern Maharashtra, Goa,

and Karnataka, India. See Fig. 28 for the distribution.

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | el 57

Sayyed et al.

nov.

Fig. 2. Map showing the type localities of four new species: Cnemaspis limayei sp. nov. indicated by yellow circle, Cnemaspis ajijae

sp. nov. indicated by blue circle, Cnemaspis amboliensis sp. nov. indicated by pink circle, Cnemaspis mahahali sp. nov. indicated

by red circle, locations in the northern Western Ghats, Maharashtra, India.

All four new species collected from parts of the north¬

ern Western Ghats Maharashtra, India (Fig. 2) form

monophyletic groups distinct from the other known spe¬

cies of Western Ghats Cnemaspis for which we could

obtain genetic data from topotypic material (Fig. 1).

Pair-wise genetic distances are provided (Table 5). Ge¬

netic distance between Cnemaspis of the Western Ghats

ranged from 2.7 to 21.0%. Since 16S rRNA has not been

used in Cnemaspis for species delimitation, there is no

comparative account of genetic divergence for delineat¬

ing species based on this marker. However, for other taxa

like frogs of the genus Indirana, Dahanukar et al. (2016)

showed a genetic gap between 1.9-2.4% genetic diver¬

gence in 16S rRNA indicating that distance more than

2.4% were reliable for species delimitation. Based on the

genetic and morphological divergence of the four popu¬

lations, we diagnose them as follows.

mm. Dorsal scales on trunk heterogeneous; granular, fee¬

bly keeled scales intermixed with large keeled depressed

scales; conical and spine-like tubercles absent on flank;

ventral scales smooth, larger than dorsal; pre-anal scales

larger than ventral; 26-27 scales across the belly between

lowest rows of dorsal scales; mental posteriorly pointed;

two pairs of postmentals, primary larger than second¬

ary, secondary postmentals touching first and second in¬

fralabials; nostrils in narrow contact with supralabial I;

seven lamellae on digit I of the manus and 9-11 on digit

IV, 7-8 on digit I of the pes, and 10-12 on digit IV. Males

with 4-5 femoral pores on each side, pre-anal pores ab¬

sent. Tail base visibly swollen, median sub-caudal scales

not enlarged; one triangular, slightly keeled post-anal,

very small tubercles along each side present in both sex¬

es; broadly acute, prominent tubercles with small keeled

scales dorsally on tail.

Systematics

Cnemaspis limayei sp. nov. (Figs. 3-6)

urn:lsid:zoobank.org:act:BBD5A08D-AEB2-4153-976B-ACCA9B3880C3

Holotype: BNHS 2454 (adult male); collected at night

on 12 February, 2015 on a tree branch near a dry stream

at Marutiwadi (16.221N, 73.475E; 132 m asl), near

Phondaghat, Sindhudurg district, Maharashtra, India.

Paratypes: BNHS 2455 (female), ZSI-WRC R/1051,

ZSI-WRC R/1052 (male) and ZSI-WRC R/1053 (fe¬

male); same locality as holotype on the tree trunk and on

the rocks of a dry stream, collected at the same place and

time as holotype.

Diagnosis: Small-sized Cnemaspis, SVL less than 31

Amphib. Reptile Conserv.

Description of Holotype: BNHS 2454 (adult male);

has an entire, original tail (Fig. 4a, b). SVL 29.72 mm;

head short (HL/SVL = 0.13), wide (HW/HL = 1.0), not

strongly depressed (HD/HL = 0.63), distinct from elon¬

gate neck; canthus rostralis not prominent; loreal slightly

inflated; snout slightly longer (E-S/HL = 0.77), longer

than eye diameter (OD/E-S = 0.30); scales on snout and

canthus rostralis large, keeled, slightly larger than those

on forehead and interorbital; scales on forehead, inter¬

orbital, and occipital smaller, slightly keeled, granular

(Fig. 6a); eye small (OD/HL = 0.23), pupil round, su-

perciliaries not elongated; ear opening small, deep, oval

shaped (0.07); eye to ear distance much greater than di¬

ameter of eye (E-E/OD = 2.0); rostral wider (1.12 mm)

than deep (0.63 mm), slightly swollen, weakly divided;

nostrils in narrow contact with supralabial I; rostral in

4 August 2018 | Volume 12 | Number 2 | el 57

Four new species of the genus Cnemaspis

Table 1. Mensural and meristic data for the type series of Cnemaspis limayei sp. nov. Abbreviations as stated in Materials and

Methods (* = regenerated tail, ** = sub-adult, ? = broken finger, - = pores not present).

Measurement (mm)

Holotype

Paratypes

BNHS 2454

ZSI-WRC R/1052

ZSI-WRC R/1051

BNHS 2455

ZSI-WRC R/1053

male

male*

male**

female

(SVL)

29.72

30.11

25.71

30.16

29.74

(TRL)

12.14

11.94

9.80

13.88

13.96

(TW)

5.45

5.47

4.53

6.23

6.37

(TL)

36.99

30.69

29.46

31.19

28.35

(TW)

2.82

2.69

2.07

2.69

2.42

(HL)

4.92

4.81

4.39

4.83

4.68

(HW)

5.00

5.09

4.96

5.07

5.06

(HD)

3.12

3.49

2.87

3.15

3.11

(FL)

3.77

3.71

3.54

3.93

3.81

(TBL)

4.37

4.43

4.26

4.84

4.71

(E-N)

3.27

3.50

3.19

3.38

3.25

(E-S)

3.83

4.17

3.87

4.02

4.15

(E-E)

2.43

2.73

2.35

2.83

2.86

(EE)

0.07

0.06

0.03

0.08

0.07

(IN)

0.94

0.82

0.80

1.00

0.99

(OD)

1.18

1.17

0.99

1.06

1.00

(10)

3.28

3.93

3.60

3.63

3.73

HE/SVE

0.13

0.16

0.17

0.16

HW/SVE

0.17

0.19

0.17

HW/HE

1.02

1.06

1.13

1.05

1.08

E-S/HE

0.78

0.87

0.88

0.83

0.89

HD/HE

0.63

0.73

0.65

0.66

E-S/HW

0.76

0.82

0.78

0.79

0.82

OD/E-S

0.31

0.28

0.26

0.24

OD/HE

0.24

0.23

0.22

0.21

EE/HE

0.01

E-E/OD

2.06

2.33

2.37

2.67

2.86

TRE/SVE

0.41

0.40

0.38

0.46

0.47

FE/SVE

0.13

0.12

0.14

0.13

TBE/SVE

0.15

0.17

0.16

TE/SVE

1.24

1.01

1.14

1.03

0.95

MVS

SupraE

9/8

9/9

9/8

8/8

8/7

InfraE

8/8

7/8

8/7

FPores

5 on right, 4

on left

5 on right, 4 on left

4 on each side

MEam R

7-8-10-10-8

7-9.10-10-9

7-8-10-10-8

PEamR

7-8-10-10-10

8-9-11-10-?

7-8-10-11-10

8-8-10-10-10

MEam E

7-8-10-10-8

7-9-11-11-9

7-8-10-10-8

7-8-10-9-8

PEam E

7-8-11-10-11

8-9-12-12-11

7-8-12-11-10

7-8-11-10-10

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

Fig. 3. Holotype male (BNHS 2454) of Cnemaspis limayei sp.

nov. in life.

Fig. 5 (above), (a) Dorsal, (b) ventral, and (c lateral view of

the mid body of Cnemaspis limayei sp. nov. Holotype (BNHS

2454).

Fig. 6 (right), (a) Dorsal, (b) ventral, and (c) lateral view of

the head, (d) ventral view of right manus, (e) ventral view of

right pes, and (f) the lower body of Cnemaspis limayei sp. nov.

Holotype (BNHS 2454).

Fig. 4. (a) Dorsal and (b) ventral view of the full body of

Cnemaspis limayei sp. nov. Holotype (BNHS 2454).

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

Fig. 7. Habitat of Cnemaspis Umayei sp. nov.

contact with supralabial I; single row of scales separates

orbit from supralabials; mental triangular, wider (1.43

mm) than deep (1.06 mm), posteriorly pointed; two pairs

of postmentals, primary larger than secondary, second¬

ary postmentals touching first and second infralabials;

single enlarged gular scale prevents posterior contact of

left and right postmentals (Fig. 6b); infralabials bordered

by row of elongated scales; supralabials to angle of jaw-

nine right, eight left; infralabials- eight left, eight right

(Fig. 6c); body relatively slender, not elongate (TRL/

SVL = 0.40), without ventrolateral folds; dorsal scales on

trunk heterogeneous, granular, feebly keeled intermixed

with large keeled depressed scales (Fig. 5a); conical and

spine-like tubercles absent on fiank (Fig. 5c); ventral

scales smooth, larger than dorsal, pre-anal scales larger

than ventral; 27 scales across the belly between lowest

rows of dorsal scales (Fig. 5b); pre-anal pores absent,

five femoral pores on right, four on left side (Fig. 6f);

fore and hind limbs relatively short, slender; forearm

and tibia short (FL/SVL = 0.12; TBL/SVL = 0.14); in¬

terdigital webbing absent. Lamellae 7-8-10-10-8 (right

manus. Fig. 6d), 7-8-10-10-10 (right pes. Fig. 6e), rela¬

tive length of digits (measurements in mm): IV (3.09) >

III (2.74) > V (2.65) > II (1.84) > I (1.36) (right manus),

IV (3.95) > III (3.16) > V (3.14) > II (2. 58) > I (1.61)

(right pes); tail sub-cylindrical, longer than snout-vent

length (TL/SVL = 1.22), base visibly swollen, median

sub-caudal scales not enlarged; triangular, slightly keeled

post-anal, very small tubercles along each side present;

broadly acute, prominent tubercles with small keeled

scales on dorsal tail.

Color in life (Fig. 3): Dorsal body brown; asymmetrical

black marks with yellow dots on head; yellow dots more

on snout than head; pale brownish-black line from nasal

to mid eye; supraciliaries yellow, alternatively black; iris

orange with thin orange line bordering pupil; pupil cir¬

cular, black; supralabials brown and yellow; lower jaw

and ventral side of throat yellow; black “W” mark poste¬

riorly yellow, present on basal part of head; black arrow¬

head shaped patch on nuchal; semicircular black marks,

posteriorly yellow, present on dorsal vertebra to sacral

vertebra; few yellow spots present on fiank; fore and

hindlimbs with brown background with pale yellow and

black patches; ventral body whitish-yellow; tail dorsally

brown, with irregular black patches, ventrally grayish.

Color pattern in preservation (Fig. 4a, b): Dorsum col¬

or changes in to brownish-grey, black marking on body

faded; ventral body color turned in to faded yellow; ven¬

tral head color changes in to yellow with scattered grey

patches; ventral side of limbs and tail turned in to grey.

Variation: Adult specimens range in the type series size

29 to 30.16 mm (Table 1). All paratypes resemble the

holotype in most respects except for the following char¬

acters: 9-11 lamellae under fourth digit of manus, 7-8

lamellae under first digit of pes and 10-12 under fourth

digit of pes. ZSI-WRC R/1051 male has four femoral

pores on each side.

Etymology: Specific epithet is a patronym in honor

of Mr. Sunil. B. Limaye, Chief Conservator of Forests

(Wildlife) Pune.

Common name: Limaye’s Day Gecko

Natnral history: This is a nocturnal species found ac¬

tive on the tree trunk above 1-3 meters above ground, on

the rock bed of a dried stream surrounded by forest (Fig.

7). Individuals were also observed (not collected) on the

walls of houses made of mud and on compound wall

structures of stone in Marutiwadi village. The population

of this species was not dense at the locality and nearby

area where the type series were collected. Gravid females

were observed in the months of October and November

at the study area. The types were found sympatrically

with Hemidactylus sp., Eutropis cf. macularia, Ahaetulla

nasuta, and Amphiesma beddomei in the same habitat.

Distribution: This species is currently known only from

the type locality at Marutiwadi (16.221N, 73.475E; 132

m asl), near Phondaghat, Sindhudurg district, Maharash¬

tra, India (Fig. 2).

Remarks: Cnemaspis Umayei is distinguished from C.

girii and C. flaviventralis by the absence of conical and

spine-like tubercles on fiank, having more femoral pores,

and pre-cloacal scales larger than ventral body scales

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | el 57

Sayyed et al.

(Table 6). Additionally, C. girii and C. flaviventralis

are reported from higher elevations (Mirza et al. 2014;

Sayyed et al. 2016), whereas C. limayei is reported at

lower elevations -132 m asl. In this study we did not ob¬

serve C. girii anywhere except from the Kaas plateau and

Chalkewadi plateau, Satara district, Maharashtra, India,

suggesting it is endemic to Satara.

Comparison: Cnemaspis limayei sp. nov. can be sepa¬

rated from all its Indian congeners based on a combina¬

tion of characters including: SVL 30.2 mm maximum in

adults (vs. SVL 61.0 mm in C. anaikattiensis, 50.6 mm

in C. beddomei, 45.1 mm in C. heteropholis, 41.7 mm

in C. kottiyoorensis, 42.3 mm in C. nilagirica, and 42.7

mm in C. sisparensis); femoral pores present in males

(vs. absent in C. assamensis, C. beddomei, C. nairi, and

C. ornata); 4-5 femoral pores on each side (vs. six in C.

heteropholis, 5-15 in C. jerdonii, 15-18 in C. littoralis,

and 7-8 in C. sisparensis)', spine-like tubercles absent on

flank (vs. present in C. assamensis, C. gracilis, C. in-

draneildasii, C. monticola, C. mysoriensis, C. nilagirica,

and C. tropidogaster)', pre-anal pores absent (vs. present

in C. adii, C. andersonii, C. australis, C. beddomei, C.

goaensis, C. gracilis, C. monticola, C. nairi, C. ornata,

C. otai, C. tropidogaster, C. wicksii, and C. yercauden-

sis)', median sub-caudal scales not enlarged (vs. enlarged

in C. adii, C. australis, C. indica, C. littoralis, C. sispa¬

rensis, and C. wynadensis)', lamellae under fourth digit

of pes 10-12 (vs. 12 in C. indraneildasii and 20-21 in

C. kottiyoorensis)', dorsal scales on trunk heterogeneous

(vs. homogeneous in C. adii, C. boiei, C. indica, C. jer¬

donii, C. kolhapurensis, C. littoralis, C. mysoriensis, C.

nilagirica, and C. wynadensis)', midventral scales 26-27

(vs. 20-22 in C. heteropholis)', two pairs of postmentals

(vs. three pairs in C. anaikattiensis)', supralabials to angle

of jaws 7-9, broadly acute shape, small tubercles inter¬

mixed with small keeled scales on tail (vs. supralabials

to angle of jaws six, dorsal scales on tail granular and

smooth in C. kottiyoorensis).

The new species is similar in general appearance to

Cnemaspis girii but differs by absence of conical tu¬

bercles on flank (vs. conical tubercles present on flank);

forehead, interorbital, and occipital with smaller slightly

keeled granular scales, larger tubercles not present (vs.

forehead and interorbital region, occipital and temporal

region with much smaller, unkeeled, granular scales in¬

termixed with larger tubercles); pre-cloacal scales larger

than ventral body scales (vs. pre-cloacal scales and ven¬

tral body scales are equal); males with 4-5 femoral pores

on each side (vs. males with four femoral pores on each

side); nine supralabials to angle of jaws (vs. eight supra¬

labials to angle of jaws); maximum SVL 31 (vs. maxi¬

mum SVL 35 mm); 10-12 lamellae under fourth digit of

pes (vs. 17-20) (Table 6); from C. flaviventralis by hav¬

ing SVL less than 31 mm (vs. maximum SVL 37 mm);

conical and spine-like tubercles absent on flank (vs. large

keeled conical tubercles present on flanks); 26-27 mid-

ventrals (vs. 28-29 midventrals) (Table 6); two pairs of

postmentals (vs. three pairs of postmentals); males with

4-5 femoral pores on each side (vs. males with three

femoral pores on each side); pre-cloacal scales larger

than ventral body scales (vs. pre-cloacal scales same as

ventral body scales).

Cnemaspis ajijae sp. nov. (Figs. 8-11)

urn:lsid:zoobank.org:act:A88992C2-BBC4-4A32-80C9-6683CllA9252

Holotype: BNHS 2456, adult male; collected by hand at

night on 15 November, 2015, on a tree trunk beside a dry

stream surrounded by dense forest, at Mahabaleshwar

(17.545N, 73.403E; 1,377 m asl), Satara district, Maha¬

rashtra, India.

Paratypes: ZSI-WRC R/1054, ZSI-WRC R/1056 (male),

and ZSI-WRC R/1055 (female) share the same data and

same locality as the holotype and ZSI-WRC R/1057

(male) and ZSI-WRC R/1058 (female), in the dense

forest at Mahabaleshwar (17.553N, 73.391E; 1,291 m

asl), Satara district, Maharashtra, India, collected on 15

November, 2015; ZSI-WRC R/1059, ZSI-WRC R/1060

(male), and BNHS 2457 (female), Panchgani (17.554N,

73.483E; 1,323 m asl), Satara district, Maharashtra, In¬

dia, collected on 29 October, 2015.

Diagnosis: A medium sized Cnemaspis, SVE less than

37 mm. Dorsal scales on trunk heterogeneous; granular,

keeled scales intermixed with large keeled, depressed

scales; conical and spine-like tubercles absent on flank;

ventral scales larger than dorsal, smooth; 29-30 scales

across belly between lowest rows of dorsal scales; three

pairs of postmentals, primary larger than others, sec¬

ondary postmentals touching first and second infralabi¬

als; third chinshield smaller than second; 7-8 lamellae

on digit I of manus and 9-12 on digit IV, 7-8 on digit I

of pes and 10-13 on digit IV; males with three or four

femoral pores, pre-anal pore absent; median row of sub-

caudals smooth, imbricate not enlarged; small triangu¬

lar tubercles along each side present in both sexes; very

small acute, prominent tubercles with small keeled scales

on dorsal tail.

Description of Holotype: BNHS 2456 (adult male);

has an entire, original tail (Fig. 9a, b). SVE 29.80 mm;

head moderately short (HE/SVE = 0.17), wide (HW/HE

= 1.03), not strongly depressed (HD/HE = 0.61), distinct

from elongate neck; canthus rostralis not prominent;

snout slightly longer (E-S/HE = 0.75), much longer than

eye diameter (OD/ E-S = 0.30); weakly keeled, granular

scales on snout and on maxilla; scales on forehead and on

interorbital granular, smaller than snout (Fig. 11a); eye

fairly small (OD/HE = 0.23), pupil round; superciliaries

not elongated; ear opening deep, circular, small (EE/HE

= 0.02); eye to ear distance much greater than diameter

of eyes (E-E/OD = 2.43); rostral wider (1.51 mm) than

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

Fig. 8. Holotype male (BNHS 2456) of Cnemaspis ajijae sp.

nov. in life.

Fig. 10 (above). Dorsal, (b) ventral, and (c) lateral view of the

mid body of Cnemaspis ajijae sp. nov. Holotype (BNHS 2456).

Fig. 11 (right), (a) Dorsal, (b) ventral, and (c) lateral view of

the head, (d) ventral view of right manus, (e) ventral view of

right pes, and (f) the lower body of Cnemaspis ajijae sp. nov.

Holotype (BNHS 2456).

Fig. 9. (a) Dorsal and (b) ventral view of the full body of

Cnemaspis ajijae sp. nov. Holotype male (BNHS 2456).

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

Fig. 12. (a) Dense evergreen forest of Mahabaleshwar, (B)

showing wall of lateritic plateau (tableland of Panchgani) and

connected forest, habitat of Cnemaspis ajijae sp. nov.

deep (0.58 mm), slightly swollen, divided; nostrils not

in contact with supralabial I; rostral in contact with su-

pralabial I; two rows of scales separate orbit from su-

pralabials; mental triangular, approximately equivalent

(1.68 mm) as long (1.14 mm); three pairs of postmen¬

tals, primary larger than others, secondary postmentals

touching to first and second infralabials, third chinshiled

smaller than second; single enlarged gular scale prevents

posterior contact of left and right postmentals (Fig. 11b);

infralabials bordered by a row of small elongated scales;

supralabials to angle of jaw- seven right, eight left; in¬

fralabials- seven left, seven right (Fig. lie); body rela¬

tively slender, not elongate (TRL/SVL = 0.43) without

ventrolateral folds; dorsal scales on trunk heterogeneous,

granular, keeled, intermixed with large keeled depressed

scales (Fig. 10a); conical and spine-like tubercles absent

on fiank (Fig. 10c); ventral scales smooth, larger than

dorsal; midbody scales across belly between the lowest

rows of dorsal scales 29 (Fig. 10b); three femoral pores

on each side, pre-anal pores absent (Fig. Ilf); fore and

hindlimbs relatively short, slender; forearm and tibia

short (FL/SVL = 0.14; TBL/SVL = 0.14); interdigital

webbing absent; lamellae 7-8-10-10-9 (right manus.

Fig. lid), 7-8-10-11-10 (right pes. Fig. lie); relative

length of digits (measurements in mm): IV (2.92) > V

(2.63) >III (2.41) >II (2.16) > I (1.84) (right manus); IV

(3. 79) > V (3.30) > III (3.05) > II (2.85) > I (1.35) (right

pes); tail sub-cylindrical, longer than snout-vent length

(TL/SVL = 1.10); tail base visibly swollen; triangular

shape post-anal small keeled tubercles along each side

present; median row of sub-caudals smooth, imbricate,

not enlarged; very small acute, prominent tubercles with

small keeled scales on dorsal tail.

Color in life (Fig. 8): Dorsum ground color grey; heart-

shaped mark on the dorsal head; semicircular dark-black

mark on the nape; vertically elongate black mark on cer¬

vical; black and brown chevron marks scattered on the

dorsal vertebrae with light orange patches; brown and

orange spots on fianks; supraciliaries brownish; pupil

circular, black surrounded by yellowish-orange color;

single brownish line with black scales from nasal to mid

eye region, similar line runs from the posterior eye to ear

opening; supralabials barred alternately with brown and

light orange; throat and ventrum of body white; ventral

view of lower and upper arm grey; original part of the tail

brown with marks of black transverse lines; ventral part

of the tail grayish.

Color pattern in alcohol preservation (Fig. 9a, b):

Dorsum brown and black marks in life faded, turned in

to brown, ventral body color turned in to yellowish-grey

with scattered grey patches and ventral tail became dark-

grey.

Variation: Adult specimens range in the type series size

28-36.23 mm (Table 2). All paratypes resemble the ho-

lotype in most respects except for the following charac¬

ters: 7-8 lamellae under first digit of manus, 9-12 under

fourth digit of manus, 7-8 lamellae under first digit of

pes, and 10-13 under fourth digit of pes. Holotype BNHS

2456 (male) has three femoral pores each side, ZSI-WRC

R/1054, ZSI-WRC R/1059, ZSI-WRC R/1056, and ZSI-

WRC R/1057 (males) have four femoral pores each side,

ZSI-WRC R/1060 has four on right, three on left. 7-8

supralabials to the angle of jaw, 7-8 infralabials to the

angle of jaw, and two pairs of postmentals in ZSI-WRC

R/1056 and ZSI-WRC R/1058.

Etymology: Specific epithet is a patronym in honor of

Mrs. Ajija Sayyed, mother of the first author.

Common name: Ajija’s Day Gecko.

Natnral history: This species can be found at night in

dense evergreen forest, as well as in the human habitation

in Mahabaleshwar. The type series (BNHS 2456 male

and ZSI-WRC R/1055 female) were collected beside a

dry stream surrounded by dense forest (Fig. 12a), speci¬

mens (ZSI-WRC R/1060 male and ZSI-WRC R/1058

female) on the wall of bus stand in Mahabaleshwar, wall

of lateritic plateau (tableland of Panchgani), and in the

jungle area nearby the town of Panchgani (Fig. 12b).

During this survey we observed several individuals of

Cnemaspis ajijae at both localities. The types were found

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | el 57

Four new species of the genus Cnemaspis

Table 2. Mensural and meristic data for the type series of Cnemaspis ajijae sp. nov. Abbreviations as stated in Materials and

Methods (* = regenerated tail, ** = sub-adult, - = pores not present).

Measurement

(mm)

Holotype

Paratypes

BNHS

2456

ZSI-WRC

R/1054

ZSI-WRC

R/1055

BNHS

2457

ZSI-WRC

R/1058

ZSI-WRC

R/1059

ZSI-WRC

R/1060

ZSI-WRC

R/1056

ZSI-WRC

R/1057

male

female*

female

female*

male

male**

male

(SVL)

29.80

28.68

36.23

35.24

32.79

28.35

28.93

24.38

29.07

(TRL)

12.92

12.48

16.02

16.66

16.97

10.68

11.18

11.15

12.56

(TW)

5.92

6.68

8.09

9.14

7.06

5.62

5.84

5.44

6.30

(TL)

32.89

31.56

23.31

36.51

25.02

26.66

30.69

25.73

32.70

(TW)

2.48

3.42

3.01

3.62

3.14

3.49

2.89

1.64

2.97

(HL)

5.27

5.08

5.36

5.85

6.42

5.23

5.10

4.76

5.20

(HW)

5.48

5.68

5.81

5.98

5.75

5.38

5.50

4.64

5.68

(HD)

3.23

3.66

3.70

3.96

4.21

3.18

3.21

2.65

3.58

(FL)

4.27

4.21

4.62

4.83

4.17

4.22

3.70

4.23

(TBL)

4.27

4.51

4.52

5.11

5.00

4.29

4.32

4.13

4.32

(E-N)

3.40

3.89

3.99

3.98

4.21

3.08

3.56

3.12

3.66

(E-S)

3.98

4.75

4.59

4.74

5.21

4.06

4.10

4.18

4.41

(E-E)

2.99

3.02

3.01

3.14

3.11

2.94

2.82

2.17

2.74

(EL)

0.12

0.10

0.11

0.12

0.11

0.10

0.09

0.07

0.10

(IN)

1.05

1.08

1.07

1.08

1.02

0.92

0.97

0.90

0.93

(OD)

1.23

1.30

1.02

1.13

1.25

1.12

1.03

0.92

1.10

(10)

3.90

3.62

3.76

4.01

3.73

3.12

3.43

2.50

3.61

HL/SVL

0.18

0.15

0.17

0.19

0.18

0.20

0.18

HW/SVL

0.18

0.19

0.16

0.17

0.19

HW/HL

1.04

1.11

1.08

1.02

0.90

1.03

1.07

0.97

1.09

E-S/HL

0.75

0.93

0.86

0.81

0.77

0.80

0.88

0.85

HD/HL

0.61

0.72

0.69

0.68

0.65

0.60

0.63

0.56

0.69

E-S/HW

0.73

0.84

0.79

0.90

0.75

0.74

0.90

0.77

OD/E-S

0.31

0.27

0.22

0.24

0.27

0.25

0.22

0.25

OD/HL

0.23

0.25

0.19

0.21

0.20

0.19

0.21

EL/HL

0.02

0.01

0.02

0.01

E-E/OD

2.43

2.32

2.95

2.78

2.48

2.62

2.73

2.36

2.49

TRL/SVL

0.43

0.44

0.47

0.52

0.38

0.39

0.45

0.43

FL/SVL

0.14

0.15

0.12

0.13

0.15

0.14

TBL/SVL

0.14

0.16

0.12

0.15

0.17

0.15

TL/SVL

1.10

0.64

1.04

0.76

0.94

1.06

1.10

MVS

SupraL

7/8

8/8

7/7

8/8

InfraL

7/7

8/8

7/7

8/8

7/7

8/7

FPores

3 on each side

4 on each side

4 on right, 3 on left

4 on each side

MLam R

7-8-10-10-9

8-10-12-12-10

7-9-11-10-8

7-9-11-11-9

7-8-11-10-10

7-9-12-12-10

8-9-12-11-9

7-8-10-9-9

7-8-10-9-8

PLam R

7-8-10-11-10

8-9-67-13-13

7-8-11-11-11

7-9-11-11-10

7-8-11-11-10

8-9-13-12-13

8-9-12-12-12

7-8-10-10-11

7-8-11-10-10

MLam L

7-8-10-10-9

8-10-13-12-10

7-8-10-10-8

7-9-11-11-9

7-8-9-10-8

8-9-12-12-10

8-9-11-11-9

7-8-10-9-9

7-8-10-10-8

PLam L

7-8-10-10-10

8-9-13-13-13

7-8-11-11-11

7-9-12-11-11

7-8-10-10-11

8-9-13-13-13

8-9-13-12-12

7-8-12-11-10

7-8-11-11-10

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

sympatrically with Hemidactylus sp., Trimeresurus mal-

abaricus, Trimeresurus gramineus, Boiga trigonata, Ly-

codon aulicus, and L. travancoricus in the same habitat

where specimens BNHS 2456 (male), ZSI-WRC R/1054

(male), and ZSI-WRC R/1055 (female) were collected.

At Panchgani, Hemidactylus maculates and Hemidacty¬

lus sp. were observed at the same habitat where BNHS

2457 (female), ZSI-WRC R/1056 (male), and ZSI-WRC

R/1057 (male) were collected.

Distribution: This species is seemingly abundant in Ma-

habaleshwar (17.545N, 73.403E; 1,377 m asl), (17.553N,

73.39IE; 1,291 m asl), and in Panchgani (17.554N,

73.483E; 1,323 m asl), Satara district, Maharashtra, In¬

dia. It is currently known only from a small area around

the type locality. See Fig. 2 for the type locality of the

species.

Remarks: Cnemaspis ajijae is distinguished from C.

girii and from C. flaviventralis by several morphologi¬

cal characters. C. ajijae can easily be distinguished from

C. girii by having maximum SVE 37 mm (vs. 35 mm);

conical and spine-like tubercles absent on flank (vs. large

keeled conical tubercles on flank); 29-30 midventrals

(vs. 26-28 midventrals); three pairs of postmentals (vs.

two pairs of postmentals); very small acute, prominent

tubercles dorsally on tail (vs. large tubercles present on

dorsal part of tail); from C. jlaviventralis by absence of

conical and spine-like tubercles on flank (vs. large keeled

conical tubercles present on flanks); 29-30 midventrals

(vs. 28-29); 7-8 supralabials (vs. 7-9); 10-13 lamellae

on digit IV of pes (vs. 10-12); small tubercles on the tail

(vs. large tubercles on the tail).

Comparison: Cnemaspis ajijae can be separated from

all its Indian congeners based on a combination of char¬

acters including: SVE 37 mm maximum in adults (vs.

SVE 61 mm in C. anaikattiensis, 50.6 mm in C. bed-

domei, 45.1 mm in C. heteropholis, 42.3 mm in C. nila-

girica, and 42.7 mm in C. sisparensis); males with femo¬

ral pores (vs. absent in C. assamensis, C. beddomei, C.

nairi, and C. ornata); males with 3-4 femoral pores (vs.

six in C. heteropholis, 5-15 in C.jerdonii, and 15-18 in

C. littoralis); pre-anal pores absent in males (vs. present

in C. adii, C. andersonii, C. australis, C. beddomei, C.

goaensis, C. gracilis, C. monticola, C. nairi, C. ornata,

C. otai, C. wicksii, and C. yercaudensis); spine-like tu¬

bercles absent on flank (vs. present in C. assamensis, C.

goaensis, C. gracilis, C. monticola, C. nilagirica, and C.

tropidogaster); dorsal scales on trunk heterogeneous (vs.

homogenous in C. adii, C. boiei, C. indica, C.jerdonii, C.

kolhapurensis, C. littoralis, C. mysoriensis, C. nilagirica,

C. sisparensis, and C. wynadensis); sub-caudal scales not

enlarged (vs. enlarged in C. kottiyoorensis, C. monticola,

and C. wynadensis)', 29-30 midventral scales (vs. 26-27

in C. anaikattiensis)', dorsal scales on both fore and hind

limbs are weakly carinate (vs. dorsal scales on both fore

Amphib. Reptile Conserv.

and hind limbs smooth in C. wicksii)', 10-13 lamellae on

fourth digit of the pes (vs. 20-21 in C. kottiyoorensis)',

nostril not in contact with supralabial (vs. nostril in con¬

tact with the first supralabial in C. anaikattiensis).

Cnemaspis ajijae can be distinguished from C. indra-

neildasii based on a combination of characters including:

scales on flank heterogeneous (vs. flank mostly homog¬

enous); spine-like tubercles absent on flank (vs. spine¬

like tubercles present on flanks); dorsal scales large (vs.

small); 29-30 midventrals (vs. 20); nostrils not in contact

with supralabial (vs. nostril connects first supralabial);

dorsal scales on limbs weakly carinate (vs. not carinate);

7-8 supralabials to angle of jaw (vs. 8-9); very small

acute shape, tubercles on the tail (vs. enlarged pointed

tubercle); from C. girii by having maximum SVE 37 mm

(vs. SVE less than 35 mm); conical and spine-like tuber¬

cles absent on flank (vs. large keeled conical tubercles on

flank); 29-30 midventrals (vs. 26-28 midventrals) (Table

6); three pairs of postmentals (vs. two pairs of postmen¬

tals); very small acute, prominent tubercles dorsally on

tail (vs. large tubercles present on dorsal part of tail);

from C. flaviventralis by absence of conical and spine¬

like tubercles on flank (vs. large keeled conical tubercles

present on flanks); 29-30 midventrals (vs. 28-29); 7-8

supralabials (vs. 7-9); 10-13 lamellae on digit IV pes (vs.

10-12) (Table 6); small tubercles on the tail (vs. large tu¬

bercles on the tail); from C. limayei by having maximum

SVE 37 mm (vs. less than 31 mm); 29-30 midventrals

(vs. 26-27); three pairs of postmentals (vs. two pairs of

postmentals); males with three or four femoral pores (vs.

4-5 femoral pores) (Table 6).

Cnemaspis amboliensis sp. nov. (Figs. 13-18)

urn:lsid:zoobank.org:act:6D80B074-DF22-478B-AD45-658640E80B6A

Holotype: BNHS 2458 (adult male); collected on 23 Oc¬

tober, 2015 at Amboli (15.960 N, 73.999 E; 735 m asl),

Sindhudurg district, Maharashtra, India.

Paratypes: BNHS 2459 (adult female), BNHS 2504,

BNHS 2506, BNHS 2507, BNHS 2508, and BNHS 2505

(all male) have the same collection data as for the holo¬

type, collected on tree trunks, rocks, inside walls of local

houses, and on stone compound walls.

Diagnosis: Medium-sized day gecko, SVE less than

32 mm; dorsal scales on trunk heterogeneous; granular,

keeled, small scales intermixed with some large keeled

scales; some conical and spine-like tubercles on flank;

ventral scales smooth, imbricate, larger than dorsal;

19-22 midbody scales across the belly; scales on snout

feebly keeled; canthus rostralis and forehead granular,

feebly keeled; rostral not swollen, medial groove absent;

gular with carinate scales, anterior gular scales smooth;

dorsal scales on forelimb and hindlimb tricarinate; tail

sub-cylindrical, ventrally swollen, one small triangular

post-anal spur along each side present in males; sub-cau-

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

Fig. 13. Holotype male (BNHS 2458) of Cnemaspis amboliensis

sp. nov. in life.

Fig. 15 (above), (a) Dorsal, (b) ventral, and (c) lateral view

of the mid body of Cnemaspis amboliensis sp. nov. Holotype

(BNHS 2458).

Fig. 16 (right), (a) Dorsal, (b) ventral, and (c) lateral view of

the head, (d) ventral view of right manus, (e) ventral view of

right pes, and (f) the lower body of Cnemaspis amboliensis sp.

nov. Holotype (BNHS 2458).

Fig. 14. (a) Dorsal and (b) ventral view of the full body of

Cnemaspis amboliensis sp. nov. Holotype (BNHS 2458).

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

■ M

. ^

Fig. 17. Showing tricarinate scales on (a) forelimb, (b) hindlimb

of Cnemaspis amboliensis sp. nov. Holotype (BNHS 2458).

dal smooth, imbricate, second and third rows each side

carinated, median row slightly enlarged; 6-7 lamellae on

digit I of manus and 10 on digit IV, 6-7 on digit I of pes

and 10-11 on digit IV; males with three or four pre-anal

pores and 3-4 femoral pores on each side of thigh.

Description of holotype: BNHS 2458 (adult male); in

good condition with an original tail (Fig. 14a, b); 29.87

mm SVL; head fairly short (HL/SVL = 0.19), wide (HW/

HL = 0.90), slightly depressed (HD/HL = 0.55), distinct

from moderately elongated neck; loreal not inflated,

canthus rostralis not prominent; snout elongated (E-S/

HL = 0.79), much longer than eye diameter (OD/E-S

= 0.24); scales on snout, canthus rostralis and forehead

granular, feebly keeled; scales on interorbital and oc¬

cipital smaller, granular with some feebly keeled (Fig.

16a); eye moderately small (OD/HL = 0.19), pupil round;

supraciliary scales slightly enlarged; ear opening small,

oval shape, higher than wide (EL/HL = 0.03); eye to ear

distance greater than diameter of eyes (E-E/OD = 1.77);

rostral smooth wider than deeper, not swollen, medial

groove absent (Fig. 16a, and Fig. 18a, c), in contact with

first supralabial; nostrils not in contact with supralabial

I; nares separated by two enlarged supranasals, three

internasal scales, medal one moderately small, extends

Fig. 18. Showing (a) rostral, (b) dorsal view of tail, (c)

arrangement of nasals scales, (d) ventral view of tail of

Cnemaspis amboliensis sp. nov. Holotype (BNHS 2458).

towards snout tip (Fig. 18a, c, and Fig. 19a); two rows

of scales separate the orbit from the supralabials; men¬

tal sub-triangular, slightly wider (1.48 mm) than longer

(1.31 mm), posteriorly not pointed; two pairs of post¬

mentals, primary postmentals separated by mental, larger

than secondary, surrounded laterally by first infralabial,

secondary in contact with first and second infralabial

(Fig. 16b and Fig. 19c); body relatively slender, not

elongate (TRL/SVL = 0.38), without ventrolateral folds;

supralabials to angle of jaw- seven right, eight left, at

midorbital position- seven right, seven left; infralabials-

seven left, seven right (Fig. 16c); dorsal scales on trunk

heterogeneous, granular keeled small scales intermixed

with some large keeled scales (Fig. 15a); neck and sa¬

crum with feebly keeled scales, lateral part of the neck

with granular, small feebly carinate scales; few conical

tubercles and spine-like tubercles present on flank (Fig.

15c); ventral scales smooth, imbricate, larger than dorsal

(Fig. 15b); 22 midbody scales across the belly between

the lowest rows of dorsal scales; gular region with some

feebly carinate scales, anterior gular scales smooth; three

pre-anal and four femoral pores on each side (Fig. 16f);

fore and hindlimbs relatively short, slender; forearm and

tibia short (FL/SVL = 0.13; TBL/SVL = 0.14); dorsal

scales of the forelimb and hindlimb tricarinate, keels well

aligned to form more or continuous lines on dorsal part

of humerus and femur, laterally carinate (Fig. 17a, b, and

Fig. 20a, c), ventrally smooth; lamellae 7-8-11-10-9

right manus (Fig. 16d), 7-9-11-11-10 right pes (Fig.

16e); IV (3.05) > III (2.47) > II (2.35) > V (2.26) > I

(1.47) (right manus); IV (4.06) > V (3.18) > III (3. 16)

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

Fig. 19. Comparison of the snout and ventral regions, (a) Snout, (c) ventral view of head of Cnemaspis

amboliensis sp. nov. Holotype (BNHS 2458), and (b) snout, (d) ventral view of head of Cnemaspis goaensis

Holotype (male) (ZSI-K 22110).

Fig. 20. Comparison of the dorsal scales of forelimb and hindlimb region, (a) Dorsal part of humerus, (c)

dorsal part of femur region of Cnemaspis amboliensis sp. nov. Holotype (BNHS 2458), and (b) dorsal part

humerus, (d) dorsal part femur region of Cnemaspis goaensis Holotype (male) (ZSI-K 22110).

> II (2.20) > I (1.33) (right pes), interdigital webbing

absent; tail longer than snout-vent length (TaL/SVL =

1.21), sub-cylindrical, ventrally swollen; small triangular

post-anal tubercles present along each side; sub-caudal

smooth, imbricate, median row slightly enlarged, sec¬

ond and third rows each side carinated (Fig. 18d, 21c);

prominent acuminate keeled tubercles present with small

keeled scales on dorsal tail (Fig. 18b, 21a).

Color in life (Fig. 13): Dorsal body brownish-yellow

including tail; faded brown line present at interorbital re¬

gion; lateral side of head and neck consists of two pale

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

Fig. 21. Comparison of the dorsal and ventral pholidosis of caudal region, (a) Dorsal, (c) ventral caudal region of Cnemaspis

amboliensis sp. nov. Holotype (BNHS 2458), and (b) dorsal, (d) ventral caudal region of Cnemaspis goaensis Holotype (male)

(ZSI-K 22110).

brown lines, one from nasal to mid eye, other posterior

age of eye to ear; semicircle in brown marking on pos¬

terior head; dark-blackish-brown notched mark with a

light-yellow patch in posterior end of it present on neck;

supraciliaries yellow; labials light yellow with some

dark-yellow spots; pupil rounded, black with surround¬

ing yellow iris; ventral arm, throat, and ventral head light

brown; venter dusty white; ventral hindlimb and ground

of tail dusty white with irregular yellow markings; mid¬

dorsal area of body brownish-yellow, with five arrow¬

head shaped markings between fore and hindlimbs; some

brown spots present in upper flank, slightly augmented

by yellow on lower flank; original part of tail brownish-

yellow, with pale brown bands.

Color pattern in alcohol preservation (Fig. 14a, b):

Dorsum ground color became brownish, brown and

black markings faded light; ventral body including tail

color turned in to grey.

Etymology: The specific epithet amboliensis refers to

the type locality Amboli, from which the type series was

collected.

Common name: Amboli Day Gecko.

Natnral history: This species was nocturnally active on

the tree trunks and rocks of the wooded area of Amboli

town (Fig. 22a) and was also found on the inside and

outside of the walls of local houses and on the stone com¬

pound walls in Amboli town (Fig. 22b). They were not

found active during the day time in the study area. Appar¬

ently healthy populations of this species can be seen dur¬

ing June-September; we have observed gravid females

in the months of September and October. The types were

found sympatric with Hemidactylus sp., Cyrtodactylus

albofasciatus, H. prashadi, Cnemaspis kolhapurensis,

C. flaviventralis, Bungarus caeruleus, Trimeresurus

malabaricus, Lycodon travancoricus, Macropisthodon

plumbicolor, Uropeltis sp., Raorchestes ghatei, Pseu-

dophilautus amboli, Indirana chiravasi, Rhacophorus

malabaricus, Xanthophryne tigerina, and Duttaphrynus

melanostictus.

Distribution: This species is currently known only from

its type locality at Amboli (15.960N, 73.999E; 735 m

asl), Sindhudurg district, Maharashtra, India. See Fig. 2

for the type locality of the species.

Variation: Adult specimens range in size from 28-32

mm (Table 3). All paratypes resemble the holotype ex¬

cept as follows: the number of lamellae on digit I of the

manus is seven in all males, female BNHS 2459 with six,

and 10 on digit IV in all the specimens, on digit I of the

pes it is seven, specimen number BNHS 2507 male with

six at left pes, female BNHS 2459 with six on left and

right pes, and 10-11 on digit IV. Holotype male BNHS

2458 has four pre-anal and four femoral pores on each

side. All specimens are almost similar with each other in

color and in external features. Mensural data for the type

series is given in Table 3.

Remarks: Cnemaspis amboliensis is distinguished from

C. goaensis by having a maximum SVL 32 mm (vs. less

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

Fig. 22. (a) Forest, (b) Amboli town, habitat of Cnemaspis

amboliensis sp. nov.

than 28.3 mm); rostral scale not divided, medial groove

absent (vs. rostral scale partially divided by a medial

groove); nares separated by two supranasals, of the three

internasal scales, middle one extends towards snout tip

(vs. nares separated by two enlarged supranasals, a single

internasal); primary postmental scale separated by men¬

tal scale (vs. primary postmental scale separated by sin¬

gle gular scale); scales on ventral surface of neck smooth

(vs. scales on ventral surface of neck feebly carinate);

dorsal scales on forelimb and hindlimb tricarinate (vs.

dorsal scales on both fore and hindlimb weakly carinate)

(Table 6).

Comparison: Cnemaspis amboliensis may be distin¬

guished from all other peninsular Indian congeners on

the basis of the following differing or non-overlapping

characters: dorsal scales on trunk heterogeneous (vs.

dorsal scales homogenous in C. adii, C. boiei, C. indi-

ca, C. indraneildasii, C. jerdonii, C. kolhapurensis, C.

littoralis, C. mysoriensis, C. nilagirica, C. sisparensis,

and C. wynadensis); males with pre-anal and femoral

pores (vs. males with femoral pores in C. flaviventralis,

C. girii, C. heteropholis, and C. kottiyoorensis; no pre-

anal or femoral pores in C. assamensis, two pre-anal

pores present which is separated by two unpored scales,

3-5 femoral pores on each side in C. gracilis., femoral

pores absent whereas pre-anal pores present in C. nairi);

spine-like tubercles present on flanks (vs. spine like tu¬

bercles absent on flanks in C. anaikattiensis, C. australis.

