Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [Special Section]: 1-4 (e113).

SHORT COMMUNICATION

Confirming the presence of Clelia equatoriana Amaral, 1924

(Squamata: Dipsadidae) in Peru

Muan C. Chavez-Arribasplata, 2 Diego Vasquez, 3 Claudia Torres, 4 Lourdes Y. Echevarria, and

5 Pablo J. Venegas

12A5 Centro de Ornilo/ogiciy Biodiversidad (CORB1D1). Cade Santa Rita 105, Urb. Los Huertos de San Antonio, Surco, Lima 33, PERU 3 Museo de

Historia Natural, UniversidadNacional Mayor de San Marcos (MUSM) Av. Arenales 1256, Lince, Lima 14, PERU

Abstract .—In 2010, Aguilar et al. (2010) reported Clelia equatoriana for northern Peru; however, no

voucher specimens or any data proving the record were mentioned. Here we confirm the presence

of C. equatoriana in Peru based on collected specimens from a recent survey conducted in Piura

Department, Peru, and provide novel data from the examination of museum specimens. Our findings

extend the known distribution of the species ca. 331 km (straight line distance) SE from previous

records in central Ecuador.

Key words. Latitude effect, subcaudals, Tabaconas Namballe, lizard, geographic distribution, range extension

Citation: Chavez-Arribasplata JC, Vasquez D, Torres C, Echevarria LY, Venegas PJ. 2016. Confirming the presence of Clelia equatoriana Amaral,

1924 (Squamata: Dipsadidae) in Peru. Amphibian & Reptile Conservation 10(1) [Special Section]: 1-4 (e113).

CommercialNoDerivatives 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: 06 November 2015; Accepted: 29 December 2015; Published: 16 February 2016

The neotropical dipsadid snake genus Clelia Fitzinger

1826 consists of relatively large snakes (total length >

two m in C. clelia and C. plumbed) that show a striking

ontogenetic color change, from orange or red hatchlings

to dark gray or black adults (Scott et al. 2006). Currently,

the genus contains seven species widely distributed in

Central and South America: C. clelia distributed from

southern Mexico to southwestern Peru; C. equatoriana

distributed from northern Costa Rica through Panama and

Colombia to Amazonian Ecuador; C. errabunda in Saint

Lucia; C. hussami from southern Minas Gerais, Brazil to

Uruguay and central Argentina; C. langeri in Santa Cruz

and Chuquisaca, Bolivia; C. plumbea from south of the

Amazon river in Brazil to Mato Grosso do Sul and Para¬

guay, and the Atlantic rainforest of Brazil; and C. scyta-

lina from Jalisco and Veracruz in Mexico to Panama, and

in South America in Colombia and Ecuador (Zaher 1996;

Pizzatto 2005; Cisneros-Heredia et al. 2007; Uetz 2015;

Reichle and Embert 2005). These snakes are known by

several common names in various countries (e.g., “mus-

surana” in Brazil, “zopilota” in Costa Rica, “chonta” in

Ecuador, “aguajemachaco” and “machacuai” in Peru,

and “cribo” in some Caribbean islands). Representatives

of this genus have the particular habit of preying on other

snakes, a behavior that has been reported several times

before for C. clelia, C. hussami, and C. plumbea (Vitt

and Vangilder 1983; Pinto and Lema 2002), and recently

in C. equatoriana (Rojas-Morales 2012). Consequently,

the genus Clelia plays an important role in regulation of

populations of other snakes, including large venomous

snakes of the Bothrops and Crotalus genera (Campbell

and Lamar 2004).

In Peru there are currently two species of Clelia

formally reported: C. clelia and C. bicolor (Dixon and

Soini 1986; Carrillo and Icochea 1995), but the latter

was re-allocated to the genus Mussurana by Zaher et al.

(2009). More recently, Aguilar et al. (2010) reported C.

equatoriana for Tabaconas Namballe National Sanctuary

(TNNS), a natural protected area located in the north of

Cajamarca department, close to the border between Ec¬

uador and Peru. However, no voucher specimen or any

additional information proving the record of C. equato-

Correspondence. Email: l juancarlos.chav@gmail.com (Corresponding author); 2 amadil41@hotmail.com; 3 amadil41@hotmail.

com; 3claprist@gmail.com; 41oiirdese.20@gmail.com; 5sancarranca@yahoo.es

Amphib. Reptile Conserv.