C. beddomei, C. ornate, C. otai, and C. yercaudensis);

sub-caudal slightly enlarged and smooth; rostral medial

groove absent (vs. no median series of enlarged sub-cau-

dals; rostral medial groove present in C. flaviventralis;

sub-caudals enlarged, keeled intermixed with smooth,

carinate scales; rostral scale partially divided by a me¬

dial groove in C. monticola; no median series of enlarged

sub-caudals in C. andersonii); males with three or four

pre-anal pores and 3-4 femoral pores on each side of the

thigh; dorsal scales on forelimb and hindlimbs tricari¬

nate, (vs. four pre-anal pores, four or five femoral pores

on each side; dorsal scales on both fore and hind limbs

smooth in C. wicksi); ventral scales smooth, imbricate;

19-22 midventrals (vs. ventral scales of the body keeled

and imbricate; 28 midventrals in C. tropidogaster); from

Cnemaspis limayei sp. nov. and Cnemaspis ajijae sp.

nov. by having conical and spine-like tubercles on flank;

presence of pre-anal pores; 19-22 midventrals (vs. coni¬

cal and spine-like tubercles absent; pre-anal pores absent

in both species; 26-27 midventrals in Cnemaspis limayei

sp. nov., 29-30 in Cnemaspis ajijae sp. nov.). New spe¬

cies is similar in size and general appearance to Cnemas¬

pis goaensis, however differs from this by rostral scale

not divided, medial groove absent (Fig. 18a and 19a) (vs.

rostral scale partially divided by a medial groove (Fig.

19b)); nares separated by two supranasals, of the three

internasal scales, middle one extends towards snout tip

(vs. nares separated by two enlarged supranasals, single

internasal); primary postmental scale separated by men¬

tal scale (vs. primary postmental scale separated by sin¬

gle gular scale); scales on ventral surface of neck smooth

(vs. scales on ventral surface of neck feebly carinate);

granular keeled small scales intermixed with some large

keeled scales dorsally; neck and sacrum with feebly

keeled scales (vs. mid-dorsal granules, mixed with large

keeled tubercles from head to sacrum); dorsal scales on

forelimb and hindlimb tricarinate (Fig. 17a, b, and 20 a,

c) (vs. dorsal scales on both fore and hindlimb weakly

carinate (Fig. 20b, d)); lamellae manus 7-8-11-10-9,

pes 7-9-11-11-10, measurement of right Angers: fourth

Anger larger than third, third larger than second, second

larger than fifth, and fifth larger than first; toes: fourth

larger than fifth, fifth larger than third, third larger than

second, and second larger than first (vs. lamellae manus

9-12-13-15-11, pes 9-12-16-16-16; measurement of

right Angers: fourth Anger equal to second, third smaller

than fourth and second, fifth smaller than third, and first

smaller than fifth; toes: second larger than first, first larger

than third, third larger than fourth, and fourth larger than

fifth); sub-caudal smooth, imbricate, median row slightly

enlarged, second and third rows each side carinated (Fig.

18d and 21c) (vs. median row of subcaudals slightly en¬

larged, smooth (Fig. 2Id)); prominent acuminate keeled

tubercles present with small keeled scales on dorsal tail

(Fig. 18b and 21a) (vs. dorsal scales on mid-tail acute,

imbricate, carinate (Fig. 21b)).

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

Table 3. Mensural and meristic data for the type series of Cnemaspis amboliensis sp. nov. Abbreviations as stated in Materials and

Methods (* = regenerated tail, ? = broken finger, - = pores not present).

Measurement

(mm)

Holotype

Paratypes

BNHS 2458

BNHS 2504

BNHS 2505

BNHS 2459

BNHS 2506

BNHS 2507

BNHS 2508

male

female

male*

male

(SVL)

29.87

28.60

29.24

31.47

28.61

28.74

29.85

(TRL)

11.52

12.24

11.91

14.52

12.37

12.17

12.54

(TrW)

6.30

5.98

6.19

7.19

5.45

6.31

6.45

(TaL)

36.27

37.30

33.69

34.78

28.88

29.98

30.45

(TaW)

2.97

2.84

2.99

2.54

3.36

2.96

3.01

(HL)

5.83

4.89

5.03

5.65

4.82

5.00

5.52

(HW)

5.26

5.10

5.13

4.88

5.15

4.64

5.04

(HD)

3.24

3.31

3.19

3.02

3.15

3.31

3.33

(FL)

4.03

3.97

4.26

4.47

4.12

4.23

4.39

(TBL)

4.39

4.32

4.37

4.74

4.80

4.64

4.63

(E-N)

3.79

3.24

3.45

3.94

3.35

3.39

3.79

(E-S)

4.66

4.17

4.29

4.63

3.98

4.40

4.64

(E-E)

2.01

2.63

2.92

2.39

2.29

2.30

2.88

(EE)

0.19

0.18

0.17

0.14

0.15

0.18

(IN)

0.90

1.02

0.99

1.00

0.99

1.01

(OD)

1.13

1.12

1.11

1.23

1.02

1.09

1.13

(10)

2.67

2.86

3.02

2.72

2.92

2.63

2.84

HE/SVE

0.20

0.17

1.17

0.18

0.17

0.18

HW/SVE

0.18

0.16

0.18

0.16

0.17

HW/HE

0.90

1.04

1.02

0.86

1.07

0.93

0.91

E-S/HE

0.80

0.85

1.84

0.82

0.83

0.88

0.84

HD/HE

0.56

0.68

0.63

0.53

0.65

0.66

0.60

E-S/HW

0.89

0.82

0.84

0.95

0.77

0.95

0.52

OD/E-S

0.24

0.27

0.26

0.27

0.26

0.25

0.24

OD/HE

0.19

0.23

0.22

0.21

0.22

0.20

EE/HE

0.03

0.04

0.03

E-E/OD

1.78

2.35

2.63

1.94

2.25

2.11

2.55

TRE/SVE

0.39

0.43

0.07

0.46

0.43

0.42

FE/SVE

0.13

0.14

0.15

0.14

0.15

TBE/SVE

0.15

0.43

0.15

0.17

0.16

TaE/SVE

1.21

1.30

1.15

1.11

1.01

1.04

1.02

MVS

SupraE

7/8

8/8

7/8

8/8

8/7

7/8

InfraE

7/7

7/8

8/7

7/6

7/8

FPores

4 on each side

3 on each side

4 on each side

3 on each side

PaPores

MEam R

7-8-11-10-9

7-8-10-10-8

7-8-11-10-8

6-8-10-10-8

7-8-10-10-9

7-9-11-10-8

7-8-10-10-9

PEam R

7-9-11-11-10

7-8-11-10-10

7-9-10-10-10

6-8-10-10-10

7-8-10-11-11

7-9-11-10-10

7-8-11-11-10

MEam E

7-9-10-10-9

7-8-10-10-8

6-8-10-10-8

7-8-11-10-8

7-8-10-10-9

7-8-11-10-9

PEam E

7-9-10-11-10

7-8-11-10-?

7-8-10-10-10

6-8-11-10-10

7-9-11-11-10

6-7-10-11-11

7-8-11-10-10

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | el 57

Four new species of the genus Cnemaspis

Cnemaspis mahabali sp. nov. (Figs. 23-26)

urn:lsid:zoobank.org:act:D25C0B90-783A-4057-9D68-278200660A0E

Holotype: BNHS 2449 (adult male) collected on 21 De¬

cember 2016 at Bhira, near Tamhini (18.454N, 73.222E;

558 m asl), Pune district, Maharashtra, India.

Paratypes: BNHS 2502 and BNHS 2450 (adult male),

BNHS 2451 and BNHS 2503 (adult female), have same

collection data as holotype on the tree trunk.

Diagnosis: A medium-sized, robust Cnemaspis with

maximum snout-vent length of 32.25 mm; dorsal scales

heterogeneous consisting of granular small scales inter¬

mixed with few large feebly keeled scales; conical and

spine-like tubercles absent on the flank; scales on neck

not keeled; ventral part of neck feebly carinate; gular

smooth; ventral scales of body smooth, imbricate, larger

than dorsal; 26 midbody scales across the belly; dorsal

scales of forelimb and hindlimb strongly keeled; ventral

scales of both limbs smooth; scales on snout, canthus ros-

tralis, and forehead granular, feebly keeled and those on

interorbital and occipital smaller, granular; rostrum with

partially dividing median groove, rostral in contact with

first supralabial; nares separated from each other by two

supranasals and a small internasal scale; nostrils not in

contact with supralabial I; nasals bordered posteriorly by

three post nasals; mental scale sub-triangular, longer than

wider, posteriorly not pointed; two pairs of postmentals,

primary postmentals separated by large median scales,

primary postmentals larger than secondary, secondary

postmentals in contact with first and second infralabial;

supralabials 8-9; infralabials 7-8; three femoral pores

on each side; 10-11 lamellae on digit IV of manus and

11-12 on digit IV of pes; extremely small post-anal spur

along each side; sub-caudal smooth, imbricate, median

row weakly enlarged; small keeled pointed tubercles

present with small, keeled scales on dorsal tail.

Description of holotype: BNHS 2449 (adult male), in

good condition with an entire tail (Fig. 24a, b). 31.06

mm SVL; head short (HL/SVL = 0.27), slightly wide

(HW/HL = 0.60), depressed (HD/HL = 0.34); snout short

(E-S/HL = 0.43), longer than eye diameter (OD/E-S =

0.27); scales on snout, canthus rostralis, forehead granu¬

lar, feebly keeled; interorbital, occipital smaller, granu¬

lar scales (Fig. 26a); eye small (OD/HE = 0.11), pupil

round; supraciliary scales slightly enlarged; ear opening

very small, higher than wide (EE/HE = 0.004); eye to

ear distance greater than diameter of eyes (E-E/OD =

2.64); rostrum broader than long, with a partially divid¬

ing median groove, rostral in contact with first supral¬

abial; nares separated from each other by two suprana¬

sals and a small internasal scale (Fig. 26a); nostrils not in

contact with supralabial I; nasals bordered posteriorly by

three post nasals (Fig. 26c); mental scale sub-triangular,

longer than wide, posteriorly not pointed; two pairs of

postmentals, primary postmentals separated by large in¬

termediate scales, primary postmentals larger than sec¬

ondary, surrounded laterally by first infralabial, second¬

ary in contact with first and second infralabial (Fig. 26b);

body relatively slender, not elongate (TRE/SVE = 0.42),

without ventrolateral folds; supralabials to angle of jaw-

eight right, eight left; infralabials- seven left, eight right

(Fig. 26c); dorsal scales on trunk heterogeneous, granu¬

lar small scales intermixed with few large feebly keeled

scales (Fig. 25a); scales on neck not keeled; scales on

ventral part of neck feebly carinate; conical and spine¬

like tubercles absent on the flank (Fig. 25c); ventral

scales smooth, imbricate, larger than dorsal (Fig. 25b);

26 midbody scales across belly between lowest rows of

dorsal scales; gular smooth; three femoral pores on each

side (Fig. 26f); fore and hindlimbs relatively short, slen¬

der; forearm and tibia short (FE/SVE = 0.12; TBE/SVE

= 0.13); dorsal scales on forelimb and hindlimb strongly

keeled; ventral scales of both limbs smooth; lamellae

7-9-11-10-9 right manus (Fig. 26d), 7-9-11-11-11

right pes (Fig. 26e); IV (2.58) > III (2.22) > II (2.04) > V

(1.97) > I (1.72) (right manus); IV (3.30) > V (2.66) > III

(2.65) > II (2.36) > I (1.61) (right pes), interdigital web¬

bing absent; tail longer than snout-vent length (TaE/ S VE

= 1.15), sub-cylindrical, ventrally swollen; extremely

small post-anal spur along each side; sub-caudal smooth,

imbricate, median row weakly enlarged (Fig. 24b); small

keeled pointed tubercles present with small, keeled scales

on dorsal tail (Fig. 24a).

Color in life (Fig. 23): Dorsal part of body brown; dark-

brown line present on canthal region connected with eye

to nasal; chevron-like single mark on interorbital area;

‘W’ mark on head; small black patch on the nuchal; five

dark-brown markings posteriorly surrounded by light-

yellow present on mid dorsal body; supraciliaries brown;

pupil circular, black with surrounding being reddish-

brown; supralabial brown with orange spots; ventral side

of body including throat grey; brown with light-yellow

stripes on both limbs and on Angers; ventral side of lower

and upper arm grayish; light-yellowish spots scattered

in upper flank; original part of tail grayish-brown with

few irregular dark-brown patches; ventral surface of tail

grayish.

Color pattern in alcohol preservation (Fig. 24a, b):

Dorsum body color became light brown; vertebral mark¬

ings became dark-brown; dorsal part of limbs and tail be¬

came light brown with dark patches; one light brown ra¬

diating line from posterior edge of eyes; venter of throat,

body and tail unpatterned yellowish-brown.

Etymology: The specific epithet is a patronym, honoring

Mr. Anil Mahabal, retired scientist of Zoological Survey

of India, Pune, Maharashtra, for his immense contribu¬

tion to Indian natural history.

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

Fig. 23. Holotype male (BNHS 2449) of Cnemaspis mahabali

sp. nov. in life.

Fig. 25. (a) Dorsal, (b) ventral, and (c) lateral view of the mid

body of Cnemaspis mahabali sp. nov. Holotype (BNHS 2449).

Fig. 26 (right), (a) Dorsal, (b) ventral, and (c) lateral view of

the head, (d) ventral view of right manus, (e) ventral view of

right pes, and (f) the lower body of Cnemaspis mahabali sp.

nov. Holotype (BNHS 2449).

Fig. 24. (a) Dorsal and (b) ventral view of the full body of

Cnemaspis mahabali sp. nov. Holotype (BNHS 2449).

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

Fig. 27. Habitat of Cnemaspis mahabali sp. nov.

Common name: Mahabal’s Day Gecko

Natural history: All specimens in the type series were

collected at night on tree trunks and on branches. This

species was not observed to be active in the day time

during the study period. This species is widely distrib¬

uted in the western parts of Maharashtra, recorded from

the coastal area to up on the hills of the Western Ghats

below the elevation 600 m from the sea level. Type local¬

ity of this gecko is Bhira, near Tamhini, Pune district,

Maharashtra, India (Fig. 27a, b), where all the specimens

were collected. The types were found sympatrically with

Hemidactylus sp., H. cf. maculates, Macropisthodon

plumbicolor, Duttaphrynus melanostictus, Raorchestes

ghatei, and Polypedates maculates.

Distribution: This species is widely distributed in most

parts of the coastal forest and hilly regions of western

Maharashtra. In this study we have reported this species

from Ratnagiri district, parts of Thane district, the hilly

area of Raigad district, and Pune district. Live speci¬

mens were examined from Devrukh, Guhagar, Chiplun,

Mulshi, Tail Baila, Patnus, Bhira, Phansad, Mangaon,

Tambadi, and Uran in Maharashtra. Type specimens were

collected from Bhira, near Tamhini (18.454N, 73.222E;

558 m asl), Pune district, Maharashtra, India. See Fig. 2

for the type locality of the species.

Variation: Adult specimens range in size from 27-33

mm (Table 4). All paratypes resemble the holotype and

all specimens are similar with each other in color and in

external features.

Remarks: Cnemaspis mahabali is distinguished from

C. girii, C. flaviventralis, and C. ajijae by several mor¬

phological characters. C. mahabali can easily be distin¬

guished from C. girii and C. flaviventralis by lacking

conical tubercles on the flanks; low count of midventrals

19-22; scales on ventral part of neck carinate; and from

C. ajijae by having few large weakly keeled scales on the

dorsal body; carinate scales on ventral part of neck; two

pairs of postmentals (Table 6).

Comparison: Cnemaspis mahabali may be distin¬

guished from all other peninsular Indian congeners on

the basis of the following differing or non-overlapping

characters: dorsal scales on trunk heterogeneous (vs. dor¬

sal scales homogenous in C. adii, C. boiei, C. indraneil-

dasii, C. indica, C.jerdonii, C. kolhapurensis, C. littora-

lis, C. mysoriensis, C. nilagirica, C. sisparensis, and C.

wynadensis); conical and spine-like tubercles absent on

the flank (vs. spine-like tubercles present on flank in C.

assamensis, C. indraneildasii, C. jerdonii, C. littoralis,

C. monticola, C. mysoriensis, C. nilagirica, and C. tropi-

dogaster, conical tubercles present on flanks in C. kotti-

yoorensis and C. flaviventralis)', males with three femoral

pores on each side (vs. males with six femoral pores in

C. heteropholis, five in C. indica, 5-15 in C. jerdonii,

15-18 in C. littoralis, 7-8 in C. sisparensis, 4-6 in C.

wynadensis', males with pre-anal as well as femoral pores

in C. andersonii, C. australis, C. goaensis, C. gracilis, C.

mysoriensis, C. otai, and C. yercaudensis', femoral pores

absent whereas pre-anal pores present in C. beddomei,

C. nairi, and C. ornata', pores absent in both sexes of

C. assamensis and C. boiei', continuous series of 24-28

pre-anal femoral pores in C. kolhapurensis)', two pairs

of postmentals (vs. three pairs of postmentals in C. aji¬

jae sp. nov. and C. anaikattiensis)', gulars scales smooth;

scales on ventral part of neck feebly carinate (vs. gulars

scales carinate in C. andersonii, scales on ventral part of

neck smooth in C. kolhapurensis and C. flaviventralis)',

differs from Cnemaspis amboliensis sp. nov. by conical

and spine-like tubercles absent on flank; 26 midventrals;

dorsal scales on forelimb and hindlimbs strongly keeled;

three femoral pores (vs. conical and spine-like tubercles

on flank; 19-22 midventrals; dorsal scales on forelimb

and hindlimb tricarinate; males with three or four pre-

anal pores and 3-4 femoral pores).

This new species is similar in size and general appear¬

ance to Cnemaspis girii, C. ajijae, and C. limayei, but

differs from these by having large feebly keeled scales

on dorsal part of body; conical tubercles absent on flank;

26 midventrals; scales on ventral part of neck feebly cari¬

nate; two pairs of postmentals; dorsal scales on forelimb

and hindlimb strongly keeled; inner surface of forelimb

and hindlimb smooth; three femoral pores on each side;

pre-anal scales same as ventral scales of the body; small

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

Table 4. Mensural and meristic data for the type series of Cnemaspis mahabali sp. nov. Abbreviations as stated in Materials and

Methods (# = juvenile, ? = broken finger, - = pores not present).

Measurement

(mm)

Holotype

Para types

BNHS 2449

BNHS 2450

BNHS 2502

BNHS 2451

BNHS 2503

male

female

female #

(SVL)

31.06

30.72

27.93

32.25

18.11

(TRL)

13.10

13.14

11.22

14.08

7.04

(TrW)

6.83

6.15

6.33

8.49

2.95

(TaL)

35.76

32.95

31.87

34.33

19.02

(TaW)

2.80

2.73

2.51

3.02

0.87

(HL)

8.63

7.70

7.71

8.02

5.51

(HW)

5.24

5.33

4.88

5.68

3.02

(HD)

2.97

3.32

3.16

3.49

1.86

(FL)

3.95

4.92

3.92

4.92

2.03

(TBL)

4.31

5.47

4.17

5.61

2.94

(E-N)

2.93

2.97

2.99

3.16

1.68

(E-S)

3.74

3.80

3.67

4.07

2.35

(E-E)

2.72

2.80

2.48

2.84

1.48

(EE)

0.04

0.05

0.04

0.05

0.02

(IN)

0.82

0.92

0.82

0.97

0.59

(OD)

1.03

1.09

1.03

1.20

0.87

(10)

3.61

3.48

3.09

3.34

2.04

HE/SVE

0.27

0.25

0.27

0.24

0.30

HW/SVE

0.16

0.17

0.16

HW/HE

0.60

0.69

0.63

0.70

0.54

E-S/HE

0.43

0.49

0.48

0.50

0.42

HD/HE

0.34

0.43

0.40

0.43

0.33

E-S/HW

0.71

0.75

0.71

0.77

OD/E-S

0.27

0.28

0.29

0.37

OD/HE

0.11

0.14

0.13

0.14

0.15

EE/HE

0.004

0.006

0.005

0.006

0.003

E-E/OD

2.64

2.56

2.40

2.36

1.70

TRE/SVE

0.42

0.40

0.43

0.38

FE/SVE

0.12

0.16

0.14

0.15

0.11

TBE/SVE

0.13

0.18

0.14

0.17

0.16

TaE/SVE

1.15

1.07

1.14

1.06

1.05

MVS

SupraE

8/8

9/9

8/8

InfraE

8/7

7/7

8/8

8/7

7/7

FPores

3 on each side

MEam R

7-9-11-10-9

8-9-10-10-9

8-8-11-11-9

8-9-10-10-9

7-8-11-10-9

PEam R

7.9.11.11.11

8-9-11-11-11

7.9.11.11.10

7-9-11-11-11

MEam E

8-8-10-11-9

8-9-10-10-9

8-8-10-11-9

8-9-11-11-9

7-8-10-10-8

PEam E

7.9.11.11.11

8-9-11-11-10

7-9-11-12-10

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

0 170 340 660 km

I-1-1-1-1-1-1-1-1

Fig. 28. Map showing distribution of Cnemaspis goaensis, in the parts of south Maharashtra, Goa and Karnataka, India. Localities

indicated by blue circles.

keeled pointed tubercles present on dorsal tail (vs. large

smooth scales on dorsal aspect; conical tubercles pres¬

ent; part of neck smooth; inner surface of forelimb and

hindlimb keeled; large pointed tubercles present on the

dorsal tail in C. girii; dorsal granular, keeled scales in¬

termixed with large keeled depressed scales; part of neck

smooth; 29-30 midventrals; three pairs of postmentals in

C. ajijae sp. nov.; dorsal scales feebly keeled; intermixed

with large keeled depressed scales; part of neck smooth;

pre-anal scales large than ventral; males with 4-5 femo¬

ral pores on each side in C. limayei (Table 6).

Discussion

In their important work on South Asian Cnemaspis, Man-

amendra-Arachchi et al. (2007) reported 34 species from

peninsular India and Sri Lanka, based on their explora¬

tion and discovery of many species in Sri Lanka. They

also predicted that the number of species was likely to in¬

crease substantially, especially with further explorations

in the Western Ghats. However, in the last few years only

five species, C. kolhapurensis, C. girii, C. kottiyooren-

sis, C. adii, and C. flaviventralis, have been described

from the Western Ghats, and only a single species from

Sri Lanka, C. kandambyi (Uetz and Hosek 2017). This

is likely due to the slow pace at which systematic sur¬

veys are being carried out, and the intensive investment

of time and money needed for accurate taxonomic work

in such a small-bodied and cryptic taxon. The discovery

of four new species in the current study is therefore not

surprising.

Our taxonomic sampling for genetic analysis is still

incomplete. Nevertheless, we show that all the proposed

species are morphologically distinct from their Indian

congeners and genetically distinct from geographically

proximate species. Two of our species, C. mahabali and

C. limayei, are closely related and form a clade with C.

girii. However, the three species can be separated based

on the presence or absence of canonical tubercles on

fianks, number of midventral scales, and the number of

lamellae under digit IV of the pes. Genetically, these spe¬

cies are separated by at least 2.8% uncorrected genetic

distance from each other.

Intraspecific genetic distances of 1.5% were observed

among the specimens of C. ajijae collected from Panch-

gani and parts of Mahabaleshwar Satara, Maharashtra.

This suggests potential cryptic diversity within C. ajijae.

Nevertheless, the specimens from these localities were

morphologically similar. Additionally, Srinivasulu et al.

(2014) provide a point locality for C. indraneildasii de¬

scribed by Bauer (2002) from Mahabaleshwar, attribut¬

ing the record to Smith (1935). They, however, do not

provide rationale for this record. Although C. ajijae from

Mahabaleshwar can be confused with C. indraneildasii,

they can easily be distinguished from one another by the

following morphological characters: spine-like tubercles

absent on flank (vs. present) and dorsal scales on trunk

heterogeneous (vs. homogenous).

With description of four new species of Cnemaspis,

the total number of species within this genus from India

is elevated to 33. The description of several new species

from the Western Ghats suggests that our understanding

of species richness within this genus is still limited, and

that undescribed diversity likely still remains. Several

species of this genus are assessed as either Near Threat¬

ened or Data Deficient by the lUCN (Srinivasulu et al.

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | el 57

Table 5. Pair-wise uncorrected genetic distance between putative Cnemaspis species and K2P distances between individual sequences.

Sayyed et al

000 0

■S

Species

-g

-s

'|,

js'

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

Table 6. Comparative account according to the original description of the genus Cnemaspis from the northern Western Ghats

and geographically close species from southern Western Ghats (? = data not available, - = pores not present), Abbreviations: see

Materials and Methods.

Species and locality

Sptub

PaPores

FPores

MVS

SupraL

SVL

Lamp IVth

Cnemaspis limayei sp. nov.

(Sindhudurg, Maharashtra)

absent

26-27

7-9

31 mm

10-12

Cnemaspis ajijae sp. nov.

(Satara, Maharashtra)

absent

3-4

29-30

7-8

37 mm

11-13

Cnemaspis amboliensis sp. nov.

(Sindhudurg, Maharashtra)

present

3-4

19-22

7-8

32 mm

10-11

Cnemaspis mahabali sp. nov.

(Pune, Maharashtra)

absent

8-9

32.25 mm

11-12

Cnemaspis kolhapurensis

(Kolhapur, Maharashtra)

absent

24-28

20-23

40 mm

10-12

Cnemaspis girii

(Satara, Maharashtra)

absent

26-28

7-8

35 mm

17-20

Cnemaspis goaensis

(Goa)

present

2-4

18-22

28.03 mm

Cnemaspis adii

(Ballari, Karnataka)

absent

22-26

8-9

35 mm

20-22

Cnemaspis heteropholis

(Uttara Kannada, Karnataka)

absent

20-22

9-8

45.01 mm

Cnemaspis flaviventralis

(Sindhudurg, Maharashtra)

absent

28-29

7-9

36.04 mm

10-12

2014). Thus, additional taxonomic and natural history

investigations are needed to determine the true richness,

species limits, and conservation status of South Asian

Cnemaspis. While one of our new species, C. amboli-

ensis, appears to be common and widespread and is thus

likely not of great conservation concern, the other three

are known from very small geographic areas and did not

appear to have abundant populations. Thus, they may be

imperiled by lUCN criteria.

Based on the morphological divergence described

here for the northern Western Ghats species, a compara¬

tive account for those and the geographically proximate

Key to the species of Cnemaspis in northern Western Ghats

1) Cnemaspis goaensis

Dorsal scales on body heterogeneous; gulars weakly carinate; pre-anal and femoral pores present in males; subcaudals slightly enlarged and

smooth (Sharma RC 1976; Manamendra-Arachchi et al. 2007).

2) Cnemaspis kolhapurensis

Dorsal scales homogenous, small and feebly keeled; no spine-like tubercles on flanks; a continuous series of 24-28 precloacal-femoral

pores (Giri et al. 2009).

3) Cnemaspis girii

Dorsal scales heterogeneous; large scales on the dorsal part of body smooth; keeled conical tubercles on flank; males with four femoral

pores (Mirza et al. 2014).

4) Cnemaspis flaviventralis

Dorsal scales heterogeneous; large keeled conical tubercles on flanks; three pairs of postmentals; without a series of enlarged median sub-

caudal scales; males with three femoral pores (Sayyed et al. 2016).

5) Cnemaspis limayei sp. nov.

Dorsal scales on body heterogeneous; conical and spine-like tubercles absent on flank, almost homogeneous; pre-anal scales large than

ventral; males with four- five femoral pores; sub-caudals not enlarged.

6) Cnemaspis ajijae sp. nov.

Dorsal scales on body heterogeneous; granular keeled scales intermixed with large keeled depressed scales; conical and spine-like tubercles

absent on flank; three pairs of postmentals; males with three or four femoral pores.

7) Cnemaspis amboliensis sp. nov.

Dorsal scales on body heterogeneous; granular, conical and spine-like tubercles on flank; scales on snout feebly keeled; dorsal scales of the

forelimb and hindlimb tricarinate; three or four pre-anal pores and three to four femoral pores on each side of the thigh.

8) Cnemaspis mahabali sp. nov.

Dorsal scales on body heterogeneous; conical and spine-like tubercles absent on the flank; scales on ventral part of neck feebly carinate;

dorsal scales of the forelimb and hindlimb strongly keeled; three femoral pores on each side.

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

Table 7. GenBank accession numbers for the DNA-sequence dataset.

Species

Locality

Voucher

16S

Cnemaspis mahabali sp. nov

Pune, Maharashtra

BNHS 2451

MHl 74353

Cnemaspis mahabali sp. nov

Pune, Maharashtra

BNHS 2502

MHl 74352

Cnemaspis mahabali sp. nov

Pune, Maharashtra

BNHS 2503

MHl 74354

Cnemaspis amboliensis sp. nov

Sindhudurg, Maharashtra

BNHS 2458

MHl 74358

Cnemaspis amboliensis sp. nov

Sindhudurg, Maharashtra

BNHS 2505

MHl 74355

Cnemaspis amboliensis sp. nov

Sindhudurg, Maharashtra

BNHS 2507

MHl 74357

Cnemaspis amboliensis sp. nov

Sindhudurg, Maharashtra

BNHS 2508

MHl 74356

Cnemaspis ajijae sp. nov

Satara, Maharashtra

ZSl WRCR/1055

KX753650

Cnemaspis ajijae sp. nov

Satara, Maharashtra

ZSl WRCR/1056

KX753651

Cnemaspis ajijae sp. nov

Satara, Maharashtra

ZSl WRCR/1057

KX753652

Cnemaspis ajijae sp. nov

Satara, Maharashtra

ZSl WRCR/1058

KX753653

Cnemaspis ajijae sp. nov

Satara, Maharashtra

ZSl WRCR/1059

KX753648

Cnemaspis ajijae sp. nov

Satara, Maharashtra

ZSl WRCR/1060

KX753649

Cnemaspis limayei sp. nov

Sindhudurg, Maharashtra

ZSl WRCR/1052

KX753646

Cnemaspis limayei sp. nov

Sindhudurg, Maharashtra

ZSl WRCR/1053

KX753647

Cnemaspis yercaudensis

Salem, Tamil Nadu

BNHS 2509

MHl 74359

Cnemaspis yercaudensis

Salem, Tamil Nadu

BNHS 2510

MHl 74360

Cnemaspis otai

Vellore, Tamil Nadu

BNHS 2511

MHl 74361

Cnemaspis otai

Vellore, Tamil Nadu

BNHS 2512

MHl 74362

Cnemaspis gracilis

Palakkad, Kerala

BNHS 2513

MHl 74369

Cnemaspis gracilis

Palakkad, Kerala

BNHS 2514

MHl 74370

Cnemaspis indica

Nilgiris, Tamil Nadu.

BNHS 2515

MHl 74365

Cnemaspis indica

Nilgiris, Tamil Nadu.

BNHS 2516

MHl 74366

Cnemaspis littoralis

Kozhikode, Kerala

BNHS 2517

MHl 74367

Cnemaspis littoralis

Kozhikode, Kerala

BNHS 2518

MHl 74368

Cnemaspis kottiyoorensis

Kannur, Kerala

BNHS 2519

MHl 74363

Cnemaspis wynadensis

Wayanad, Kerala

BNHS 2520

MHl 74364

Cnemaspis indraneildasii

Uttara Kannada, Karnataka

BNHS 2460

KX753656

Cnemaspis indraneildasii

Uttara Kannada, Karnataka

BNHS 2461

KX753657

Cnemaspis indraneildasii

Uttara Kannada, Karnataka

BNHS 2462

KX753658

Cnemaspis indraneildasii

Uttara Kannada, Karnataka

BNHS 2463

KX753659

Cnemaspis goaensis

Kolhapur, Maharashtra

CnKh 33

MHl 74375

Cnemaspis goaensis

Kolhapur, Maharashtra

ChKh 34

MHl 74376

Cnemaspis goaensis

Kolhapur, Maharashtra

CnKo 48

MHl 74377

Cnemaspis goaensis

Kolhapur, Maharashtra

CnKo 49

MHl 74378

Cnemaspis goaensis

Shimoga, Karnataka

CnlnAr 1

MH174371

Cnemaspis goaensis

Shimoga, Karnataka

CnInAr 2

MHl 74372

Cnemaspis goaensis

Shimoga, Karnataka

CnInAl

MHl 74373

Cnemaspis goaensis

Shimoga, Karnataka

CnInA2

MHl 74374

Cnemaspis flaviventralis

Sindhudurg, Maharashtra

ZSl WRCR/1042

KX269819

Cnemaspis jlaviventralis

Sindhudurg, Maharashtra

ZSl WRCR/1043

KX269820

Cnemaspis girii

Satara, Maharashtra

BNHS 2445

KX269823

Cnemaspis girii

Satara, Maharashtra

BNHS 2446

KX269824

Cnemaspis kolhapurensis

Sindhudurg, Maharashtra

BNHS 2447

KX269821

Cnemaspis kolhapurensis

Sindhudurg, Maharashtra

BNHS 2448

KX269822

Cnemaspis heteropholis

Shimoga, Karnataka

BNHS 2466

KX753660

Cnemaspis add

Ballari, Karnataka

BNHS 2464

KX753654

Cnemaspis add

Ballari, Karnataka

BNHS 2465

KX753655

Cnemaspis goaensis

Goa

ZSl WRCR/1044

KX269825

Cnemaspis goaensis

Goa

ZSl WRCR/1045

KX269826

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Four new species of the genus Cnemaspis

species from the southern Western Ghats is provided

(Table 6). Furthermore, we provide a key to the northern

Western Ghats species as follows:

Appendix

Cnemaspis goaensis: Holotype (male) ZSI-K 22110,

ZSI-K 22213-22216 (four paratypes); “ca. 3 km S. of

Forest Rest House, Canacona (Poinguinim), Goa ” Also,

two specimens of Cnemaspis goaensis, ZSI R/1044 and

ZSI R/1045, were collected outside the protected area

near the type locality of the species, for examination and

used for genetic analysis.

Cnemaspis goaensis: CnKh 33, ChKh 34, CnKo 48, and

CnKo 49 collected from the human habitation at Kolha¬

pur, district Maharashtra, for examination and used for

genetic analysis.

Cnemaspis indraneildasii: BNHS 2460 and BNHS 2461

collected from Gund and specimens BNHS 2462 and

BNHS 2463 from Dandeli, Karnataka. CnInAr land Cnl-

nAr 2 collected from Agumbe road, specimens CnInA 1

and CnInA 2 collected near Agumbe, Shimoga district,

Karnataka. Collected for examination and used for ge¬

netic analysis.

Cnemaspis adii: BNHS 2464 and BNHS 2465 collected

from the Hampi, Karnataka type locality of the species,

for examination and used for genetic analysis.

Cnemaspis yercaudensis: BNHS 2509 and BNHS 2510

collected from the Yercaud town, Tamil Nadu, for exami¬

nation and used for genetic analysis.

Cnemaspis otai: BNHS 2511 and BNHS 2512 collected

from the Vellore fort, Tamil Nadu, for examination and

used for genetic analysis.

Cnemaspis gracilis: Voucher specimen (male) BNHS

1182; Goa. Also, two specimens of Cnemaspis gracilis,

BNHS 2513 and BNHS 2514, collected from the Palak-

kad, Kerala, for examination and used for genetic analy¬

sis.

Cnemaspis indica: Voucher specimens BNHS 1252-10

(male) and BNHS 1252-1 (female); Nilgiris, Tamil Nadu.

Also, two specimens of Cnemaspis indica, BNHS 2515

and BNHS 2516, collected from Ooty, Tamil Nadu, for

examination and used for genetic analysis.

Cnemaspis girii: Holotype (male) BNHS 2299; Kaas

plateau, Satara district, Maharashtra; paratypes BNHS

2081 (male) and BNHS 2078 (female); other details

same as holotype. Also, two specimens of Cnemaspis

girii, BNHS 2445 and BNHS 2446, collected from the

type locality of the species for examination and used for

genetic analysis.

Cnemaspis flaviventralis: Holotype (male) BNHS 2442,

Paratypes (male) BNHS 2443, ZSI-WRC R/1039, ZSI-

WRC R/1042, (female) BNHS 2444, ZSI-WRC R/1040,

ZSI-WRC R/1041, ZSI-WRC R/1043, examined and

Amphib. Reptile Conserv.

(male) ZSI-WRCR/1042, (female) ZSI-WRCR/1043

used for genetic analysis. All specimens were collected

from Amboli Sindhudurg district, Maharashtra.

Cnemaspis littoralis: Voucher specimen (male) BNHS

1150; Nilambur, Malabar. Also, two specimens of Cne¬

maspis littoralis, BNHS 2517 and BNHS 2518, collected

from the Kozhikode, Kerala, for examination and used

for genetic analysis.

Cnemaspis kolhapurensis: Holotype (male) BNHS

1855; Dajipur, Kolhapur district, Maharashtra. Also, two

specimens of Cnemaspis kolhapurensis, BNHS 2447 and

BNHS 2448, collected from Amboli, Sindhudurg district,

Maharashtra, for examination and used for genetic analy¬

sis.

Cnemaspis heteropholis: BNHS 2466 collected from

the Shimoga, Karnataka, for examination and used for

genetic analysis.

Cnemaspis kottiyoorensis: BNHS 2519, collected from

the Kannur, Kerala, for examination and used for genetic

analysis.

Cnemaspis wynadensis: Voucher specimen BNHS 1042

(male) and BNHS 1043 (male); Mannarghat, Palghat,

Kerala. Also, one specimen of Cnemaspis wynadensis,

BNHS 2520, collected from the \ythiri, Kerala, for ex¬

amination and used for genetic analysis.

The specimens used for new species described in this

paper:

Cnemaspis limayei sp. nov.: ZSI WRC R/1052 and ZSI

WRC R/1053, collected from the Sindhudurg district,

Maharashtra, used for genetic analysis.

Cnemaspis ajijae sp. nov.: ZSI WRC R/1055, ZSI WRC

R/1056, ZSI WRC R/1057, ZSI WRC R/1058, ZSI WRC

R/1059, and ZSI WRC R/1060, collected from the Sa¬

tara, Maharashtra, used for genetic analysis.

Cnemaspis amboliensis sp. nov.: BNHS 2458, BNHS

2505, BNHS 2507, and BNHS 2508, collected from

Sindhudurg, Maharashtra, used for genetic analysis.

Cnemaspis mahabali sp. nov.: BNHS 2451, BNHS

2502, and BNHS 2503, collected from Pune, Maharash¬

tra, used for genetic analysis.

The source of morphological comparison data for C. adii

was taken from Srinivasulu et al. (2015); for C. australis,

C. monticola, C. nilagirica, C. beddomei, C. boiei, C. or-

nata, C. andersonii, C. jerdonii, C. wicksii, C. goaensis,

and C. sisparensis from Manamendra-Arachchi et al.

(2007); for C. indraneildasii from Bauer (2002); for

C. heteropholis from Bauer (2002) and Ganesh et al.

(2011); for C. otai and C. yercaudensis from Das and

Bauer (2000); for C. anaikattiensis from Mukherjee et al.

(2005); for C. mysoriensis from Giri et al. (2009); for C.

kottiyoorensis from Cyriac and Umesh (2014); for C. as-

samensis from Das and Sengupta (2000); for C. nairi and

August 2018 | Volume 12 | Number 2 | e157

Sayyed et al.

C. tropidogaster from Inger, Marx, and Koshy (1984);

for C. kolhapurensis from Giri et al. (2009); and for C.

girii from Mirza et al (2014).

Acknowledgements. —The authors are thankful to

the following: Hemant Ghate and Robert Powell for their

support and help in the work; Neelesh Dahanukar for

the molecular work and his support in the work; Raj go-

pal Path for his support in the work; Anilkumar Khaire

for inspiration; Principal Chief Conservator of Forests

(Wildlife) and Chief Wildlife Warden, Maharashtra State

for granting the Permit to AS, (D-22(8)AVL/CR-94 (14-

15)7638/2015-16); Forest Departments of Tamil Nadu,

and Kerala for the Permit to RD (WL10-41691/2014 and

94/2009/ & WLD). We are thankful to Patrick Campbell,

Natural History Museum, London; Deepak Apte, Direc¬

tor and Rahul Khot, museum curator, Bombay Natural

History Society, Mumbai for granting access to speci¬

mens and registration of the type specimens, and Vithoba

Hegde, senior field assistant, Priya Wareker, research

fellow, and Sham Jadhav, assistant, for their help; the

Officer In-charge and Shrikant Jadhav, scientist. Zoo¬

logical Survey of India, Western Regional Center, Pune,

for their help and the registration of the type specimens;

Rajesh Sanap, Vivek Sharma, Zeeshan Mirza, Harshal

Bhosle, and Amod Zhambre for the motivation. Field

work would not have been possible without the help of

Kaka Bhise in Amboli and Vinod Shrivardhankar in Bhi-

ra. Thanks to Abhijit Nale, Kapil Taple, Rahul Thombre,

Mangesh Karve, Aman Adsul, Kiran Ahire, Devendra

Bhosle, Ashutosh Suryavanshi, Satyajit Gujar, Nitesh

Anandan, Chaitanya Shukla, and Hrishikesh Awale,

members of WLPRS. RAP was funded by US NSF grant

DEB-1441719.

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Sammlung im zoologischen Museum der kaiserlichen

Akademie der Wissenschaften zu St. Petersburg. Me-

moires de TAcademie imperiale des Sciences de St.

Petersbourg 35: 1-72.

Uetz P, Hosek J, editors. 2017. The Reptile Database.

Available: http://www.reptile-database.org [Ac¬

cessed: 22 May 2017].

Amit Sayyed is a head researcher at the Wildlife Protection and Research Society, India. He is

working on the faunal diversity and conservation of reptiles; his main interests have been in the

taxonomy of geckos and frogs. He has published several papers on natural history, faunal diversity,

and descriptions of new species. Amit is the author of three books; Amazing Creature of the Earth

(Snakes of Maharashtra, Goa and Karnataka), Butterflies and Spiders of The Western Ghats, and

Dangerous Bite and First Aid. His PhD focused on wildlife and wildlife conservation. He plans to

pursue his further studies on phylogenetic systematics, taxonomy, and natural history of the Indian

Cnemaspis.

Alex Pyron is an Associate Professor of Biology at George Washington University. He works on

theoretical and applied methods in statistical phylogenetics, using reptiles and amphibians as model

groups.

Dr. Dileepkumar R is currently Principal Investigator under Young Investigator’s Programmer in

Biotechnology in the Centre for Venom Informatics, University of Kerala. He is also co-investigating

projects in the area of venomics supported by KSCSTE, Govt, of Kerala. His research broadly

encompasses the venomics, venom gland transcriptomics, and genomics of venomous snakes. His

ongoing projects are centered on understanding the venom composition of venomous species in the

animal kingdom. His publications, book chapters, etc., focus on snake taxonomy, venomics, and

applications of medical technologies.

Amphib. Reptile Conserv.

August 2018 | Volume 12 | Number 2 | e157

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

12(2) [General Section]: 30-40 (el58).

Diversity of anurans in forest fragments of

southwestern Ethiopia: The case of the Yayu Coffee Forest

Biosphere Reserve (YCFBR)

^Tilahun Mulatu and ^Abebe Getahun

'Chair of Ecosystem Planning and Management, Ethiopian Institute of Architecture, Building Construction and City Development, Addis Ababa

University, P.O. Box 518, Addis Ababa, ETHIOPIA ^Department of Zoological Sciences, College of Natural Sciences, Addis Ababa University, Addis

Ababa, ETHIOPIA

Abstract .—^Amphibia is the least studied group of vertebrates in Ethiopia. Besides contributions from a few

expeditions in the past, the effort given to amphibian studies among academicians and researchers has been

very minimal until recently. This is the cause for the lack of detailed information on amphibians, even in known

protected areas. Despite the huge role of forest fragments in southwestern Ethiopia for conserving amphibian

diversity, the focus given for amphibian conservation within these forest fragments is almost insignificant.

The increasing rate of deforestation and fungal infection are threatening the survival of anurans (tailless

amphibians) of the region. The current study focused on determining the anuran (tailless amphibian) diversity

in a recently recognized reserve, the Yayu Coffee Forest Biosphere Reserve (YCFBR), in southwestern Ethiopia.

A total of 101 individuals, from ten different species (nearly 6.5% of the total number of known species in the

country), was collected from four different habitats including forests, stream sides, swamps, and temporary

ponds, with a greater number collected or observed in swampy areas. As expected, the wet season collection

yielded a greater number of collected individuals than the dry season collection. These results indicated the

potential of the reserve for conserving regional amphibian fauna. Future conservation measures should focus

on reducing the extent of deforestation, particularly in protected areas, while also estimating the magnitude of

fungal infection among diverse anuran species of the region.