February 2016 | Volume 10 | Number 1 | e113

Chavez-Arribasplata et al.

85“0'0"W 80“0’0"W 75 o 0'0"W 70°Q'0”W

- 1 - 1 - — I —-- “^1 -

85’Q'0 M W 80°0'0"W 75 o 0 , 0"W 70°Q'a"W

Fig. 1. Map of Isthmian Central America and northwestern

South America showing the locality records of Clelia equato¬

riana (circles). Black circles are records by Zaher (1996), red

circle is Quebrada Molleton and blue circle is El Sauce.

riana in Peru was provided. In fact, this record was in a

small handbook produced by the WWF, which was in¬

tended for public awareness, rather than being a formal

scientific report. We examined several specimens of the

genus Clelia in the Herpetology Collection of Museo de

Historia Natural de la Universidad Nacional Mayor de

San Marcos (MUSM). We found a specimen assigned

to C. equatoriana (MUSM 24981) collected on a survey

made in April 2003. Even though not clearly stated, we

suspect that this was the specimen in which the Aguilar

et al. (2010) record was based. MUSM 24981 is an adult

female from El Sauce Forest (-5.17°S, -79.16°W, 1,500

m), Namballe District, San Ignacio Province, Cajamarca

Department, Peru (Fig. 1). A recent survey conducted

in the montane forests of Piura Department provided

us with two additional specimens, which were depos¬

ited in the herpetological collection of Centro de Omi-

tologia y Biodiversidad (CORBIDI), Lima, Peru (COR-

BIDI 14869 and 14875) (Fig. 2). These specimens were

found in August 2014 at Quebrada Molleton (-4.99°S,

-79.37°W, 2,222 m), Pena Rica village, in Carmen de la

Frontera District, Huancabamba Province, Piura Depart¬

ment, Peru (Fig. 1). Both specimens are juvenile males

that were found hiding under a log on the side of a stream

in a secondary forest.

All examined specimens agree with the description

of C. equatoriana by Zaher (1996) in having 17-17-17

dorsal scale rows, as well as the other characters pre¬

sented in Table 1. However, specimens from Quebrada

Molleton show a lower number of subcaudals (60-69)

than the range described for males of Clelia equatoriana

(75-80 in males) by Zaher (1996). Interestingly, a similar

segmental pattern of variation is found in the subcaudals

for other Dipsadidae species: Atractus carrioni and A.

gigas (Passos et al. 2010, 2013). Both species have their

southernmost records in the same region and similar el¬

evations to the records of C. equatoriana reported herein

(Piura and Cajamarca departments). In the case of both

Atractus species, the authors attribute the observed varia¬

tion to a possible latitude effect in somitogenesis, which

leads to the increase of the number of segmental counts

in hotter and more humid localities towards the equator.

Nevertheless, additional specimens need to be examined

to test whether this latitudinal effect holds across differ¬

ent elevational gradients and Dipsadidae genera.

According to Zaher (1996), the southernmost record

of Clelia equatoriana is in Bucay, Guayas Province, Ec¬

uador. Records from El Sauce Forest and Pena Rica in

TNNS extend the known distributional range of C. equa¬

toriana by ca. 331 km (straight line distance) SE. These

records for Cajamarca and Piura confirm that the distri¬

bution of this species can be more austral than previously

thought and supports the importance of protected areas

such as TNNS in the conservation of this species in Peru.

Acknowledgments. —We thank J. Cordova for allow¬

ing access to the herpetology collection at MUSM. We

also thank K. Siu-Ting for her valuable review and com-

Fig. 2. Individuals of Clelia equatoriana from Quebrada Mol¬

leton, Piura, Peru: CORBIDI 14869 (A) and 14875 (B).

Amphib. Reptile Conserv.

February 2016 | Volume 10 | Number 1 | e113

Confirming the presence of Clelia equatoriana in Peru

ments on a previous version of this manuscript. We are

especially grateful to Nature and Culture International,

World Land Trust, and the Gerencia de Recursos Natura-

les del Gobierno Regional de Piura for funding our held

work.

Literature Cited

Aguilar C, Dobiey M, Venegas P. 2010. Reptiles y anh-

bios del santuario. Pp. 89-96 In: Conociendo el san-

tuario national Tabaconas Namballe. Editors, Mena

JL, Valdivia G. World Wildlife Fund - Ohcina del

Programa Peru, Lima.