Keywords. Amphibians, biodiversity, conservation, deforestation, fungal infection, survey

Citation: Mulatu T, Getahun A. 2018. Diversity of anurans in forest fragments of southwestern Ethiopia: The case of the Yayu Coffee Forest Biosphere

Reserve (YCFBR). Amphibian & Reptiie Conservation 12(2) [General Section]: 30-40 (el58).

mercialNoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium,

provided the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized

publication credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptiie Conservation-, official journal website

<amphibian-reptiie-conservation.org>.

Received: 23 April 2016; Accepted: 15 February 2018; Published: 1 September 2018

Introduction

Amphibians are the least studied group of vertebrates in

Ethiopia (Largen and Spawls 2010). This, according to

Mengistu et al. (2013), is due to the fact that amphibians

provide no economic value to the society, the society’s

negative attitude towards them, and the increasing focus

among academicians and researchers on large and more

visible mammals than on the small and mainly nocturnal

amphibians. A significant fraction of the known Ethio¬

pian amphibians is endemic to the country, comprising

around 40% of total species (Mengistu et al. 2013). How¬

ever, the few studies on amphibians that have been con¬

ducted in the past were mostly collecting expeditions and

did not provide further information on the ecology and

systematics of amphibians.

CorrGSpondonCG. 'tilahunmulatu@yahoo.com (Corresponding author)

Different scholars from the United Kingdom, Italy, and

Germany were involved in these expeditions that took

place between 1839 and 1939, resulting in the collection

of a large number of amphibians. According to Eargen

(2001), the collected specimens were placed in differ¬

ent museums in Europe, including: Museum National

d’Histoire Naturelle in Paris, Natural History Museum

in Eondon, Museum Koenig in Bonn, and the Eiverpool

Museum. After 1939, a collaborative work of Yalden,

Eargen, and Morris identified the collected specimens.

The Harenna forest expedition in 1986 was the most suc¬

cessful expedition resulting in the largest number of am¬

phibians ever collected (Eargen 2001). This expedition

suggests the existence of vast, yet undescribed amphib¬

ian fauna in the country that needs further study on distri¬

bution, diversity, and abundance in the different regions

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 58

Mulatu and Getahun

□□ Boundary of Ethiopia

★ Collection sites

• Localities

- Rivers

‘ I Boundary of YCFBR

Elevation

I I -189 - 500

[3 500 -1,000

I i,ooi; -1,500

I 1,501 - 2,000

I 2,001 - 2,500

I 2,501 - 3,000

I 3,001 - 4,000

Fig. 1. Location of study area.

of the country. There have been few amphibian studies in

the country, such as by Roman Kassahun (2009) in Bale

Mountains National Park (BMNP) and Wondwosen Tito

(2009) in Abijatta - Shalla Lakes and Awash National

Park, Teme (2016) in Wombera district of Awi zone, and

NABU (2017) in Kaffa biosphere reserve. The study on

the distribution of Ptychadena (Mengistu 2012; Freilich

et al. 2014), Leptopelis spp. (Weinsheimer et al. 2010;

Mengistu 2012), Xenopus (Evans et al. 2011), Afrixalus

spp. (Mertens et al. 2016) and Phrynobatrachus (Zimkus

2008; Zimkus et al. 2010) were few taxon-based amphib¬

ian studies in the country. A new dimension of research

on amphibians of Ethiopia i.e., their susceptibility to fun¬

gal infection, started in 2008 that surveyed selected parts

of Ethiopia for any incidence of fungal infection (Gower

et al. 2012). Despite the growing number of research fo¬

cused on ecology of amphibians, data on status and dis¬

tribution of amphibians in Ethiopia is still insufficient es¬

pecially when compared with relatively well-studied bird

and mammalian taxa.

Amphibians are strongly associated with wetlands

and forest ecosystems, and the forest refuge in south¬

western Ethiopia hosts one of the most suitable habitats

for amphibians (Mengistu 2012). A continuous study on

the status of amphibians and their suitable habitats is nec¬

essary to update knowledge of their distribution and to

understand their responses to environmental change. The

Yayu Coffee Forest Biosphere Reserve (YCFBR) is situ¬

ated in Ilu Abba Bora Zone of the Oromia Regional State,

southwestern Ethiopia (ECFF 2012). It is the center for

Coffea arabica which is globally a highly popular cof¬

fee species. The reserve is an important place not only

for the conservation of (wild species of) Coffea arabica,

but also the vast biodiversity living in the forest reserve.

The reserve also includes afro-montane biodiversity

hotspots, important bird areas, archaeological sites, ritual

sites, caves, and waterfalls (ECFF 2012). The Yayu Cof¬

fee Forest obtained its Forest Biosphere Reserve status in

2010, together with 12 other forest fragments throughout

the world (UNESCO Press Release 2010). The reserve is

divided into core, buffer, and transition zones based on

the extent of anthropogenic disturbances in the reserve

(Gole 2003). The present study is focused on the anuran

(tailless amphibian) diversity and relative abundance in

Yayu Woreda, southwestern Ethiopia. This study aimed

at providing the first data set of anurans in the reserve

and will thus contribute as a basis for future amphibian

studies in the reserve.

Methods and Materials

Study area

YCFBR (Fig. 1) is located between 8°42” to 8°44’23”N

and 35°20’31” to 36°18’20”E, and it covers 167,021 ha

(Gole 2003). The forest is characterized by rolling topog¬

raphy and it is dissected by small streams and two major

rivers, Geba and Dogi. There is continuous forest cover

along the rivers. The land frequently changes from fiat

surface plateaus to very steep slopes and valley bottoms

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 58

Diversity of anurans in forest fragments of southwestern Ethiopia

within a short distance. The elevation in the whole reserve

ranges from 1,100 to 2,337 meters above sea level (Gole

2003). The forest type in the reserve is predominantly

afromontane rainforest and considered to be a transition

between lowland and montane forest types (Gole et al.

2008). Three plant community types exist within the re¬

serve including C. Arabica-Cassipourea malosana, Ar-

gomuellera macrophylla-Celtis africana, and Dracaena

fragrans-Telclea noblis communities (Gole et al. 2008).

Samples for the recent study were collected from 10 col¬

lection sites distributed in four localities in the reserve

including Yayu, Elemo, Hurumu, and Nopa (Table 1).

According to the climate data for ten years, obtained

from National Meteorology Agency (2012), the mean an¬

nual temperature of the study area is 23.76 °C and the

mean annual rainfall is 1,625 mm. As it can be seen in

Fig. 2, the rainfall pattern is uni-modal and it reaches its

highest between May and September (wet season) and its

lowest between November and March (dry season) while

there is a small amount of rainfall in April and October,

although this shows variation year to year.

Anuran sampling

A preliminary survey of the study area was conducted for

a week from September 20-26, 2011. Information from

local people and an opportunistic night survey were used

to locate important amphibian habitats in the study area.

A total of 10 sampling sites from among these diverse

habitats were selected for collecting species data.

The data collection took place at selected dates during

core dry season which is between January 3-19, 2012

and wet season which is between June 28-July 15, 2012.

In order to encounter as many anuran species as possible,

factors like time of the day were considered. The data

collection was conducted in the time interval between

6:00 AM and 9:00 AM for day time and 6:00 PM to 9:00

PM for night time collection. A time-constrained visual

Table 1. List of species, habitat data, and man-hours spent per

collection site. (SWP- Swamp, PND- Pond, FOR- Forest, STS-

Streamside, RUN- Rock underneath, Breg- Amietophrynus

regularis, Hvir- Hyperolius viridiflavus, Hnas- Hyperolius

nasutus, Fobs- Paracassina obscura, Lreg- Leptopelis

ragazzii, Xcli- Xenopus clivii, Cbec- Conraua beccarii, Panc-

Ptychadena anchiatae, Pneu- Ptychadena neumanni, Pnat-

Phrynobatrachus natalensis).

Habitat data

'.fi

SWP

PND

FOR

Fanoshishe SWP

Elemo Haro SWP

Fani SWP

Kersa STS

Hurumu SWP

Beriche SWP

Lege Ferengi FOR

SWP

Mechibe STR

RUN

Gombo

Yayo

Mesengo

Geba

Man- Hour spent

per habitat

3= ^

1490 m 2

1490 m 1

1599 m 1

1267 m 1

1952 m r

1959 m 1

1941 m 1

1763 m r

1763 m 2

1819m 1

1680 m r

1680 m 1

1665 m 1

3 3 „ y y

3 3 „ y ..

3 3 .. y ..

3 3 y y

3 3

Species list

.. J .... J J ..

.. J J J .. .. ..

.. .. J .. J J ..

y „ .. „ y ..

„ „ J .... J ..

.. y „ „

.. .. .. J J

y „ .. ..

- - .. .. J J

y y - -

and acoustic encounter survey was used for field record¬

ing of anuran species.

A combination of visual and acoustic encounter sur¬

veys was conducted for sampling anurans in terrestrial

and semi-aquatic habitats, following Lambert (2002).

Ponds, streams, and river banks were sampled using

seine netting (Lambert 2002). Randomized walk de¬

sign within a constrained area was used for sampling

swamps and forest habitats (Lambert 2002). Due to the

smaller size of swamps, the search was done throughout

the entire area. The search for anurans in each habitat

was time-constrained, and it was conducted with three

people, spending one hour per collection site; a total of

three man-hours were spent for each collection site. Col¬

lections were done both during the day and night.

Anuran species encountered were identified to the

lowest taxonomic level possible. A picture and voucher

specimen were taken in those cases where it was not pos¬

sible to identify the species in the field. The field speci¬

mens collected for later identification were kept inside

the laboratory for graduate programs in Ecological and

Systematic Zoology, Addis Ababa University.

Results and Discussion

Diversity of amphibian habitats

The study area comprises a number of wetlands and riv¬

ers (Fig. 3). There are also a number of streams flowing

into these rivers. Additionally, the valley bottom which is

formed as a result of rugged topography of the study area

contributes to the existence of numerous small to large

sized swamps and ponds (Gole et al. 2009). Gole et al.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 58

Mulatu and Getahun

Forest

Pond

Fig. 3. Types of wetlands in YCFBR.

(2009) indicated that wetlands in the study area consist of

at least two physiognomically different vegetation types:

riparian forest along the rivers and streams, and the tree¬

less swamp vegetation with stagnant or slowly moving

water.

River/stream sides were found in two of the ten col¬

lection sites in the study area. The rivers flow year-round,

while the streams dry up during the dry season. Differ¬

ent plant vegetations that grow following the river and

stream lines potentially serve as a shelter for the adult

frogs.

There are a number of swampy areas in the study site.

Swamps are the most prevalent kind of wetlands in the

study area since they are found in eight of the ten col¬

lection sites. The amount of water varies in the various

swamps in the study site. The level of water shows great

variation between seasons as they get saturated during

wet season and dried up during dry season. Few swamps

formed close to built-up areas and road sides. The major¬

ity of the swamps are dominated by herbs while few of

them are dominated by grass. Main disturbances in these

habitats include drainage of water for household utiliza¬

tion and removal of vegetation for making thatching.

The majority of the ponds in the study area are tempo¬

rary ponds which are completely dried up during the dry

season. During wet season, it is easier to get temporary

ponds since the water level increases during this period.

Swamp

Ponds were found in only one of the ten collection sites

in the study area. Human activities near these ponds in¬

clude using pond water for household utilization and for

their cattle, agricultural, and sand mining activities near

the ponds, and removal of pond vegetation for making

thatching.

The forests of the reserve also serve as suitable habi¬

tat for amphibians (Gole et al. 2009). The forest in the

study area is divided into core, buffer, and transitional

zones. Two of the ten collection sites comprise forest

habitats, and these habitats are dominated by wild coffee

populations, though other plant species also exist in these

forests. Possible anuran microhabitats in these forests in¬

clude tree leaves, underneath rocks, and rotten logs.

Species accounts

Amietophrynus regularis Reuss, 1833

Amietophrynus regularis, which is the most widespread

species of Amietophrynus in Ethiopia, is abundant in the

central and western plateau of the county between 400-

2,500 meters, and its distribution overlaps with the distri¬

bution of two other species of Amietophrynus (A. kerin-

yagae and A. asmarae) in most of its distribution ranges

(Tandy et al. 1982; Largen 2001). However, according to

Tandy et al. (1982), habitats in southwestern Ethiopia are

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 58

Diversity of anurans in forest fragments of southwestern Ethiopia

0 10 20 30 40 50 60 70 80 90 100

Man-Hour

Fig. 4. Species cumulative curve.

exclusively comprised of A. regularis. During the current

survey, the species was collected from one locality at the

elevation of 1,490 meters. Deeply pitted parotid glands,

distinct longitudinal ridges on the tarsus, and glands be¬

neath the forearm forming a row of separate tubercles

were observed on field-captured individuals. These char¬

acteristics are diagnostic for the species (Largen 2001).

During the dry season, no individual from the species

was collected from any of the possible habitats. Howev¬

er, two individuals were observed calling from a swampy

area that was shared by P. neumanni and P. anchiatae,

and one individual was observed in swampy roadsides.

Hyperolius viridiflavus Dumeril and Bibron, 1841

Largen (2001) indicated that a molecular study for the

species resulted in three varieties which are the typical

viridiflavus variety, the pachydermus variety and destefa-

nii variety. The viridiflavus variety is common in south¬

western Ethiopia especially in the vicinity of Jimma in

the elevational range between 1,200-2,400 m (Largen

1842; Largen 2001). During the current survey, the spe¬

cies was found in elevational range between 1,400-1,900

m. During the dry season, no individuals of the species

were observed and advertising calls were not heard for

the species during the entire survey. During wet season,

a large aggregate of individuals from the species was ob¬

served on the vegetation that grows around temporary

ponds. The relatively unique advertisement calls of Hy¬

perolius spp. (Channing et al. 2002) help to easily deter¬

mine the presence of the species.

Hyperolius nasutus Gunther, 1865

This small sized and slender species of Hyperolius was

reported to exist in southwestern Ethiopia in elevational

range between 500-2,000 m (Largen 2001; Channing et

al. 2002) (Fig. 5C). It exhibits a uniform color pattern

which is green coloration with silvery white dorso-lateral

stripes similar to the findings of Largen (2001). During

the current survey, the species was observed in the eleva¬

tional range between 1,400-1,900 m. The species prefers

pond vegetations and it also shares its breeding site with

H. viridiflavous as Largen (2001) discussed. During the

dry season, no individuals were observed for this species

and the possible habitats for this species (i.e., pond and

stream vegetations) were significantly dried up during

this period. During wet season, a good number of this

species was observed near pond vegetations. The specific

calls of the species were used for their identification dur¬

ing the survey.

Paracassina obscura Boulenger, 1895

Fig. 5. Selected species encountered in YCFBR. (A) Leptopelis ragazzii, (B) Paracassina obscura, (C) Hyperolius nasutus,

(D) Xenopus clivii, (E) Ptychadena anchiatae, (F) Conraua beccarii.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 58

Mulatu and Getahun

Paracassina obscura (Fig. 5B) is one of the least stud¬

ied species of anura despite being endemic to Ethiopian

highlands. Largen (2001) indicated that the species ex¬

ists to the west of rift valley, unlike its relative P. koun-

hiensis (distributed to eastern rift valley), in the eleva-

tional range between 820-3,000 m. During the current

survey, the species was observed in the elevational range

between 1,400-1,900 m. The species was observed in

home gardens and farm lands. Individuals were heard

calling from these habitats during both the day and night

time. A bright-yellow patch at the groin which is clearly

surrounded by a dark pigment was observed on captured

individuals. This character differentiates P. obscura from

its relative P. kounhiensis. During the dry season, no sin¬

gle individual for the species was observed from all sites.

During the wet season, the species were abundantly ob¬

served in home gardens, forest clearings, swampy areas

and abandoned farmlands. It has a species-specific call

which is weak and stays for a second. This call is like

a sound that comes out from water droppings as Largen

(2001) explained.

Leptopelis ragazzii Boulenger, 1896

Identification of the various species of the genus Lep¬

topelis (especially L. susanae, L. ragazzii, and L. van-

nutellii) using only morphological characters is almost

impossible (Mengistu et al. 2012). Consulting former

distribution of the species is more reliable for identifying

the species (Weinsheimer et al. 2010). L. ragazzii (Fig.

5A), which is a small sized species, is known to exist in

southwestern Ethiopia in the elevational range between

1,900-3,100 m (Largen 2001). During the current survey,

the species was collected in the elevation range between

l, 490-1,680 m. During wet season, a greater number of

individuals was caught than during dry season collection.

Xenopus clivii Peracca, 1898

Largen (2001) discussed the existence of two species of

in Ethiopia, A c/m/andA largeni.X. clivii (¥\g.

5D), which is the widespread species of Xenopus, is the

one that was reported to exist in southwestern Ethiopia

(Evans et al. 2011). Largen (2001) indicated that the spe¬

cies exists in the elevational range between 1,900-2,750

m. During the current survey, the species was observed

in the elevational range between 1,400-1,900 m. Dorso-

ventrally fiattened body with dorsal eyes and the digits

that are extensively webbed were observed in collected

individuals. This is an adaptation for entirely aquatic ex¬

istence. The species has a very narrow habitat require¬

ment since it only exists in permanent and temporary

ponds. Tentacles that surround their eyes and the black

claw on their metatarsal tubercle, that differentiate this

species from its relative (A largeni), were observed in

the current collection. The adult individuals for the spe¬

cies were collected only from one collection site while

the tadpoles were observed in temporary ponds in other

collection sites. During the dry season, a good number of

individuals were collected, while the number decreased

during the wet season.

Conraua beccarii Boulenger, 1911

Conraua beccarii (Fig. 5F), the largest anuran species

in eastern Africa, was reported to exist in southwestern

Ethiopia in the elevational range between 300-2,500 m

(Largen 2001). During the current survey, the species

was collected from one collection site at elevation of

1,665 m taking refuge under rock crevices over which

fast moving stream water fiows. The hidden nature of

the habitat and the fast-fiowing stream water were bar¬

riers for catching individuals. Dorsal eyes, a dorso-ven-

trally fiattened body especially near the head region, and

digits that are significantly webbed like Xenopus were

observed in caught individuals. During the dry season,

three adult individuals and many tadpoles and froglets

were observed under rock crevices. However, during the

wet season no individual tadpole or froglet was observed

while the number of adult individuals does not show sig¬

nificant variation from the dry season.

Ptychadena anchiatae Bocage, 1868

Largen (2001) discussed that P. anchiatae (Fig. 5E) is

a widespread species of Ptychadena like its relative P.

neumanni, and he also indicated that the species exists

in southwestern Ethiopia below 1,800 m. During the cur¬

rent survey, the species was only recorded from one lo¬

cality at the elevation of 1,400 m. The species shares it

breeding site with its relative {P. neumanni) and it was

difficult to differentiate them by their calls when they

coexist. The species has extended webbing on the digits

and a pale triangle on the anterior region of the head to

differentiate it morphologically from P. neumanni. The

female is significantly larger than the male and like other

species of Ptychadena the male is characterize by the vo¬

cal sac. During the dry season, no individuals from the

species were caught while few individuals were caught

during the wet season.

Ptychadena neumanni Ahl, 1924

Ptychadena neumanni, which is the most widespread

species of anura throughout the country, is also reported

to exist in southwestern Ethiopia (Largen 2001). Recent¬

ly, Freilich et al. (2014) and Smith et al. (2017) indicated

that climatic variabilities and geologic events in Ethiopia

contributed for adaptive speciation within the species.

Largen (2001) indicated that the species exists in the

elevational range between 1,800-3,800 m though some

specimens were collected in lowland with the elevation

of 820 m in southwest Ethiopia. During the current sur¬

vey, the species was recorded in the elevational range

between 1,400-1,900 m. The species was cosmopolitan.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | e158

Diversity of anurans in forest fragments of southwestern Ethiopia

existing in almost all types of habitats (i.e., swamps, pond

edges, stream sides, and forest clearings), and it was also

collected from all collection sites. The species has re¬

stricted webbing on its digits that differentiate it from its

relative (P. anchiatae) (Largen 2001). However, it shares

many characteristics with P. erlargeri, which make it dif¬

ficult to differentiate the two species using morphology

(Mengistu et al. 2012). Wet season collection yields more

individuals from the species than dry season collection.

Phrynobatrachus natalensis Smith, 1849

Phrynobatrachus natalensis, which is a small sized

species, was reported to be as equally well-distributed

as Ptychadena neumanni throughout Ethiopia in the el-

evational range between 300-2,200 m (Largen 2001;

Schick et al. 2010; Zimkus and Schick, 2010; Zimkus et

al. 2010). During the current survey, the species was ob¬

served only in a single locality at the elevation of 1,680

m. The species possesses large oval marks on the anterior

region of its dorsum that are arranged in an X-shaped

configuration and dark pigmentation on the gular region

as Largen (2001) discussed. This feature differentiates it

from its relative {P. minutus) which was not collected in

the current study though it has a possible existence in

southwestern Ethiopia (Largen 2001; Schick et al. 2010).

This could arise due to the higher probability of the spe¬

cies to exist above 2,000 m than P. natalensis. During the

dry season, many individuals for the species were col¬

lected from a swamp near to a slow-moving stream. Dur¬

ing wet season, no individuals were observed from any

of the collection sites. The species produces an abrupt

species-specific call that has high strength and spans very

short. It shares its habitat with P. neumanni.

Species Diversity and Relative Abundance

A total of 101 individuals that belong to four families and

ten species were collected, both during the wet and dry

season, investing a search effort of 96 man-hours in total

for all the collection sites (Table 2, Fig. 4). The major¬

ity of the collected individuals belong to one species {P.

neumanni) that comprises around 31% of all collected

individuals, while B. regularis is the least represented in

this collection (3%). As expected wet season collection

resulted in significantly larger number of anurans than

dry season collection (t = -2.6, df = 18, P < 0.05) and

the species collected in both seasons were distributed

between an elevational gradient of 1,400 and 1,900 m.

However, a species-wise comparison between the two

seasons shows a different result for Phrynobatrachus na¬

talensis and Conraua beccarii. In the case of P. natalen¬

sis, no individual was collected in the wet season and for

C. beccarii the same number of individuals was collected

between the two seasons (Table 2).

Largen (2001) reported the existence of around 31

anuran species taking refuge inside forests of southwest-

Table 2. Wet and dry season diversity and abundance of anuran

species in YCFBR.

Abundance

Family

Species

Dry

Wet

Total

Bufonidae

Bufo regularis

Hyperoliidae

Hyperolius viridiflavous

Hyperolius nasutus

Parakassina obscura

Leptopelis ragazzii

Pipidae

Xenopus clivii

Ranidae

Conraua beccarii

Ptychadena anchiatae

Ptychadena neumanni

Phrynobatrachus natalensis

Total

101

ern Ethiopia, however in the current research we were

able to observe 10 anuran species. A potential reason for

a reduced number of species in the current study is that

some of the species reported by Largen (2001) includ¬

ing Bufo steindachneri, Hemisus marmoratus, Hypero-

lius balfouri, Hyperolius kivuensis, Kassina senegalen-

sis, Leptopelis bocagii, Haplobatrachus occipitalis, and

Ptychadena mascareniensis are essentially lowland sa¬

vanna species (Largen and Spawls 2010) with reduced

potential of occurrence in transitional afromontane rain¬

forest forest of YCFBR. The behavior of some species

as being highly secretive {Hemisus microscaphus and

Leptopelis gramineus) and species with extremely rare

distribution in southwestern Ethiopia {Ptychadena pum-

ilio, Bufo petoni, and B. maculatus) could be the reason

for not observing these species with the 96 man-hours

invested in the field. The unresolved and overlapping

taxonomic identification key of the two species of Lep-

topelsis {L. ragazzii and L. vannutellii) and Ptychadena

{P neumanni and P. erlargeri) (Weinsheimer et al. 2010;

Mengistu 2012; Freilich et al. 2014) seems to be the

reason for no representation of L. vannutellii and P. er¬

largeri in the current observation indicating the absence

of these to be non-genuine. Despite the fact thdit Afrixalus

spp. {A. clarkeorum, A. enseticola, and A. quadrivittatus)

have significant portions of their distributions in south¬

western Ethiopia (Largen 1974; Mertens et al. 2016),

we were not able to observe a single individual that we

are certain of identifying as Afrixalus spp. However, a

number of immature individuals we observed have close

resemblance with Afrixalus spp. giving us a clue that the

Afrixalus spp. potentially shares habitats With Hyperolius

spp. in our study site.

Most surprisingly two other species {Afrana angolen-

sis and Phrynobatrachus minutus) are not observed in

the current study despite, as indicated by Largen (2001),

their habitat requirement and geographic distribution

matches our study area. Hence, it seems our results do

not confirm a genuine absence of P. minutus from our

study area. The more infrequent occurrence of P. minutus

in comparison with P. natalensis (Largen 2001) could be

the reason for no observation of the species. However,

lack of observation of A. angolensis, which is abundant

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | e158

Mulatu and Getahun

and frequently observed in its natural habitat (Largen and

Spawls 2010), is in contrast to the findings of previous

studies including Largen (2001) and Largen and Spawls

(2010). The fact that a recent inventory of anurans in oth¬

er coffee forests in southern Ethiopia (NABU 2017) and

southwestern Ethiopia (Mertens et al. 2016), with similar

physiognomy and climatic features with YCFBR, also

lacking observation of the same species could indicate

that the known distribution range of the species needs to

be revised with additional field observations.

In general, the Yayu Coffee Forest Biosphere Reserve

(YCFBR) comprises diverse wetlands including swamp,

stream side and temporary ponds comprised within and

around the vast forested areas. Such abundant and diver¬

sified wetlands are the result of the rugged topography

of the study area (Gole et al. 2009). Considering the fact

that the reserve is the only internationally recognized

biosphere reserve in southwestern Ethiopia and still

comprises 10 species and 101 individuals of amphibians

which is comparable to what is found in other biodiver¬

sity hotspots and protected areas in the country (Kassun

2009; Tito 2009; Teme et al. 2016), it can be concluded

that the reserve contributes immensely to the conserva¬

tion of amphibians in the country. According to EWNHS

(1996), there are natural forest priority areas in south¬

western Ethiopia, namely Syllem-Wangas, Sheko, Yeki,

and Godere that have similar climate and fioral composi¬

tion with YCFBR. However, there is scant information

on herpetofauna of these naturally forested priority areas

and hence the findings of our research can also be an indi¬

cator for the possible existence of even greater diversity

and abundance of anurans in these forest priority areas.

Potential threats for the survival of anuran species

Amphibian population decline was first recognized as

a global phenomenon in the early 1990s (Hayes et al.

2010). Nowadays, amphibians have been repeatedly re¬

ported to decline at an alarming rate by many authors

(Pechmann and Wilbur 1994; GAA 2004; Stuart et al.

2004; Blaustein and Bancroft 2007; Gallant et al. 2007;

Hayes et al. 2010), and these same authors also discussed

this global decline of amphibians together with appropri¬

ate measures which could be taken to minimize the prob¬

lem. In Ethiopia, the main factor triggering the loss of

amphibians from different ecosystems is habitat degra¬

dation and deforestation (Mengistu 2012), though other

unstudied factors like climate change and pollution from

agrochemicals and disease (Gower et al. 2012) could also

play their own role for the declining number of amphib¬

ians in the country.

The chain of forests in southwestern Ethiopia, the

largest tract of forest next to the one found in the Hare-

na forest of Bale Mountains (Williams et al. 2004), is

known for remarkably unique species that are not shared

by similar habitats in East and Central Africa (Yalden et

al. 1996). Williams et al. (2004) indicated that the arid

and semi-arid belts stretching from southern Sudan to

northern Kenya is insulating the forest fragments, serv¬

ing as an effective barrier to the forest-dwelling species

of the Guineo-Congolian forest block. This elevates the

relevance of YCFBR for conserving the typical herpeto¬

fauna of southwestern Ethiopia. However, a significant

decline of forested areas in southwestern Ethiopia due to

the ever-increasing deforestation (Reusing 2000; Dessie

and Kleman 2007) can pose serious problems threatening

the survival of not yet well-studied anuran species of the

region. Studies indicate recent expansion of coffee farms,

plantations, and agricultural fields into former natural

forest areas in response to the ever-increasing population

of the region (Reid et al. 2000; Denboba 2005; Tadesse et

al. 2014) and coal mining (Suleman 2016) to be the main

reasons for deforestation.

Weinsheim et al. (2010) stressed that deforestation is

higher in the highlands of Ethiopia where greater amphib¬

ian endemism is recorded. As also indicated by Eoader et

al. (2014), it is the long-term persistence of highland for¬

ested habitats in Ethiopia which contributes to increasing

the diversity of anuran species. Besides, the discovery

of pathogenic chytrid fungus {Batrachochytridium den-

drobatidis) on anuran samples from the highlands of

Ethiopia particularly in Bale Mountains (Gower et al.

2011) could indicate the potential existence of the same

pathogen in highland afromontane forests in southwest¬

ern Ethiopia as the pathogen is highly epidemic in moist

highland (Pounds et al. 2006). This potentially risks the

survival of the anuran species taking refuge inside forests

of southwestern Ethiopia. However, the study by Gower

et al. (2011) asserts no lethal impacts of the fungus on

selected anuran species in Bale Mountains, different an¬

uran species have variable levels of susceptibility to the

fungus (Savage and Zamudio 2011) and hence further

study on its impacts on all anuran species in highlands

afromontane forest including YCFBR is recommended

to estimate the real impact of the fungus.

The combined effects of deforestation and sporadic

pathogenic fungus are the main threats for anuran species

of southwestern Ethiopia, asserting that future conserva¬

tion efforts should be targeting minimizing agricultural

expansion and halting the spread of fungal infection.

Avoiding the expansion of agriculture is often very chal¬

lenging as it demands local farm holders to entirely de¬

pend on coffee plantation, which is harvested at only one

time of the year and is not always reliable to support the

livelihood of the small farm holder. Fortunately, the live¬

lihood of local people in and around YCFBR is not de¬

pendent on crop cultivation, offering a good opportunity

to augment forest biodiversity conservation (Nischalke

et al. 2017). Besides, the implemented nature conserva¬

tion approaches in the reserve are reconciled with social

development strategies and hence they demand the active

participation of local community in the management and

protection of their natural resources for their own ben¬

efit (Gole et al. 2003; Senbeta et al. 2007). YCFBR has

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | e158

Diversity of anurans in forest fragments of southwestern Ethiopia

huge potential for supporting local communities through

provision of non-forest tree products (Asfaw and Etefa

2017). Hence, the recognition of the forest in 2012 as

a biosphere reserve through UNESCO’s Man and the

Biosphere Program was the right move to reconcile lo¬

cal development and nature conservation. A recent study,

Beyene (2014), also showed that the recognition of the

forest as a biosphere reserve contributes to reducing the

extent of deforestation and hence strengthens biodiver¬

sity conservation efforts in the region.

Conclusion

Forest fragments in southwestern Ethiopia are the rem¬

nants of three-decade long deforestation that took place

throughout Ethiopia. However, the same history of de¬

forestation seems to reappear on these forest fragments

as we have witnessed a recent expansion of agricultural

areas and coal mining sites into formerly forested areas.

Such trends will deteriorate the yet unstudied biodiver¬

sity comprised within these forest fragments. Called by

addressing this issue, the Yayu Coffee Forest Biosphere

Reserve (YCFBR) was established in 2010 as part of

UNESCO’s Man and the Biosphere Program.

While YCFBR has huge potential for conserving bio¬

diversity, only few researchers were focused on uncover¬

ing the rich biodiversity of the reserve. Particularly, when

it comes to amphibians or anurans, the focus is very

limited due to the negative attitude of the general pub¬

lic toward amphibians and the economic worthlessness

of amphibians. Considering the anuran species diversity

in the reserve, YCFBR can contribute to conserving the

amphibian diversity of southwestern Ethiopia. The af-

romontane forests in the reserve, which has no equivalent

in the rest of Africa, serve as refugia for typical anuran

diversity of southwestern Ethiopia.

Future conservation efforts should focus on under¬

standing the status and distribution of anurans or am¬

phibians in forest fragments of southwestern Ethiopia.

This includes detailed study on the responses of anuran

species to deforestation and their susceptibility for the

sporadic Bd {Batrachochytridium dendrobatidis) infec¬

tion. Altogether, this will contribute to reducing the de¬

clining number of anuran species in the country.

Acknowledgements. —^We are thankful to the Re¬

search Publication Office (RPO), Addis Ababa Univer¬

sity for funding all the expenses to conduct this research.

Further gratitude is forwarded to Abebe Amha (Ph.D.),

Stephan Boissinot (Ph.D.), and Xenia Freilich (Ph.D.) for

their willingness to give us their professional advice on

proper field collection of amphibians, and Neftali Sillero

(Ph.D.) and Abebe Getahun (Ph.D.) for their willingness

to revise this manuscript and give professional comments

thereof Further gratitude is forwarded to Kelly Eevitker

for proof reading the English of our manuscript.

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Tilahun Mulatu is a biologist and ecologist with special interest in spatial ecology. He grew up in

the capital of Ethiopia, Addis Ababa. He obtained his first degree in applied biology in August 2007

after completing a three-year Bachelor degree program in the Department of Applied Biology, Addis

Ababa University. After his graduation he served as a high school biology instructor until March

2009, when he was recmited to serve in the Department of Biology at Addis Ababa University as

a graduate assistant. From March 10, 2010 - September 2012, he served in the same department

as an assistant lecturer. Tilahun left for Poitiers, France in September 2012 to receive his Master’s

degree in Applied Ecology from the University of Poitiers. As his Master’s program involved

three other universities, he also moved to the University of East Anglia, United Kingdom, and the

University of Coimbra, Portugal while attending his Master’s degree. He obtained this degree in

September 2014. He started working under the chair of ecosystem planning and management at

the Ethiopian institute of Architecture, Building Construction and City Development (EiABC),

Addis Ababa University, as a lecturer in June 2015.

Abebe Getahun is a Professor of Aquatic Biology in the Department of Zoological Sciences,

Addis Ababa University, Ethiopia. He obtained his B.Sc. in Biology and M.Sc. in Zoology from

the Addis Ababa University, Ethiopia, and M.Phil. and Ph.D. from the City University of New

York, USA, in Ecology, Evolution, and Behavior. He has conducted extensive research on the

diversity, distribution, and utilization of fishes of Ethiopia including on aquaculture/aquaponics.

He has authored or co-authored over 60 articles and a Guide Book on Fishes of Lake Tana. He is

teaching undergraduate and post graduate (M.Sc. and Ph.D.) courses, advising Ph.D., M.Sc., and

undergraduate students, and is also involved in research projects.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 58

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptiie Conservation

12(2) [General Section]: 41-82 (el59).

Snakes of Angola: An annotated checklist

^’^William R. Branch

'Research Associate, Department of Zoology, P O Box 77000, Nelson Mandela University, Port Elizabeth 6031, SOUTH AFRICA ^National

Geographic Okavango Wilderness Project, Wild Bird Trust, SOUTH AFRICA

Abstract —The first annotated checklist for over 120 years is presented of the snakes of Angola, Africa. It

details the snakes currently recorded from Angola (including the Cabinda enclave), and summarizes the

literature documenting their description, provenance, and often tortuous taxonomic history. The species

are assigned, with comment where appropriate, to higher taxonomic groupings based on modern snake

phylogenetic studies, and the need or potential for further studies are noted. In 1895 Jose Vicente Barboza

du Bocage recorded 71 snakes in his monographic treatment of the Angolan herpetofauna, and subsequently

Monard (1937) added 10 additional species. This review documents the 122 snakes currently recorded from

Angola, and lists a further seven that have been recorded in close proximity to the Angolan border and which

can be expected to occur in Angola, albeit marginally. Cryptic diversity identified in taxa such as the Boaedon

capensis-fuiiginosus-iineatus complex and Phiiothamnus semivariegatus complex indicate more species can

be expected. Relative to southern Africa, the Angolan snake fauna contains a higher proportion of colubrid

snakes, enhanced particularly by diverse arboreal species of the Congo Basin. There are relatively few endemic

snakes (5.4%), and most inhabit the mesic grasslands of the escarpment and adjacent highlands. None are

obviously threatened, although records of the endemic Angolan adder (Bitis heraidica) remain scarce and the

species may require directed surveys to assess its conservation status.

Resumo. —Este trabalho apresenta a primeira lista comentada de cobras de Angola, Africa. Esta lista enumera

as especies atualmente registadas em Angola (incluindo o enclave de Cabinda) e resume a bibliografia que

documenta a sua descrigao, origem, e historia taxonomica, muitas vezes sinuosa. As especies sao atribuidas,

com comentarios quando se justifica, a grupos taxonomicos superiores, com base em estudos filogeneticos

recentes, e a necessidade ou potencial para mais estudos e tambem referida. Em 1895, Jose Vicente Barboza

du Bocage registou 71 especies de cobras no seu estudo monografico da herpetofauna angolana. Mais tarde,

Monard (1937) adicionou 10 especies. Esta revisao documenta as 122 especies atualmente registadas para

Angola, e lista outras sete que foram tambem encontradas perto das fronteiras angolanas e cuja ocorrencia em

Angola, embora de mode marginal, pode ser expectavel. A diversidade criptica identificada em taxons como

o complexo Boaedon capensis-fuiiginosus-iineatus e o complexo Phiiothamnus semivariegatus indicam que

mais especies podem ser esperadas. Comparativamente com a Africa Austral, a fauna de cobras de Angola tern

uma maior proporgao de colubrideos, aumentada particularmente por diversas especies arboricolas da Bacia

do Congo. Ha relativamente poucas (5,4%) especies endemicas, e a maioria ocupa as pastagens de altitude

da escarpa e o planalto adjacente. Nenhuma especie se encontra ameagada de forma evidente, embora os

registos da vibora angolana endemica {Bitis heraidica) continuem a ser escassos, e a especie possa requerer

levantamentos dirigidos para que o seu estatuto de conservagao seja avaliado.

Keywords. Bocage, taxonomy, Africa, diversity, endemicity, conservation

Citation: Branch W. 2018. Snakes of Angola: An annotated checklist. Amphibian & Reptile Conservation 12(2) [General Section]: 41-82 (e159).

atives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided the

original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication credit

sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation-, official journal website <amphibian-reptile-

conservation. org>.

Received: 28 February 2018; Accepted: 31 May 2018; Published: 5 September 2018

Introduction

The need for national summaries of biodiversity and the

selection of suitable areas for protection are of increas¬

ing urgency in the face of exploding human populations

and their demand for natural resources. However, bio¬

diversity surveys in Angola were severely curtailed in

the post-colonial era, and for nearly 60 years studies on

Angolan biodiversity remained almost quiescent. Reptile

studies were particularly curtailed and as a consequence

knowledge of the status and distribution of many Ango¬

lan snake species remains poorly known. This is under¬

standable as the last monographic review was published

over 120 years ago (Bocage 1895). Nearly 50 years later

Monard (1937) prepared an updated overview incorpo¬

rating new material (Monard 1931), but overlooked a

Corr6Spond6nC6. ' williamroybranch@gmail.com (Corresponding Author)

Amphib. Reptile Conserv.

41 September 2018 | Volume 12 | Number 2 | e159

Branch

number of regional reports (e.g., Schmidt 1933; Parker

1936), and many others have subsequently appeared

(e.g., Mertens 1938; Bogert 1940; Hellmich 1957a;

1957b; FitzSimons 1959). Summaries of snakes in the

collection of the Museu do Dundu in extreme north¬

east Angola (Laurent 1950, 1954; Tys van den Auden-

aerde 1967) added numerous records of snakes from the

Congo Basin. Others were added by Laurent (1964),

who also described the extensive collections of reptiles

from northeast and southwest Angola collected by An¬

tonio Barros de Machado, then Director of the Museu

do Dundu. In the last 10 years increasing access to An¬

gola, coupled with awareness of the urgent need to docu¬

ment the remaining wildlife and to revitalize the Angolan

protected area network, has led to increasing numbers

of herpetological surveys (Huntley 2009; Ceriaco et al.

2014, 2016; Ernst et al. 2014; Huntley 2015; Conradie et

al. 2016,2017; Conradie and Branch 2017; Branch 2018)

and the concomittent discovery of new species (amphib¬

ians - Conradie et al. 2012a, 2013; reptiles - Conradie et

al. 2012b; Stanley et al. 2016; Branch et al. 2017). Re¬

gional (Branch et al. 2012; Ceriaco et al. 2014a, 2016a;

Baptista et al. 2018a) and even national (Oliveira 2017)

summaries are increasingly appearing, and this checklist

forms part of these developments.

All these publications included important taxonomic

insights and new records of Angolan snakes, but usu¬

ally listed only generalized localities for the species with

little discussion of habitat associations, although Parker

(1936), Monard (1937), and Hellmich (1957b) attempted

to place the herpetofauna within an early biogeographic

framework. Most, however, were working with museum

collections, many collected by explorers or local people

and with little habitat insight or even detailed locality

data. These deficiencies all limit their usefulness in con¬

structing biogeographic patterns, or in gaining insight

into the conservation of the listed species. At the out¬

set it is important to stress aspects of Bocage’s (1895)

monograph, particularly with respect to its coverage and

the taxonomic milieu in which it was written. His work

summarizes nearly 30 years of study on the Angolan her¬

petofauna, but also includes discussion of some species

from the adjacent Congo area, particularly between the

Congo River mouth and the Bight. His discussions in¬

clude material from Cabinda Province (formerly Portu¬

guese Congo), which was established as a protectorate of

Angola in 1885, but only fully incorporated into Angola

by the mid-1920s.

Although Bocage described many new genera and

species from Angola, many were subsequently synony-

mized under other names. In part, this was because his

papers before the mid-1880s were during the early stages

of his development as a taxonomist, and he was not as

familiar with the peripheral literature. More importantly

he was not based at a major European museum such as

those in Eondon, Paris, or Berlin, and Portugal was also

no longer a dominant colonial power. Albert Gunther

and George Boulenger at the British Museum were fa¬

miliar with a cosmopolitan herpetofauna rather than just

African, and perhaps were less impressed by novelty

than Bocage. Certainly, Bocage deferred to Boulenger’s

judgement when the latter synonymized many of Bo¬

cage’s new taxa, often without comment in his publica¬

tions and his monumental catalogue of snakes (Bouleng¬

er 1883-1886). Bocage did, however, continue to discuss

variation within Boulenger’s composite species and sub¬

sequently, as modern taxonomy developed new tools and

insight into evolutionary relationships, some of Bocage’s

names were revived (e.g., Afrotyphlops angolensis from

Typhlops punctatus and Phliothamnus angolensis from

P. irregularis). In some of his species accounts Bocage

used varieties (var.), e.g., Typhlops punctatus var. line-

olatus, which in many respects is an outdated equivalent

of subspecies. The latter itself has been under criticism

as lineage-based species definitions become increasingly

prevalent (Frost and Hillis 1990) and the subspecies con¬

cept considered by many redundant. Acceptance of this

in the modern taxonomic approach is reflected in recent

revisions in which no new subspecies of southern Afri¬

can snakes have been described in the last 40 years, and

many earlier subspecies proposed in particular by Eau-

rent and Broadley in the 1960s-1980s have been either

rejected or raised to full species. Many of the ‘varieties’

Bocage noted have subsequently been re-assessed, some

rejected, some becoming full species.

The online Reptile Database (Uetz et al. 2018) has

revolutionized public access to biodiversity documenta¬

tion. It provides a wealth of information for every cur¬

rently recognized reptile species, summarizing details of

the original description, locality of type material, gener¬

alized synonymies, relevant literature, etc. It is a ‘home

base’ for systematic studies and the generation of region¬

al checklists. Understandably, however, it is not with¬

out problems. A search on Reptile Database (Uetz et al.

2018) currently generates a list of 267 Angolan reptiles,

including 122 snakes. Unfortunately, it is inaccurate in a

number of respects, as some species are included that are

unknown from the country: e.g., Calabaria reinhardti,

Aparallactus guentheri, and Hemirhagerrhis nototae-

nia, and even the South American Micrurus bogerti and

Madagascan Compsophis boulengeri. Other species are

duplicated and listed under historical and current assign¬

ments: e.g., Atheris ansiolepis (= Atheris squamigera)

and Bitis peringueyi (= Bitis heraldica).

As a first small step towards a modern synthesis of the

Angolan herpetofauna an up-to-date annotated checklist

of the snakes of Angola is summarized below. It builds

on the summaries in Bocage (1895) and Monard (1937),

and on a modern electronic database (Uetz et al. 2018),

but includes details of taxonomic changes, literature con¬

flicts, and new discoveries. It does not fully cover the his¬

torical literature, or directly re-assess the identification of

historical museum material. It also makes no attempt to

map the distributions of Angolan snakes, as other initia¬

tives are currently involved in these tasks (Marques et al.

2014). Instead it serves as a working document for Afri-

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

can and Angolan researchers, established and new, who

want to understand the diversity of Angolan snakes and

the development of our knowledge of them. The check¬

list details the 122 snakes currently reeorded from An¬

gola (ineluding the Cabinda enelave), and summarizes

the literature documenting their description, provenance,

and often tortuous taxonomic history. The species are as¬

signed, with eomment where appropriate, to higher taxo-

nomie groupings based on modern snake phylogenetic

studies, and the need or potential for further studies are

noted. For the historical literature authors of only origi¬

nal records are noted, and historical place names have

been updated to their modern name or spelling at the first

occurrence and the updated name subsequently used. Ad¬

ditional new snake distribution records for Angola will

be published elsewhere (Branch et al, in prep.). Gazet¬

teer of most historic Angolan place names mentioned in

early faunal accounts is included in Crawford-Cabral and

Mesquitella (1989).

Comments on the Checklist

The higher-level taxonomy adopted in the checklist re-

fiects recent developments in our understanding of phy¬

logenetic relationships of African snakes. Recent taxo¬

nomic insights into higher nomenclatural categories are

included in the introductory text to the various sections

below, and many are updated versions of those presented

elsewhere for South African snakes (Braneh and Bauer

2014). Vidal et al. (2009) summarized increasing un¬

derstanding of the early history and phylogenetic rela¬

tionships of snakes. At the outset it should be stressed

that snakes are a subset of lizards but that this is rarely

refiected in current taxonomic hierarchies where snakes

are afforded similar subordinal status (Serpentes) to that

of all other lizards (Sauria). This historical anomaly is

slowly being addressed and a suitable nomenclatural hi¬

erarchy developed that addresses these relationships and

their attendant complexity (e.g., Pyron et al. 2013). It has

become increasingly evident that snakes originated on

West Gondwana, that part of the supereontinent compris¬

ing South America and Africa. Among extant lineages,

the deepest divergencies are found between what have

been termed the Amerophidia and Afrophidia (Vidal

et al. 2009). The monophyly of this division has been

supported in subsequent molecular phylogenies (e.g.,

Reynolds et al. 2014), and also reproductive anatomy

(Seigel et al. 2011), but awaits full aeeeptance (Hisang

et al. 2015). The division oeeurred 106 (116-97) Ma,

probably in association with continental breakup. Most

(-85%) living snakes are afrophidians and are now glob¬

ally distributed, having initially dispersed out of Afriea

through Laurasia or on the ‘Indian raff. Most basal af-

rophidian families (Henophidia) diverged in the Creta¬

ceous (104-70 Ma), while advanced afrophidian families

(Caenophidia) diverged in the early Cenozoic, 63-33 Ma

(Vidal et al. 2009).

The -3,500 living snakes display an evolutionary trend

of increasing gape size, from fossorial seoleeophidians

(locally represented by the blind snakes, Typhlopidae,

and thread snakes, Leptotyphlopidae) to ecologically di¬

verse alethinophidians, large-mouthed lineages that feed

and swallow large prey (also more descriptively called

macrostomatans). Among the latter, the Henophidia (py¬

thons and relatives) comprise relictual lineages scattered

throughout tropieal and subtropieal regions, and includ¬

ing all but one of the world’s largest (>5 m) snakes. The

Caenophidia comprise the great majority of living snakes

(-2,700 spp.), divided among numerous families, and in¬

eluding all the venomous species.

The use of subspecies (previously races or variet¬

ies) has been declining in the 2P^ Century (Uetz and

Stylianou 2018), and no snake subspecies have been

described in greater southern Afriea (the subcontinent

and all the countries immediately bordering to the north)

since two South Africa races of Lycodonomorphus lae-

vissimus (Raw 1973).

SCOLECOPHIDIA

Blind snakes (Typhlopidae) and thread snakes (Lepto¬

typhlopidae) and their non-Afriean relatives are aneient

lineages. Due to their burrowing life style they are mor¬

phologically conservative and have thus been a source of

great taxonomic confusion in studies based on traditional

morphology. Fortunately, reeent detailed morphological

studies by Broadley and Wallach on both Afriean fami¬

lies (Broadly and Watson 1976; Broadley and Broadley

1999; Broadley and Wallach 2007a, b, 2009) has brought

relative stability to speeies boundaries in both. However,

modern moleeular phylogenies have not only revealed

the antiquity of these lineages, but also deep divergences

between them (Adalsteinsson et al. 2009; Hedges et al.

2014; Pyron and Wallach 2014; Nagy et al. 2015). As

a consequence, higher taxonomic relationships within

seoleeophidians have been in upheaval with numerous

new genera, and the shuffling of species between these

as taxon sampling inereases. In leptotyphlopids numer¬

ous eryptie species have been signaled (Adalsteinsson

et al. 2009), and studies on Australian typhlopids have

shown similar diversity (Martin et al 2013). Despite the

eurrent taxonomic upheaval, deeper insight into both

phylogenetie relationships and biogeographic patterns

continue to emerge (Vidal et al. 2010; Nagy et al. 2015),

with seoleeophidians believed to undergo an initial di-

versifieation following the separation of East and West

Gondwana and isolation on Indigascar, the eombined

India and Madagascar (Vidal et al. 2010). Studies on An¬

golan scolecophidions are in their infancy and it is likely

that further taxonomic diversity awaits diseovery. West¬

ern arid leptotyphlopids of Southern Afriean were plaeed

in the genus Namibiana by Adalsteinsson et al. (2009),

whilst Broadley and Wallaeh (2009) placed most Ango¬

lan typhlopids in the new genus Afrotyphlops, as well as

reviving Letheobia for the majority of the small, pink,

attenuate forms.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | e159

Branch

Family: Leptotyphlopidae

Shaba Thread Snake

Leptotyphlops kafubi (Boulenger 1919)

Glaucoma kafubi Boulenger, 1919. Descriptions d’Ophidian et

d’un Batracien nouveaux de Congo. Rev. Zool. Afr., Bruxelles

7(2); 186.

Revived from synonymy of Leptotyphlops nigricans by

Broadley and Broadley (1999). Unknown from Angola

by Bocage (1895), but subsequently recorded by Laurent

(1964, as Leptotyphlops emini emini) from Lagoa Calun-

do and Luisavo Falls (= Quedas do Luisavo). No fresh

material has been recorded.

Peter’s Thread Snake

Leptotyphlops scutifrons (Peters 1854)

Stenostoma scutifrons Peters, 1854. Diagnosen neuer Batrach-

ier, welche zusammen mit der fruher (24 Juli und 17 August)

gegebenen Ubersicht der Schlangen und Eidechsen mitgetheilt

warden. Ber. Bekanntmach. Geeignet. Verhandl. Kdnigl.-Bre¬

uss. Akad. Wiss., Berlin 1854: 621.

Bocage’s (1895) records from Catumbela and Novo Re¬

dondo (=Sumbe) were reassigned to Nambiana latifrons

(Broadley and Broadley 1999). Bocage’s (1895) material

from Duque de Braganca (= Calandula), Huila, Biballa

(= Bibala), Capangombe, Caconda and Cahata, and Fer¬

reira’s (1904) from Cazengo and Zembe were destroyed

and their current assignment remains problematic. The

presence of this species in Angola is currently confirmed

by only a single specimen from Chitau, north of Kuito

(Broadley and Broadley 1999), however, Adalsteinsson

et al. (2009) demonstrated that numerous cryptic lineages

are currently subsumed under L. scutifrons (type locality

Sena, Mozambique), and it is unlikely that Angolan ma¬

terial is conspecific with Mozambique material.

Damara Thread Snake

Namibiana labialis (Stemfeld 1908)

Glaucoma labialis Stemfeld, 1908. Neue und ungeniigend

bekannte afrikanische Schlangen. S. Ber. Ges. Naturforsch.

Fre unde Berlin 4: 92.

The first Angolan specimen was collected in May 1954

by Dr Charles Koch at Miranda, Cunene Province, An¬

gola (1614Dd, 16°47’S, 14°57’E). This was documented

by Broadley and Broadley (1999), but overlooked in sub¬

sequent reviews of the region (Ceriaco et al. 2016a). No

other Angolan material has been recorded. Adalsteinsson

et al. (2009) placed the species and others in the new ge¬

nus Namibiana.

Benguela Thread Snake

Namibiana latifrons (Stemfeld 1908)

Glaucoma latifrons Stemfeld, 1908. Neue und ungeniigend

bekannte afrikanische Schlangen. S. Ber. Ges. Naturforsch.

Freunde Berlin 4: 94.

This species has caused confusion, and continues to

be mis-assigned to L. scutifrons (Ceriaco et al. 2016a).

Stemfeld (1908) noted that material identified as Glau¬

coma scutifrons by Boulenger was different from Peters

(1854) type for the species, and therefore proposed the

new name Glaucoma latifrons for this material. Unfor¬

tunately, he gave no type locality. Broadley and Watson

(1976) noted that Boulenger’s knowledge of scutifrons

was based solely on two specimens from Benguela, iden¬

tified as such by Peters (1865). To stabilize the situation,

they designated the Benguela material as the types of

Leptotyphlops latifrons, and also reassigned Bocage’s

(1895) records of Stenosoma scutifrons from Catumbe¬

la and N’Gunza (= Novo Redondo) to L. latifrons. The

species is endemic to the coastal region of southwestern

Angola.

Angolan Beaked Thread Snake

Namibiana rostrata (Bocage 1886)

Stenostoma rostratum Bocage, 1886. Typhlopiens nouveaux de

la Faune africaine. Jorn. Sci. Math. Phys. Nat., Lisboa 11: 173.

The only specimen known to Bocage (1886) was his type

of Stenosoma rostrata sent by Anchieta from “Humbe,

sur les bords du Cunene.” Broadley and Broadley (1999)

noted additional material, including another thread snake

collected in May 1954 by Dr Charles Koch at Vane e

Lombe, Namibe Province, Angola (1513Ad, 15°30’S,

13°30’E), two from west of Huila, and a problematic

record from “Euanda.” Many old specimens were listed

from the home base of the collector, rather than from

where they were actually collected. Fresh material from

Euanda is required to confirm if the species extends as

far north.

Family: Typhlopidae

Angolan Blind Snake

Afrotyphlops angolensis (Bocage 1866)

Onychocephalus angolensis Bocage, 1866. Lista dos reptis das

possessoes portuguezas d’Africa occidental que existem no

Museu Lisboa. Jorn. Sci. Math. Phys. Nat., Lisboa 1: 46, 65.

First described by Bocage (1866a) as Onychocephalus

angolensis nov. sp.?, but without a diagnosis and there¬

fore a nomen nudum. It was soon correctly described by

Bocage (1866b) from “districto do Duque de Braganga,

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

situado na latitude de Loanda, porem umas 75 leguas

para o interior, portuguezas Africa occidental.” He con¬

fusingly treated it as Typhlops congicus (Boettger 1887)

in his monograph (Bocage 1895) even though his own

name had priority. Parker (1936, as T. punctatus inter-

medius) recorded it from Congulu and Quirimbo, and

Laurent (1954, finally as T. angolensis) from Dondo.

Laurent (1964) described Dundo material as the subspe¬

cies T. angolensis adolfi, but this is no longer recognized

(Broadley and Wallach 2009).

Angolan Giant Blind Snake

Afrotyphlops anomalus (Bocage 1873)

Onychocephalus anomalus Bocage, 1873. Reptiles nouveaux

de I’interieur de Mossamedes. Jorn. Sci. Math. Phys. Nat., Lis¬

boa 4; 248.

Described by Bocage (1873a) from “Huilla, finterieur

de Mossamedes,” (= Huila, Huila Province, not “Moga-

medes [= Namibe]” as recorded by Ceriaco et al. 2016a).

Other localities are given in Bocage (1895) and Monard

(1937). Broadley and Wallach (2009) discussed the var¬

ied taxonomic history of the species and placed it in a

new genus (Megatyphlops) that was itself subsequently

subsumed within the current genus (Hedges et al. 2014).

The species appears to occur on the high plateau and has

recently been recorded at Lubango and other inland lo¬

calities.

Blotched Blind Snake

Afrotyphlops congestus (Dumeril and Bibron 1844)

Onychocephalus congestus Dumeril and Bibron, 1844. Erpe-

tologie Generale ou Histoire Naturelle Complete des Reptiles.

Vol. 6. Libr. Encyclopedique Roret, Paris: 334.

A northern Congo Basin species that extends south to

Cabinda, and is known only from the type of Typhlops

{Onychocephalus) crassatus Peters, 1881 collected at

“Chinchoxo” (= Landana, Cabinda). Its complicated

taxonomic history is reviewed by Broadley and Wallach

(2009).

Lined Blind Snake

Afrotyphlops lineolatus (Jan 1864)

Typhlops {Ophthalmidion) lineolatus Jan, 1864. Iconographie

generale des ophidiens. 9. Livraison. J.B. Bailiere etFils, Paris:

24.

First recorded from Angola by Bocage (1893) when he

described Typhlops boulengeri (type-locality “Quin-

dumbo, dans Finterieur de Benguella, Angola”). Later

corrected to Typhlops punctatus lineolata (Bocage

1895), and described again as Typhlops bocagei by Fer¬

reira (1904) from “Cabicula, Bom Jesus (margens do

Quanza).” Additional material was noted from Cazengo

(Ferreira 1903), Rio Luinha (Ferreira 1906), and Dun¬

do Laurent (1954, 1964). Broadley and Wallach (2009)

designated a lectotype (BMNH 1946.1.11.18) for T

boulengeri Bocage, 1893. Ceriaco et al. (2014b) noted a

syntype (MHNFCP 017434) from Angola donated to the

Porto museum from Lisbon, and confirmed the identity

of the specimen. However, Bocage’s original material

from “Quindumbo” (= Chindumbo - Crawford-Cabral

and Mesquitela 1989) is now lost, and a range extend¬

ing to Benguela Province does not conform to the spe¬

cies’ known habiat (forest) and distribution (Broadley

and Wallach 2009). The only other Angolan localities

given for the species by Broadley and Wallach (2009)

are Cazengo (Ferreira 1903) and Rio Luinha (Ferreira

1906), both in Cuanza Norte Province. Bocage’s (1893)

Chindumbo locality should be treated with caution un¬

less confirmed by new material. Ceriaco et al. (2014b)

signaled the possible rediscovered of one of the syntypes

of Typhlops bocagei Ferreira, 1904, but have not subse¬

quently discussed its significance with respect to its cur¬

rent taxonomic status.

Schmidt’s Blind Snake

Afrotyphlops schmidti (Laurent 1956)

Typhlops schmidti LauYQnt, 1956. Laurent RF. 1956. Contribu¬

tion a Fherpetologie de la region des Grandes Lacs de F Afrique

centrale. Ann. Mus. Roy. Congo Beige (Sci. Zool.) 48: 71.

Described from “Nyunzu, Terr. D’Albertville,” Demo¬

cratic Republic of the Congo (DRC) (Laurent 1956),

the species is mainly restricted to southern DRC and ad¬

jacent northern Zambia, with only two records for An¬

gola; Calundo and Cazombo, Moxico Province (Laurent

1964).

Schlegel’s Blind Snake

Afrotyphlops schlegelii (Bianconi 1847)

Typhylops [sic] schlegelii Bianconi, 1849. Lettera al Dottore

Filippo de-Filippi, Professore di Zoologia a Torino sopra al-

cune nuove specie di rettili del Mozambico. Nuovi Ann. Sci.

Nat., Bologna (ser. 2) 10: 106.

All country records are restricted to localities above the

escarpment in southwest Angola. Bocage was confused

by this species and described it as a new species on three

different occasions: Onychocephalus petersii described

from “Biballa” (= Bibala, Namibe Province) (Bocage

1873a), Typhlops {Onychocephalus) humbo described

from Quissange (Bocage 1886), and Typhlops hottento-

tus from “Humbe” (Bocage 1893). Broadley and Wallach

(2009) discuss this taxonomic confusion and summarize

recent records.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Branch

Fig. 1. Afrotyphlops mucrosa, Lungue Bungue River, Mexico

Province {Photo: Werner Conradie).

Giant Blind Snake

Afrotyphlops mucruso (Peters 1854)

Onychocephalus mucruso Peters, 1854. Diagnosen neuer Ba-

trachier, welche zusammen mit der frilher (24. Juli und 17.

August) gegebenen Ubersicht der Schlangen und Eidechsen

mitgetheilt werden. Per Bekanntmach Geeignet. Verhandl.

Kdnigl.-Preuss. Akad. Wiss., Berlin 1854: 621.

A sister species to the previous species and long considered

an eastern subspecies. Broadley and Wallach (2009) map

five localities in northwest Angola but give no voucher

details. Laurent (1964) listed Typhlops schlegeli mucruso

from Chicapa, Calonda and Camissombo in Lunda Prov¬

ince, and these may be the source (in part) of these records.

It extends further south in the poorly surveyed eastern re¬

gion of the country (Fig. 1.; Conradie April 2018).

Leopoldville Beaked Blind Snake

Letheobia praeocularis (Stejneger 1894)

Typhlops praeocularis Stejneger, 1894. Description of a new

species of blind snake (Typhlopidae) from the Congo Free

State. Proc. US Natl. Mus. 16: 709.

Known in Angola from a single record from Dundo (Lau¬

rent 1964, as Typhlops praeocularis lundensis). Roux-

Esteve (1974) rejected the race lundensis and transferred

the species to Rhinotyphlops. Binomials were retained by

Broadley and Wallach (2007), who transferred the spe¬

cies again to Letheobia.

HENOPHIDIA

Family: Pythonidae

Pythons form part of the Henophidia, along with boas

and their relatives. They are restricted to the Old World

with about 40 species in eight genera, most within Aus¬

tralasia and with only four in Africa, three of which occur

in Angola. The Calabar Burrowing Python (Calabaria

reinhardtii) is no longer included within the Pythonidae

as it has greater affinities with boas, and is placed in a

monotypic subfamily (Calabariinae) within an enlarged

Boidae (Pyron et al. 2014). It is restricted to forest habi¬

tat in West Africa and the Congo Basin, but is unknown

from Angola, although it may extend south into Cabinda.

Amphib. Reptile Conserv.

Namib Dwarf Python

Python anchietae (Bocage 1887)

Python anchietae Bocage, 1887. Sur un Python nouveau

d’Afrique. Jorn. Sci. Math. Phys. Nat., Lisboa 12: 87.

Bocage (1887a) described the dwarf python from “Ca-

tumbella” (= Catumbela, Benguela Province). Additional

Angolan specimens are noted from Hanha (Bogert 1940)

and 18 km from Lobito to Hanha (Laurent 1964). Dur¬

ing field work in Angola (1974), Wulf Haacke collected

the only record from the inland plateau at Viriambundo,

Huila Province (15°33’S, 14°03’E, 1,288 m a.s.L). Re¬

stricted mainly to the coastal plain of southwest Angola,

with a more extensive distribution in northern Namibia.

Southern African Python

Python natalensis (Smith 1840)

Python natalensis Smith, 1840. Illustrations of the Zoology of

South Africa, Reptilia. Smith, Elder, and Co., Fondon: 3, pi. 9.

Angolan pythons examined by Bocage (1895) were

mostly from southern Angola and conformed to P. na¬

talensis. He thus referred them to P. natalensis, but he

was cautious whether they were “a species apart or... a

simple variety of P. Sebae\ For most of the 20* Century

African pythons were treated as a monotypic P. sebae,

although Monard (1931) noted that his material from

Ebanga, Chimporo and Vila da Ponte (= Cuvango) had

the characteristics of natalensis. Broadley (1984) revived

P. s. natalensis as a subspecies for southern populations,

to which he referred material from Bocage (1895), Bo¬

gert (1940), and Monard (1937). He considered the

Kwanza River to be the northern boundary for the spe¬

cies in the west. Eater he raised P. s. natalensis to a full

species (Broadley 1999).

Fig. 2. Python natalensis, bottom of Feba Pass, Namibe Prov¬

ince.

Northern African Python

Python sebae (Gmelin 1789)

Coluber Sebae Gmelin in Finnaeus, 1789. Carol! a Finne Sys-

tema naturae. 13. ed., Tom 1 Pars 3. G. E. Beer, Fipsiae: 1118.

September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

Bocage (1895) did not refer any Angolan pythons to P.

sebae. Broadley (1984), however, considered the species

to enter northern Angola, reaching as far south as Am-

briz on the coast. Both Laurent (1954, 1964) and Tys van

den Audenaerde (1967) recorded pythons from Dundo,

which Broadley (1984) also referred to P. sebae. Broad¬

ley (1984) showed that scalation features overlapped

considerably between sebae and natalensis, and that

the most distinctive features between the putative taxa

were the extent of scale fragmentation on the crown of

the head and head color pattern. There are no Angolan

localities where parapatry occurs between the two taxa

(Broadley 1984).

Fig. 3. Python sebae, Cabesa da Cobra, Soyo {Photo: Warren

Klein).

CAENOPHIDIA

Within Africa most snakes are part of the Caenophidian

radiation, i.e., the ‘higher’ snakes, and include a suite of

snake families that comprise the Colubroidea. Knowledge

of snake relationships has changed drastically in the new

millennium, fuelled by developments in genetic sequenc¬

ing, computer assessment and modeling of relationships,

and increasing gene and taxon sampling. Numerous new

arrangements and higher taxonomic categories have been

proposed, and increasing concensus is being reached.

Branch and Bauer (2014) gave a summary with an Af¬

rican perspective, but this has been modified by recent

phylogenetic updates (e.g., Hsiang et al. 2015; Figueroa

et al. 2016). The concept of the Colubroidea proposed by

Vidal et al. (2007, 2010), which is restricted to a clade of

snakes that is sister to the Elapoidea (Elapidae + Eamp-

rophiidae) of Kelly et al. (2008) is maintained. It includes

various families previously treated as subfamilies within

a more inclusive Colubridae (e.g., Natricidae and other

non-African families), and the Colubroidea is therefore

equivalent to previous usage of the Colubridae. Pyron et

al. (2011) give fuller discussion and a confiicting treat¬

ment. A major difference between confiicting arrange¬

ments is that basal caenophidian lineages such as the

Viperidae are included, along with other diverse snakes,

within the Colubroidea of Pyron et al. (2011), but not

within the restricted usage of Vidal et al. (2007, 2010).

The families Natricidae and Colubridae, the latter includ¬

ing the recently recognized subfamily Grayinae (Pyron et

al. 2011), are the only representatives of the Colubroidea

in Angola. It should be stressed, however, that the higher-

level relationships of snakes remain unsettled, and un¬

derstanding of the snake tree of life remains incomplete

(Figueroa et al. 2016) and that the arrangement adopted

here is likely to change again.

Family: Colubridae

Relationships within the African colubrid radiation re¬

main unresolved. Based on cranial features Bourgeois

(1968) recognized a subfamily Boiginae that included

the genera Boiga (restricted now to Asia as the two Afri¬

can species were transferred to Toxicodryas), Telescopus,

Crotaphopeltis, and Dipsadoboa. Subsequent molecular

data (Gravlund 2001; Kelly et al. 2003; Figuerao et al.

2016) also support the inclusion of Dasypeltis within the

Boiginae. Bourgeois (1968) erected two other subfami¬

lies: the Dispholidinae including diverse tree snakes of

the genera Thrasops, Rhamnophis, Dispholidus, Thelot-

ornis and Xyelodontophis (the latter now synonymized

with Thelotornis, Eimermacher 2012); and the Philo-

thamninae, including the genera Philothamnus and Hap-

sidophrys. The latter has not been supported by molecular

data although Figuerao et al. (2016) found these genera

were sister to Bourgeois Dispholidinae. Broadley and

Wallach (2002) recognized Bourgeios subfamily as the

tribe Dispholidini, and the diverse colubrids treated as

subfamilies by Bourgeois (1968) are perhaps best treated

as tribes (Boigini, Dispholidini) within the Colubridae.

Relatively few African colubrids have been included in

phylogenies, usually with only single species representa¬

tives of the diverse genera, and fuller resolution of their

relationships await fuller taxon sampling.

Subfamily: Colubrinae

White-lipped Snake

Crotaphopeltis hotamboeia (Eaurenti 1768)

Coronella hotamboeiaLsLUYQnti, 1768. Specimen medicum, ex-

hibens synopsin reptilium emendatam cum experimentis circa

venena et antidota reptilium austracorum, quod authoritate et

consensu. Vienna, Joan. Thomae: 58.

Bocage (1895) considered the species (as Crotaphopeltis

rufescens) to be common throughout much of Angola,

and most recent surveys (e.g., Parker 1936; Hellmich

1957; Ceriaco et al. 2016b; Conradie et al. 2016, 2017)

confirm this, although it appears rare in northeast Angola

(Eaurent 1950).

Barotse Water Snake

Crotaphopeltis barotseensis (Broadley 1968)

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Crotaphopeltis barotseensis Broadley, 1968. A new species

of Crotaphopeltis (Serpentes; Colubridae) from Barotseland,

Zambia. Fieldiana. Zoology 51(10): 136.

A poorly known snake, still known in Zambia only from

the type locality (Kalabo). All other records are restricted

to the Okavango Delta (Rasmussen 1997), with the ex¬

ception of the first records for Angola collected during

the National Geographic Okavango Wilderness Project

survey of the headwaters of the Cuito River (Conradie et

al. 2017; Conradie and Branch 2017).

Fig. 4. Crotaphopeltis barotseensis, Lake Saliakembo, Cuando

Cubango {Photo: Werner Conradie).

Confusing Egg-Eater

Dasylepis confusa (Trape and Mane 2006)

et al. 2017; Conradie and Branch 2017) conform to this

species, as does the illustration of D. scabra from Can-

gandala in Ceriaco et al. (2016b) as it has the ‘5E’ color

pattern (Gans 1959) that Trape and Mane (2006) refer to

D. confusa. Further studies are required to resolve the

species’ distribution in Angola and further south.

Palm Egg-Eater

Dasypeltis palmarum (Eeach 1818)

Coluber palmarum Leach, 1818. in Tuckey, Narrative of an ex¬

pedition to explore the river Zaire, usually called the Congo, in

South Africa, in 1816. London, J. Murray: 408.

Bocage (1895) listed uniform-colored egg-eaters, which

he referred to var. palmarum from Ambaca, Catumbela,

Dombe (= Dombe Grande), Quissange and Quindumbo,

and also from Landana in Cabinda (Peters 1877). Bocage

(1897) later listed “Dous exemplares da var. palmarum’'

in material collected by Achietae at Hanha, and Bou-

lenger (1905) referred material from Punga Andonga (=

Pungo Andongo) to D. scabra var. palmarum. Laurent

(1964) also recorded a uniform colored snake from Dun-

do that may well have been D. palmarum. Various Dasy¬

peltis species with blotched patterns occasionally have

uniform-colored individuals, e.g., D. scabra (Branch

1998), D. atra and D. medici Spawls et al. (2018), and

the status of uniform-colored Angolan egg-eaters and

their assignment to D. palmarum needs further study.

Dasypeltis confusa Trape and Mane, 2006. Le genre Dasypeltis

Wagler (Serpentes : Colubridae) en Afrique de I’Ouest: descrip¬

tion de trois especes et d’une sous-espece nouvelles. Bull. Soc.

Herp., France 119: 28.

Fig. 5. Dasylepis confusa, Cuanavale River Source, Cuando

Cubango {Photo: Werner Conradie).

This recently described species was described from Sen¬

egal, with disjunct records as far east as Cameroon, West

Africa (Trape and Mane 2006). Bates (2013) recorded

additional material, and extended the range as far south

as Gabon, but Bates et al. (2012) and Bates and Broadley

(2018) noted that D. confusa extends in to Angola. Recent

material collected during the NGOWP surveys (Conradie

Fig. 6. Dasypeltis palmarum, Soyo {Photo: Warren Klein).

Rhombic Egg-Eater

Dasypeltis scabra (hmn2iQ\\s 1758)

Coluber scaber Linnaeus, 1758. Systema naturae per regna

tria naturae, secundum classes, ordines, genera, species, cum

characteribus, differentiis, synonymis, locis. Tomus 1. Editio

decima, reformata. Laurentii Salvii, Holmiae. 10* Edition: 223.

Bocage (1895) considered egg-eaters to comprise a “single

species of the genus Dasypeltis" and with “several varieties

more or less distinct in their colors” occurring in Angola.

However, as his material was destroyed in the Lisbon fire

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Snakes of Angola: An annotated checklist

it is now difficult to assign his records to the diverse spe¬

cies now recognized or recently described. Subsequent

authors, e.g., Parker (1936), Laurent (1954, 1964), Tys

van den Audenaerde (1967), Ceriaeo et al. (2016) have

reported new Angolan material (but see D. confusa and

D. plamarum). Bates and Broadley (2018) elevated D.

loveridgei Mertens 1954 to a full species based on Namib¬

ian material, but whether it extends into southern Angola

remains uncertain.

Shreve’s Tree Snake

Dipsadoboa shrevei (Loveridge 1932)

Crotaphopeltis shrevei Loveridge, 1932. New opisthoglyphous

snakes of the genera Crotaphopeltis and Trimerorhinus from

Angola and Kenya Colony. Proc. Biol Soc. Washington 45; 83.

Described by Loveridge (1932) as Crotaphopeltis schrev-

cz, on a single specimen from “Missao de Dondi, Bella

Vista” (Missao do Dondi, Huambo, 12°32’S, 16°15’E).

Only two other Angolan specimens are known (Chitau,

Schmidt 1933; Lagoa Calundo, Moxico Province, Laurent

1964). It is more common in northern Zambia, extending

into southeastern DRC. Tanzanian records (Rasmussen

1986; Spawls et al. 2018) from Arusha usually refer to as

D. s. kageleri are particularly problematic, which possibly

deserves specific status (Branch et al. submitted).

Punctate Boomslang

Dispholidus typus punctatus (Laurent 1955)

Dispholidus typus punctatus Laurent, 1955. Diagnoses prelimi-

naires des quelques Serpents venimeux. Rev. Zool. Bot. Afr 51:

129.

Bocage first recorded the boomslang (as Bucephalus

capensis) in Angola from Dondo, and later (Bocage 1895)

noted that the species was very abundant in the highlands,

but absent from the eoastal region. He listed numerous

localities, and others are given by Laurent (1950, 1954,

1964), Tys van den Audenaerde (1967), Peters (1881),

Loveridge (1936), Bogert (1940), Managas (1973, 1981),

Ceriaeo et al. (2016b), etc. Boomslang display both onto-

gentic and sexual dimorphic color change, compounded

by regional eolor phases. This has resulted in numerous

taxonomic names, treated by various authors as synonyms,

species or subspecies. Males of the most widespread color

form in Angola have a dark body with numerous yellow

blotehes, and were refered to Dispholidus typus punctatus

based on material from Dundo by Laurent (1954). In a mo¬

lecular phylogeny of the Boomslang, Eimemacher (2012)

identified at least four separate clades that he eoncluded

represented distinet speeies, including D. viridis from

southern Africa, north and west of the Great Escarpment,

and D. punctatus from Angola, northern Zambia and ad-

jaeent DRC, but deferred taxonomic revival of these spe¬

cies pending elarifieation of additional putative new taxa

in East Africa.

Green Boomslang

Dispholidus typus viridis (Smith 1838)

Bucephalus viridis Smith, 1838. Illustration Zoology of South

Africa pi. 3.

Bocage (1882) referred material from Caconda to Bu¬

cephalus capensis, var. viridis Smith 1838. Eater, how¬

ever, he noted a green boomslang only from Humbe (Bo-

eage 1895), with other color forms, particularly punctuate,

oecurring at other loealities, but also at Humbe. Dispho¬

lidus typus viridis was recently revived by Broadley and

Blaylock (2003), following a molecular phylogeny of the

genus (Eimemacher 2012).

Emerald Snake

Hapsidophrys smaragdina (Schlegel 1837)

Dendrophis smaragdina Schlegel, 1837. Essai sur la physiono-

mie des serpens. Partie Descriptive. La Haye (J. Kips, J. HZ. et

W. P. van Stockum): 237.

It was known to Bocage (1895) only from Cabinda (Lan-

dana). Subsequently, Ferreira (1903) recorded the first An¬

golan specimen from N’Dalla-Tando (= N’Dalatando) and

Hellmich (1957) added a large series from Piri-Dembos (=

Piri). Eaurent (1954, 1964) and Tys van den Audenaerde

(1967) recorded the first records from northeast Angola

(Dundo).

Angolan Green Snake

Philothamnus angolensis (Bocage 1882)

Philothamnus angolensis Bocage, 1882. Notice sur les especes

du genre "'Philothamnus’" qui se trouvent au Museum de Lis-

bonne. Jorn. Sc. List. 9(33): 7.

Most Angolan material has been referred to Chlorophis

(= Philothamnus) irregularis: e.g., Peters (1881), Bocage

(1895), Ferreira (1903), Parker (1936), Monard (1937), Bo¬

gert (1940), Hellmich (1957), and Eaurent (1964). Although

Hughes (1985) resolved confusion concerning the content

of Philothamnus irregularis and restricted this name to West

African populations, reviving Bocage’s P. angolensis for

southern populations, this has sometimes been overlooked

(e.g., Ceriaeo et al 2014; Ceriaeo et al. 2016b).

Striped Green Snake

Philothamnus dorsalis (Bocage 1866)

Leptophis dorsalis Bocage, 1866. Lista dos reptis das pos-

sessoes portuguezas d’Africa occidental que existem no Museu

Lisboa. Jorn. Sci. Math. Phys. Nat., Lisboa 1: 48.

When naming Leptophis dorsalis Bocage (1866) gave the

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September 2018 | Volume 12 | Number 2 | el 59

Branch

ill-defined type locality “Molembo, Afrique occidentale,”

which he later emended (Bocage 1882) to “Molembo de la

Cote de Loango an nord du Zaire” (= Malembo, Cabinda

Prov., ext. NW Angola, 05°20’S, 12°irE). He later (Bo¬

cage 1895) listed a number of Angolan localities (Rio Dande,

Loanda, Benguela, Catumbela, and Pungo-Andongo). Those

south of the Kwanza River are likely to have been confused

with P. omatus (e.g., Catumbela), or their origin given as the

collector’s home base (e.g., Benguela). Additional material

was recorded (often as P. semivariegatus dorsalis, Loveridge

1958) from Dondo and Libolo (= Calulo) (Hellmich 1957).

Fig. 7. Philothamnus dorsalis, Soyo {Photo: Warren Klein).

Emerald Green Snake

Philothamnus heterodermus (Hallowell 1857)

Chlorophis heterodermus Hallowell, 1857. Notes of a collec¬

tion of reptiles from the Gaboon country, West Africa, recently

presented to the Academy of Natural Sciences of Philadelphia,

by Dr. Henry A. Ford. Proc. Acad. Nat. Sci. Philadelphia 9; 54.

Bocage (1895) knew Philothamnus herterodermus only

from the Congo, but it was subsequently recorded for An¬

gola from: Congulu (= Fazenda Congulo, Parker (1936),

Piri (Hellmich 1957b), Dundo (Tys van den Audenaerde

1967), and Capanda Dam (Ceriaco et al. 2014).

Slender Green Snake

Philothamnus heterolepidotus (Gunther 1863)

Ahaetulla heterolepidota Gunther, 1863. On some species of

tree-snakes (Ahaetulla). Ann. Mag. Nat. Hist. (3) 11: 286.

This elegant species was known from Angola to Bocage

(1866, 1879, 1882, 1887b), and by 1895 he knew it from

varied localities (Dondo, Duque de Braganca. Quibala,

Caconda and Cassange). Additional records include

Muita River, Cameia, Mexico and Dundo (Eaurent 1950,

1954, 1964; Tys van den Audenaerde 1967).

Southeastern Green Snake

Philothamnus hoplogaster (Gunther 1863)

Ahaetulla hoplogaster Gunther, 1863. On some species of tree-

snakes (Ahaetulla). Ann. Mag. Nat. Hist. (3) 11: 284.

Bocage (1895) did not include P. hoplogaster in his

monograph, but listed material that he had earlier (Bo¬

cage 1887b) referred to P. hoplogaster from St Salvador

du Congo (= M’banza-Kongo) as P. irregularis. The sta¬

tus of this specimen remains unresolved. Eaurent (1964)

listed Angolan material from Dundo, Alto Chicapa and

Camissombo, but whether this material is correctly iden¬

tified also remains unknown. Eoveridge (1958) did not

list the species from Angola or discuss any material from

the country. In contrast, Hughes (1985) mapped seven

Angolan records (without M’banza-Kongo), but cited no

authority for these records. A snake from north of Sauri-

mo, Eunda Sul Province shows genetic monophyly with

P. hoplogaster but deep divergence (Englebrecht et al.

2018). This is provisionally referred to this species, but

is being investigated further. .

Gunther’s Green Snake

Philothamnus nitidus (Gunther 1863)

Ahaetulla nitida Gunther, 1863. On some species of tree-snakes

{Ahaetulla). Ann. Mag. Nat. Hist. (3) 11: 286.

The only Angolan records are from Dundo (Eaurent

1964; Tys van den Audenaerde 1967). Eoveridge (1958)

treated P. nitidus and P. dorsalis (see above) as subspe¬

cies of P. semivariegatus, but Eaurent (1960) revived P.

nitidus as a valid species and described DRC material as

a new race, P. n. loveridgei, that Hughes (1985) contin¬

ued to recognize. However, a molecular phyogeny of the

genus (Englebrecht et al. 2018) confirmed specific status

of P nitidus but only intra-specific divergence between

the two putative subspecies. We therefore reject P. n. lov¬

eridgei and P. nitidus reverts to binomials.

Ornate Green Snake

Philothamnus ornatus (Bocage 1872)

Philothamnus ornatus Bocage, 1872. Diagnoses de quelques

especes nouvelles de Reptiles d’Afrique occidentale. Jorn. Sci.

Lisbon 4: 80.

Bocage (1972) described his new species on three speci¬

mens “from Cacheu, on the coast of Guinea, the other two

collected at Huilla by Mr. d’Anchieta” and was therefore

probably composite, with the Guinea specimens refer¬

able to P. dorsalis, which also has a dorsal stripe. No-

menclatural problems were avoided when Bogert (1940)

restricted the type locality to Huilla (= Huila), and dis¬

cussed another specimen from Huambo. Additional ma¬

terial was recorded from Bela Vista (Hellmich 1957),

and obtained during the National Geographic Okavango

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Snakes of Angola: An annotated checklist

Wilderness Project survey of the headwaters of the Cuito Hook-nosed Snake

River (Conradie et al. 2016, 2017). Scaphiophis albopunctatus (Peters 1870)

Spotted Bush Snake

Philothamnus semivariegatus (Smith 1840)

Dendrophis {Philothamnus) semivariegata Smith, 1840. Illus¬

trations of the zoology of South Africa, Reptilia. Smith, Elder,

and Co., London, 2.

Scaphiophis albopunctatus Peters, 1870. Eine Mitteilung uber

neue Amphibien (Hemidactylus, Urosaura, Tropidolepisma,

Geophis, Uriechis, Scaphiophis, Hoplocephlaus, Rana, Ento-

mogossus, Cystignathus, Hylodes, Arthroleptis, Phyllobates,

Cophomantis) des Koniglich-zoologischen Museums. Monats-

ber Akad. Wiss., Berlin 1870: 644.

Bocage (1882b) first described Philothamnus smithii,

based on diverse material from Portuguse Guinea and

Angola. He was cautious referring southern Angolan ma¬

terial from Capangombe and Maconjo to his new species,

but on coloration considered Angolan material from Ca-

tumbela, Huila and Humbe to be similar to material from

Bissau. He later (Bocage 1895) referred all Angolan ma¬

terial to P. semivariegatus. Loveridge (1958) restricted

the name P. smithii to Portuguse Guinea, and Trape and

Balde (2014) revived smithii as a West African subspe¬

cies of P. semivariegatus, extending from Guinea to Ni¬

ger (Trape and Mane 2015). They noted that “Molecular

studies suggest that this taxon could deserve full species

status.” Subsequent Angolan material from Dundo was

referred to P. semivariegatus (Laurent 1954, 1964). Re¬

cent Angolan material, including some included in a re¬

cent molecular phylogeny of the genus (Englebrecht et

al. 2018), confirmed cryptic diversity within P. semiva¬

riegatus.

Fig. 8. Philothamnus semivariegatus, Humpata, Huila.

Large-eyed Green Treesnake

Rhamnophis aethiopissa (Gunther 1862)

Rhamnophis aethiopissa Gunther, 1862. On new species of

snakes in the collection of the British Museum. Ann. Mag. Nat.

Hist. (3) 9; 129.

In his monograph Bocage (1895) did not consider Rham¬

nophis aethiopissa Gunther described from “West Af¬

rica,” as it was then unknown from either Congo or An¬

gola. It is a rare arboreal snake of closed canopy forest,

and the first Angolan recorded was from Piri (Hellmich

1957).

Bocage (1895) did not know this eastern savannah spe¬

cies from Angola. The only Angolan record is from Mui-

ta River (Laurent 1950).

Damara Tiger Snake

Telescopusfinkeldeyi (Haacke 2013)

Telescopus finkeldeyi Haacke, 2013. Description of a new Ti¬

ger Snake (Colubridae, Telescopus) from south-western Africa.

Zootaxa 3737(3); 281.

A recently described species (Haacke 2013) mainly re¬

stricted to Damaraland, Namibia, entering the southwest

arid region of Angola. Haacke (2013) recorded a single

Angolan record from just north of Namibe and an addi¬

tional record from Espinheira (Fig. 19.; Branch Decem¬

ber 2012).

Fig. 9. Telescopus finkeldeyi, Espinheira, Namibe.

Western Tiger Snake

Telescopus semiannulatus polystictus (Mertens 1954)

Telescopus semiannulatus polystictus Mertens, 1954. Neue

Schlangenrassen aus Sudwest- und Sudafrika. Zool. Anz. 152;

215.

Bocage (1895) noted that this snake (as Crotaphopeltis

semiannulatus) was rare in Angola, and was known to

him only from Gambos, Humbe, and Quissange. Addi¬

tional specimens were recorded from Calulo (Hellmich

1957) and recently from coastal Namibe. All these speci¬

mens are referred to the western subspecies T. s. polyst¬

ictus, which probably deserves specific status.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Branch

Fig. 10. Telescopus semiannulatuspolystictus, Lucira, Namibe.

Yellow-throated Treesnake

Thrasops flavigularis (Hallowell 1852)

Dendrophis flavigularis Hallowell, 1852. On a new genus and

two new species of African serpents. Proc. Acad. Nat. Sci. Phil¬

adelphia 1852: 205.

Bocage (1895) knew the species in Angola only from

Cabinda (Landana), although it was subsequently re¬

corded from Piri (Hellmich 1957).

Jackson’s Treesnake

Thrasops jacksoni (Gunther 1895)

Oates’ Vine Snake

Thelotornis capensis oatesi (Gunther 1881)

Dryiophis oatesi Gunther, 1881. In Oates, Matabeleland and

the Victoria Falls. London.

Bocage (1895) discussed this species under the name

Dryiophis kirtlandii var. oatesi, and knew that it was

“frequently seen in the highlands of Angola to the south

of the Kwuanza.” Additional material has been recorded

(as Thelotornis capensis) from Hanha (Bogert 1940),

Chitado (Hellmich 1957), Alto Chicapa (Laurent 1964),

and Longa (Conradie et al 2016). Loveridge (1953) re¬

vived oatesii as a subspecies of T kirtlandii, later trans¬

ferred to the southern species T capensis by Broadley

(1979a).

Forest Vine Snake

Thelotornis kirtlandii (Hallowell 1844)

Leptophis kirtlandii Hallowell, 1844. Descriptions of new spe¬

cies of African reptiles. Proc. Acad. Nat. Sci. Philadelphia

1844: 62.

Known to Bocage (1895) in Angola only from Calan-

dula. Subsequent material recorded from Caconda and

Quirimbo (Parker 1936), Piri-Dembos (Hellmich 1957);

Dundo (Laurent 1964; Tys van den Audenaerde 1967)

and He Bena-Mai, Rio Luachimo (Laurent 1954).

Fig. 11. Thelotornis kirtlandii, Soyo {Photo: Warren Klein).

Thrasops jacksonii Gunther, 1895. Notice of Reptiles and Ba-

trachians collected in the eastern half of tropical Africa. Ann.

Mag. Nat. Hist. (6) 15: 528.

Bocage (1895) did not know the species from Angola,

although it was subsequently recorded from Dundo (Tys

van den Audenaerde 1967). Broadley and Wallach (2002)

reviewed the genus, and did not include Angola in the

range of T jacksoni, although in their map they plot a lo¬

cality at Calandula. The recent discovery of the species in

Gabon (Carlino and Pauwels 2013) and also from Soyo,

Angola (Fig. 12. Klein April 2011) indicates that the spe¬

cies may occur in sympatry with T flavigularis in the

forests of Cabinda and even northwestern Angola. The

status of both species in the region needs confirmation.

Fig. 12. Thrasops jacksoni, Soyo {Photo: Warren Klein).

Blanding’s Treesnake

Toxicodryas blandingii (Hallowell 1844)

Dipsas blandingii Hallowell, 1844. Description of new species

of African reptiles. Proc. Acad. Nat. Sci. Philadelphia 1844:

170.

This large, nocturnal snake has been recorded infre¬

quently from Angola. Bocage (1895) had no records for

Angola and the first documented record for the country

was based on a specimen from Congula (Parker 1936).

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Snakes of Angola: An annotated checklist

Subsequently Hellmich (1957) recorded material from

Piri and Laurent (1964) from Dundo.