Campbell J, Lamar W. 2004. The Venomous Reptiles of

the Western Hemisphere. Two-volume set. Cornell

University Press. Ithaca, New York, USA. 976 p.

Carrillo N, Icochea J. 1995. Lista taxonomica preliminar

de los reptiles vivientes del Peru. Publicaciones del

Mnseo de Historia Natural UNMSM (A) 49: 1-27.

Cisneros-Heredia D, Kuch U, Freire A, Wtister W. 2007.

Reptilia, Squamata, Colubridae, Clelia clelia : Range

extensions and new provincial records from Ecuador.

Check List 3(3): 280-281.

Dixon J, Soini P. 1986. The Reptiles of the Upper Ama¬

zon Basin, Iqnitos Region, Peru. Milwaukee Public

Museum. Milwaukee, Wisconsin, USA. 154 p.

Passos P, Dobiey M, Venegas PJ. 2010. Variation and

natural history notes on giant groundsnake Atractus

gigas (Serpentes: Dipsadidae). South American Jour¬

nal of Herpetology 5(2): 73-82.

Passos P, Echevarria LY, Venegas PJ. 2013. Morphologi¬

cal variation of Atractus carrioni (Serpentes: Dipsadi¬

dae). South American Journal of Herpetology 8(2):

109-120.

Pinto C, Lema T. 2002. Comportamento alimentar e die-

ta de serpentes, generos Boiruna e Clelia (Serpentes,

Colubridae). Iheringia, Serie Zoologia, Porto Alegre

92(2): 9-19.

Pizzatto L, 2005. Body size, reproductive biology and

abundance of the rare Pseudoboini snakes genera

Clelia and Boiruna (Serpentes, Colubridae) in Brazil.

Phyllomedusa 4(2): 111-122.

Rojas-Morales J. 2012. Snakes of an urban-rural land¬

scape in the central Andes of Colombia: Species com¬

position, distribution and natural history. Phlyllomedu-

sa 11:135-154.

Scott N, Giraudo A, Schrocchi G, Aquino A, Cacciali P,

Motte M. 2006. The genera Boiruna and Clelia (Ser¬

pentes: Pseudoboini) in Paraguay and Argentina. Pa-

peis Avulsos de Zoologia 46(9): 77-105.

Uetz P. 2015. The Reptile Database. Available: http://

www.reptile-database.org. [Accessed: 31 July 2015],

Vitt L, Vangilder L. 1983. Ecology of a snake community

in northeastern Brazil. Amphibia-Reptilia 4: 273-296.

Zaher H. 1996. A new genus and species of Pseudoboine

snake, with a revision of the genus Clelia (Serpentes,

Xenodontinae). Bolletino Museo Regionale di Scienze

Naturali 14: 289-337.

Zaher H, Gobbi-Grazziotin F, Cadle JE, Murphy RW, de

Moura-Leite JC ,Bonatto SL. 2009. Molecular phy-

logeny of advanced snakes (Serpentes, Caenophidia)

with an emphasis on South American Xenodontines

a revised classification and descriptions of new taxa.

Pape is Avulsos de Zoologia 49( 11): 115-153.

Table 1. Morphometric characters (in cm) and scale counts of Clelia equatoriana specimens (MUSM 24981, CORBIDI 14869, and

CORB1D1 14875) compared to mean measurements and scale counts for C. equatoriana and C. clelia data from Zaher (1996). (*)

tail incomplete.

MUSM

CORBIDI

Character

24981

14869

14875

Clelia equatoriana

Clelia clelia

(female)

(male)

Total length (cm)

136.5

34.2

157.5 max

225 max

Tail length (cm)

5.7

17.5 max

40 max

Dorsal rows

17-17-17

17-19-17

19-19-17

Ventrals

211

220

204

202-207 (male)

200-217 (female)

201-230 (male)

218-244 (female)

Subcaudals

57*

75-80 (male)

54-64 (female)

81-98 (male)

70-91 (female)

Loreal presence

present

Preoculars

Postoculars

2 + 3

temporals

2 + 2/2 + 3

2 + 3

2 + 2

2 + 3

1+3 rarely

2 + 2 rarely

Supralabials

Infralabials

Amphib. Reptile Conserv.