Powdered Treesnake

Toxicodryaspulverulenta (Fischer 1856)

Dipsas pulverulenta Fischer, 1856. Neue Schlangen des Ham-

burgischen Naturhistorischen Museums. Abhandl. Nat. Ver

Hamburg 3(4): 81.

As with Blanding’s Tree Snake, Bocage (1895) knew

of no Angolan material, and again Parker (1936) and

Hellmich (1957) recorded this species from Congulu and

Piri, respectively. No additional material is known.

Subfamily: Grayinae

Ornate Water Snake

Grayia ornata (Bocage 1866)

Macrophis ornatus Bocage, 1866. Reptiles nouveaux ou peu

connus recueillis dans les possessions portugaises de f Afrique

occidental, que se trouvent au Museum de Lisbonne. Jam. Sci.

Math Phys. Nat., Lisboa 1: 47.

graph, although it was soon synonymized with G. smithii

by Gunther (1895). Bocage (1895) noted that it must be

rare and confined to north of the Kwanza. The few subse¬

quent specimens confirm its rarity: Cambondo (Ferreira

1904), Dundo and Luachimo River (Laurent 1964).

Thollon’s Water Snake

Grayia tholloni (Mocquard 1897)

Grayia tholloni Mocquard, 1897. Sur une collection de Reptiles

recueillis par M. Haug, a Lambarene. Bull. Soc. Philom., Paris

(8)9; 11.

Bocage (1895) had no records for Angola and the only

documented record for the country remains Laurent’s

(1964) record from Rio Muita, Lunda Norte.

Family: Natricidae

The Natricidae is a recent coloniser of Africa, and has a

reltively low diversity in sub-Saharan Africa with only

five genera (Afronatrix, Heliophis, Hydraethops, Limno-

phis, md Natriciteres) and 11 species. All are semiaquat-

ic, feeding on frogs and fish.

Bocage (1866) described Macrophis ornatus by from

Duque de Braganca (= Calandula), and it was transferred

to Grayia by Sternfeld (1917). Little additional material

has been recorded: Dundo (Laurent 1954, 1964), Lagoa

Carumbo (Branch and Conradie 2015).

Fig. 13. Grayia ornata, Lagoa Cammba, Lunde Norte.

Smith’s Water Snake

Grayia smithii (Leach 1818)

Coluber smythii Leach, 1818. In Tuckey: Narrative of an expe¬

dition to explore the river Zaire, usually called the Congo, in

South Africa, in 1816. London, J. Murray; 409.

Bocage (1866) described Grayia triangularis based on

“I’exemplaire d’Angola, recueilli a Rio Dande par M.

Banyures.” He continued to use this name in his mono-

Bangweulu Swamp Snake

Limnophis bangweolicus (Mertens 1936)

Helicops bangweolicus Mertens, 1936. Fine neue Natter der

Gattung Helicops aus Inner-Afrika. Zoologischer Anzeiger 114;

284.

Mertens’ (1936) species L. bangweolicus was described

from Lake Bangweulu, northern Zambia, and treated as

a synonym of Gunther’s L. bicolor by de Witte (1953).

It was revived as a subspecies by Laurent (1964), who

found snakes referable to both taxa in northeast An¬

gola, recording L. bicolor bangweolicus from Calundo,

Moxico. Broadley (1991a) found the species in close

proximity in the Ikelenge pedicle in northwest Zambia,

and suggested that the two races should be treated as full

species. This was supported by additional material from

the region (Haagner et al. 2000), which also supported a

dietary difference between the two species. Conradie et

al (2016, 2017) record additional material in the Angolan

Okavango catchment.

Striped Swamp Snake

Limnophis bicolor (Gunther 1865)

Limnophis bicolor Gunther, 1865. Fourth account of new Spe¬

cies of Snakes in the Collection of the British Museum. Ann.

Mag. Nat. Hist. (3) 15; 97.

Gunther (1865) described Limnophis bicolor based on

two specimens sent by Bayao in 1864 from Calandula.

Bocage (1866, 1879) discussed additional specimens

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sent by Anchieta from Huila, Caconda, Quindumbo, and

Cahata, and Boulenger (1893) transferred the species to

Helicops. Bocage (1895) discussed the species (as Heli-

cops bicolor) and noted that it was found only on the high

plateau. Later specimens were recorded from Hanha (Bo¬

cage 1896) and numerous localities on the southern high

plateau (Monard 1931, 1937), and Bela Vista (Hellmich

1957). After reassessing the taxonomic status of L. bi¬

color (see above), Laurent (1964) recorded specimens of

L. b. bicolor from Alto Chilo and Alto Chicapa. Branch

and Conradie (2015) recorded the species around Lagoa

Carumba, Lunda Norte, where it was commonly caught

in fish traps.

Fig. 14. Limnophis bicolor, Lagoa Carumba, Lunde Norte.

Western Forest Marsh Snake

Natriciteres bipostocularis (Broadley 1962)

Natriciteres variegata bipostocularis Broadley, 1962. Serpen-

tes, Colubridae, Natriciteres olivacea bipostocularis n. subsp.

Occ. Pap. Nat. Mus. South Rhodesia 3 (26B): 785.

The status of Marsh snakes in Angola remains problem¬

atic. Although Bocage listed two species, Natriciteres fu-

liginoides and N. olivaceus, in his monograph, only the

latter was known to him from Angola, based on Peter’s

(1882) record from Malanje and Anchieta’s record from

Punga Andongo (also noted by Boulenger 1905). Later

records included Dondo (Hellmich (1957) and Dundo

and Muita (Laurent 1950, 1954). Subsequently, Broad¬

ley (1966a) referred Peter’s Malanje record and another

from Bela Vista (Hellmich 1956, as Natriciteres oliva¬

cea uluguruensis) to N variegata bipostocularis, which

was described from northeastern Zambia and extended

west through Katanga to the highlands of central Ango¬

la (Broadley 1966b). It is now treated as a full species

(Broadley et al. 2003). There remains a large disjunction

between the main range of N. bipostocularis in Zambia

and DRC and the isolated records in western Angola, al¬

though the eastern regions of the country remain poorly

surveyed.

Olive Marsh Snake

Natriciteres olivacea (Peters 1854)

Coronella olivacea Peters, 1854. Diagnosen neuer Batrachier,

welche zusammen mit der fruher (24. Juli und 17. August)

gegebenen Ubersicht der Schlangen und Eidechsen mitgetheilt

werden. Ber. Bekanntmach. Geeignet. Verhandl. Kdnigl.-Pre-

uss. Akad. Wiss., Berlin 1854; 622.

Bocage (1895) knew this small marsh snake (as Mizodon

olivaceus) from relatively few localities, e.g., Pungo-An-

dongo and Malange (= Malanje), and considered that its

range south was limited by the Kwanza River. Additional

material includes: “Angola,” a specimen from the Vernay

Lang Angola expedition without further details (Bogert

1840), Dundo and Muita (Laurent 1954), and Dondo

(Hellmich 1957).

Family: Lamprophiidae

Early molecular studies helped clarify interfamilial re¬

lationships within advanced snakes (Vidal and Hedges

2002a; Kelly et al. 2003; Lawson et al. 2005; Vidal et al.

2007), and highlighted the existence of a major clade (El-

apoidea, Vidal et al. 2007) that included elapids (cobras,

mambas, sea snakes, etc.) and a large and diverse radia¬

tion of mostly African and Malagacy snakes. The latter

radiation has been treated as the Eamprophiidae (Vidal et

al. 2009). Pyron et al. (2011) noted that they considered

“the most difficult aspect of higher-level colubroid tax¬

onomy to be Eamprophiidae, the assemblage of mostly

African snakes related to Elapidae.” The Eamprophiidae

envisaged by Vidal et al. (2007) initially included only

four subfamilies: the Psammophiinae, Atractaspidinae,

Eamprophiinae, and Pseudoxyrhophiinae. Kelly et al.

(2008) treated these as full families, and also proposed

the additional families Prosymnidae and Pseudaspididae.

Subsequent studies (e.g., Pyron et al. 2011; Figueroa et al.

2016) have basically retained these groupings as subfam¬

ilies (but with additional, sometimes non-African mem¬

bers of various subfamilies). The Pseudoxyrhophiinae

include numerous Malagasy genera with a number of

species also found in the Comoros Islands. Surprisingly,

the African genera Duberria, Amplorhinus (and probably

Montaspis) are also included in this subfamily, but none

occur in Angola.

Subfamily: Atractaspidinae

The atractaspidines are sometimes raised to a full fam¬

ily (Figueroa et al. 2016), with a reduced Atractaspidi¬

nae containing only Atractaspis and South African

Homoroselaps, and all other genera placed in a new

subfamily, the Aparallacinae. This arrangement is not ad¬

opted here, where the Atractaspidinae is retained within

the Eamprophiidae and contains all genera traditionally

placed in that group. A suite of genera have usually been

assigned to the atractaspidines (Amblyodipsas, Aparal-

lactus, Atractaspis, Brachyophis, Chilorhinophis, El-

apotinus, Homoroselaps, Hypoptophis, Macrelaps,

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Snakes of Angola: An annotated checklist

Poecilopholis, Polemon, Xenocalamus), which are dis¬

tributed broadly in Africa but with a limited occurrence

of some genera in the Middle East. The monophyly of

atractaspidines is well supported by both morphological

(McDowell 1968; Underwood and Kochva 1993; Zaher

1999) and molecular data (Nagy et al. 2005; Vidal and

Hedges 2007; Portillo et al. 2018). Figueroa et al. (2016)

proposed that Xenocalamus be synonymized with Am-

blyodipsas, but based this on very limited taxon sampling

of these genera. A more comprehensive molecular analy¬

sis of the subfamily (Portillo et al. 2018) included 158

individuals from six of eight aparallactine genera, and

revealed numerous cryptic taxa, as well as the need for a

number of generic readjustments to retain monophyletic

clades that continue to include generic status for hot\\ Xe¬

nocalamus and Amblyodipsas, but with adjusted species

content.

Bibron’s Burrowing Asp

Atractaspis bibronii (Smith 1849)

Atractaspis bibronii Smith, 1849. Illustrations of the Zoology

of South Africa. 3 (Reptiles). Smith, Elder, and Co., London:

51.

Bocage (1895) knew the species from Catumbela, Ben-

guela and Dombe Grande, the only places from which

Anchieta sent material, and on this basis noted that “cette

espece, qui parait affectionner en Angola la zone litto-

rale.” Additional material (as Atractaspis bibroni rostra-

ta) was recorded from Dundo (Eaurent 1950,1954,1964;

Tys van den Audenaerde 1967).

Congo Burrowing Asp

Atractaspis congica (Peters 1877)

Atractaspis congica Peters, 1877. Ubersicht der Amphibien

aus Chinchoxo (Westafrika), welche von der Africanischen Ge-

sellschaft dem Berliner zoologischen Museum ubergeben sind.

Monatsber. konigl. Akad. Wiss. Berlin. 1877 (October); 616.

Bocage (1873) did not recognize the first Angolan record

sent to him from Huilla by Anchieta, as a new species

and mistakenly referred it to Atractaspis aterrima. Pe¬

ters (1877) described the species a few years later from

Eandana, Cabinda. He later (Peters 1881) record another

specimen from Cuango. Bocage (1895) concluded that

the species lived in the highlands of the interior, as he had

received material from Quibula, Quindumbo, Galanga,

Caconda, and Huila from Anchieta. Eater additions in¬

cluded: Cazengo (Ferreira 1904), Calandula, Golungo

Alto (Boulenger 1905), Bimbe (Monard 1937), Entre

Rios, Piri, Bela Vista, Alto Cubal (Hellmich 1957), Alto

Cuilo (as A. c. congica), and Calundo, Moxico (A. c.

orientalis) (Eaurent 1964), and show that it has a wider

distribution than known to Bocage (1895). The status of

Eaurent’s subspecies of A. congica, e.g., A. c. orientalis

Eaurent, 1945 from Katanga and northern Zambia, and

A. c. lelupi Eaurent, 1950 from Katanga remain problem¬

atic, although Wallach et al. (2014) treated A. lelupi as a

full species.

Fig. 15. Atractaspis congica, Soyo {Photo: Warren Klein).

Southern Reticulate Burrowing Asp

Atractaspis reticulata heterochilus (Boulenger 1901)

Atractaspis heterochilus Boulenger, 1901. Materiaux pour la

faune du Congo. Batraciens et reptiles nouveaux. Ann. Mus.

Congo Beige, Zool. (sect. C. ser. 1) 2: 13.

Hellmich’s (1957) record of A. reticulata heterochilus

from Piri-Dembos (= Piri) is the only record of this spe¬

cies from Angola, and needs re-examination to confirm

it is not misidentified. The race is recorded from Camer¬

oon to Gabon and so may extend through Cabinda further

south.

Subfamily: Aparallactinae

Common Purple-glossed Snake

Amblyodipsas polylepis (Bocage 1873)

Calamelaps polylepis Bocage, 1873. Melanges erpetologiques.

II. Sur quelques reptiles et batraciens nouveaux, rares ou peu

connus d‘Afrique occidentale. Jorn. Acad. Sci., Lisboa 4: 216.

Bocage’s (1873) description of Calamelaps polylepis

was based on a snake from Dondo, and later material was

added from Quissange and Humbe (Bocage 1895), Ca-

bicula (Ferreira 1904), and Cazengo (Boulenger 1905).

Broadley (1971a) when revising the genus added no fur¬

ther Angolan material, but recognized an East African

race {A. p. hildebrandtii) whose status has not been re¬

assessed.

Kalahari Purple-glossed Snake

Amblyodipsas ventrimaculata (Roux 1907)

Rhinocalamus ventrimaculata Roux, 1907. Sur quelques Rep¬

tiles sud-africains. Rev. suisse Zool. 15: 78.

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September 2018 | Volume 12 | Number 2 | el 59

Branch

The presence of this Kalahari species was first recorded

in Angola from material collected during NGOWP sur¬

veys (Conradie et al. 2017; Conradie and Branch 2017)

and from Bicuar National Park (Baptista et af, in prep.).

Fig. 16. Amhlyodipsas ventrimaculata, Cuito River Source,

Cuando Cubango {Photo: Werner Conradie).

Cape Centipede Eater

Aparallactus capensis (Smith 1849)

Aparallactus capensis Smith, 1849. Illustrations of the Zoology

of South Africa. 3 (Reptiles). Smith, Elder, and Co., London,

16.

Bocage (1895) recorded the species (as Urechis capen¬

sis) from Sumbe, Bibala and Gambos, but discussed vari¬

ation among this small sample (5 specimens). Boulenger

(1895) considered Bocage’s material to be composite and

refered some to A. guentheri and the others to two new

species, A. bocagii and A. punctatoUneatus, but confus¬

ingly without allocating to which of Bocage’s specimens/

localities these names applied! The type localities for these

new species therefore by default became simply ‘Ango¬

la’. Loveridge (1944) revised the genus and assigned Bo¬

cage’s Quindumbo specimen to true A. c. capensis, and

his other material from Bibala, Gambos and Sumbe to A.

c. bocagii. Boulenger’s A. puntatolineatus was relegated

to the synonymy of A. c. capensis by Loveridge (1944),

but treated as a subspecies, A. capensis puntatolineatus,

by De Witte and Laurent (1947). Laurent (1954) record¬

ed A. c. punctatoUneatus from Dundo and Sombo, and

Broadley (1961) continued to recognized, c. puntatolin¬

eatus as a northwestern race. However, after discussing

in detail morphological variation in all subspecies Broad¬

ley (1966a) rejected them all and later formally returned

A. capensis to bionomials (Broadley 1983). However, a

recent molecular phylogeny of the Aparallactinae (Por¬

tillo et al. 2018) noted deep divergence between various

A. capensis populations, for which some of Boulenger’s

names may be available. Branch and McCarthy (1992)

recorded a specimen near Cuito Cuanavale with a blunt

head and low labials counts, but that was otherwise typi¬

cal for A. capensis. Apart from this specimen, no recent

material has been collected and the status of Angolan A.

capensis, particularly western bocagii (currently in the

synonymy of A. capensis), is unresolved.

Wilson’s burrowing snake

Hypoptophis wilsoni (Boulnger 1908)

Hypoptophis wilsoni Boulnger, 1908. Description of three new

snakes from Africa. Ann. Mag. Nat. Hist. (8) 2; 93.

It is known in Angola from only a single record of H.

wilsoni katangae from Dundo (Laurent 1964). De Witte

and Laurent (1947) differentiated H. w. katangae from

typical H. w. wilsoni by its lower ventral and subcaudal

counts and nasal condition. Broadley (1966a) noted that

Zambian material was intermediate between the two

poorly defined races and subsequently reverted to bino¬

mials (Broadley 1998a; Broadley et al. 2003). No recent

material has been available for genetic assessment.

Collared Snake-Eater

Polemon collaris (Peters 1881)

Microsoma collare Peters, 1881. Zwei neue von Herrn Major

von Mechow wahrend seiner letzten Expedition nach West-

Afrika entdeckte Schlangen und eine Ubersicht der von ihm

mitgebrachten herpetologischen Sammlung. Sitzungsber. Ges.

naturf. Freunde, Berlin 1881(9); 148.

Peters (1881) described Microsoma collare from “Ma-

cange, Cuango, West-Afrika.” However, Peters (1881)

interchanged the spellings Macange and Malange fre¬

quently in the paper, and Crawford-Cabral and Mes-

quitella (1989) listed Malange as a variant of Malanje.

Wallach et al. (2014) corrected the type locality to

Malanje, Malanje Province, northern Angola (9°33’S,

16°20’E). Crawford-Cabral and Mesquitela (1989), who

prepared a summary of all published records of Angolan

terrestrial vertebrates (1784-1974), do not discuss any of

Peters’ reptile publications, and aslo list “Macanje” as a

version of Maconge, Mo^amedes (= Namibe Province,

15°01’S, 13°12’E). However, this cannot refer to Peters’

locality as Polemon collaris is a forest species. Bocage

(1887b) referred a specimen from Cazengo to this spe¬

cies, and later two from Quindumbo (Bocage 1895). Ad¬

ditional material was noted form Gulango Alto (Ferreira

1904), Entre Rios and Bela Vista (Hellmich 1957 - as

Miodon gabonensis, then a senior synonym of M. col¬

laris, which was later revalidated as a full species, Bogert

194). A recent specimen, confirmed by genetic mono-

phyly (Portillo et al. 2018) was collected from northeast

Angola.

Bi-colored Quill-snouted Snake

Xenocalamus bicolor machadoi (Laurent 1954)

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September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

Xenocalamus bicolor machadoi Laurent, 1954. Reptiles et

batraciens de la region de Dundo (Angola) (Deuxieme note).

Companhia de Diamantes de Angola (Diamang), Servifos Cul-

turais, Publicagoes Culturais, No. 2: 45.

First recorded from Angola by Boulenger (1905) on a

specimen from between Benguela and Bihe (= Bie Prov¬

ince) collected by Anchieta, but mistakenly assigned

to Xenocalamus mechowii, Peters. Laurent (1954) de¬

scribed Xenocalamus bicolor machadoi from Dundo, in¬

cluding Boulenger’s (1905) material. Broadley (1971a)

reviewed the genus and recognized four subspecies in

X. bicolor and two in X. mechowii (see below). Genetic

material is required to re-assess the status of these races.

Elongate Quill-snouted Snake

Xenocalamus mechowii (Peters 1881)

Xenocalamus mechowii Peters, 1881. Zwei neue von Herrn

Major von Mechow wahrend seiner letzten Expedition nach

West-Afrika entdeckte Schlangen und eine Ubersicht der von

ihm mitgebrachten herpetologischen Sammlung. Sitzungsber.

Ges. naturf. Freunde, Berlin 1881(9): 147.

Described by Peters (1881) from “Macange, West-

Afrika.” Wallach et al. (2014) corrected the type local¬

ity to Malanje (see above). Bocage (1895) overlooked

Peters (1881) description and did not discuss the genus

in Angola. Witte and Laurent (1947) described X. m.

inornatus from northern Namibia, and Laurent (1954)

noted the second Angolan specimen of X. m. mechowi

from Sombo. Broadley (1971a) continued to recognize

both subspecies, but had no additional Angolan material.

Branch and McCarthy (1992) recorded the first Angolan

record of the southern race from near Lupire. The status

of the X. mechowii subspecies has not been genetically

assessed.

Subfamily: Lamprophiinae

This assemblage of African snakes (equivalent to the

Lamprophiidae of Kelly et al. 2008) includes a basic

division between wolf snakes and their relatives (Lyco-

phidion, Chamaelycus, Hormonotus, Mehelya, Goni-

onotophis, Limaformsa, etc.) and house snakes and their

relatives (Boaedon, Bothrophthalmus, Lycodonomor-

phus, etc.). Kelly et al. (2011a) restricted Lamprophis

for a group of four South African snakes, and revived

Boaedon for all other African house snakes (albeit that

not all taxa had been assessed). In addition, numerous

additional cryptic taxa within southern Boaedon popula¬

tions were identified, but not described pending further

studies. The presence of paraphyletic genera within file

snakes was also identified (Kelly et al. 2011a), but the in¬

clusion of Mehelya within Gonionotophis to maintain ge¬

neric monophyly was premature. Fuller taxon sampling

has resulted in the revival of Mehelya (but with reduced

content), the description of two new genera (Limafor-

Amphib. Reptile Conserv.

mosa and Gracililima), and reduced content for Goni¬

onotophis, now restricted to the majority of the dwarf

file snake species (Broadley et al. 2018). Unresolved,

although the subject of ongoing investigation, are spe¬

cies boundaries and phylogenentic relathionships within

house snakes (Boaedon) and their aquatic relatives (Ly-

codonomorphus).

House Snake

Boaedon capensis-fuliginosus-lineatus complex

(Boie 1827)

Resolution of the taxonomic status of these house snakes

remains one of the most persistent and challeging prob¬

lems in African herpetology (e.g., Roux-Esteve and

Guibe 1965; Thorpe and McCarthy 1978; Hughes 1997).

Numerous studies have juggled the nomenclature, with

various names and generic assignments proposed, as well

as new species described (Greenbaum et al. 2015; Trape

and Medannikov 2016). Kelly et al. (2011a) resolved

most generic affiliations although adjustments have oc¬

curred (Greenbaum et al. 2015) or are probably required

(Lycodonomorphus (?) subtaeniatus, see below). Lamp¬

rophis is now restricted to a few South African endem¬

ics, with all other names/species by genetic monophyly

or default being transferred to Boaedon. House snakes

are common throughout Angola, but none were included

in the above studies, and neither were many other Afri¬

can populations or even putative species (e.g., mentalis,

erlangeri, arabicus, bedriagae, etc.), and the status of

all remains unresolved. The names Alopecion variega-

tum Bocage, 1867 and Boaedon lineatus var. angolen-

sis Bocage, 1895, currently considered synonyms of B.

lineatus, remain unresolved. In addition, the Coastal

House Snake (B. littoralis) recently described (Trape and

Mediannikov 2016) from the coastal region of southern

Gabon and Republic of Congo, may extend at least into

Cabinda. It is probable that many different house snake

species currently occur in Angola, but their distributions

and diagnostic features, as well as the allocation of ex¬

isting names and description of new species, are under

investigation (Hallermann et al, in prep.). In addition, it

is likely that a number of species from adjacent regions,

e.g. B. radfordi, are likely to enter the northern forested

habitats.

Fig. 17 Boaedon capensis-fuliginosus-lineatus complex, near

Menongue, Cuando Cubango.

September 2018 | Volume 12 | Number 2 | el 59

Branch

Olive House Snake

Boaedon olivaceus (Dumeril 1856)

Holuropholis olivaceus Dumeril, 1856. Note sur les reptiles du

Gabon. Revue et Magasin de Zoologie Pure et Appliquee, Paris

(2) 8: 466.

Bocage (1895) specifically stated that he knew of no

Angolan material. It was subsequently recorded from

Dundo (Laurent 1954; Tys van den Audenaerde 1967),

but the recent description of a sister species, B. radfordii,

from the Albertine Rift (Greenbaum et al. 2015), means

that the relationship of Dundo material to this new spe¬

cies requires assessment.

Red-Black Striped House Snake

Bothrophthalmus lineatus (Peters 1863)

Elaphis {Bothrophthalmus) lineatus Peters, 1863. fiber einige

neue oder weniger bekannte Schlangenarten des zoologischen

Museums zu Berlin. Monatsb. Kdnigl. Akad. Wiss., Berlin

1863; 287.

Not known to Bocage (1895) from Angola, although it

was subsequently recorded from Dundo (Laurent 1950,

1954, 1964; Tys van den Audenaerde 1967). This re¬

mains the only Angolan locality. Plain western popula¬

tions, previously know as B. 1. brunnaeus, are now treat¬

ed as a full species (Pauwels et al. 2006), and may extend

south to Cabinda.

Parker’s Banded Snake

Chamaelycus parkeri (Angel 1934)

Oophilositum parkeri Amgel, 1934. Remarques sur le genre

Oophilositum Parker (Colubride aglyphe) et description d’une

espece nouvelle. Bull. Soc. Zool., France 59: 417.

Parker (1936) recorded Oophilositum parkeri from Fa¬

zenda Congulu, and this remains the only known Ango¬

lan material. The species was transferred to Chamaelycus

by de Witte (1963). Parker’s (1936) material creates a

significant disjunction in the species’ range, which else¬

where is restricted to DRC and Congo Brazzaville (Trape

and Roux-Esteve 1995).

Yellow Forest Snake

Hormonotus modestus (Dumeril, Bibron and Dumeril

1854)

Lamprophis modestus Dumeril, Bibron and Dumeril, 1854. Er-

petologie generale ou Histoire Naturelle complete des Reptiles.

Vol. 7 (partie 1). Paris, 429.

A characteristic species of the Congo forest known from

Angola from a single specimen from scarp forest habitat

at Congula (Parker 1936).

Brussaux’s Dwarf File Snake

Gonionotophis brusseauii (Mocquard 1889)

Gonionotus brussauxi Mocquard, 1889. Sur une collection de

reptiles du Congo. Bull. Soc. Philomath., Paris (8) 1: 146.

This small file snake was not known to Bocage (1895)

from Angola. Eaurent (1954) documented the only An¬

golan record from Dundo.

Common File Snake

Limaformosa capensis (Smith 1847)

Heterolepis capensis Smith, 1847. Illustrations of the zoology

of South Africa, Reptilia. Smith, Elder, and Co., London: 55.

Bocage (1895) did not know this species from Angola.

Monard (1937) signalled the first record (as Mehelya

capensis capensis) from Huambo, and additional mate¬

rial was recorded from Rio des Ganguelles (Angel 1923),

‘Rives du la Calundo’, 105 km E Euso, Mexico (Eaurent

1964), and 14 km north Mapupa (Branch and McCar¬

thy 1992). Despite Eaurent’s records, Haacke (1981) and

Broadley (1983) continued to exclude Angola from the

range of M. capensis. The few known records are all re¬

stricted to the eastern regions of Angola. In a molecular

analysis Kelly et al. (2011a) showed that Gonionotop¬

his brussauxi (Mocquard 1889; type species) was nested

within Mehelya, and as the former generic name had

priority, the latter and all other file snakes were placed

in Gonionotophis. Eanza and Broadley (2016) also re¬

assessed northern populations related to G. capensis,

with the revival of G. chanleri and G. savorgnani as full

species. Moreover, a more recent phylogeny with greater

taxon sampling confirmed monophyletic clades within

Gonionotophis sensu lato, and led to the revival of Me¬

helya (but restricted to M. poensis, M. stenophthalmus,

G. egbensis, G. gabouensis (Trape and Mane 2005), and

G laurenti); a more traditional content for Gonionotop¬

his (including the dwarf species G brussami, G. grantii

and provisionally G klingi); the description of a new ge¬

nus, Gracililima for G. nyassae; and a new genus Lima¬

formosa for the remaining file snakes of the G. capensis

complex (Broadley et al. 2018).

Savorgan’s File Snake

Limaformosa savorgani (Moquard 1887)

Heterolepis Savorgnani Mocquard, 1887. Du genre Heterolepis

et des especes qui le composent, dont trois nouvelles. Bull. Soc.

Philom., Paris (7) 11: 27.

Bocage knew no file snakes from Angola and reported

only Heterolepis bicarinatus from the Congo (Bocage

1866). He later (Bocage 1895) corrected this identifica¬

tion to H. guirali. Eoveridge (1939) undertook the last

revision of file snakes and placed H. guirali in the synon-

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Snakes of Angola: An annotated checklist

ymy of Mehelya capensis savorgani. Lanza and Broad-

ley (2016) revived M. savorgani as a full species, and

included northern Angola in the species’ range, but ex¬

amined no Angolan material or gave citations for Ango¬

lan localities. It was recently transferred to a new genus

by Broadley et al. (2018).

Fig. 18. Limaformosa savorgani, Soyo {Photo: Warren Klein).

Vernay’s File Snake

Limaformosa vernayi (Bogert 1940)

Mehelya vernayi Bogert, 1940. Herpetological results of the

Vemay Angola Expedition. I. Snakes, including an arrangement

of the African Colubridae. Bull. Amer. Mus. Nat. Hist. 77: 28.

This poorly known file snake was described from Hanha

by Bogert (1940). Haacke (1981) reviewed file snakes in

the western arid region, and noted few additional records

(<10) for northern Namibia, but no new material from

Angola. It was recently transferred to a new genus by

Broadley et al. (2018).

Fig. 19. Mehelya poensis, Lagoa Cammba, Lunde Norte.

Equatorial File Snake

Mehelya poensis (Smith 1849)

Heterolepis poensis Smith, 1849. Illustrations of the zoology

of South Africa, Reptilia. Smith, Elder, and Co., London: 55.

Bocage (1895) did not discuss M. poensis from either

Angola or the Congo region. Its presence in Angola was

first signaled from Cabicula (Cazengo) (Ferreira 1904).

Additional material was recorded from Piri (Hellmich

1957); Dundo (Laurent 1950), Muita River (Tys van den

Audenaerde 1967), and Lagoa Carumbo (Branch and

Conradie 2015). Recently placed in an expanded Goni-

onotophis (Kelly et al 2011a), but now returned to a re¬

duced Mehelya (Broadley et al. 2018).

White-bellied Water Snake

Lycodonomorphus (?) subtaeniatus (Laurent 1954)

Lycodonomorphus subtaeniatus Laurent, 1954. Reptiles et

batraciens de la region de Dundo (Angola) (Deuxieme note).

Companhia de Diamantes de Angola (Diamang), Servigos Cul-

turais, Publicagoes Culturais, No. 23: 38.

Laurent (1954) described Lycodonomorphus subtaenia¬

tus based on a specimen from Keseki (DRC) and a series

of 12 paratypes, including four from Dundo, which re¬

main the only Angolan material. At the same time Lau¬

rent (1954) also described the subspecies L. s. upembae

from Nyonga (DRC). The type series for both taxa in¬

cluded material previously identified as Boaedon, i.e.,

B. virgatus and B. lineatus, respectively (Laurent 1952;

De Witte 1933). It was thus not unexpected that when

preparing a molecular phylogeny of house snakes of the

description of their new species B. radfordii, Greenbaum

et al. (2015) found L. s. upembae embedded within Boae¬

don, to which it was transferred. No new material was

available to assess the generic relationships of L. sub¬

taeniatus, but the current placement within Lycodono¬

morphus is problematic and its relationship to B. virgatus

with which has previously been confused should be in¬

vestigated.

Hellmich’s Wolf Snake

Lycophidion hellmichi (Laurent 1964)

Lycophidion hellmichi Laurent, 1964. Reptiles et batraciens de

I’Angola (troisieme note). Companhia de Di amantes de Ango¬

la (Diamang), Servigos Culturais, Museu do Dundo (Angola),

No. 67: 95.

Laurent (1964) described Lycophidion hellmichi from

“Kapolopopo, desert de Mossamedes,” and included the

L. c. capense (Hellmich 1957, Entre Rios) in the syn¬

onym, which was the first record of the species in Ango¬

la. He later (Laurent 1968) assigned a number of Namib¬

ian specimens to L. hellmichi, which Broadley (1991b)

later realized were a new species, L. namibianum. The

only remaining Nambian specimen of L. hellmichi is

from the Kaokoveld (Broadley 1991b), and Broadley

(1996) recorded another Angolan specimen from Quis-

sange. It therefore appears to be known from only three

specimens.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Branch

Flat Wolf Snake

Lycophidion laterale (Hallowell 1857)

Lycophidion laterale Hallowell, 1857. Notes of a collection of

reptiles from the Gaboon country, West Africa, recently pre¬

sented to the Academy of Natural Sciences of Philadelphia, by

Dr. Henry A. Ford. Proc. Acad. Nat. Sci. Philadelphia 9: 58.

A dwarf West African species, ranging from Ivory Coast

to Uganda, and south to Angola. It was known to Bocage

(1866,1895) only from Molembo (Cabinda), but later re¬

corded from north of the Kwanza River (Ferreira 1903)

and from N’Dalatando (Monard 1937).

Speckled Wolf Snake

Lycophidion meleagre (Boulenger 1893)

Lycophidium meleagris Boulenger, 1893. Catalogue of the

snakes in the British Museum (Nat. Hist.) I. London (Taylor

and Francis); 337.

A small wolf snake described from Ambriz and Am-

brizete (= N’zeto) in coastal northern Angola (Bou¬

lenger, 1893). These remained the only material known

to Bocage (1895). Additional material was recorded

from Cabiri (Ferreira 1904), Libolo-Luati (= Calu-

lo) (Hellmich 1957), Landana (Cabinda) and Luanda

(Broadley 1996). Specimens referred to this species have

been recorded over 3,000 km away in coastal northern

Tanzania (Broadley 1996), thus creating a considerable

zoogeographic anomaly. The assignment of the Tanza¬

nian material needs to be tested for genetic monophyly.

Spotted Wolf Snake

Lycophidion multimaculatum (Boettger 1888)

Lycophidion capense multimaculata Boettger, 1888. Materi-

alien zur Fauna des unteren Congo. IT Reptilien und Batrachier.

Per. Senck. Ges. 1887: 67.

Bocage (1895) referred Angolan material to two forms of

L. capense, one from the Congo (St. Salvador), Cabinda

and Calandula (that may be referrable to L. meleagris) and

wolf snakes from the southern parts of Angola (Galanga,

inland from Namibe, Caconda) that he referred to var.

multimaculata (Boettger). Additional records are record¬

ed as L. capense from Malanje (Peters 1881), Entre-Rios,

and Piri (Hellmich 1957); and as L. c. multimaculatum

from Dundo, Alto Cuilo, Cazombo, Macondo, Calonda

(Laurent 1964). Broadley (1991b) reviewed Namibian

wolf snakes, describing L. namibianum (see below), and

raised L. multimaculatum to a full species, recording it

as far south as the Caprivi. Branch and McCarthy (1992)

recorded the species from Cuito Cuanavale. Broadley

(1996) reviewed all the Angolan species, mapped their

distributions, and no longer considered L. capense to oc¬

cur in the country.

Namib Wolf Snake

Lycophidion namibianum (Broadley 1991)

Lycophidion namibianum Broadley, 1991. A review of the Na¬

mibian snakes of the genus Lycophidion (Serpentes: Colubri-

dae), with the description of a new endemic species. Annals

Transvaal Mus. 35 (14); 210.

This attractive wolf snake was described by Broadley

(1991b) from northern Namibia, and included many Na¬

mibian specimens previously confused with Laurent’s L.

hellmichi (Broadley 1991). The only Angola record was

collected at Espinheira (Branch et al, in prep.).

Fig. 20. Lycophidion namibianum, Espinheira, Namibe.

Ornate Wolf Snake

Lycophidion ornatum (Parker 1936)

Lycophidion ornatum Parker, 1936. Dr. Karl Jordan’s expedi¬

tion to South-West Africa and Angola: Herpetological collec¬

tions. Novit. Zool. (London) 40: 122.

Described from two specimens from scarp forest at Fa¬

zenda Congulu (Parker 1936), which remain the only

known Angolan records. Elsewhere it extends through

DRC to south Sudan (Broadley 1996).

Subfamily: Psammophiinae

The psammophiine genera {Dipsina, Hemirhagerrhis,

Malpolon, Psammophis, Psammophylax, Rhamphio-

phis) are distributed throughout Africa, the Middle East,

south-central Asia, and southern Europe (Branch 1998;

Kelly et al. 2008), with Mimophis restricted to Mada¬

gascar. Their monophyly is supported by morphological

and molecular data (Cadle 1994; Brandstatter 1996; Za-

her 1999; Vidal and Hedges 2002a; Kelly et al. 2008).

Two recent genera have been synonymized: Dromophis

with Psammophis (Kelly et al. 2008) and Rhagerhis with

Malpolon (Figueroa et al. 2016). Figueroa et al. (2016)

consider Asian Psammodynastes to also be part of the

Psammophiidae, but this species has a bifurcate, heav¬

ily ornamented hemipenis (see Fig. 17 in Zaher 1999)

that contrasts with the well established synapomorphy of

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September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

the simple, tubular and unadorned hemipenes of psam-

mophines. We therefore continue to exclude Psammody-

nastes from the Psammophidae.

Viperine Rock Snake

Hemirhagerrhis viperina (Bocage 1873)

PsammophyJax viperinus Bocage 1873. Bocage JVB. 1873.

Melanges erpetologiques. II. Sur quelques reptiles et batraciens

nouveaux, rares ou peu connus d‘Afrique occidentale. Jorn.

Acad. Sci., Lisboa 4: 222.

Bocage (1873) described Psammophylax viperinus from

Dombe Grande, and later added further Anchieta mate¬

rial from Humbe, Maconjo, and Capangombe (Bocage

1895), but deferred to Boulenger in treating this material

as Psammophylax nototaenia. Bogert (1940) added new

records from the Vernay Lang Angola expedition from

Hanha, Huambo, and Munhino, and revived it as a sub¬

species, H. nototaenia vipernua, for all Angolan mate¬

rial. Laurent (1964) recorded a specimen from Humpata.

Broadley (2000) revived H. viperina again as a full spe¬

cies, and noted no H. nototaenia from Angola.

Angolan Sand Snake

Psammophis angolensis (Bocage 1872)

Amphiophis angolensis Bocage, 1872. Diagnoses de quelques

especes nouvelles de Reptiles d’Afrique occidentale. Jorn. Sci.,

Lisbon 4: 82.

This dwarf psammophine was described by Bocage

(1872) from “Dondo, (interieur d’Angola).” Wallach

et al. (2014) discuss confusion over the correct loca¬

tion of Bocage’s type locality “Dondo,” which has been

presented in various forms; e.g., Danda, Loanda Distr.

(Loveridge 1940; Broadley 1962), Donda, Loanda Distr.

(FitzSimons 1962; Auerbach 1987), and Dondo, Luanda

Distr. (Crawford-Cabral and Mesquitela 1989). They

note that it was correctly located by Loveridge (1957)

and Hellmich (1957a) as Dondo, north bank of Kwanza

River, SW Cuanza Norte Distr., NW Angola (09°4rS,

14°26’E, elevation 50 m). Additional material was not¬

ed (Bocage 1895) from Caconda, Quindumbo, Humbe,

Sumbe, Pungo-Andongo and Ambrizette (=N’Zeto), and

later from Dundo, Cameia, and Lagoa Calundo (Laurent

1964).

Ansorge’s Sand Snake

Psammophis ansorgii (Boulenger 1905)

Psammophis ansorgii Boulenger, 1905. A list of the batrachi-

ans and reptiles collected by Dr. W. J. Ansorge in Angola with

descriptions of new species. Ann. Mag. Nat. Hist. (7) 16; 113.

Boulenger (1905) described Psammophis ansorgii on a

snake collected by Anchieta between “Benguella to Bihe,

Angola.” Hellmich (1957) recorded the species from

Bela Vista, but of his seven specimens, five were just

heads. However, this new material allowed him to note

that the preocular was, at most, only in narrow contact

with the frontal, and he therefore removed the species

from the synonymy of P. jallae, where it had been placed

by Loveridge (1940). This was followed by Broadley

(1977, 2002), who noted no new material. Branch et al

(2018a) discussed new material and phylogenetic rela¬

tionships, and noted that it is one of the few snakes en¬

demic to Angola.

Fig. 21. Psammophis ansorgii, Tundavala, Huila {Photo: Ninda

Baptist a).

Jalla’s Sand Snake

Psammophis jallae (Peracca 1896)

Psammophis jallae Peracca, 1896. Retili et Anfibi raccolti a

Kazungula e sulla strada da Kazungula a Buluwaio dal Rev.

Luigi Jalla, Missionario Valdese nelf alto Zambese. Boll. Mus.

Zool. Comp. Anat. Univ. Torino 11(255); 2.

Loveridge (1940) removed P. jallae from the synonymy

of P. crucifer, and Broadley (1977, 2002) discussed ad¬

ditional material, although noting that it remained known

from Angola only from the type of Psammophis rohani

Angel, 1921 (Lumuna River). A second Angolan record

was noted by Conradie et al. (2017).

Leopard Sand Snake

Psammophis leopardinus (Bocage 1887)

Psammophis sibilans var. leopardinus Bocage 1887. Melanges

erpetologiques. 1. Reptiles et Batraciens du Congo. If Rep¬

tiles de Dahomey. III. Reptiles de file du Prince. IV. Reptiles

et Batraciens de Quissange (Benguella) envoyes par M. J.

d’Anchieta. Jorn. Sci. Math. Phys. Nat., Lisboa 11; 206.

Bocage (1887b) was confronted with diverse color pat¬

terns and scalation in sand snakes, and like many sub¬

sequent researchers (Boulenger 1895; Loveridge 1940;

Broadley 1977, 2002) found it difficult to resolve species

boundaries. As was customary at the time he used variet¬

ies to characterize intraspecific grouping. Some of these

have subsequently been demonstrated, both morphologi¬

cally (Broadley 1977, 2002) and genetically (Kelly et al.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Branch

2008) to be valid species. One such is this species, first

described by Bocage (1887) as a variety of P. sibilans

(now restricted to North Africa), eventually as a western

subspecies of P. brevirostris (Broadley, 1977), before be¬

ing raised to a full species (Broadley 2002). The latter

lists numerous localities for western Angola, including

the type locality Catumbela.

Namib Sand Snake

Psammophis namibensis (Broadley 1975)

Psammophis leightoni namibensis Broadley, 1975. A review of

Psammophis leightoni and Psammophis notosticus in southern

Africa (Serpentes; Colubridae). Arnoldia 7(13); 9.

Some early Bocage Psammophis material from Mossa-

medes (= Namibe, MBL 1809) and Rio Curoca (MBL

1810) was studied by Broadley before the Lisbon fire.

He assigned them to a new taxon, described initially as a

western arid subspecies (Broadley 1975) of P. leightoni,

but later treated as a full species, P. namibensis (Broad¬

ley 2002). Additional material, collected by Haacke from

Cunene Forde 15 km NE, Iona Res, Foz do Cunene and

Pico de Acezevedo, was also assigned to this species

(Broadley 2002). It has a much greater distribution in the

western arid region, through Namibia to South Africa.

Karoo Sand Snake

Psammophis notostictus (Peters 1867)

Psammophis moniliger var. notostictus Peters 1867. Peters,

Wilhem Carl Hartwig 1867. Uber eine Sammlung von Fled-

erthieren und Amphibien aus Otjimbingue in Sudwestafrica,

welche Hr. Missionar Hahn dem zoologischen Museum zuge-

sandt hat. Monatsber. konigl. Akad. Wiss., Berlin 1867 (April):

237.

Bocage (1887) described Psammophis sibilans, var.

nova, stenocephalus from “Finterieur de Mossamedes”

(= Rio Curoca), but this was later synonymized with P.

notostictus by Broadley (1977) and in Angola is known

from only a few localities in Namibe Province, but ex¬

tending south to the Karoo of South Africa. Broadley

(2002) noted material from Rio Sau Nicolau (= Rio Ben-

tiaba), and Ceriaco et al. (2016a) from Espinheira and

Pico de Azevedo.

Mozambique Grass Snake

Psammophis mossambicus (Peters 1882)

Psammophis sibilans var. mossambica Peters 1882. Natur-

wissenschaftliche Reise nach Mossambique auf Befehl seiner

Majestat es Konigs Friedrich Wilhelm IV. in den Jahren 1842

bis 1848 ausgefefiihrt von Wilhelm C. Peters. Zoologie III.

Amphibien. Berlin (Reimer), 122.

The large sand snakes of the Psammophis sibilans-

philippsi-mossambicus complex have remained a recur¬

rent taxonomic problem among African snakes for over

100 years; see discussion and shifting nomenclature in

Bocage (1887, 1895), Boulenger (1895), Eoveridge

(1940), Broadley (1977, 2002), Hughes (1999), and

Brandstatter (1995, 1996). Most Angolan Psammophis

have previously been included in varied taxa within this

complex. Broadley (2002) lists numerous localities for

this species, which is widespread in savannah and sec¬

ondary habitats in Angola. Whether all Angolan large

sand snakes are referable to P. mossambicus, or whether

northwest populations have affinities with West African

P. phillippsi, requires fresh material and genetic assess¬

ment.

Western Strip-bellied Sand Snake

Psammophis subtaeniatus (Peters 1882)

Psammophis sibilans var. subtaeniata Peters 1882. Naturwis-

senschaftliche Reise nach Mossambique auf Befehl seiner

Majestat es Konigs Friedrich Wilhelm IV. in den Jahren 1842

bis 1848 ausgefefiihrt von Wilhelm C. Peters. Zoologie III.

Amphibien. Berlin (Reimer), 121.

Bocage (1895) recognized five forms of Psammophis

sibilans (vars. A-E), of which var. A he contrasted with

Peters’ var. subtaeniata. He noted that his specimens

from Rio Bengo, Catumbela, Bibala, Maconjo, Humbe

and Cunene, all collected by Anchieta, looked like var.

subtaeniata. Broadley (2002) list numerous localities

for P. subtaeniatus in Angola, including many from the

Eisbon Museum subsequently lost in the fire. The spe¬

cies is restricted to the semi-arid scrubland and mopane

woodland, above and below the escarpment in southwest

Angola. Broadley (2002) includes Psammophis bocagii

Boulenger, 1895 as a synonym.

Western Sand Snake

Psammophis trigrammus (Gunther 1865)

Psammophis trigrammus Gunther, 1865. Fourth account of

new Species of Snakes in the Collection of the British Museum.

Ann. Mag. Nat. Hist. (3) 15; 95.

For many years this snake was known only from the type

locality (“Sao Nicolau, Mossamedes”). Bocage (1887)

seemed to consider it distinct, but only as a variety of

Psammophis sibilans and not identical to the other variet¬

ies he described at the time (e.g., stenocephalus and leop-

ardinus). Eater he seems to have overlooked the species

as he did not discuss it in his monograph (Bocage 1895).

This may be why Monard (1937) also overlooked it, as

its taxonomic status has never been challenged. Broad¬

ley (2002) plotted the limited extenstion into southwest

Angola.

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September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

Zambezi Sand Snake

Psammophis zambiensis (Hughes and Wade 2000)

Psammophis zambiensis Hughes, 2000. On the African leopard

whip snake, Psammophis leopardinus Bocage, 1887 (Serpen-

tes, Colubridae), with the description of a new species from

Zambia. Bull. nat. Hist. Mus. Land (Zool.) 68(2); 75.

Only recently described (Hughes and Wade 2002),

the species was first reported from Angola during the

NGOWP surveys in the headwater region of the Ango¬

lan Okavango catchment (Conradie et al. 2017; Conradie

and Branch 2017).

Fig. 22. Psammophis zambiensis, Cuanavale River Source, Cu-

ando Cubango {Photo: Werner Conradie).

Bocage (1873) described Psammophylax ocellatus on

the basis of an adult snake from “I’interieur de Mossa-

medes (Gambos).” Additional material was recorded (as

P. rhombeatus) from Humbe (Bocage 1895; Boulenger

1896) and Tundavala (Baptista et al. 2018a). Broadley

(1977) reviewed the Angolan material and revived P. r

ocellatus as a northern subspecies. Branch et al. (2018a)

extending the known range to the Chela escarpment re¬

gion and also validated P. ocellatus as a full species.

Fig. 23. Psammophylax ocellatus, Humpata, Huila.

Striped Skaapstekker

Psammophylax tritaeniatus (Gunther 1868)

Striped Beaked Skaapstekker

Psammophylax acutus (Gunther 1888)

Psammophis acutus Gunther, 1888. Contribution to the knowl¬

edge of snakes of tropical Africa. Ann. Mag. Nat. Hist. (6) 1:

321.

First recorded from Angola by Bocage (1873) from

material sent by Capello and Ivens from Cassange (=

Baixa de Cassange). Unfortunatey he misidentified it as

Rhagerhis tritaeniata and later, after Gunther (1888) had

described it as a new species on a specimen from Pungo

Andongo, apologized to the collectors (Bocage 1895)

and noted more specimens from Caconda and Huila. Ad¬

ditional material was noted from Benguela to Bie (Bou¬

lenger 1905), Bela Vista (Hellmich 1957), Alto Chicapa,

Alto Cuilo, and Dundo (Laurent 1964). Broadley (1971c)

revised the species, which was later transferred to Psam¬

mophylax (Kelly et al. 2008).

Huila Skaapstekker

Psammophylax ocellatus (Bocage 1873)

Psammophylax ocellatus Bocage 1873. Melanges erpe-

tologiques. II. Sur quelques reptiles et batraciens nouveaux,

rares ou peu connus d‘Afrique occidentale. Jorn. Acad. Sci.,

Lisboa 4: 221.

Rhagerrhis tritaeniatus Gunther, 1868. Sixth account of new

species of snakes in the collection of the British Museum. Ann.

Mag. Nat. Hist. (4) 1: 423.

First recorded from Dondo by Bocage (1873), and later

from the Rio Kwanza, Quissange, Cahata, Quindumho,

Caconda, Huila, Gambos, and Humbe by Bocage (1895),

who noted it was one of the most common and wide¬

spread snakes on the highlands of Angola. Additional

records from Cuvango, Mupanda, Vila da Ponte (= Cu-

vango) (Monard 1937), Capelongo (Bogert 1940), Alto

Cubal (Hellmich 1957), Cazombo, Forte Roqadas (Lau¬

rent 1964), and Calombe (Manaqas, 1973). Broadley

(1977) reviewed the genus.

Subfamily: Prosymninae

A small subfamily containing only the African genus

Prosymna, which currently includes 16 species. It is

well-differentiated genetically (Kelly et al. 2008) and

morphologically (Broadley 1980). They are small terres¬

trial snakes with an exclusive diet of reptile eggs (Broad¬

ley 1979b).

Zambezi Shovel-snout Snake

Prosymna ambigua (Bocage 1873)

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September 2018 | Volume 12 | Number 2 | el 59

Branch

Prosymna ambiguus Bocage, 1873. Melanges erpetologiques.

II. Sur quelques reptiles et batraciens nouveaux, rares on pen

connus d‘Afrique occidentale. Jorn. Acad. Sci., Lisboa 4: 218.

Bocage (1873) described Prosymna ambiguous on a

small juvenile from Calandula, which remained the only

Angolan specimen known to him (Bocage 1895). Lau¬

rent (1954) reviewed Prosymna ambigua and described

three new subspecies, including P. a. brevis based on an

extensive series from Dundo and Sombe, and Sandoa in

DRC. This subspecies was subsequently synonymized

by Broadley (1980) with typical P. a. ambigua, who con¬

tinued to recognize P. a. bocagii Boulenger, 1897 for a

northern race in the Congo basin. Recent material was

collected from Cangandala (Ceriaco et al. 2016b).

Fig. 24. Prosymna ambigua, 20 km W Lola, Namibe.

Angola Shovel-snout Snake

Prosymna angolensis (Boulenger 1915)

Prosymna angolensis Boulenger, 1915. A list of the snakes of

the Belgian and Portuguese Congo, northern Rhodesia, and An¬

gola. Proc. Zool. Soc., London 1915: 208.

Boulenger (1915) gave a terse description of Prosymna

angolensis, noting only that he referred snakes discussed

by Bocage as Prosymna frontalis (1895, p. 98, pL, fig.

2) to his new species and gave the type locality simply

as “Angola.” Loveridge 1958 cleared up the mess and

designated “Huila, 15°5’S, 13°30’E, Angola” as the type

locality. Broadley (1980) assigned Bocage’s inland fron¬

talis material from Bibala, Caconda, and Maconjo to P.

angolensis, as well other material, including an unusual

coastal record (Mossamedes) that may be either a mis-

identified P. frontalis or the locality of the collector rather

than that of the specimen. Additional records from Bela

Vista (Hellmich 1957), and southeast Angola (Conradie

et al. 2017; Conradie and Branch 2017), and all indicate

that it is a species of the escarpment foothills and inland

plateau.

South-west Shovel-snout Snake

Prosymna frontalis (Peters 1867)

Temnorhynchus frontalis Peters, 1867. Uber eine Sammlung

von Flederthieren und Amphibien aus Otjimbingue in Sudwe-

stafrica, welche Hr. Missionar Hahn dem zoologischen Muse¬

um zugesandt hat. Monatsber. konigl. Akad. Wiss. Berlin. 1867

(April): 236.

Fig. 25. Prosymna frontalis, Espinheira, Namibe.

Peters (1867) described Temnorhynchus frontalis from

“Otjimbingue, Siidwestafrika” (= Otjimbingwe, SE

Erongo Distr., cen. Namibia), and Bocage (1882) noted

the first record of the species in Angola, based on mate¬

rial collected by Anchieta from Bibala and Mossamedes.

He later referred inland material sent by Anchieta from

Qiussange, Quibula, Quindumbo, Caconda, Huila, Ma¬

conjo and Bibala to this species (Bocage 1895), but

records above the escarpment were incorporated into

Boulenger’s (1915) description of P. angolensis. This re¬

stricted P. frontalis to the arid habitats below the escarp¬

ment in southwest Angola.

Fig. 26. Prosymna visseri, Espinheira, Namibe.

Visser’s Shovel-snout Snake

Prosymna visseri (FitzSimons 1959)

Prosymna visseri FitzSimons, 1959. Some new reptiles from

southern Africa and southern Angola. Annals Transvaal Mus.

23: 406.

Prosymna visseri was one of the first new reptiles that

Charles Koch collected incidentally whilst researching

the tenebrionid beetle fauna of the Angolan Namib re¬

gion. Described by FitzSimons (1959) from Caraculo,

Haacke collected several others during his field work in

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Snakes of Angola: An annotated checklist

southwest Angola, but the species was known from only

three specimens at the time of Broadley’s (1980) revi¬

sion. It was later described from Namibia (McLachlan

1987), from where all subsequent specimens have come

(Bauer et al. 2000). All Angolan records are from arid

habitats in the coastal southwest, but the species extends

further inland in the Kaokoveld, Namibia.

Subfamily: Pseudaspinae

This small subfamily currently includes only two species

in monotypic genera. Although Pyron et al. (2011) ex¬

panded the content of Pseudaspidinae to include African

Buhoma and Asian Psammodynastes, this was not sup¬

ported by Figueroa et al. (2016), who rather associated

the latter with the Psammophiinae (but see above).

Mole Snake

Pseudaspis cana (Linnaeus 1758)

Coluber canus Linnaeus, 1758. Systema naturae per regna tria

naturae, secundum classes, ordines, genera, species, cum char-

acteribus, differentiis, synonymis, locis. Tomus I. Editio deci-

ma, reformata. Laurentii Salvii, Holmiae. 10* Edition; 221.

First recorded from Angola by Bocage (1882) when de¬

scribing a new genus and species Ophirhina anchietae

from Caconda, in the interior of Benguela. Bocage soon

realized that his ‘new’ species was already known, and

discussed further material in his monograph (Bocage

1895) as Pseudaspis cana. Monard (1937) recorded ma¬

terial from Cuvango and Sangueve, and Bogert (1940)

from Mombolo.

Fig. 27. Pythonodipsas carinata, Baba, Namibe.

without locality details. FitzSimons (1962) and Broadley

(1983) both utilized Koch’s record in species maps and

noted the distribution to include southern Angola.

Family: Elapidae

The taxonomy of African elapids has changed consider¬

ably in recent years, particularly among cobras (Naja),

although the generic relationships of allied species have

also been affected and numerous new species have been

described (Broadley 1968, 1995; Broadley and Wiister

2004; Wiister and Broadley 2003, 2007; Wallach et al.

2009; Ceriaco et al. 2017; Wiister et al. 2018). Water co¬

bras (Boulengerina) were demonstrated to be closely re¬

lated to the Forest Cobra (Naja melanoleuca) and there¬

fore synonymized WithNaja (Nagy et al. 2005; Wiister et

al. 2007). Wallach et al. (2009) proposed four subgenera

within Naja, with the subgenus Naja restricted to Asia,

and with the three other subgenera used for African co¬

bras, and Angolan species assigned to various subgen¬

era: i.e., Naja (Boulengerina) melanoleuca, N (Uraeus)

anchietae, N. (Afronaja) mossambica, N. (Afronaja)

nigricollis, and N. (Afronaja) n. nigricincta. Moreover,

a recent revisions of forest cobras (Ceriaco et al. 2017;

Wiister et al. 2018) recognize a suite of five species, with

typical N. (B.) melanoleuca entering northern Angola and

the revived N. (B.) subfulva occuring in central Angola.

Fig. 28. Aspidelaps lubricus cowlesi, 70 km N Namibe, Na¬

mibe.

Coral Shield Cobra

Aspidelaps lubricus cowlesi (Bogert 1940)

Western-keeled Snake

Pythonodipsas carinata (Gilnther 1868)

Pythonodipsas carinata Gunther 1868. Sixth account of new

species of snakes in the collection of the British Museum. Ann.

Mag. Nat. Hist. (4) 1; 426.

First collected in September 1956 in Angola by Koch

from Cima, Rio Giraul, Mossamedes and included in

an updated map for the species (Branch et al. 1997), but

Aspidelaps lubricus cowlesi Bogert, 1940. Herpetological re¬

sults of the Vernay Angola Expedition. I. Snakes, including an

arrangement of the African Colubridae. Bull. Amer. Mus. Nat.

Hist. 77: 94.

Bogert (1940) described Aspidelaps lubricus cowlesi

from a snake collected from Munhino (101 km east of

Namibe, via railroad). Originally considered endemic to

Angola, Mertens (1971) extended its range into the Kao¬

koveld. Broadley and Baldwin (2006) relegated it to the

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synonymy of the northern Namibia subspecies A. 1. in-

fuscatus, citing intermediates between A. 1. cowlesi and

A. 1. infuscatus. Unusually they still presented an account

for A. 1. cowlesi even though synonymizing it. Moreover,

no morphological data was presented to show that even

A. 1. infuscata was a valid taxon, or a molecular data pre¬

sented to support the new taxonomic arrangement.

Fig. 29. Dendroaspis jamesoni, Gabela, Cuanza Sul.

Jameson’s Mamba

Dendroaspis jamesoni (Traill 1843)

Elaps jamesoni Traill, 1843. Description of the Flaps jamesoni,

a new species of serpent from Demerara. Edinburgh New. Phil.

J. 34 (67): 53.

The identification of historical records of Angolan mam¬

bas is complicated by confusion between green and black

mambas in the early literature. Prior to 1946 the Black

Mamba {D. polylepis) was considered a juvenile of the

southern Green Mamba {D. angusticeps), and recognition

of the Black mamba as a separate species was only con¬

firmed by FitzSimons (1946). Bocage (1888) discussed

the identification of mambas, reducing the number of

species then known from seven to three, whilst also de¬

scribing a new, overlooked species from Angola, D. ne-

glectus (hence the unusual name). He gave detailed scale

counts of the material he examined, and also presented

a diagnostic key to the species recognized. From this it

is evident that he applied the existing names wrongly:

His D. jamesoni is now identified as D. viridis; his D.

angusticeps is really D. polylepis, and his new species

D. neglectus was already known as D. jamesoni. This

arrangement continued to be refiected in his monograph

(1895), where two species of mamba were correctly con¬

sidered to occur in Angola, both unfortunately incorrect¬

ly named. Dendroaspis jamesoni has been recorded from

Pungo-Andonga (the type of Dendroaspis welwitschii

Gunther, 1865, which Bocage considered a synynoym of

his own D. neglectus even though Gunther’s name had

priority). As D. neglectus its was recorded from north of

the Kwanza River (Bocage 1888) and from N’Dalatando

(Ferreira 1903); and recorded correctly as Dendroaspis

jamesoni jamesoni from Piri, Bela Vista (Hellmich 1957)

and Dundo (Laurent 1954), although the status of the

eastern subspecies D. j. kaimosae needs genetic assess¬

ment. Recent material was discussed by Vaz Pinto and

Branch (2015).

Black Mamba

Dendroaspis polylepis (Gunther 1864)

Dendraspis polylepis Gunther, 1864. Report on a collection of

reptiles and fishes made by Dr. Kirk in the Zambesi and Nyassa

Regions. Proc. Zool. Soc., London 1864: 310.

All early records (up to 1946) from Angola were dis¬

cussed under 79. angusticeps (Peters 1881; Bocage 1888,

1895; Monard 1937; Bogert 1940), until D. polylepis was

shown to be a valid species (FitzSimons 1946). The spe¬

cies is nowhere common, but is relatively well-known in

the central and southern regions. Bocage (1895) recorded

it from the “hauts-plateaux de I’interieur d’Angola,” al¬

though Bogert (1940) recorded it from Hanha.

Gunther’s Garter Snake

Elapsoidea guentherii (Bocage 1866)

Elapsoidea guntherii Bocage, 1866. Lista dos reptis das pos-

sessoes portuguezas d’Africa occidental que existem no Museu

Lisboa. Jorn. Sci. Math. Phys. Nat., Lisboa 1: 50.

Bocage (1866) described both the genus and species

Elapsoidea guentherii in one of his first herpetological

papers. The description was based on material sent by

Anchieta from Cabinda, and led to the long association

beween these two icons of Angolan herpetology. Another

adult from Bissau was included in the description, and

to avoid confusion Parker (1949) later restricted the type

locality to Cabinda. Additional material was discussed

by Bocage (1895), Loveridge (1936), Bogert (1940),

Hellmich (1957), Laurent (1964), and Broadley (1971b)

who summarized its range from the northern parts of An¬

gola, through Zambia to Zimbabwe.

Angolan Garter Snake

Elapsoidea semiannulata semiannulata (Bocage 1882)

Elapsoidea semi-annulata Bocage, 1882. Reptiles rares ou

nouveaux d’Angola. Journ. Sci., Lisboa 8(32): 303.

After describing the previous species, Bocage (1882) de¬

scribed Elapsoidea semiannulata from additional mate¬

rial from Caconda, but in his monograph (Bocage 1895)

treated his new species as E. guentheri var. semiannulata.

Laurent (1964) described Elapsoidea decosteri huilen-

sis from Humpata, which was relegated to the synonym

of E. s. semiannulata by (Broadley 1971b). However,

Broadley (1971b) also treated a species described by

Werner (1897) from Ghana as a northern race, Elapsoi¬

dea semiannulata moebiusi, that extended to Gabon but

was unrecorded from Cabinda or northern Angola. All of

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Snakes of Angola: An annotated checklist

Bocage’s northern localities (1866, 1895, 1897) were re¬

stricted to Bissau, but Broadley (1998) reassessed some

of Laurent’s Congo-Kinshasa material and reassigned it

to E. s. moebiusi. Although Broadley (1998) described

the range of E. s. moebiusi as extending into northern An¬

gola, he neither mapped nor noted any localities support¬

ing this claim. His closest locality was a poorly defined

“Bas Congo” (Broadley 1998), and the race remains

unknown from Angola, including Cabinda. Haacke and

Finkeldey (1967) recorded the first record of E. s. semi-

annulata from southern Africa. Broadley (1971b) sum¬

marized and mapped the species in Angola, and recog¬

nized s. boulengeri Boettger, 1895, from Mozambique

as an eastern race, which he later raised to a full spe¬

cies (Broadley 1998). This is unrecorded from Angola,

although known from adjacent regions in Namibia and

Zambia, and parapatry between E. s. semiannulata and

E. boulengeri occurs in the Caprivi area.

Anchieta’s Cobra

Naja (Uraeus) anchietae (Bocage 1879)

Naja anchietaQ Bocage, 1879. Reptiles et batraciens nouveaux

d’Angola. J. Acad. Sci., Lisbon 7: 89.

As with the previous species, Bocage (1879) also de¬

scribed Naja anchietae from snakes sent from Caconda.

Mertens (1937) relegated it to a subspecies of the Egyp¬

tian Cobra {N. haje anchietae), Broadley (1995) referred

southern populations of N. haje to the Snouted Cobra (N.

annulifera), retaining N. a. anchietae as a western race.

Finally, Broadley and Wiister (2004) revalidated Bo¬

cage’s original name. Angolan specimens have thus been

decribed under various names. Broadley and Wiirster

(2004) refer Bocage’s (1895) N. haje material to N. an¬

chietae. Known records were summarized and mapped

in Broadley (1995) and Broadley and Wiister (2004), and

its range was extended north to Capanda Dam by Ceriaco

et al. (2014a).

Fig. 30. Naja annulata, Lagoa Cammbo, Lunde Norte.

Banded Water Cobra

Naja (Boulengerina) annulata (Peters, 1876)

Naja annulata Buchholz and Peters in Peters, 1876. Fine zweite

Mittheilung uber die von Hrn. Professor Dr. R. Buchholz in

Westafrica gesammelten Amphibien. Monatsber. konigl. Akad.

Wiss. Berlin 1876 (February): 119.

Bocage (1985) mentioned Naja annulata from the Con¬

go, but knew of no Angolan records. The first and only

record for the country came from Lagoa Carumbo region

(Branch and Conradie 2012). Wiister et al. (2018) noted

thdii Aspidelaps bocagei Sauvage, 1884 (type locality:

Gabon and Majumba) is not a synonym of N. melano-

leuca, as recorded by various authors, but instead of Naja

annulata.

Fig. 31. Naja melanoleuca, Soyo {Photo: Warren Klein).

Central African Forest Cobra

Naja {Boulengerina) melanoleuca (Hallowell 1857)

Naja haje var. melanoleuca Hallowell 1857. Notes of a collec¬

tion of reptiles from the Gaboon country. West Africa, recently

presented to the Academy of Natural Sciences of Philadelphia,

by Dr. Henry A. Ford. Proc. Acad. Nat. Sci. Philadelphia 9: 61.

Bocage (1895) had a very different understanding of spe¬

cies boundaries within African cobras compared with

modern nomenclature, but the loss of so much of his

material has made assignment of his records to modern

species difficult. Much of his cobra material was grouped

as varieties under the spitting cobra N. nigricollis, e.g.,

Naja nigricolls var. melanoleuca. Fortunately, Broadley

(1974) examined this material before it was destroyed in

the Lisbon Museum fire (1978) and corrected the identity

of the individual apecimens of which none was refer-

rable to true Naja melanoleaca (Hallowell). To stabilize

taxonomy Broadley (1974) designated MBL 1972 from

“Caconda” as the lectotype of Bocage’s (1895) Naja ni¬

gricolls var. melanoleuca, and treated it as a synonym

of N. nigricollis. Whether Bocage knew true forest co¬

bras from Angola remains debatable, but subsequent re¬

cords confirming its presence are from Pungo Andongo

(Boulenger 1905), Piri, Calulo, Sangeunge (Hellmich

1957), Dundo (Laurent 1954, 1964) and Soyo (Wiister

et al. 2018). Genetic material from the latter validated

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September 2018 | Volume 12 | Number 2 | el 59

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the presense of true N. melanoleuca in northwest Angola

(Wiister et al. 2018). Laurent (1964) considered that true

N. melanoleuca in northeast Angola was probably con¬

fined to forest galleries with N. subfulva living in second¬

ary forests and the intervening savannas.

Brown Forest Cobra

Naja {Boulengerina) subfulva (Laurent 1955)

Naja melanoleuca subfulva Laurent, 1955. Diagnoses prelimi-

naires des quelques Serpents venimeux. Rev. Zool. Bot. Afr. 51:

127-139.

The taxonomic status of the different populations of the

forest cobra has long been problematic. Don Broadley

(in litt., July 2013), in conjunction with Wolfgang Wiister

and colleagues, recognized at least six species-level taxa

within the forest cobra complex, including an endemic

species on Sao Tome. Description of these cryptic taxa

was delayed by Broadley’s illness, and the Sao Tome

population subsequently described as Naja {Boulengeri¬

na) peroescobari (Ceriaco et al. 2017). The delay also led

to some nomenclatural confusion for other forest cobra

populations, with various authors prematurely adopting

some of the taxonomic findings. Broadley and Blaylock

(2013) referred southern populations to Laurent’s (1955)

savannah-inhabiting subspecies N. m. subfulva, and not¬

ed that unpublished molecular data indicated it deserved

specific status. They also used subgenera within Naja as

proposed by Wallach et al. (2009), but some authors sub¬

sequently elevated these subgenera to full genera, e.g.,

Wallach et al. (2014) and Ceriaco et al. (2016b), even

though the intention of using subgenera was to avoid

medical issues possibly arising from nomenclatural

changes in the names of medically important snakes.

Chirio and Ineich (2006) and Ceriaco et al. (2017) also re¬

vived N. subfulva, the latter based on genetic divergence

of Mozambique material. Wiister et al. (2018) mapped

populations of N. subfulva from large parts of Angola.

However, the assignment was presumably based on his¬

torical aspects, as no analysis was presented for Angolan

N. subfluva morphology or genetic divergence compared

with other populations. The status and distribution of N.

subfulva in Angola therefore requires further study.

Mozambique Cobra

Naja (Afronaja) mossambica (Peters 1854)

Naja mossambica Peters, 1854. Diagnosen neuer Batrachier,

welche zusammen mit der frilher (24. Juli und 17. August)

gegebenen Ubersicht der Schlangen und Eidechsen mitgetheilt

werden. Ber. Bekanntmach Geeignet. Verhandl. Konigl.-Bre¬

uss. Akad. Wiss., Berlin 1854: 625.

Broadley (1974) first signaled the presence of N mos¬

sambica in Angola when he identified a specimen from

Amphib. Reptile Conserv.

Maconjo (MBL 1964, now lost) that Bocage (1895) had

referred to N. nigricollis var. fasciata. Broadley (1968)

treated mossambica as a full species after finding sym-

patry between N. nigricollis and N. mossambica in Zam¬

bia. The species is poorly known in Angola and the few

records are restricted to the southern provinces (Conradie

et al. 2016).

Western Barred Spitting Cobra

Naja {Afronaja) nigricincta nigricincta (Bogert 1940)

Naja nigricollis nigricincta Bogert, 1940. Herpetological re¬

sults of the Vernay Angola Expedition. I. Snakes, including an

arrangement of the African Colubridae. Bull. Amer. Mus. Nat.

Hist. 77: 89.

Broadley (1974), after examination of Bocage’s original

material, assigned Bocage’s (1895) N nigricollis vax.fas-

ciata to Bogert’s N. nigricollis nigricinta, and designated

MBL 1968 (now lost) from Benguela as the lectotype.

He was also unable to find Bocage’s var. fasciata from

Dondo, and this record may be in error. Molecular stud¬

ies later supported the elevation of N. nigricinta to a full

species, with a southern subspecies N. n. woodi (Wuster

et al. 2007). However, no Angolan material was includ¬

ed in this analysis and the status of Namibian material

needs confirmation, as the type locality for N nigricincta

is “Munhino (101 km east of Mossamedes via railroad,”

and nominotypical Angolan N. nigricinta have a different

color pattern to those in Namibia.

Fig. 32. Naja nigricincta, 40 km N Caracul, Namibe.

Black Spitting Cobra

Naja {Afronaja) nigricollis (Reinhardt 1843)

Naja nigricollis Reinhardt, 1843. Beskrivelse af nogle nye

Slangearter. Danske Vidensk. Selsk. Afhandl. 10: 369.

Broadley (1968) had reinstated N. mossambica as a full

species after confirming sympatry with N. nigricollis in

eastern Zambia. He treated pallida, katiensis, nigricincta

and woodi as subspecies of N. mossambica, whilst vari¬

ous other names, e.g., craw shay i, occidentalis, and atri-

68 September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

ceps were considered synonyms of N. nigricollis. Later,

Broadley (1974) assigned Bocage’s (1895) N. nigricollis

var. occidentalis to N. nigricollis nigricollis, and desig¬

nated MBL 1963 (now lost) from Dondo as the lectotype.

He also noted that Naja nigricollis var. melanoleuca Bo-

cage, 1895, described from Angola, was preoccupied by

N. melanoleuca (Hallowell). Finding additional sympat-

ry between N. mossambica and N. nigricollis nigricincta

in Angola, he treated nigricincta and the all-black woodi

from western South Africa and southern Namibia as sub¬

species of N. nigricollis. Following a molecular analysis

of African spitting cobras, Wuster et al. (2007) validated

N. nigricinta as a valid species, with A. nigricincta woodi

as a southern race. The Black Spitting Cobra, N. nigricol¬

lis, therefore reverted to binomials. It is widespread in

Angola, but generally absent from closed-canopy forest.

Gold’s Tree Cobra

Pseudohaje goldii (Boulenger 1895)

Najagoldii Boulenger, 1895. On some new or little-known rep¬

tiles obtained by W. H. Crosse Esq. on the Niger. Ann. Mag.

Nat. Hist. (6) 16: 34.

Parker (1935, as Naja goldii) recorded the first examples

of this arboreal cobra from south of the Congo River and

for Angola. The specimens were collected from remnant

scarp forest near Quirimbo. The only other historical An¬

golan material is from Peri (Hellmich 1957) and Dundo

(Laurent 1950, 1954).

Family: Viperidae

Compared with other snake families, African Viperidae

have had relatively little taxonomic change, with vari¬

ous new species described (e.g.. Bids harenna, Gower et

al. 2016; Causus rasmusseni Broadley, 2014) or species

boundaries readjusted (e.g., revival of Bids rhinoceros,

Lenk et al. 1999) but few generic re-arrangements. With

the exception of confusion over night adder identifica¬

tions (see below), few taxonomic changes have affected

Angolan vipers. As with the genus Naja, vipers of the ge¬

nus Bids had a number of subgenera proposed, and these

are adopted in the following species accounts.

Variable Bush Viper

Atheris squamigera (Hallowell 1854)

Echis squamigera Hallowell, 1854. Descriptions of new rep¬

tiles from Guinea. Proc. Acad. Nat. Sci. Philadelphia 1854:

193.

Bocage (1895) knew of no Angolan material except

Peters’ (1881) record from Cuango. Later material

was recorded from Gulongo Alto (Ferreira 1904), Piri

(Hellmich 1957), and Dundo and Luachimo River (Lau¬

rent 1954, 1964). Atheris squamigera is widespread in

the Congo Basin, and a highly variable species. Laurent

(1964) only tentatively attributed four specimens from

the Dundo region to A. squamigera, as he believed that

there were two sympatric species in the lower Congo,

and that A. anisolepis may be valid. Recognition of Moc-

quard’s anisolepis had been problematic, placed first in

the synonymy of A. squamigera by Boulenger (1896),

revived as a subspecies by Bogert (1940), treated again

as a full species by Broadley (1998b), until finally again

synonymized by Lawson and Ustach (2000).

Puff Adder

Bids (Bids) arietans (Merrem 1820)

Vipera (Echidna) arietans Merrem. 1820. Versuch eines Sys¬

tems der Amphibien I (Tentamen Systematis Amphibiorum). J.

C. Kriegeri, Marburg: 152.

Although Bocage (1895) noted that the species was com¬

mon in Angola, there are few records from the southwest

region. Gunther’s (1865) record from Moqamedes (= Na-

mibe) may have reflected the collector’s home base.

Homed Adder

Bids (Calechidna) caudalis (Smith 1839)

Vipera (Cerastes) caudalis Smith, 1839. Illustrations of the Zo¬

ology of South Africa, Reptilia. Smith, Elder, and Co., Eondon:

Although common through the western arid regions of

southern Africa (Branch 1998) there are few Angolan

records of the species. Bocage (1867) first signalled its

presence (as Cerastes caudalis) with a specimen from

Namibe collected by Anchieta. Subsequent records were

added by Bocage (1895, Capangombe, Rio Curoca), and

Laurent (1964, “35 km south of Namibe”).

Fig. 33 Bids caudalis, Baba, Namibe.

Gaboon Adder

Bids (Macrocerastes) gabonica (Dumeril, Bibron and

Dumeril 1854)

Echidna gabonica Dumeril, Bibron and Dumeril 1854. Erpetol-

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Branch

ogie generale ou histoire naturelle complete des reptiles. Tome

septieme. Deuxieme partie, comprenant Thistoire des serpents

venimeux. Paris, Librairie Encyclopedique de Roret; 1428.

At the time of his monograph Bocage (1895) knew this

iconic species only from Landana, Cabinda, and consid¬

ered that the species was unlikely to occur south of the

Kwanza River. However, a year later he reported two

specimens from Hanha collected by Anchieta (Bocage

1896). Subsequently others have been recorded: Ferreira

(1903), Laurent (1950, 1954, 1964), Tys van den Aude-

naerde (1967), Oliveira et al. (2016), and Conradie et al.

(2017). It is widespread in Angola in suitable forest edge

habitat.

Angolan Adder

Bitis (?) heraldica (Bocage 1889)

Vipera heraldica Bocage, 1889. Melanges erpetologiques. II.

Sur une vipere apparemment nouvelle d’Angola. Journ. Sci.,

Lisboa (2) 2: 127.

Bocage (1889) described this endemic Angolan snake

from “sur les bords de la riviere Calae, Tun des afflu¬

ents du Cunene, entre le 13 et le 14 parallele a Lest de

Caconda, Angola” (= Calue River, a tributary of the Cu¬

nene River, east of Caconda, Huila District, Angola).

Soon after its description the species was incorrectly

synonymized with the Bitis peringueyi, a dwarf adder

from Namib Desert dune habitat (Boulenger 1896), lead¬

ing to over 50 years of confusion. Early records of 'B.

peringueyf in southern Angola were all based on Bou¬

lenger’s synonymization of B. heraldica: i.e., a female

from “Between Benguela and Bie” (Boulenger 1905),

one from Caluquembe (Monard 1937), and another from

“Mombolo, Angola” from the Vernay, Lang, Boulton, ex¬

pedition of 1925 (Bogert 1940). The specific status of Bi¬

tis heraldica was revalidated by Mertens (1958) follow¬

ing the collection of a series of Bitis heraldica from Piri

(Hellmich 1957) and the species confirmed as endemic to

the Angolan highlands. Due to the absence of any fresh

genetic material the subgenera status remains uncertain.

Rhinoceros Viper

Bitis {Macrocerastes) nasicornis (Shaw 1802)

Coluber nasicornis Shaw, 1802. General Zoology or System¬

atic Natural History, vol. 3, Pt 1. Thomas Davison, London: 94.

Unknown to Bocage (1895) from Angola or Congo, it

was first recorded from Angola from scarp forest at Quir-

imbo (Parker 1936). That it could be regionally common

is shown by Hellmich’s (1957) astonishing record of 53

specimens from Piri. Recent material was recorded from

Uige (Ernst et al. 2016).

Two-lined Night Adder

Causus bilineatus (Boulenger 1905)

Causus bilineatus Boulenger, 1905. A list of the batrachians

and reptiles collected by Dr. W. J. Ansorge in Angola with de¬

scriptions of new species. Ann. Mag. Nat. Hist. (7) 16: 114.

Night adders in Angola have been a source of great con¬

fusion, with Bocage (1895) and most subsequent authors

recognizing only two species in Angola, C. rhombea-

tus and C. resimus. Bocage (1895), however, did note

well-marked adders with lateral stripes from Calandula,

Quissange, Caconda, and Huila, but made no taxonomic

descision. Boulenger (1905) on receiving additional ma¬

terial from Anchieta noted these same features and pro¬

posed the name bilineatus. This name was overlooked by

subsequent authors until Eaurent (1955) described Cau¬

sus lineatus for material from DRC. Eater, when look¬

ing at material from Dundo that he realized was refer¬

able to Boulanger’s bilineatus and also conspecific with

his DRC material, Eaurent (1964) used trinomials and

treated his DRC material as the subpecies C. bilineatus

lineatus and Angolan material from Calundo, Moxico,

as nominotypic C. b. bilineatus. Broadley (1968), based

on unpublished analysis in his Ph.D. thesis (Broadley

1966a), synonymized C. b. lineatus with C. bilineatus,

and this was supported by Rasmussen (2005), who re¬

viewed C. bilineatus, re-assessed and mapped Angolan

night adders, and corrected many early misidentifications

by Hellmich (1957) and Eaurent (1964). He noted that C.

bilineatus occurred in sympatry with C. rhombeatus in

the Benguela-Bie area, Caconda, and Chitau, and with C.

rhombeatus and C. maculatus at Piri.

Angola Green Night Adder

Causus resimus (Peters 1862)

Heterophis resimus Peters, 1862. fiber die von dem so frilh in

Afrika verstorbenen Freiherrn von Barnim und Dr. Hartmann

auf ihrer Reise durch Aegypten, Nubien und dem Sennar gesa-

mmelten Amphibien. Monatsber. Akad. Wiss., Berlin 1862: 277.

Bocage (1895) tentatively proposed the name C. resimus,

var. angolensis for night adders from several localities

in Angola, including Rio Dande, Rio Bengo, Cazengo,

Sumbe, Quissange, Rio Chimha, Bibala, and Maconjo.

Additional records of C. resimus have also been re¬

ported from Cazengo, Caculo, Cabicula (Ferreira 1904),

Quirimbo, Fazenda Congulo (Parker 1936), and Hanha

(Bogert 1940). Few of these have been accepted by Ras¬

mussen (2005). The Green Night Adder is currently dis¬

tributed in four isolated populations around the Congo

Basin, with the most isolated being that recorded from

Angolan scarp forest refugia. The taxonomic status of the

Angolan population, and the applicability of Bocage’s

(1895) C. resimus, var. angolensis or C. nasalis Stej-

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

neger, 1893, remain unresolved. A preliminary molecu¬

lar phylogeny of Causus (Tolley et al. in prep.) indicates

cryptic diversity within C. resimus, and supports the

distinctiveness of Angolan material. Rasmussen (2005)

mapped 13 localities for C. resimus in Angola, including

one from Cabinda.

Rasmussen’s Night Adder

Causus cf rasmusseni (Broadley 2014)

Causus rasmusseni Broadley, 2014. A new species of Causus

Lichtenstein from the Congo/Zambezi watershed in north¬

western Zambia (Reptilia: Squamata: Viperidae). Arnoldia

Zimbabwe 10 (29); 342.

Broadley (2014) described C. rasmusseni based on four

specimens from northern Zambia. It is weakly differenti¬

ated from sympatric C. rhombeatus by having slightly

fewer ventrals in males (130-132, versus 134-150 in

Zambian C. rhombeatus) and reduced blotches or uni¬

form dorsal coloration. Although the un-patterned dor¬

sal pattern and low ventral counts of a specimen from

Rio Longa (Conradie et al. 2016) conform to Broadley’s

concept of C. rasmusseni, we caution acceptance of its

presence in Angola, and indeed its specific status. A pre¬

liminary molecular phylogeny of night adders (Tolley et

al, in prep.) supports such caution.

Fig. 34. Causus cf rasumusseni, Rio Longa, Cuando Cubango.

Rhombic Night Adder

Causus rhombeatus (Lichtenstein 1823)

Sepedon rhombeata Lichtenstein, 1823. Verzeichniss der Dou-

bletten des zoologischen Museums der Konigl. Universitat zu

Berlin nebst Beschreibung vieler bisher unbekannter Arten von

Saugethieren, Vogeln, Amphibien und Fischen. Konigl. Preuss.

Akad. Wiss./ T. Trautwein, Berlin; 106.

Bocage consistently used this name (Bocage 1879, 1880,

1895) and considered it widespread in the interior of

Angola. Additional material was noted from Golungo

Alto (Ferreira 1904), MtMoco, Quirimbo (Parker 1936),

Calundo, Dundo, and Cossa (Laurent 1964). Bocage

(1895) considered material from Calandula, Quissange,

Caconda, and Huila formed “une variete bien caracte-

risee du C. rhombeatus’’’’ which was later described as

C. bilineatus by Boulenger (1905). Rasmussen (2005)

mapped the species in Angola, and corrected misidenti-

fied material (Hellmich 1957, Laurent 1964), following

confusion with C. maculatus in northern populations.

West African Night Adder

Causus maculatus (Hallowell 1842)

Distichurus maculatus Hallowell, 1842. Description of a new

genus of Serpents from Western Africa. J. Acad. not. Sci. Phila¬

delphia 8; 337.

This species was confused with C. rhombeatus by Bo¬

cage, and the first material from Angola was noted by

Laurent (1964) from Dundo. Rasmussen (2005) noted

the importance of the lateral oblique scale row number in

distinguishing between the two species, corrected earlier

misindentifications, and showed that C. maculatus oc¬

curs in sympatry with both C. rhombeatus and C. bilinea¬

tus at Piri. It is restricted to the northern parts of Angola.

Lichtenstein’s Night Adder

Causus Uchtensteini (Jan 1859)

Aspidelaps lichtensteinii Jan, 1859. Additions et rectifications

aux Plan et Prodrome de I’lconographie descriptive des Ophi-

diens. Rev. Mag. Zool. 11; 511.

Laurent (1964) recorded the first and only specimens of

C. Uchtensteini in Angola from Dundo and the Lukashi

River, 50 km east Dundo. In the Dundo region it occurs

in sympatry with both C. rhombeatus and C. maculatus

(Rasmussen 2005).

Species not confirmed for Angola that may

occur

Hallowell’s House Snake

Boaedon virgatus (Hallowell 1854)

Coelopeltis virgata Hallowell, 1854. Remarks on the geograph¬

ical distribution of reptiles, with descriptions of several species

supposed to be new, and corrections of former papers. Proc.

Acad. Nat. Sci. Philadelphia 1854; 98-105.

Known from both Congo (Brazzaville) and Gabon (Pau-

wels and Vande weghe 2008), and therefore likely to oc¬

cur in forested habitats in Cabinda.

Plain Striped House Snake

Bothrophthalmus brunneus (Gunther 1863)

Bothrophthalmus lineatus brunneus Gunther, 1863. Third ac¬

count of new species of snakes in the collection of the British

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Branch

Museum. Ann. Mag. Nat. Hist. (3) 12; 348.

Not known to Bocage (1895) from Angola. Although

Bothrophthalmus lineatus was recorded from Dundo

(Laurent 1950, 1954, 1964; Tys van den Audenaerde

1967), the plain western subspecies B. 1. brunnaeus,

from Cameroon to Gabon, is now treated as a full species

(Pauwels and Vande weghe 2008). It is possible that B.

brunnaeus may extend south to the Cabinda forests, and

those in Angola just south of the border.

Mopane Racer

Mopanveldophis zebrinus (Broadley and Schatti 2000)

Broadley, D.G. and Schatti, B. 2000. Anew species of Coluber

from northern Namibia (Reptilia: Serpentes). Madoqua 19(2):

171.

This unusual and rare snake was described (Broadley and

Schatti 2000) from a single specimen collected near Rua-

cana on the Cunene River, western Owamboland, Na¬

mibia (17°25’S, 14°10’E). It is known from only three

other specimens from the Kaokoveld (Bauer et al. 2001),

and from the Kunene River mouth (Cunningham et al.

2018), and was provisionally considered to form part of

a Trans-Kunene mopaneveld fauna, and that it may there¬

fore occur in southern Angola. Even at the time of its

description, however, its inclusion in the genus Coluber

was provisional as the genus was undergoing reassess¬

ment and division, particularly the African representa¬

tives (e.g., Schatti and Charvet 2003). Schatti and Utiger

(2001) erected a monotypic genus for the unusual So-

cotran racer, Hemerophis socotrae, but deferred a desci-

sion on C. zebrinus as its phylogenetic relationships were

unknown. However, it was subsequently prematurely

placed in Hemerophis (Wallach et al. 2014), creating a

3,000 km+ zoogeographic enigma, before being finally

placed in the monotypic Mopaneveldophis (Figueroa et

al. 2016). This generic name is as non-euphonious as it

is a misnomer, as Mopane veld has a much wider dis¬

tribution than that of the snake, and there is as yet no

confirmation that this attractive small racer is restricted

to mopaneveld.

Bark Snake

Hemirhaggheris nototaenia (Gunther 1864)

Coronella nototaenia Gunther, 1864. Contribution to the

knowledge of snakes of tropical Africa. Ann. Mag. Nat. Hist.

(6) 1: 309.

Hemirhaggheris in Africa was reviewed by Broadley

and Hughes (2000), who showed that all historical An¬

golan records of H. nototaenia (e.g., Bocage 1895, Mo-

nard 1937) were confused with H. viperina. The species

may enter extreme eastern Angola as it is recorded from

Caprivi (Broadley and Hughes 2000), the adjacent Ike-

Amphib. Reptile Conserv.

lenge pedicel in extreme northwest Zambia (Broadley

1991a), and Ngonye Falls, southwest Zambia (Pietersen

et al. 2017).

Eined Grass Snake

Psammophis lineatus (Dumeril, Bibron and Dumeril

1854)

Dryophylax lineatus Dumeril, Bibron and Dumeril 1854. Erpe-

tologie generale ou histoire naturelle complete des reptiles. Tome

septieme. Deuxieme partie, comprenant I’histoire des serpents

venimeux. Paris, Librairie Encyclopedique de Roret: 1124.

Its presence in Angola is based on a single record (as

Dromophis lineatus, Bogert 1940) from “Angola,”

(AMNH 50611, Vemay, Eang, Boulton 1925). The

specimen lacks detailed locality data, and members of

the Vernay-Eang Angola expedition did not visit eastern

or northern Angola, although they visited other areas in

Africa after the expedition (Hill and Carter 1940). The

species is known from savannah habitats in adjacent

countries (Hughes 2004), but Bogerf s record should be

treated with caution until the discovery of additional ma¬

terial. Dromophis was synonymized with Psammophis

(Kelly et al. 2008).

Boulenger’s Garter Snake

Elapsoidea boulengeri (Boettger 1895)

Elapsoidea boulengeri Boettger, 1895. Zwei neue Reptilien

vom Zambesi. Zoo/. Anz. 18: 62.

Broadley (1998) mapped parapatry between Elapsoi¬

dea boulengeri and E. s. semiannulata from the eastern

Caprivi and it is possible that E. boulengeri enters ex¬

treme southeast Angola.

Peringuey’s Adder

Bitisperingueyi (Boulenger 1888)

Viper a peringueyi Boulenger, 1888. On new or little known

South African reptiles. Ann. Mag. Nat. Hist. (6) 2: 141.

Haacke (1975) when reviewing the small adders of the

western arid of southern Africa recorded no B. peringueyi

from Angola, and no confirmed records have subsequent¬

ly been recorded. Despite this, the species continued to

be incorrectly listed for the country (e.g.. Branch 1998;

Dobiey and Vogel 2007; Uetz and Hozek 2017). Possi¬

bly suitable habitat occurs in the small extension of the

Namib Desert into extreme southwest Angola, but there

remain no records.

Discussion

The diversity and composition of snake families in An¬

gola basically refiects that of Sub-Saharan Africa and

September 2018 | Volume 12 | Number 2 | el 59

Snakes of Angola: An annotated checklist

particularly the adjacent subcontinent. There is relatively

low diversity in primitive groups such as scolecophidi-

ans (Typhlopidae and Leptotyphlopidae) and particularly

haenophidians (Pythonidae). The main venomous fami¬

lies Elapidae and Viperidae are well-represented in both

Angola and the subcontinent, but with more tropical rep¬

resentatives in Angola (e.g., the elapids Pseudohaje goldi

and Naja annulata, and viperids Causus lichtensteini, C.

maculatus, Athens squamigera, and Bids nasicornis). In

both regions cobras {Naja, Hemachatus) dominate the el-

apid fauna. Among viperids, however, both regions have

local but different radiations of small viperids. Small ad¬

ders of the Bids atropos-cornuta (7-10 species) and Bids

caudalis-schneideri (4-6 species) complexes in southern

Africa may be regionally common, highly endemic and

taxonomically difficult (Branch 1997, 1999; Kelly et al.

2011b). Only a single component of this radiation. Bi¬

ds caudalis, enters Angola (the phylogenetic affinities

of B. heraldica remain unresolved), whereas night ad¬

ders {Causus) are marginally present in the subcontinent

but in Angola are their most diverse anywhere in Africa

(Rasmussen 2005).

The dominant African snake family is the Lamprophi-

idae which appears to have originated in Africa, and

within which some lineages subsequently radiated into

Arabia and Asia. It appears to have been a rapid radia¬

tion, and untangling the relationships and even content

of the many subfamilies if proving difficult (e.g., Kelly

et al. 2008; Pyron et al. 2011; Figuerio et al. 2016) and

a consequently unstable higher-level classification. The

Atractaspidinae (here included within lamprophids, but

sometimes treated as a separate family; Figuerio et al.

2016), Famprophiinae. Prosyminae and Psammophinae

form important lamprophid radiations in Sub-Saharan

Africa, and together also form the dominant component

of the Angolan snake fauna (37 species). As with elapids

and viperids a number of Congo Basin species enter the

northern forests, including some currently known from

very few Angolan specimens, e.g., Lycodonomorphus (?)

subtaeniatus, Chamaelycus parkeri, Boaedon cf odva-

ceous, Bothrophthalmus lineatus, etc. Perhaps the great¬

est difference between South Africa and Angola is re-

fiected in the greater diversity of colubrids (Colubridae)

in Angola (29 species). These include numerous tropical

Congo Basin snakes that enter the northern and scarp

forests, and of particular interest are the Congo Basin

species Toxicodryas blandingii, T. pulverulenta, Rham-

nophis aethiopissa, Philothamnus niddus, DasypeUs pal-

marum, etc. The family is considered of Asian origin and

to have entered and subsequently radiated in Africa.

Although a number of new Angolan lizards have been

described (Haacke 2008; Conradie et al. 2012; Stanley

et al. 2016) or identified (e.g.. Branch et al. 2017) since

the end of civil hostilities and the start of modern biodi¬

versity surveys, no new snakes have yet been described.

It is therefore unsurprising that snake diversity in An¬

gola appears to be the most well-known component of

the country’s reptile fauna. However, the distributions

of snakes in Angola remain poorly-known, particularly

those of forest-adapted species in tropical forest asso¬

ciated with the Congo Basin along the northern border,

and with the evergreen forest isolates associated with the

escarpment along the western edge of the country. An¬

gola harbours the second highest level of vertebrate en¬

demism associated with the disjunct components of the

African Great Escarpment (Clark et al. 2011). Studies on

the phylogenetic relationships of the snakes associated

with these isolated forest populations are needed to con¬

firm their conspecificity with northern populations, and

to understand the consequences and timing of the dis¬

junctions between these forests. Similarly, the montane

grassland habitats associated with the Humpata plataeu

of the escarpment, particularly in the Tundavala region,

includes numerous regional reptile endemics (Baptista et

al. 2018a), including two psammophine snakes (Branch

et al. 2018).

As noted in the Introduction, this checklist is a first

step towards stimulating increased interest in the herpe-

tofauna of Angola. Bocage’s early stuides, culminating

in his monographic summary of Angola’s herpetological

riches, placed the country among the few African coun¬

tries with a well-studied herpetofauna during the early

colonial period. Sadly, this was followed by a century of

relative neglect, but with an early scientific revival in the

1970s that was nipped in the bud by civil confiict. How¬

ever, overviews of both reptile (Branch et al. 2018b) and

amphibian diversity (Baptista et al. 2018b) are included

in an updated synopsis of the countries’ biodiversity

(Huntley et al. 2018), and finally with peace and welcom¬

ing borders, studies on Angolan biodiversity are entering

the new Millenium.

Acknowledgements. —My colleagues Pedro Vaz

Pinto, Ninda Baptista and Werner Conradie, with whom

I work on the Angolan herpetofauna, have made my for¬

ays into that beautiful country both as exciting as it has

been scientifically productive, for which I thank them.

Although they are not co-authors on this particular sum¬

mary, they have reviewed this article fully and made

numerous corrections and useful comments, as did my

colleague Colin Tilbury. Any inaccuracies or oversights,

however, obviously remain my own. Some of my field¬

work in Angola was funded either through National Geo¬

graphic Exploration Grants (Branch 2011) or as part of

the National Geographic Okavango Wilderness Project

(National Geographic Society grant number EC07IS¬

IS). I also want to thank Werner Conradie, Ninda Bap¬

tista and Warren Klein for the use of their photos.

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London-born Bill Branch was employed as Curator of Herpetology at the Port Elizabeth Museum for over 30 years (1979-2011), and

although now retired remains Curator Emeritus Herpetology. His herpetological studies have concentrated mainly on the systematics,

phylogenetic relationships, and conservation of African reptiles, but he has been involved in numerous other studies on the reproduction

and diet of African snakes. He has published over 300 scientific articles, as well as numerous popular articles and books. The latter include:

South African Red Data Book of Reptiles and Amphibians (1988), Dangerous Snakes ofAfrica (1995, with Steve Spawls), Field Guide to

the Reptiles of Southern Africa (1998), Tortoises, Terrapins and Turtles of Africa (2008), and Atlas and Red Data Book of the Reptiles of

South Africa, Lesotho and Swaziland (multi-authored, 2014), as well as smaller photographic guides. In 2004 he was the 4'*' recipient of

the “Exceptional Contribution to Herpetology” award of the Herpetological Association of Africa. He has undertaken field work in over 16

African countries, and described nearly 50 species, including geckos, lacertids, chameleons, cordylids, tortoises, adders, and frogs.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 59

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

12(2) [General Section]: 83-89 (el60).

New distribution records, observations on natural

history, and notes on reproduction of the poorly known

Sudanese Unicorn Chameleon (Chamaeleonidae: Trioceros

conirostratus) from Uganda, Africa

^Daniel F. Hughes, ^Daniel G. Blackburn, ^Lukwago Wilber, and "^Mathias Behangana

' Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Drive, Urbana, Illinois 61801, USA ^Department

of Biology, Electron Microscopy Center, Trinity College, Hartford, Connecticut 06106, USA ^ Department of Environmental Management, School of

Forestry, Makerere University, Kampala, UGANDA ‘^Herpetology Division, Makerere University, Kampala, UGANDA

Abstract —^We provide data derived from nearly four months of field surveys on the distribution, natural

history, and habitat of the poorly known Sudanese Unicorn Chameleon (Trioceros conirostratus) from Northern

Region, Uganda, Africa. Our study also provides the first description of the reproductive mode and an estimate

of the litter size for T. conirostratus. Multiple individuals of T. conirostratus were detected from mid-high

elevation wooded-grassland and closed-forest habitats in six Central Forest Reserves across northeastern

Uganda during surveys conducted in 2015 and 2016. Trioceros conirostratus is viviparous as evidenced by

the presence of well-developed embryos that lacked eggshells in the oviducts. Twelve embryos were present

in one of the females. Adult males were smaller on average than adult females. The presence of variously

sized Juveniles with non-gravid and gravid adult females during surveys at the same site suggested that this

species might exhibit asynchronous reproduction. We observed a possible mechanism for predator deterrence

in this species from repulsive material stored in temporal pouches. Our results greatly expand the distribution,

and significantly add to the knowledge on the reproductive biology and natural history of T. conirostratus in

Uganda.

Keywords. Conservation, Central Forest Reserve, East Africa, Karamoja, live-bearing, Sauria, morphology

Citation: Hughes DF, Blackburn DG, Wilber L, Behangana M. 2018. New distribution records, observations on natural history, and notes on

reproduction of the poorly known Sudanese Unicorn Chameleon (Chamaeleonidae: Trioceros conirostratus) from Uganda, Africa. Amphibian & Reptile

Conservation 12(2) [General Section]: 83-89 (el60).

NoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided

the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication

credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation] official journal website <amphibian-

reptile-conservation.org>.

Received: 20 March 2017; Accepted: 10 February 2018; Published: 9 September 2018

Introduction

The Sudanese Unicom Chameleon {Trioceros conirostra¬

tus Tilbury, 1998) was described on the basis of a single

male specimen collected from 1,050 m at Lomoriti in the

Imatong Mountains of southern South Sudan (Tilbury

2010). Since its description in 1998, few observations of

this species have been made, including just two verifiable

extensions of its geographic range to the Loima Hills

(1,400 m) and Mtelo Massif (1,900-2,300 m), both sites

in northwestern Kenya (Kofeny 2006; Stipala et al. 2011,

2012). The apparent disjunct geographic distribution

of T. conirostratus prompted speculation on its occur¬

rence at similar elevations between the known localities

in southern South Sudan and northwestern Kenya (Sti¬

pala 2014a, b; Spawls et al. 2014). However, vouchered

CorrGSpondonCG. Mfhughes@illinois.edu (Corresponding author)

specimens have yet to be obtained from potential sites

between the documented populations of T. conirostratus.

The original description of T. conirostratus provided

almost no information about its natural or life history,

and thus, knowledge of its habitat preferences and ecol¬

ogy is derived from just a few Kenyan samples (Stipala

2014a). For example, this species was originally posited

to be a forest specialist because the Imatong Mountains

possess large swaths of broad-leafed forest (Tilbury

2010). However, specimens collected from the Mtelo

Massif in northwestern Kenya were found on shrubs of

a disturbed agricultural area, and the surrounding habitat

consisted of xeric-adapted woodland tree species (Stipa¬

la 2014a). Reproduction in T. conirostratus is unknown,

and no empirical investigations have been undertaken to

date (Tilbury 2010); however, it has been conjectured to

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 60

Hughes et al.

UGANDA -

Fig. 1. Locations of Central Forest Reserves (CFR) where the Sudanese Unicom Chameleon (Trioceros conirostratus) was found

during surveys from 2015-2016, Northern Region, Uganda.

be viviparous because of its phylogenetic affinities to the

live-bearing T. bitaeniatus species group (Tilbury and

Tolley 2009; Stipala 2014a).

In this report, we provide the first formal records of

T. conirostratus from six Central Forest Reserves (CFR)

in northeastern Uganda that fill critical gaps in the distri¬

bution for this species. In addition, we describe the re¬

productive mode, report on the litter size, and describe

a predator deterrent mechanism of this species. Lastly,

we present information on body sizes, color patterns, and

habitats of T. conirostratus from these recently discov¬

ered Ugandan populations.

Methods and Materials

We conducted nearly four months of fieldwork in

Northern Region, Uganda during May-July 2015 and

July-August 2016, throughout which we surveyed the

herpetofauna at several protected areas in the region, in¬

cluding six montane CFRs associated with the Karamoja

Sub-region. At each CFR, we spent an average of three

days and four nights conducting diurnal and nocturnal

visual-encounter surveys. Our team, with help from for¬

est reserve managers, Uganda People’s Defence Force

soldiers, local police, guides, and villagers searched for

chameleons in various natural forested and non-forested

habitats at each site. Chameleons were most often en¬

countered on sleeping perches at night in various types

of vegetation and located with the aid of artificial lights.

Fewer chameleons were encountered during diurnal

searches. Notes were taken on GPS coordinates, ecol¬

ogy, behavior, date, time, sex, and basic habitat details

for each collected specimen. Collected chameleons were

humanely euthanized, tissue samples taken from the hind

limb or liver and stored in 99% ethanol, and specimens

were later fixed in 10% buffered formalin. On comple¬

tion of each expedition, with permits from the proper

authorities (CITES, UWA, UNCST, and UMAAIF), the

specimens and tissue samples were transferred to the

University of Texas at El Paso’s Biodiversity Collections

in the United States.

A single gravid female T. conirostratus collected from

Morongole CFR was set aside for an assessment of its

reproductive mode. This female specimen was eutha¬

nized, and then its oviducts were removed immediately

and placed in a separate vial filled with 10% buffered

formalin. Criteria for assessing its reproductive mode

were adopted from the literature (Blackburn 1993), and

embryonic development was assessed in accord with

the Dufaure and Hubert (1961) system (Porter 1972), as

modified for chameleons by Andrews (2007).

Results

We found individuals of T. conirostratus from several

localities within six CFRs across northeastern Uganda:

Agoro-Agu, Kadam, Moroto, Morongole, Napak, and

Orom (Fig. 1; Table 1). Most individuals were detected

in wooded-grasslands and adjacent agricultural fields,

and far fewer individuals were found in closed forests.

Further, we detected more individuals in semi-disturbed

to disturbed grassland-associated areas than in pristine

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 60

Observations on the Sudanese Unicorn Chameleon

Table 1. Collection information for the Sudanese Unicorn Chameleon {Trioceros conirostratus) during surveys from 2015-2016 in

Northern Region, Uganda, Africa. CFR - Central Forest Reserve; + - detected but not collected.

Locality

Coordinates

Elevation (m)

Date

Class (no. individuals)

Agoro-Agu CFR, Lamwo District

N03.81357, E32.94207

2,134

5 July 2015

JV(8)

Kadam CFR, Nakapiripirit District

N01.79864, E34.74146

2,140

31 May 2015

M(1),F(4), JV(9)

Morongole CFR, Kaabong District

N03.80740, E34.02559

2,461

23 July 2016

M (5), F (5), JV (±)

Moroto CFR, Moroto District

N02.51234, E34.70190

1,969

4 June 2015

M(4),F(3), JV(1)

N02.52847, E34.72872

2,582

5 June 2015

M (2), F (3), JV (4)

Napak CFR, Napak District

N02.04033, E34.27148

1,875

14 July 2016

M(4),F(6), JV(1)

Orom CFR, Kitgum District

N03.40338, E33.55560

1,304

18 July 2016

M (3), F (6), JV (±)

forested habitats. However, we do not know whether the

fewer encounters in closed forests were due to actual or

apparent rarity of T. conirostratus in this habitat type.

At most sites during nearly all surveys, we encountered

small juveniles, sub-adults, and adults of both sexes (Ta¬

ble 1). Save for the rostral appendage in males, the gen¬

eral appearance of males and females was similar across

populations, yet color patterns were variable (Fig. 2-3).

The mean body size of adult males, 66.19 ± 6.09

mm (range 57.87-78.03 mm; n = 14), was significant¬

ly smaller than that of adult females, 70.97 ± 6.47 mm

(range 57.57-83.58 mm; n = 23) (t = -2.23, df = 35, P

= 0.03). The mean tail length of adult males, 66.27 ±7.8

mm (range 54.51-80.94 mm; n = 14) was about 3 mm

longer, but not significantly different, from that of adult

females, 63.36 ± 7.95 mm (range 46.33-79.8 mm; n =

23) (t = 1.09, df = 35, P = 0.28). On average, the ratio

of male tail length to body size was roughly proportional

(mean TL/SVL = 1.0 ± 0.05; range 0.93-1.12; n = 14),

whereas this ratio in females was skewed towards body

size (mean TL/SVL = 0.89 ± 0.07; range 0.78-1.03; n =

23). Mean body size of juveniles was 30.87 ± 4.97 mm

(range 22.13^2.22 mm; n = 2\) and mean tail length

was 27.22 ± 5.19 mm (range 18.18-39.01 mm; n = 21),

with the average ratio of tail length to body size favor¬

ing body size in juveniles (mean TL/SVL = 0.88 ± 0.07;

range 0.77-0.99; n = 21). The smallest juvenile for

which an emerging rostral horn could be seen was 31.7

mm SVL and 23.23 mm TL.

Threatened individuals of T. conirostratus (both sex¬

es and juveniles) would open their mouths, yet, rarely

would they bite when handled. Rather, it seemed that

opening the mouth served a dual purpose: intimidate and

expose temporal pouches (i.e., comer of jaw) (Fig. 4).

These pouches were often filled with a yellow/orange or

brown material that had an astringent odor and was simi¬

lar in consistency to partially digested food (Fig. 4A).

When handled, chameleons would open their mouths,

expose the pouches, and extrude this substance. Simulta¬

neously, chameleons would thrash their heads from side

to side; an action that often distributed the substance onto

the collector’s hand (Fig. 4B). The foul-smelling scent

from the smeared material remained on the collector for

ca. 24 hours post-washing with soap.

We found moderately well-developed embryos within

the oviducts of one female specimen (Field no. DFH 975:

SVL 66.96 mm and TL 62.23) (Fig. 5). A litter size of 12

embryos was found, with six embryos in each oviduct.

The embryos were of stage 35 (sensu Andrews 2007),

with the following characteristics: a heavily pigmented

ocular choroid, an indented external auditory meatus,

cervical flexure in the process of disappearing, mandible

extends to the tip of the snout, well-developed forelimbs

and hindlimbs with fully zygodactylous feet, digits are

prominently outlined, and connected by interdigital web¬

bing with slightly concave margins (Fig. 5). The embryos

were surrounded by fetal membranes that lay in apposi¬

tion to the uterine lining, with a chorioallantoic placenta

positioned dorsally, and a yolk sac placenta ventrally. A

very thin and transparent vestige of an eggshell mem¬

brane could sometimes be discerned at the placental

interface, yet it showed no trace of calcification. This

gravid specimen had a pigmented peritoneum.

Discussion

We documented the presence of T. conirostratus from

a series of isolated volcanic mountains in Northern Re¬

gion, Uganda, Africa. The new localities fill in critical

gaps for the geographic range of T. conirostratus. Im¬

portantly, we provide empirical evidence to support a

single unsubstantiated report of this species on social

media that ostensibly originated from an undisclosed lo¬

cality in Uganda at 1,800 m (Stipala 2014b). We found

that T. conirostratus can be found in suitable montane

habitat at an elevation range from 1,304 m to 2,582 m

in northeastern Uganda. Our field surveys indicate that

T. conirostratus can occupy both wooded-grassland and

closed-forest habitats, and although it was much more

common in the former, we do not know if this apparent

habitat preference is an artefact of sampling or not. This

species seems to tolerate anthropogenic disturbances,

largely in the form of slash and burn agricultural farming.

In general, male T. conirostratus tended to be smaller and

have longer tails than females, and this form of sexual

dimorphism is shared with members of the T. bitaeniatus

group and many other chameleons (Tilbury 2010).

Temporal pouch material was recently characterized

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 60

Hughes et al.

Male

Female

Habitat

Fig. 2. Representatives of the Sudanese Unicorn Chameleon {Trioceros conirostratus) and local habitats from the three “southern”

Central Forest Reserves (CFR) surveyed in Northern Region, Uganda, Africa. Male and female from Kadam CFR (top row), male

and female from Moroto CFR (middle row), and male and female from Napak CFR (bottom row).

Male ■

■ Female ■

Habitat

Fig. 3. Representatives of the Sudanese Unicom Chameleon {Trioceros conirostratus) and local habitats from the three “northern”

Central Forest Reserves (CFR) surveyed in Northern Region, Uganda, Africa. Sub-adult male and female from Orom CFR (top

row), male and female from Morongole CFR (middle row), and juvenile male and juvenile female from Agoro-Agu CFR (bottom

row).

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el60

Observations on the Sudanese Unicorn Chameleon

Fig. 4. Examples of Sudanese Unicom Chameleons {Trioceros

conirostratus) displaying a threatened posture with the mouth

open. (A) Threatened male from Morongole Central Forest

Reserve (CFR) displaying a temporal pouch filled with brown

odiferous material (in-detail); (B) Another threatened male

from Morongole CFR demonstrating an empty temporal pouch

depleted after being handled (in-detail).

from the Jackson’s Chameleon {Trioceros jacksonii), and

it was found to contain volatile and odiferous compounds

derived from those found in prey items and/or sloughed

skin (Freest et al. 2016). Freest et al. (2016) speculated

that this pouch material was involved with insect luring,

and while this is likely true, the behavior we observed in

T. conirostratus seemed more for predator deterrence. We

also observed an analogous behavior and foul-smelling

substance in three other species of chameleons: T. ellioti,

T johnstoni, and Kinyongia xenorhina from Western Re¬

gion, Uganda (D.F. Hughes, personal observation).

The reproductive mode of T. conirostratus was deter¬

mined to be viviparous. The embryos were at stage 35,

which lies somewhat beyond the stage of oviposition in

typical oviparous squamates (Blackburn 1995). In addi¬

tion, no eggshell was present; rather, dissection revealed

a thin, barely-visible vestige of the shell membrane that

lacked any trace of calcification. This feature is indicative

of viviparity, because in oviparous squamates, an opaque

eggshell surrounds embryos of mid-stage and beyond.

Given the absence of an eggshell, the chorioallantois and

yolk sac formed placentas in conjunction with the uterine

Fig. 5. A developing embryo of the Sudanese Unicorn

Chameleon {Trioceros conirostratus), removed from a female

oviduct. Scale bar = 1 mm.

lining, and their topography conformed to that of typi¬

cal viviparous lizards (Stewart and Blackburn 2014). The

association of viviparity with a pigmented adult perito¬

neum is consistent with that of other live-bearing Trioc¬

eros (see Tilbury et al. 2006). The litter size (12 embryos)

lies within the range of other viviparous species of the T

bitaeniatus group. Reports on litter size are available for

several other members of this species group, including

T ellioti (4-12 young: Leptien 1989), T hoehnelii (7-18

young: Spawls et al. 2002), and T iacksonii (7-28 young:

Spawls et al. 2002).

We encountered small Juveniles (< 30 mm SVL),

as well as gravid and non-gravid adult females of T

conirostratus during the same surveys, and we suggest

that this may be indicative of asynchronous reproduc¬

tion, which is common among live-bearing chameleon

species of the South African genus Bradypodion (Tolley

and Burger 2007; Tolley and Jackson 2014) and other

viviparous Trioceros species (e.g., T bitaeniatus (Necas

1999)). More investigation is warranted to determine the

ecological variables associated with the timing of repro¬

duction in T conirostratus populations in Uganda.

Conservation

Frior to our surveys in 2015, T conirostratus had not

been recorded from Uganda and thus was not included

on the country’s national checklist of reptiles (Behan-

gana 2015). We consider illegal wildlife trade to be the

primary conservation threat facing T conirostratus in

Uganda, in large part because this species was consid¬

ered rare prior to our surveys, and thereby highly cov¬

eted by chameleon hobbyists. For instance, a wildlife

trafficker had already harvested this species before we

first documented its presence in Uganda. In May of 2015,

local sources indicated to our team that an international

animal dealer purchased 100 live T conirostratus several

months prior to our arrival at the Kadam Central For¬

est Reserve (CFR). We were advised that the chameleons

were collected by the indigenous people and purchased

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | e160

Hughes et al.

at Uganda shillings 10,000 ($2.70 USD) by a local inter¬

preter on behalf of the trader. The local interpreter in turn

received Uganda shillings 80,000 ($21.60 USD) per live

chameleon. We discovered that numerous chameleons

perished during the collection process and many more

died in captivity while amassing the trader’s full request.

The lUCN Red List considers T. conirostratus as

Least Concern (Stipala 2014b), and our surveys general¬

ly support this conservation assessment. The populations

we encountered in Uganda seemed tolerant of agricultur¬

al and fire-based anthropogenic disturbances. However,

the conservation of T. conirostratus is complicated by

the fact that its lUCN assessment was completed prior to

our discovery of this species in Uganda. Furthermore, the

demand for pet chameleons is often met, in part, by the

export of wild animals from several East African coun¬

tries, including Uganda (Carpenter et al. 2004; Jenkins

et al. 2014). According to the CITES Trade Database,

over 50,000 live chameleons have been exported from

Uganda since 2000, including several species that do not

even occur in the country (e.g., Chamaeleo senegalensis

(Tilbury 2010)). Indeed, because of the general interest

for pet chameleons, it is reasonable to think that a trade

in this species could develop from Uganda. Based on

our surveys, T. conirostratus is now formally included

in the checklist of Uganda reptiles, which serves as an

important first-step towards the sustainable trade in this

species (Jenkins et al. 2014). Yet, if collection pressure

for this species becomes unsustainable, breeding farms

located within the species range may help to mitigate

threats from overharvesting while providing a boost to

local economies (Otieno 2015).

We do, however, recommend that the governing bod¬

ies in Uganda use caution when setting export quotas and

licensing international trade permits in T. conirostratus

for three important reasons: 1) this species is currently

known only from protected areas in Uganda; 2) the risk

of trading in populations with an unknown conservation

status or of dubious origin is high for Uganda; and 3) it

has not yet been fully evaluated whether any of these iso¬

lated populations represent a cryptic species that could be

endemic to Uganda.

Acknowledgements. —^We thank the Uganda Na¬

tional Council of Science and Technology (UNCST) for

granting us the research permit to conduct our ongoing

Herpetofaunal Conservation Assessment of Uganda. We

also thank the Uganda Wildlife Authority (UWA) and

the National Forest Authority (NFA) for the necessary

permits to work in the Protected Areas (PAs) under their

jurisdiction. We are indebted to Mr. James Eutalo, Com¬

missioner of Wildlife Conservation and CITES Authority

for Uganda, and Mr. Aggrey Rwetsiba, Senior Monitor¬

ing and Research Coordinator for UWA, and other staff

under their supervision, that tirelessly worked hard to

make sure we received the correct documents in a timely

manner. We are further indebted to the UWA and NFA

field staff that assisted us with access, protection, and ex¬

cellent companionship to carry out our research in the

various PAs. We thank Dr. Daniel Aleper for outstand¬

ing accommodations and guidance while in Moroto. Our

most sincere gratitude is owed to the indigenous cultures

of Uganda for contributing vital local knowledge on the

fauna and flora during our surveys, including the Kar-

amajong, Tepeth, Kadam, Dodoth, Ik, and Pokot peoples.

Easily, we owe thanks to the members of the armed forces

of Uganda People’s Defence Force (UPDF) and Uganda

Police Officers (UPO) that accompanied us at sites with

heightened security concerns.

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don, United Kingdom. 544 p.

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V. 2014. The Kenya Reptile Atlas: A Free Source of

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Raton, Florida, USA. 748 p.

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Wilkinson P, Godley B, Evans MR. 2011. A new spe¬

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ley B, Nyamache J, Evans MR. 2012. A new species

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meleons in the Highlands of Kenya. Jan Stipala, Sin¬

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cluding the Chameleons of Europe, the Middle East

and Asia. Edition Chimaira, Frankfurt am Main, Ger¬

many. 831 p.

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Africa. Struik Publishers, Cape Town, South Africa.

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Daniel Hughes is a post-doctoral researcher at the University of Illinois at Urbana-Champaign (USA). He

earned his B.Sc. in Zoology from Humboldt State University (2011), M.Sc. in Biology from Shippensburg

University (2013), and Ph.D. in Ecology and Evolutionary Biology from the University of Texas at El Paso

(2018). His research focuses on the ecology and evolution of reptiles and amphibians, with an emphasis on

Afromontane chameleons and the conservation Uganda’s herpetofauna.

Daniel Blackburn is a professor of biology at Trinity College (Hartford, CT, USA), where he teaches courses

in zoology and evolution. He earned his M.Sc. and Ph.D. in zoology from Cornell University. His research

focuses on reptile placentation and the evolution of vertebrate viviparity.

Lukwago Wilber received his Master’s degree in Environment and Natural Resources from Makerere

University in 2017. His research focuses on the ecology of reptiles and amphibians in Africa. His forestry

background aids him to correlate vegetative changes in forest habitats to herpetological diversity. He

currently works with the Uganda National Roads Authority (UNRA) as a Senior Environment Officer and as

an Ecologist on all road development projects.

Mathias Behangana received his Ph.D. in Environment and Natural Resources in 2010, and M.Sc. in

Zoology in 1997 from Makerere University. He trained as an environmental/wildlife ecologist and has over

23 years of experience in research and monitoring biodiversity with a focus on amphibian and reptilian fauna

of Africa. He currently works as the Founding Director for NICE-Planet Eimited, a local NGO.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 60

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptiie Conservation

12(2) [General Section]: 90-97 (el61).

Enumeration of Herpetofaunal assemblage of Surajpur

Wetland, National Capital Region (India)

Nasim Ahmad Ansari

Wildlife Institute of India, Chandrabani, Dehradim -248001, Uttarakhand, INDIA

Abstract —Surajpur wetland is an important wetland in the National Capital Region, India, known for its rich

biodiversity. The present study was conducted from March 2010 to February 2013 to record the herpetofaunal

diversity at the study area by applying standard methods and survey techniques. During the study period, a

total of 19 species of herpetofauna belonging to 14 families and three orders were recorded. It comprised of six

species of amphibians belonging to five families and 13 species of reptiles belonging to nine families. Family

Dicroglossidae (Amphibians) and Colubridae (Reptiles) recorded maximum two and three species respectively.

The relative abundance analysis showed that, among 19 species of herpetofauna, eight were common, four

were uncommon, and seven were rarely recorded in the study area. The Indian Garden Lizard Caiotes versicoior

was most common during the study period. Of the 19 species recorded, 10 species are Least Concern and nine

species are under Not Evaluated category in the lUCN Red List, while four species are listed in Schedule-1 of

the Indian Wildlife (Protection) Act, 1972.

Keywords. Amphibians, reptiles, biodiversity, conservation, abundance, survey

Citation: Ansari NA. 2018. Enumeration of herpetofaunal assemblage of Surajpur Wetland, National Capital Region (India). Amphibian & Reptile

Conservation 12(2) [General Section]: 90-97 (el 61).

atives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided the

original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication credit

sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation-, official journal website <amphibian-reptile-

conservation. org>.

Received: 14 June 2017; Accepted: 11 June 2018; Published: 20 September 2018

Introduction

Amphibians and reptiles, collectively known as herpeto¬

fauna, comprise the highest proportion of threatened spe¬

cies among vertebrates in the world (Baillie et al. 2010;

Bohm et al. 2013), and are found in a diverse range of

habitats and microhabitats, from deserts to grass-lands,

from forests to oceans, and from hills to our households.

They are declining rapidly in both numbers and range in

recent decades due to anthropogenic pressures like direct

killing, habitat destruction, road killing, pesticides, dis¬

eases, and climate change (Stuart et al. 2004; Rodrigues

et al. 2010). More than 9,700 species of reptiles and

6,800 species of amphibians are reported globally (Les-

barreres et al. 2014).

India hosts rich herpetofaunal diversity; about 518

species of reptiles and 342 species of amphibians, includ¬

ing 66% of amphibians and 37% of reptiles are reported

to be endemic to India (Aengals et al. 2011; Dinesh et al.

2013). Herpetofaunal diversity studies have mostly con¬

centrated in the Western Ghats (Chandramouli and Ga-

nesh 2011; Nath et al. 2012; Ramesh et al. 2013; Vasan-

thi et al. 2014) and Central India (Ishaque and Sarsavan

2014; Narayana et al. 2014; Yadav et al. 2014; Fellow

2015; Radav and Yankanchi 2015; Rout et al. 2015; So-

lanki et al. 2015), with very few studies in northern India

(Das et al. 2012; Kanaujia and Kumar 2013; Singh and

Banyal 2013; Prasad et al. 2018). Studies on the herpe¬

tofauna have been made by several authors, but there is

no such study in Surajpur Lake to address the conserva¬

tion of herpetofauna. In context of this, this study was

made to explore the diversity of herpetofauna at Surajpur

wetland and to discuss the conservation and management

implications in context of results, hitherto unreported.

Methods and Materials

The present study was conducted at Surajpur Lake

(28°3L425’N, 77°29’714’E), an urban wetland located

in district Gautam Budh Nagar, Uttar Pradesh under Na¬

tional Capital Region, India, which falls under the Gan-

getic Plain Biogeographic Zone (Rodger et al. 2002). The

study area was located at an elevation of 184.7 meters

above mean sea level (Fig. 1). Surajpur Lake has been

protected under reserve forest and spreads over an area

of 308 hectares. The lake is mainly rain-fed, and other

sources for water recharge are Hawaliya drain, which is

attached to Hindon River, and Tilapta irrigation canal.

Corr6Spond6nC6. 'dr.ansari.nasim@gmail.com (Corresponding author)

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 61

Ansari

KJ^ochia Khurd V9llgrgre

Blvuda Vilage

Tustyana

Vlllago

Bhdral

PalTQleum

Pohc© Line

GoJony

Dsvia

Village

Yamaha

Motors

Surajpur Village

1 ——

1NI*)Ia ' ..

I ^ '

--iLAE-:?

CTP ■ ■

ri;

1 \.

. ^1

1 ;

4—-li-

ij",-

■ — —r

Habitat

Wetland

Woodland

Grassland

H Marshland

Area (Ha)

152

Percentage (%)

10.39

49.35

31.16

9.09

Fig. 1. Map of the study area showing terrestrial and aquatic habitats.

KEY

Wetland Boundary

Protected Area Boundary

Metal Road

Main Gate

Canal-Perennial

Canal-Seasonal

Nature Trail

Matchan

Watch Tower

Visitor Zone

Range office (Dadri) Forest Nursery

Bridge

The mean monthly maximum and minimum temperature

ranges between 17 °C to 41 °C and 6 °C to 30 °C, re¬

spectively, with the highest temperature observed during

June and the lowest during January. The study area has

been characterized into various major habitats: wetland,

marshland, grassland and woodland. These major habi¬

tats have been further categorized in micro habitats on

the basis of dominant vegetation, water availability, and

soil type (Ansari and Ram 2016).

Data was collected in predominant terrestrial and

aquatic habitats of Surajpur wetland. Ad-libitum records

were maintained (Altmann 1974) on a monthly basis

from March 2010 to February 2013 (total 36 surveys

during 36 months). Extensive active surveys were made

by direct search technique, visual encounter methodol¬

ogy (Campbell and Christman 1982; Heyer et al. 1994;

Sutherland 1996) on all available microhabitats, mainly

in leaf litter, under rocks, fallen and decaying logs, tree

bark, grass clumps, on shrubs, on herbs, in tree holes,

alongside forest nature trails, edges on wetland, marshy

areas, and under water, between 0800-1600 hours. Op¬

portunistic diurnal and nocturnal searches (1800-2000

hours) were also conducted along the nature trails, inside

forest and open areas.

All species encountered were identified up to species

level by consulting standard field guides such as Dan¬

iel (2002) and Datta (1997), and conservation status has

been assigned according to lUCN Red List (lUCN 2016)

and the Indian Wildlife (Protection) Act (1972). Nomen¬

clature and taxonomic arrangement in the text follows

Frost (2009) for amphibians, and Aengals et al. (2011)

for reptiles. The relative abundance categories were as¬

signed as common (>16 times), uncommon (six to 15

times), and rare (one to five times), based on sighting

frequencies (Walmiki et al. 2012). The photographic re¬

cords were maintained by using Panasonic DMC FZ35

digital camera with close-up mode and were deposited to

WWF- India Secretariat.

Results

During the study period, a total of 19 species of herpe-

tofauna belonging to 14 families and three orders were

recorded, of which amphibians represented six species

belonging to five families, and reptiles represented 13

species belonging to nine families (Table 1). The rela¬

tive abundance analysis showed that, among 19 species

of herpetofauna, eight were common, four were uncom¬

mon, and seven species were rarely recorded in the study

area.

Among amphibians, the family Dicroglossidae re¬

corded maximum two species (Indian Bullfrog Hoploba-

trachus tigerinus and Skittering Frog Euphlyctis cyano-

phlyctis), followed by Bufonidae (Asian Common Toad

Duttaphrynus melanostictus), Microhylidae (Ornament¬

ed Pygmy Frog Microhyla ornate), Ranidae (Field Frog

Fejervarya limnocharis), and Rhacophoridae (Common

Tree Frog Polypedates maculatus) with one species each.

Asian Common Toad Duttaphrynus melanostictus was

commonly seen in monsoon in wetland areas in calling

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 61

Enumeration of Herpetofaunal assemblage of Surajpur Wetland

Table 1. List of Herpetofauna recorded in Surajpur wetland.

Abundance

Family Common name Scientific name Habitat status+ Conservation status

lUCN# IW(P)Act*

AMPHIBIANS

Order: Anura

Bufonidae

Asian Common Toad

Duttaphryniis melanostictiis (Schneider

1799)

Marshland

Dicroglossidae

Indian Bullfrog

Hoplobatrachus tigerinus (Daudin 1803)

Marshland

Dicroglossidae

Skittering Frog

Euphlyctis cyanophlyctis (Schneider 1799)

Marshland

Microhylidae

Ornamented Pygmy Frog

Microhyla ornata (Dumeril and Bibron

1841)

Marshland

Not Fisted

Ranidae

Field Frog

Fejervarya limnocharis (Gravenhorst 1829)

Marshland

Rhacophoridae

Common Tree Frog

Polypedates maculatus (Gray 1830)

Woodland

Not Fisted

REPTILES

Order: Testudines

Bataguridae

Indian Roofed Turtle

Pangshura tectum (Gray 1830)

Wetland

Trionychidae

Indian Flapshell Turtle

Lissemys punctata (Bonnaterre 1789)

Wetland

Order: Squamata (Sub-order Sauria)

Agamidae

Indian Garden Fizard

Calotes versicolor (Daudin 1812)

Woodland

Gekkonidae

Yellow Green House

Gecko

Hemidactylus flaviviridis (Rtipell 1835)

Woodland

Not Fisted

Scincidae

Common Keeled Skink

Eutropis carinata (Schneider 1801)

Grassland

Scincidae

Spotted Supple Skink

Lygosoma punctata (Gmelin 1799)

Grassland

Varanidae

Bengal Monitor

Varanus bengalensis (Daudin 1802)

Grassland

Order: Squamata (Sub-order Serpentes)

Boidae

Red Sand Boa

Eryx johnii (Russell 1801)

Woodland

Colubridae

Indian Ratsnake

Ptyas mucosa (Finnaeus 1758)

Woodland,

Grassland

Colubridae

Common Wolf Snake

Lycodon aulicus (Finnaeus 1754)

Woodland,

Grassland

Colubridae

Checkered Keelback

Xenochrophis piscator (Schneider 1799)

Wetland

Elapidae

Common Indian Krait

Bungarus caeruleus (Schneider 1801)

Woodland

Elapidae

Spectacled cobra

Naja naja (Finnaeus 1758)

Woodland

mode, whereas Indian Bullfrog Hoplobatrachus tigeri-

nus was mostly solitary and nocturnal in nature, and in¬

habited holes and bushes near permanent water sources.

Skittering Frog Euphlyctis cyanophlyctis was often seen

at the edge of wetland with their eyes above the water,

and seen commonly round the year. Ornamented Pygmy

Frog Microhyla ornata was observed while calling in

aggregation in monsoon, not very common in the study

area. Frog species were mostly documented in the edges

of wetland, marshland, and occasionally in grassland

habitats.

Among reptiles, family Colubridae recorded maxi¬

mum three species (Indian Ratsnake Ptyas mucosa.

Common Wolf Snake Lycodon aulicus, and Checkered

KQQlhackXenochrophispiscator), followed by Scincidae

(Common Keeled Skink Eutropis carinata and Spotted

Supple Skink Lygosoma punctata) and Elapidae (Com¬

mon Indian Krait Bungarus caeruleus and Spectacled

cobra Naja naja) with two species each. The rest of the

families, Bataguridae (Indian Roofed Turtle Pangshura

tecta), Trionychidae (Indian Flapshell Turtle Lissemys

punctata), Agamidae (Indian Garden Lizard Calotes

versicolor), Gekkonidae (Yellow Green House Gecko

Hemidactylus flaviviridis), Varanidae (Bengal Monitor

Varanus bengalensis), and Boidae (Red Sand Boa Eryx

johnii), recorded one species each. The photographic re¬

cords of the most common species are represented in Fig.

2. Indian Flapshell Turtle Lissemys punctata was seen

commonly in the wetland area whereas Indian Roofed

Turtle Pangshura tectum was seen only twice during the

study period. Indian Garden Lizard Calotes versicolor

was one of the most commonly sighted herpetofauna re¬

corded during the study period in the terrestrial habitats,

whereas Yellow Green House Gecko Hemidactylus fla¬

viviridis was seen commonly in huts of forest watchers.

Common Keeled Skink Eutropis carinata was recorded

only twice during the study period in the wet grassland

area, whereas Spotted Supple Skink Lygosoma punctata

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | e161

Ansari

Fig. 2. (A-O) Photographs of selected Amphibians and Reptiles of Surajpur Lake.

AMPHIBIANS: ORDER ANURA

Fig. 2A. Asian Common Toad Duttaphrynus melanostictus.

Fig. 2C. Skittering Frog Euphlyctis cyanophlyctis.

Fig. 2D. Skittering Frog Euphlyctis cyanophlyctis.

Fig. 2B. Eloplobatrachus tigerinus.

REPTILES: ORDER TESTUDINES

Fig. 2E. Indian Roofed Turtle Pangshura tectum.

Fig. 2F. Indian Flapshell Turtle Lissemys punctata.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 61

Enumeration of Herpetofaunal assemblage of Surajpur Wetland

Fig. 2. (A-O) Photographs of selected Amphibians and Reptiles of Surajpur Lake.

REPTILES: ORDER SQUAMATA

Fig. 2G. Indian Garden Lizard Calotes versicolor.

Fig. 2H. Indian Garden Lizard Calotes versicolor.

Fig. 21. Common Keeled Skink Eutropis carinata.

Fig. 2 J. Spotted Supple Skink Lygosoma punctata.

Fig. 2K. Bengal Monitor Varanus bengalensis.

Fig. 2L. Red Sand Boa Eryx johnii.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | e161

Ansari

Fig. 2. (A-O) Photographs of selected Amphibians and Reptiles of Surajpur Lake.

REPTILES: ORDER SQUAMATA

Fig. 2M. Indian Ratsnake Ptyas mucosa.

Fig. 2N. Checkered Keelback Xenochrophis piscator.

Fig. 20. Checkered Keelback Xenochrophis piscator.

and Bengal Monitor Varanus bengalensis were seen oc¬

casionally in the study area. Among snakes, Checkered

Keelback Xenochrophis piscator was recorded very of¬

ten in the edges of wetland habitat followed by Red Sand

Boa Eryx johnii, which was seen occasionally in wood¬

land habitat, whereas Common Wolf Snake Lycodon

aulicus. Common Indian Krait Bungarus caeruleus, and

Spectacled Cobra Naja naja were recorded seen rarely

during the study period. Among reptiles, turtles were

mostly documented in the edges of wetland, marshland,

and occasionally in grassland habitats, whereas other

reptiles, including lizards and snakes, were recorded in

woodland and grassland habitats in the study area.

According to the lUCN Red List Criteria (lUCN

2016), 10 species were listed as Least Concern (LC) and

nine species as Not Evaluated (NE). According to the In¬

dian Wildlife (Protection) Act (1972), four species have

been listed in Schedule I (Indian Roofed Turtle Pangshu-

ra tectum. Spotted Supple Skink Lygosoma punctata,

Indian Flapshell Turtle Lissemys punctate, and Bengal

Monitor Varanus bengalensis), three species in Sched¬

ule II (Indian Ratsnake Ptyas mucosa. Checkered Keel¬

back Xenochrophis piscator, and Spectacled cobra Naja

naja), three species were not listed (Ornamented Pygmy

Frog Microhyla ornate. Common Tree Frog Polypedates

maculates, and Yellow Green House Gecko Hemidacty-

lus flaviviridis), while the other nine species were listed

in Schedule IV.

Discussion

The present communication highlights results of the first

systematic survey of herpetofauna in Surajpur wetland.

The study provides baseline information on the diversity

of herpetofaunal communities in Surajpur wetland. The

inclusion of smaller vertebrates in management plans for

any particular region is necessary for overall conserva¬

tion of biodiversity at the local as well as the landscape

level (Pawar et al. 2007). The present study observed 19

herpetofaunal species, which is a first significant scien¬

tific contribution in Surajpur wetland. National Capital

Region, India. Some similar studies have been done in

Northern India. Das et al. (2012) reported 53 species

of herpetofauna from Katemiaghat wildlife sanctuary,

spread over an area of 400 km^ in Terai forest landscape.

Singh and Banyal (2013) reported only six species of

herpetofauna from Khajjiar Fake (Himachal Pradesh),

which is spread over an area of 20.69 km^ in Himalayan

Fandscape. Kanaujia and Kumar (2013) listed 24 spe¬

cies of amphibians from Uttar Pradesh. The present study

indicates that the species count at Surajpur wetland will

be likely to increase with additional detailed explorations

and systematic work.

Amphibians and reptiles are good ecological indica¬

tors, and in recent decades there has been a dramatic

decrease in their populations (Singh and Banyal 2013).

Habitat loss and fragmentation are likely the most seri¬

ous threats to herpetofauna, while roads, pesticides, in¬

fectious diseases, and climate change are other threats

(Fesbarreres et al. 2014). Awareness programs are need¬

ed to make people acquainted with herpetofauna and

their importance for a balanced ecosystem. Snake bite

management is another issue which must be taken up

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | e161

Enumeration of Herpetofaunal assemblage of Surajpur Wetland

more seriously among local communities. Illegal hunting

and poaching of turtles in the area by local communities

needs to be taken up seriously by the Forest Department

for the conservation of these highly threatened reptiles

and management of the area. Training is required at vari¬

ous levels for various target groups like school students,

local communities, visitors, and frontline staff Aware¬

ness programs may include identification of common

herpetofauna species, their importance, protection mea¬

sures, and government interventions.

Surajpur wetland area is very important in biodi¬

versity conservation, as it provides an opportunity to

conserve and preserve the native fiora, fauna, and bio¬

diversity amidst a densely populated urban area without

hindering the development of social and economic struc¬

tures (Bura et al. 2013). The urban and industrial devel¬

opment across the Greater Noida city which is resulting

in habitat destruction of herpetofauna is a matter of great

concern. This small piece of marshy land with stagnant

water has a very rich diversity of herpetofauna, creating

a small biodiversity hotspot. This area should therefore

be conserved and kept pollution-free across the city lim¬

its, as it supports a good congregation of aquatic/semi-

aquatic vertebrates. Further investigations are necessary

for utilizing this group of vertebrates as indicator species

for the management of various habitats in the study area.

Acknowledgements. —I would like to thank Uttar

Pradesh Forest Department for granting the permission

to carry out this study. I would like to express our sincere

gratitude to World Wide Fund for Nature-India for sup¬

porting this study, and anonymous referees for reviewing

the manuscript.

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Dr. Nasim Ahmad Ansari works at Wildlife Institute of India (Dehradun) and is associated

with Protected Area Network, Wildlife Management and Conservation Education Department.

He is involved in evaluation of Protected Areas, Biodiversity Finance Assessments, and

Mitigation of Human Wildlife Conflict. Earlier, he had worked for the Surajpur Wetland

Conservation Project of WWF-India from 2009 to 2013. He was awarded his Ph.D. in 2016

in Forestry and Environmental Science from Kumaun University Nainital, Uttarakhand, for

his thesis, ‘A study on bird communities and its relationship with habitat stmcture in Surajpur

Wetland, Uttar Pradesh, India’. He has participated in various National and International

Scientific Conferences and has several publications to his credit.

Amphib. Reptile Conserv.

September 2018 | Volume 12 | Number 2 | el 61

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

12(2) [General Section]: 98-105 (el62).

Range extension and highest eievationai popuiations of

Matrix tessellata in Slovakia

Simona Gezova and ^Daniel Jablonski

^Department of Zoology, Comenius University in Bratislava, Mlynskd dolina, Ilkovicova 6, 842 15, Bratislava, SLOVAKIA

Abstract —The Dice Snake, Matrix tessellata (Laurenti 1768), is one of five snake species living in Slovakia.

Because this species is understudied, there is little known about its distribution in this country. Slovakia

represents the northern limit of its occurrence in Europe. In the context of published and unpublished

distribution records and our personal database, we report the first records of this species from the upper

Vah River in the Liptovska Basin representing the species range extension in Slovakia. The newly discovered

population also represents the highest altitudinal record for N. tessellata in the country. We discuss possible

reasons for their occurrence in this region.

Keywords. Dice Snake, Natricidae, distribution, highest elevation, new records. Central Europe

Citation: Gezova S, Jablonski D. 2018. Range extension and highest eievationai populations of Natrix tessellata in Slovakia. Amphibian & Reptile

Conservation 12(2) [General Section]: 98-105 (el62).

mercialNoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium,

provided the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized

publication credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation-, official journal website

<amphibian-reptile-conservation.org>.

Received: 13 January 2018; Accepted: 21 April 2018; Published: 3 October 2018

Introduction

The Dice Snake, Natrix tessellata (Laurenti 1768), is a

well-known snake species with a wide distribution, rang¬

ing from Central Asia and northeastern Africa to Central

Europe (Gruschwitz et al. 1999; Mebert 2011 and lit¬

erature therein). Throughout this huge area, nine well-

supported and highly divergent lineages were detected

which suggests that this snake has experienced a com¬

plex radiation history (Guicking et al. 2009; Guicking

and Joger 2011). Central Europe is inhabited by a single

“European lineage” which probably originated from gla¬

cial refugial populations in the Balkan Peninsula and his¬

torically expanded north along the Danube river system

during the Holocene (Atlantikum: Guicking et al. 2009;

Guicking and Joger 2011; Vlcek et al. 2011). Today, in

central-eastern Europe, N. tessellata is a rare thermophil¬

ic species with a semiaquatic lifestyle. It prefers suitable

habitats in relatively warmer river valleys, small streams

and water reservoirs that are well exposed to solar radia¬

tion and contain some undamaged natural vegetation. In

particular, sites with rocky slopes, rubble, or dry walls,

some even near roads and railways, are suitable for over¬

wintering, oviposition, daily shelter, and thermoregula¬

tion (Rehak 1992a; Mebert 2011 and literature therein).

This species has a wide altitudinal range in Europe east

of the Caucasus from sea level to >1,000 m asl in mostly

southern regions (Rehak 1992a), with the highest record

at 1,475 m at Livinallongo, Venetia, Italy (Bruno and

Maugeri 1990, cit. in Gruschwitz et al. 1999).

The Dice Snake is a protected species in Slovakia

(Vulnerable, according to Kautman et al. 2001). Its habi¬

tat corresponds to those generally known for this species

in Central Europe north of the Alps (e.g., Gruschwitz et

al. 1999; Mebert 2011) with a maximum elevation up to

400 m asl (Lac 1968; Rehak 1992a). Although Slova¬

kia represents the regional northern border of the Dice

Snake, there is a lack of faunistic research on this species

in this country. The occurrence of N. tessellata here is

probably relatively continuous, but it is more common

to observe this species in southern and central regions,

where it is associated with the main rivers (Danube, low¬

er and middle Vah, Hron, Slana, Hornad, Torysa, and Bo-

drog; Lac and Lechovic 1964; Lac 1968; Rehak 1992a).

Occurrence and dispersion of the Dice Snake to northern

and upper parts of Slovakia along river systems have not

been clearly proven so far. In the river basin of Vah the

historically most northern findings were recorded near

the village of Horne Srnie (Lac and Lechovic 1964) and

in Zilina - Hricov Reservoir (Dobsinsky, pers. comm.).

Regional northern and probably isolated populations

were recently discovered in close proximity to Slovak lo¬

cations in north-eastern parts of the Czech Republic and

southern Poland (Vlcek et al. 2010, 2011). However, the

Corr6Spond6nC6. 'daniel.jablonski@balcanica.cz (Corresponding author)

Amphib. Reptile Conserv.

October 2018 | Volume 12 | Number 2 | el 62

Gezova and Jablonski

o Surveyed localities without presence of Natrix tessellata

Published and unpublished records

Northernmost record of the species from Zilina-Hridov

Questionable record from Poprad city

8 10 km

Vysne Matejkovo

Hungary

is. 1 2

Ruzomberok

Fig. 1. Distribution and range extension of Natrix tessellata

in Slovakia. (A) Locations surveyed in the vicinity of

Ruzomberok and Liptovska Basin. (B) Records of the species

in the country. Black line shows cut out area of new locations

while (A) is the same area but enlarged. 1 - Ruzomberok, 2 -

Liskova, 3 - Ruzomberok - Rybarpole, 4 - Liptovska Tepla, 5 -

Liptovska Mara, 6 - Ruzomberok, 7 - Hrboltova, 8 - Hubova,

9 - Stankovany, 10 - Krafovany, 11 - \^sne Matejkovo.

overall situation in Slovakia is not completely understood

and insufficient information is available about the current

distribution and habitat preference of N. tessellata.

Methods and Materials

In this work, we provide an update on the distribution of

N. tessellata in Slovakia with the first record of this spe¬

cies from upper Vah River (Fig. 1). We conducted seven

field trips (2014-2017) to the region between Krafovany

town and Liptovska Mara Dam to find new records of

the species (see Table 1, Fig. lA). To present the current

distribution of the species we combined the unpublished

authors’ database with published records of N. tessellata

from Slovakia (Fig. IB).

Basic morphometric and meristic data, presented in

Table 2, were taken from a few individuals from two lo¬

cations (see Table 1, loc. 2, 3). A caliper was used for

head measurements - head length (HL), head width

(HW), and mouth length (ML), and a tape band was ap¬

plied to record body measurements - body length (SVL),

tail length (TL), and total length (TotL). Several meris¬

tic characteristics were also taken: number of cephalic

scales - preocular (PREOC), postocular (POSTOC),

supralabial (SUPL), and sublabial (SUBL), also includ¬

ing the number of ventral (VENT) and subcaudal scales

(SUBC), and dorsal scale rows (DORS). Anomalies like

split ventral scales, fused subcaudal scales, inserted ce¬

phalic scales etc., as well as coloration of the ventral side

of the body (white, yellow, or orange) and on the upper

side of the head (spotted or not) were also recorded. All

individuals were photographed and then released in the

same location.

Results

Based on approximately 300 observations from 70 local¬

ities, N. tessellata has a large and continuous distribution

along main river systems across all southern and central

parts of Slovakia (Fig. 1). Herein described populations

in upper Vah River represent a regional northern limit of

the main distribution range (Fig. IB).

The presence of the Dice Snake along the upper

Vah River was first documented by photography near

Ruzomberok town (49.080°N, 19.317°E; 477 m asl; loc.

1) , where a juvenile was observed on 21 May, 2013 at

the river bank of Vah and channel system of the compa¬

ny Mondi Packaging Ruzomberok (Dobrota 2013, pers.

comm.; Fig. 2D). Employees of the company reported

to us the observation of individuals as early as 20 April,

2013. Since then, more juveniles and subadults ofV. tes¬

sellata were observed by the employees in different parts

of the company and the vicinity of the Vah River.

During our field trips we confirmed existence of a re¬

productive population with both adult and juvenile indi¬

viduals. On 19 July, 2017, we recorded 14 individuals

of different ages and sexes in two new locations near

Ruzomberok (Fig. 3). The first location was Eiskova

(49.084°N, 19.344°E; 482 m asl; loc. 2) where eight indi¬

viduals (juveniles, subadults, and adults) were observed

along or in the Vah River. At the second location, Ry¬

barpole (49.087°N, 19.296°E; 473 m asl; loc. 3), only

juveniles and one subadult were observed.

Some individuals from these populations had a low¬

er number of supralabial scales (SUPE, listed in Table

2) when compared with individuals from other parts in

Slovakia and the Czech Republic. Aside from this char¬

acteristic, none of the studied individuals have a sig¬

nificantly low or high number of other morphometric or

meristic characteristics. On the other hand, we noticed

some anomalies such as fused sublabial, inserted ventral,

or fused subcaudal scales. In terms of all morphometric

and meristic characteristics and ventral body coloration

taken during field trips, we did not notice any important

differences.

To obtain a better overview on situation of Dice

Snakes in the upper Vah River, we asked local natural¬

ists and fisherman for information. We obtained photo¬

graphic evidence of an adult female Dice Snake in situ

at Eiptovska Tepla (49.096°N, 19.410°E, 501 m asl; loc.

4; Bircek 2017, pers. comm.) about six km east from

location 2, Eiskova. This author also noticed one live

Amphib. Reptile Conserv.

October 2018 | Volume 12 | Number 2 | el 62

Range extension and highest elevational populations of the Dice Snake

Fig. 2. Locations and habitat of Matrix tessellata near Ruzomberok. (A) Location in Ruzomberok where the first individuals were

observed. (B) (C) (D) View on the habitat near Liskova village.

adult male and one dead adult individual near the dam

Liptovska Mara (the largest dam in Slovakia; 49.093°N,

19.486°E; 554 m asl; loc. 5). We had attempted to find

this species in Liptovska Mara during July 2017 but were

unsuccessful. However, as these locations are close to

each other, we expect the Dice Snake to occur throughout

this part of the river. The locations described herein rep¬

resent the currently highest elevations for N. tessellata

in Slovakia. We also surveyed apparently suitable and

lower locations between Ruzomberok and to the west as

far as Krafovany where any Dice Snakes were recorded.

The newly discovered population of N. tessellata in

Slovakia is located 72 km eastwards from its previously

northernmost known records (Zilina - Hricov Reservoir,

personal observations). Banks on one side of the Hricov

Reservoir are not accessible to people because of the

steep slope, but on the other side of the shore there are

some suitable places even for overwintering. Moreover,

small ponds are in the vicinity of the reservoir, but we

still cannot confirm the presence of a stable population

here.

The newly discovered population inhabits a locally

regulated river flowing from east to west, which is also a

tributary of the Danube River that ultimately drains into

the Black Sea. Around Ruzomberok the river is approxi¬

mately 40-60 m wide, 1-2 m deep with an average flow

rate of about 30 m^s *. The river flow is partially modi¬

fied with preserved natural parameters of riverbed, where

only the most damaged parts of the shore are repaired

with stones or alternatively reinforced. The bottom of the

river has unchanged stony and muddy parameters with

gravel and stony base. Shore vegetation is continuous

and predominantly intact {Alnus sp., Salix sp., Populus

sp.) and with maximum surface shading of about 22%.

To the east, the climate of the upper Vah River as far as

Liptovska Mara Dam is typically moderate with an aver¬

age annual air temperature of 6 °C. During the hottest

month of the year (July) average temperature can vary

between 16-17 °C. On only about 29 days per year does

the temperature reach more than 25 °C. Average annual

precipitation for this location is approximately 711 mm

with the highest precipitation amounts in July (Samaj

and Valovic 1981). In the studied region (Liptovska Ba¬

sin) there are known thermal springs that may affect local

microclimate. The presence of prey is neccessary for N.

tessellata occurrence. Ichthyologically, the region from

Zilina to Liptovska Mara Dam forms a foothill river zone

with the occurrence of the following flsh species: Barbus

barbus (Linnaeus 1758), Hucho hucho (Linnaeus 1758),

Leuciscus cephalus (Linnaeus 1758), L. leuciscus (Lin¬

naeus 1758), Oncorhynchus my kiss (Walbaum 1792),

Perea fluviatilis (Linnaeus 1758), Thymallus thymallus

(Linnaeus 1758), Salmo trutta (Linnaeus 1758), and oth¬

ers (Muzlk 2012). Another snake species N. matrix (Lin¬

naeus 1758) lives here in syntopy with N. tessellata. On

the bank of the river Lacerta agilis (Linnaeus 1758),

Amphib. Reptile Conserv.

100

October 2018 | Volume 12 | Number 2 | el62

Gezova and Jablonski

Fig. 3. Individuals of Matrix tessellata from the location in Liskova. (A) Adult female dorsal view. (B) Same individual in ventral

view. (C) Overall view on juvenile individual. (D) Detail of the head on the same individual.

Coronella austriaca (Laurenti 1768), and Viper a berus

(Linnaeus 1758) were also observed.

Discussion

The Diee Snake is known for its variation in the number

of preoeular, postocular, ventral, and subcaudal scales

(Lanka 1978). There is a tendency to have a lower num¬

ber of cephalic scales in Central Europe than in eastern

regions of N. tessellata (Mebert 2011). Lanka (1978),

Rehak (1989), and Moravec (2015) noticed that the most

common number of preocular scales is two for speci¬

mens from the Czech Republic. In our case eight indi¬

viduals from total 14 studied had two preocular scales

at least on the one side of the head, although one indi¬

vidual from Liskova had four preoculars (Table 2), which

is less common in Slovakia or in the Czech Republic.

Normally, three or four postocular scales are the most

common for Dice Snakes from Central Europe. Rehak

(1989) found the presence of three and four postoculars

in the Czech Republic, but in Eiskova we counted five

postoculars in one individual that can also be presented

(Moravec 2015). In two individuals from Rybarpole we

noticed six supralabials. According to Eanka (1978) it is

not an anomaly to have such a small number of scales.

A lower number of supralabials can be caused by fusing

some scales together (Moravec 2015).

The Dice Snake is usually mentioned in Slovak (and

former Czechoslovak) literature as a common species

around freshwater habitats, but only few locations are

given in detail (e.g., Eac and Eechovic 1964; Eac 1968;

Eabanc 1972; Rehak 1992a; Uhrin et al. 1996, pers.

comm.; Smolinsky 2004; Majsky 2009; Eac et al. 2017).

Overall evaluation of published and unpublished sources

showed that N. tessellata ranges from Zahorska lowland

through southern and central Slovakia to eastern parts of

the country. Data from peripheral eastern and western

parts of the country have not been verified recently.

Our data showed that the species was recorded along

the river tributaries of Morava (near the confiuence

with the Danube), Danube, Eittle Danube, Zitava, Vah,

Nitra, Hron, Ipef, Rimava, Blh, Slana, Muran, Bodva,

Homad, Eaborec, and Bodrog, which corresponds

with records reported in earlier literature (Eac and

Eechovic 1964; Eac 1968; Eac et al. 2017), but we also

extend the range knowledge with the new observations

of this species (Fig. IB). Our new records are not the

northernmost in Slovakia (or in the Carpathians) as there

are observations (Dobsinsky, pers. comm.) from Zilina-

Hricov Reservoir. All northern locations presented in the

paper from Slovakia might be colonized by the species

naturally. Increasing temperatures, appropriate regional

temperature conditions, presence of structural elements

like dry stone walls, deep rock mounds (offer shelter

for digestion, ecdysis etc.; Carlsson et al. 2011), railway

track constructions near water source, and reduced shore

Amphib. Reptile Conserv.

101

October 2018 | Volume 12 | Number 2 | el62

Range extension and highest elevational populations of the Dice Snake

Table 1. An overview of Matrix tessellata records and surveyed locations in the vicinity of Ruzomberok city. The numbers of

locations correspond with Fig. lA.

Locality number

Locality name

Coordinates

Elevation (m)

Observation

Source

Ruzomberok

49.080

19.317

477

juveniles,

subadults

Dobrota 2013,

pers. comm.

Liskova

49.084

19.344

482

juveniles,

subadults,

adults

This study

Ruzomberok -

Rybarpole

49.087

19.296

473

juveniles,

subadults

This study

Liptovska Tepla

49.096

19.410

501

one adult

Bircek 2017,

pers. comm.

Liptovska Mara

49.093

19.486

554

adults

Bircek 2017,

pers. comm.

Ruzomberok

49.087

19.302

471

This study

Hrboltova

49.101

19.243

464

This study

Hubova

49.121

19.188

449

This study

Stankovany

49.144

19.172

437

This study

Krafovany

49.153

19.139

429

This study

Vysne

Matejkovo

48.992

19.283

570

one adult

Hriadel, pers.

comm.

vegetation represent elements that provide suitable

sites for embryogenesis, ovipositing, thermoregulation,

hibernation, and protecting against predators upon spring

emergence (Conelli et al. 2011; Neumann and Mebert

2011; Strugariu et al. 2011). According to presence

of these factors there can be found more suitable sites

along Vah River for N. tessellata in future. We can still

discuss observation of the species from Zilina - Hricov

Reservoir. They probably do not form a reproductive

population because stable presence of these snakes here

is not well confirmed. Therefore, we should pay attention

to these northern observations and take better effort for

field work, especially in springtime, to explain the origin

of the population living on the upper Vah River.

Our new records from the upper Vah River region in¬

crease the altitudinal distribution of N. tessellata in Slo¬

vakia above 500 m asl (Table 1). So far, the upper limit

presented by Rehak (1992a) shows 400 m asl but most of

the findings come from lower elevations. As is suggested

by data from eastern Ukraine, Romania, or Austria, this

species is able to colonize suitable valleys on the hill

sides up to 1,000 m asl (Rehak 1992a). This should be

studied in more detail but it seems that the limiting factor

for distribution of N. tessellata in Slovakia is most likely

a combination of elevation, local climate, and places for

overwintering. In particular the lack of places for over¬

wintering is characteristic for several regions of western

or southwestern Slovakia where suitable river habitats

are presented (wide slopes allowing an easy access to the

water, shallow waters to forage fish, variable character

of banks), however the species has never been recorded

there (Lac 1968; Rehak 1992a; Kautman, pers. comm.).

In Central Europe, N. tessellata prefers shores where sed¬

iments, groups of stones, growing or fallen trees, and dif¬

ferent small dams create many places with shallow water,

open access to water, sunny areas, and shelters under the

ground. These parts of the shore are necessary for per¬

manent occurrence of the species in or near river valleys

(Moravec 2015).

We assume that the population observed along the

upper Vah River near Ruzomberok is autochthonous

and/or is partially formed by individuals migrating

from lower parts of the river. This is consistent with the

inhabited biotope and with the finding of individuals in

different age stages (juveniles and adults). Moreover,

we recorded an interesting museum specimen of N.

tessellata from higher elevation than Ruzomberok,

collected on 30 April, 1938, leg. J. Jakublk, Vysm

Matejkovo, Revuca stream (48.992°N, 19.283°E, 570

m asl; loc. 11). This record is located approximately 11

km south of Ruzomberok (Hriadel, pers. comm.). This

finding was previously stored in the Eiptov Museum in

Ruzomberok (Eac and Eechovic 1964), but the museum

specimen has now been removed from the collection, so

it is not possible to verify the record in detail.

However, why there is an established population is

unclear. Hypothetically, we could suggest that this repro¬

ducing population in the upper Vah River could have a

connection with the presence of geothermal waters and

thermal springs that may affect local microclimatic con¬

ditions. The Dice Snake occupies a wide variety of water

systems (Mebert 2011 and literature therein). As is dis¬

cussed in Mebert and Masroor (2013), the presence of N.

tessellata in high elevations of Pakistan may have a local

Amphib. Reptile Conserv.

102

October 2018 | Volume 12 | Number 2 | el 62

Gezova and Jablonski

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connection with thermal springs (see Wall 1911). Similar

records come from Romania and Hungary where indi¬

viduals frequently inhabit natural thermal springs (Grus-

chwitz et al. 1999; Strugariu et al. 2011). Therefore, there

is a possibility that the presence of thermal springs near

Vah River in the Liptov Basin could provide suitable

eonditions for the speeies oecurrenee and reproduction.

As is presented by Vlcek et al. (2010), the presence of

dark mullock (waste rock acquired in the course of coal

mining) is one of the ecological reasons that allows oe-

eurrenee of isolated populations of this thermophilie spe¬

cies in the northern area of the Czeeh Republic. This rock

absorbs and accumulates heat and creates an optimal mi¬

croclimate.

Due to expansion ability along climatically beneficial

water courses, Diee Snakes may eolonize sites even fur¬

ther north. The territory of Slovakia is characterized by

structured geomorphology with warm river valleys, sep¬

arated by high mountains (e.g., Fatra-Tatra area), where

the hypsometric temperature gradient reaches significant

differences during the day. Open warm valleys probably

play a historical role in the eolonization of northern and

upper Slovak regions for other thermophilous reptiles as

Zamenis longissimus (Laurenti 1768) or Podarcis mura-

lis (Laurenti 1768). Both species were observed in north¬

ern Slovak regions (e.g., Kminiak 1992; Rehak 1992b;

Astalos 2002). Moreover, the range of this speeies was

farther to the north in the Lower Pleistocene (see oc¬

currence of N. cf tessellata from Polish Silesia; Ivanov

1997) and probably also during warm periods after the

Last Glaeial Maximum (Vlcek et al. 2011).

In terms of the finding of N. tessellata in Ruzomberok

there should be no problem for individuals to migrate

along the riverbank up Liptovska Mara Dam. Dice Snakes

ean travel along a stretch parallel to the shoreline of 100-

500 m in a few days, and max. up to ~1,000 m (Neumann

and Mebert 2011; Velensky et al. 2011), and by crawl¬

ing and swimming can travel even 33 km downstream

(Vlcek et al. 2011). In Switzerland Conelli et al. (2011)

and in the Czeeh Republie Velensky et al. (2011) record¬

ed N. tessellata individuals overcame great movements

in the summer, but in spring and autumn they increased

migratory distances to and from their hibernacula. For

example, in Orava (northernmost region in Slovakia),

we recorded anonymous observation of the species near

Oravsky Podzamok. The published record of the dead in¬

dividual of the species found near Poprad eity (elevation

almost 700 m) is probably a case of artifieial introduetion

(Rindos and Jablonski 2014). However, we cannot ex¬

clude a case of natural dispersion into this region because

surroundings of Poprad River meet ecological require¬

ments for oceurrence of Diee Snakes in Central Europe.

Average daily temperature during the hottest month of

the year (July) in the last ten years was 17.4 °C in Poprad

(18.6 °C in Ruzomberok; Slovak Hydrometeorological

Institute 2018). Although elevational difference between

these two cities is approximately 150-200 m, no popula-

Amphib. Reptile Conserv.

103

October 2018 | Volume 12 | Number 2 | el 62

Range extension and highest elevational populations of the Dice Snake

tion of N. tessellata is confirmed in Poprad. On the other

hand, and in view of herein described records from up¬

per Vah River and Liptovska Mara Dam (approximately

70 km from Poprad city), we eannot exclude migration

along the Sub-Tatra Basin. In view of these records, sub¬

sequent mapping of N. tessellata along the Vah River and

other rivers in the country together with genetic research

is therefore needed.

Acknowledgements. —^We thank A. Bircek, M. Do-

brota, P. Dobsinsky, P. Havas, D. Jandzik, J. Kautman,

M. Pivarci, M. Rindos, L. Svecova, M. Uhrin, and P.

Vlcek for their information about field observations of

the Dice Snake in Slovakia, P. Hriadel from Liptov Mu¬

seum in Ruzomberok for his valuable information re¬

garding the museum speeimen of the species, D. Grufa

and M. Meszaros for their help during the fieldwork, and

our reviewers for their suggestions that improved the first

version of the manuscript. We also thank Z. Snopkova

from Slovak Hydrometeorologieal Institute (SHMU) for

providing meteorological data. For English corrections

we would like to thank P. Gillatt and S.R. Goldberg. The

work was supported by the Slovak Research and Devel¬

opment Agency under the eontraet No. APVV-15-0147.

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Vlcek P, Zavadil V, Jablonski D, Mebert K. 2011. Dice

Snake (Matrix tessellata) in the Baltic Sea Drainage

Basin (Karvinsko District in Silesia, Czech Republic).

Mertensiella 18: 177-187.

Wall F. 1911. Reptiles collected in Chitral. The Journal of

the Bombay Natural History Society 21(1): 132-145.

Simona Gezova is a Master’s student in the Department of Zoology at Comenius University

in Bratislava, Slovakia. She has experience mostly with European herpetofauna. Her main

interests are taxonomy, morphology, osteology, and biogeography of the genus Matrix. She

is currently working on the evolutionary history of cryptic lineage of the Matrix tessellata

complex from the Balkan Peninsula. She likes traveling, herping, and photography.

Daniel Jablonski (www.danieljablonski.com) is currently a researcher at the Comenius

University in Bratislava, Slovakia. He has been interested in amphibians and reptiles since

early childhood. His research interests concern evolutionary and historical biogeography,

questions relating to the origin and distribution of genetic diversity and its conservation in

natural populations of amphibians and reptiles. His special focus is placed in the Balkan

Peninsula, and Central and Southeast Asia, some of the most important evolutionary areas in

the world. He loves traveling and photography.

Amphib. Reptile Conserv.

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October 2018 | Volume 12 | Number 2 | el62

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

12(2) [General Section]: 106-111 (e163).

New sites of the endangered Marmaris Salamander,

Lyciasalamandra flavimembris (Mutz and Steinfartz 1995),

(Caudata: Salamandridae) from Mugla, Turkey

^Dilara Arslan, ^Qagda§ Ya§ar, ^Akin izgin, ^Cihan §en, and ^"^Kerim Qigek

'Akdeniz Konima Dernegi, Mediterranean Conservation Society, Izmir, TURKEY ^Orhaniye Inci Narin Yerlici Secondary School, Marmaris, Mugla,

TURKEY ^Section of Zoology, Department of Biology, Faculty of Science, Ege University, TR-35100, Bornova, Izmir, TURKEY

Abstract — Reported are seven new sites of Lyciasalamandra flavimembris found in southeastern Anatolia,

Turkey. These data extend the species’ distribution range by 45 km in the southwest creating a total species’ area

of 115 km^. We compared morphological and color-pattern characteristics from the new sites with previously

published data. The new populations are considered to be L. f. flavimembris.

Keywords. Amphibians, conservation, distribution, Lycian salamander, range extension, Anatolia

Citation: Arslan D, Ya§arQ, izgin A, §en C, Qigek K. 2018. New sites of the endangered Marmaris Salamander, Lyciasalamandra flavimembris (Mutz

and Steinfartz 1995), (Caudata: Salamandridae) from Mugla, Turkey. Amphibian & Reptile Conservation 12(2) [General Section]: 106-111 (e163).

NoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided

the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication

credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation; official journal website <amphibian-

reptile-conservation.org>.

Received: 05 March 2018; Accepted: 17 July 2018; Published: 18 December 2018

Introduction

Lycian salamanders (genus Lyciasalamandra) distribu¬

tion range extends from Greece in the south to the south¬

west of Turkey and covers some islands including Kas-

tellorizon, Meyisti, Kekova, and Carpathos (Ba§oglu et

al. 1994; Veith and Steinfartz 2004; Franzen et al. 2008;

Sparreboom, 2014). There are seven validated species

[Lyciasalamandra luschani, L. atifi, L. antalyana, L. bil-

lae, L. fazilae, L. flavimembris in Mediterranean Turkey

and L. helverseni in Greece] (Sparreboom 2014; Veith et

al. 2016). They inhabit Mediterranean-type shrub vegeta¬

tion and rocky limestone outcrops (Veith and Steinfartz

2004; Sparreboom 2014) and are threatened by habitat

loss and fragmentation. Lycian salamanders are endan¬

gered species due to their patchy distribution covering a

limited surface area (Kaska et al. 2009).

The Marmaris Salamander, Lyciasalamandra flavi¬

membris (Mutz and Steinfartz 1995) is listed as endan¬

gered by the lUCN Red List given that its habitat covers

less than 5,000 km^. It is threatened by habitat loss and

fragmentation caused by forest fires, and over-collection

for scientific purposes (Kaska et al. 2009). A new sub¬

species L. f ilgazi was recently discovered in Kotekli,

province of Mugla based on coloration and pattern char¬

acteristics and morphometric measurements (Uziim et

al. 2015). During our research project on conservation

activities of the Marmaris Salamander (in Marmaris and

Ula provinces of Mugla, southeastern Anatolia, Turkey)

between 2017 and 2018, we detected seven new locali¬

ties of Lyciasalamandra flavimembris.

Methods and Materials

The study site designed 10 km x 10 km Universal Trans¬

verse Mercator (UTM) grids for determining actual and

possible habitats of the species and all grids were visited

three times between February 2017 and March 2018. Vi¬

sual encounter surveys were used to detect potential sites

both during day and night times. During the daytime sur¬

vey, two observers searched by checking under stones,

rocks, outcrops and during the night surveys we observed

in all suitable habitats. Global Positioning System (GPS)

points were recorded for most localities (Garmin GPS-

map 62s). The locations that did not have coordinate data

were obtained by using Google Earth vers.7.1.2 (Google,

Inc.). All records were geo-referenced into the WGS-84

coordinate system and then checked and visualized with

ArcGIS vers. 10.1 (ESRI). The records obtained from

our field studies and the scientific literature (Baran and

Atatiir 1986; Ba§oglu et al. 1994; Mutz and Steinfartz

1995; Uziim et al. 2015; Go^men and Kari§ 2017) were

entered into the UTM grid maps.

Measurements were made in the field and individuals

Corr6Spond6nCG. ‘^kerim.cicek@ege.edu.tr (or) kerim.cicek@hotmail.com

Amphib. Reptile Conserv.

106

December 2018 | Volume 12 | Number 2 | e163

Arslan et al.

Legend

* Nn/ Lgc9Hi4if

^ L. r. n»vim«inh'ia iKjwvrti L«aaliii44|i

I L f iig«< (i^riQ^n LjK4I<«s.]

I HJCN Red List DoInbiilKin

[ I UTHiirKJl

Fig. 1. Distribution of the Marmaris Salamander. Solid red circle denotes known sites of L.f. flavimembris, solid red star shows new

recorded locations, and yellow squares show known sites of L. f ilgazi. Previously recorded localities of L. f flavimembris, and L.

f ilgazi was noted by Baran and Atatur 1986, Ba§oglu et al. 1994, Mutz and Steinfartz 1995, Uzum et al. 2015, and Goqmen and

Kari§ 2017.

were released in capture location. Used were the follow¬

ing morphometric measurements and ratios: total body

length (TBL): tip of snout to tip of tail; rostrum-anus

length (RA): tip of snout to posterior end of the cloaca

opening; length of trunk (LT): length from gular fold

to the anterior edge of cloaca opening; tail length (TL):

length from the posterior end of the cloaca opening to

the tip of tail; head and body length (HBL): length from

snout to the anterior end of the cloaca opening; nostril-

eye distance (NED); distance between nostrils (DBN);

eye diameter (ED); head length (HE): distance from the

snout to the gular fold; head width (HW); parotid length

(PE); parotid width (PW); fore limb length (FEE); hind

limb length (HEE): distance between fore and hind limbs

length (DFHE). Ratios used were: HW/HE, TE/TBE,

PW/PE, and NED/HE (e.g., Uziim et al. 2015; Goqmen

and Kari§2017). Measurements were made using a digi¬

tal caliper (Mitutoyo) with an accuracy of 0.01 mm. Mor¬

phometric measurements were then compared to pub¬

lished data (Mutz and Steinfartz 1995; Uziim et al. 2015;

Gogmen and Kari§ 2017). Mean values were provided

with their standard deviations using the PAST statistical

package (Hammer et al. 2001).

Results and Discussion

During the fieldwork carried out between February 2017

and March 2018 in Marmaris and Ula province of Mugla,

discovered were seven new localities for the species

(Aricilar [1], Turung [2], two km west of Turgut Waterfall

[3], Selimiye [4], Sogiitkoy [5], Ta§lica Village [6,7], Fig.

1) for the Marmaris Salamander [between 14 March 2017

and 17 February 2018] (Table 1).

Individuals were observed under rocky limestone out¬

crops and stones. The newly discovered populations (Fig.

2) extend the species’ distribution range 45 km south,

creating a new total distribution area of 115 km^. The

vegetation of the new sites consists of masques scrub¬

lands in Selimiye [3], Sogiitkoy [4], and Ta§lica Village

and habitats close to pine forests (Pinus brutia) Aricilar

[1], Turung [2], Turgut Waterfall [7] (Fig. 2). There is

no difference between previous elevations and the new

localities.

We observed 43 individuals (seven juveniles, 17

males, and 19 females) and measured 14 individuals

(two juveniles, four males, and eight females). The av¬

erage measurement for total body length was 111.82

Amphib. Reptile Conserv.

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December 2018 | Volume 12 | Number 2 | el 63

New sites of the Marmaris Salamander {Lyciasalamandra flavimembris)

Table 1. Geographic and some climatic information of the newly discovered sites.

Sites

Latitude

Longitude

Elevation

(m)

No. of observed individuals

Temp.

(°C)

Hum.

(%)

[1] Aricilar

37°6’

28°35’

650

6 indiv. (1 juveniles, 3 males,

2 females)

[2] Turun?

36°47’

28°12’

14 indiv. (4 juveniles, 6 males,

4 females)

[3] Turgut Waterfall

36°43’

28°7’

4 indiv. (1 male, 3 females)

[4] Selimiye

36°4rN

28°6’E

10 indiv. (1 juvenile, 5 males,

4 females) 6 indiv. measured

(1 juvenile, 3 males, 2 females)

[5] Sogutkoy

36°40’

28°6’E

150

5 indiv. (1 juvenile, 4 females)

4 species (1 juvenile, 3 females)

[6] Ta§lica

36°38’

28°6’

240

2 indiv. (1 male, 1 female)

[7] Ta§lica

36°37’N

28°6’E

240

2 indiv. (1 male, 1 female)

mm (range=104-125) for males and 114.65 mm (range

85-143) for females (Table 2). The largest specimen was

150 mm long (Franzen et al. 2008). These lengths were

slightly less than the averages found by Mutz and Stein-

fartz (1995) with 125.9 mm for males and 144.8 mm for

females, depending on the location. Uziim et al. (2015)

described L. f. ilgazi and L. f. flavimembris subspecies

and found the mean of TBL of L. f. flavimembris to be

132.49 mm (115.23-147.88) and Gb9men and Kari§

(2017) found the mean of TBL of L. f. flavimembris to

be 121.13 mm (102.00-139.00) for males and 113.21

mm (90.00-134.00) for females. Our results are in ac¬

cordance with the relevant literature.

The color patterns of all new populations showed

similarities with the subspecies L. f. flavimembris. The

dorsum ground color of the individuals is dark purplish-

brown with small irregular scattered silver-white spots.

They have a dark head with yellow eyelids and parotids.

The venter is separated from the dorsum by an unpig-

mented discontinuous line along the flanks. The tail and

the extremities are yellow to light brown-orange with

whitish spots as in mentioned literature (Steinfartz and

Mutz 1999; Sparreboom 2014, Fig. 3).

We continue our fieldwork to monitor population

trends and habitat preferences of the Marmaris Salaman¬

der. This work will be used to promote awareness-raising

activities and conservation of the species. Despite these

efforts, conservation actions are frequently hampered

and delayed by taxonomic instability that makes regional

and international cooperation difficult due to misunder¬

standings concerning the names of priority species (Isaac

et al. 2004). The Lycian salamanders have suffered from

Fig. 2. General view of new site habitats. [A]. Aricilar, [B,C]. Selimiye [D]. Ta§lica.

Amphib. Reptile Conserv.

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December 2018 | Volume 12 | Number 2 | el63

Arslan et al.

Fig. 3. Specimens from new sites. A. the right-side female, the middle male, left side juvenile from Aricilar [1]; B. A male from

Turung [2]; C. A male from Selimiye [4] D. A juvenile from Sogiitkoy [5],

this situation with three new recognized species {L. ir-

fani, L. arikani, and L. yehudahi (Go^men et al. 2011;

Go 9 men and Akman 2012) being changed to subspecies

of L. hillae (Veith et al. 2016) over the last six years.

The species has also suffered due to scientific collection

for taxonomic studies and the pet trade, reducing popu¬

lation sizes (UNEP-WCMC 2008). We hope that future

faunal and taxonomic studies on Lycian salamanders will

be designed to involve low/no specimen collections. All

Lycian salamanders are listed lUCN Red List due to its

extent of occurrence less than 5,000 km^, limited distri¬

bution, and declining habitat loss and quality. The distri¬

bution area of Marmaris salamanders with new localities

are about 100 km^. We also observed tourist activities,

urbanization, pet trade, and environmental pollution

which are further threats to populations. Conservation

and ecological studies are urgently necessary to help sus¬

tain the species.

Table 2. Summary of body measurements and related statistics (mm). \n = the number of specimens, SE = standard error of mean,

Min.-Max. = extreme values, SD = standard deviation, and other character abbreviations are given in the Materials and Methods

section]

Juveniles {n =

Males (n = 4)

Females (n =

Characters

Mean

Min

Max

Mean

Min

Max

Mean

Min

Max

9.5

3.50

6.0

13.0

4.95

13.0

1.47

10.1

17.0

2.95

12.8

1.00

7.7

16.0

2.82

40.4

1.90

38.5

42.3

2.69

61.7

2.00

57.5

66.0

4.00

61.2

3.37

47.0

76.0

9.54

30.5

3.00

27.5

33.5

4.24

49.3

2.16

44.7

54.1

4.33

53.0

3.14

35.6

67.0

8.89

20.6

2.60

18.0

23.2

3.68

35.3

1.84

30.6

39.6

3.68

36.0

2.19

26.6

43.0

6.20

5.5

4.55

0.9

10.0

6.43

13.7

0.51

12.2

14.4

1.01

11.5

0.58

10.1

14.9

1.64

5.0

1.05

3.9

6.0

1.48

8.1

0.46

7.0

9.0

.92

7.0

0.72

3.4

9.2

2.03

4.0

2.00

2.0

6.0

2.83

3.8

0.68

2.5

5.0

1.36

2.8

0.69

0.2

7.0

1.96

NED

3.0

0.00

3.0

0.41

0.82

3.5

0.48

1.37

DBN

2.7

2.31

0.4

5.0

3.27

2.3

0.95

0.6

5.0

1.89

3.4

0.70

0.4

6.0

1.97

4.5

1.50

2.12

3.9

1.02

2.04

3.5

0.27

0.75

FEE

12.5

0.50

0.71

20.8

1.65

3.30

19.4

1.66

4.69

HEE

15.0

1.00

1.41

27.3

0.48

0.96

20.7

1.93

5.47

TBE

69.0

3.00

66.0

72.0

4.24

111.8

4.97

104.0

125.0

9.94

114.7

6.72

85.0

143.0

19.02

Amphib. Reptile Conserv.

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December 2018 | Volume 12 | Number 2 | el63

New sites of the Marmaris Salamander {Lyciasalamandra flavimembris)

Acknowledgements. —This study is part of a project

supported by Rufford Foundation and Akdeniz Koruma

Dernegi (www.akdenizkoruma.org.tr/). We are grateful

to these organizations and the Rufford Foundation (Ruf¬

ford Small Grants) for their generous financial support.

We thank Lisa Emoul (Tour du Valat) for valuable com¬

ments and reviewing the English.

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Arslan et al.

Dhilara Arslan graduated with a biological sciences degree from Ege University (Izmir, Turkey) in

2015 and a Masters (Ege University), specializing in ecology. Dilara’s master thesis was “Population

Status and Conservation of Testudo graeca in Gediz Delta.” Currently, her interests are the ecology

of Turkish amphibians and reptiles, focusing on turtles and tortoises. She works at Akdeniz Koruma

Dernegi and is a project leader of “Conservation Activities of Endangered Marmaris Salamander

{Lyciasalamandra flavimembris) from Mugla, Turkey” which was supported by a Rufford Small

Grant. In addition, she works with Kerim ^i^ek at Ege University and Anthony Olivier of Tour Du

Valat Research Center (France) on the ecology of amphibians and reptiles.

Cagda^ Ya^ar earned a biological sciences degree from Ege University (Izmir, Turkey) in 2012. He

is presently working on a Masters degree in zoology mapping the herpetofauna of Turkey and the

creation of species distribution models using current and future scenarios.

Akin izgin is an undergraduate student from Dokuz Eylill University, Izmir, Turkey.

* Cihan §len gratuated from Dokuz Eyliil University Education Faculty in 2006 and began work as an

Internet Technology teacher that same year. He continues to work at the same school, Orhaniye Inci

Narin Yerlici Secondary School. His interests include agriculture, endangered species, health, and

other nature subjects. He is active in management of the Egitimsen Teacher Union, Marmaris Focal

Seeds Association, and the Marmaris International Short Film Festival.

Kerim is a herpetologist focused on taxonomy, biogeography, ecology, and conservation of

Turkish amphibians and freshwater reptiles. He earned his B.S. in biology from Dumlupinar University

in 2000 and his M.S. in zoology from Ege University (Izmir, Turkey) in 2005. Kerim’s Masters thesis

was the food composition of the Marsh Frog in Central Anatolia. He completed his Ph.D. dissertation at

Ege University in 2009, on the population dynamics of the Uludag Frog. Kerim is currently employed

at Ege University, has authored or co-authored over 90 peer-reviewed scientific publications, and is

co-editor for the journal Biharean Biologist.

Amphib. Reptile Conserv.

111 December 2018 | Volume 12 | Number 2 | e163