February 2016 | Volume 10 | Number 1 | e113

Chavez-Arribasplata et al.

Juan C. Chavez-Arribasplata is the manager of the reptile collection of Centro de Ornitologia y Biodiver-

sidad (CORBID1). He graduated as a biologist from the Universidad Nacional de Trujillo in 2012. For his

undergraduate thesis, he studied the ecological characters of lizards in the Manu National Park. Currently his

research interests are the ecology and taxonomy of reptiles in Peru, focusing on snakes. He is working with Dr.

Paola Carrasco of Centro de Zoologia Aplicada, Instituto de Diversidady Ecologia Animal (CONICET-UNC),

Cordova, Argentina on the taxonomy and systematics of the viperidae from Peru.

Diego V. Vasquez graduated from Universidad Nacional de Piura in 2005. He is an Associate Researcher at

Centro de Ornitologia y Biodiversidad (CORBIDI). For his undergraduate thesis Diego worked on the amphib¬

ian fauna of the Cuyas Cloud Forest, Piura, Peru. Diego now works as a field herpetologist for several herpeto-

logical inventories and environmental assessments for CORBIDE

Claudia Torres graduated with a biological sciences degree from Universidad Nacional Mayor de San Marcos

(UNMSM), Lima Peru, in 2002. She is studying for her Masters in Zoology with specialization in systemat¬

ics. Currently, she is an associated member at Department of Herpetology at the Natural History Museum San

Marcos (MUSM) in Lima, which also investigates the diversity of amphibians and reptiles of southern Peru.

Lourdes Y. Echevarria graduated in biological sciences from Universidad Nacional Agraria La Molina, Lima,

Peru, in 2014. As a student, she collaborated constantly in the order and management of the herpetological

collections of Centro de Ornitologia y Biodiversidad, Lima, developing a great interest in reptiles, especially

lizards. For her undergraduate thesis, Lourdes worked on the “Review of the current taxonomic status of Pe¬

tr acola ventriniaculata (Cercosaurini: Gymnophthalmidae) using morphological and ecological evidence.” She

worked as a researcher of the Museo de Zoologia (QCAZ), Pontificia Universidad Catolica del Ecuador in Quito

during 2015. Lourdes is preparing a monograph on the systematics of the Petracola ventriniaculata complex

based on the results of her undergraduate thesis, as well as other papers about taxonomy of lizards and snakes.

Pablo J. Venegas graduated in Veterinary Medicine from Universidad Nacional Pedro Ruiz Gallo, Lambayeque,

Peru, in 2005. He is currently curator of the Herpetological Collection of Centro de Ornitologia y Biodiversidad

(CORBIDI). Pablo worked as a researcher of the Museo de Zoologia QCAZ, Pontificia Universidad Catolica

del Ecuador in Quito during 2015. His current research interest is focused on the diversity and conservation of

the Neotropical herpetofauna with an emphasis on Peru and Ecuador. He has published more than 40 scientific

papers on taxonomy and systematics of Peruvian and Ecuadorian amphibians and reptiles.

Amphib. Reptile Conserv.

February 2016 | Volume 10 | Number 1 | e113

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [Special Section]: 5-12 (e115).

On the distribution and conservation of two “Lost World”

tepui summit endemic frogs, Stefania ginesi Rivero, 1968 and

S. safeties Seharis, Ayarzagiiena, and Gorzula, 1997

1 ’ 3 Philippe J. R. Kok, 14 Valerio G. Russo, ^Sebastian Ratz, and 26 Fabien Aubret

'Amphibian Evolution Lab, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, BELGIUM 2 Station dEcologie Experimentale du CNRS a

Moulis, USR 2936, 09200 Moults, FRANCE

Abstract .—It has been suggested that the inability to migrate in response to climate change is a key

threat to tepui summit biota. Tepui summit organisms might thus seriously be threatened by global

warming, and there is an urgent need to accurately evaluate their taxonomic status and distributions.

We investigated phylogenetic relationships among several populations of Stefania ginesi and

S. satelles, two endemic species reported from some isolated tepui summits, and we examined

their IUCN conservation status. Molecular phylogenetic analysis and preliminary morphological

assessment indicate that both species are actually restricted to single tepui summits and that five

candidate species are involved under these names. We advocate upgrading the conservation status

of S. ginesi from Least Concern to Endangered, and that of S. satelles from Near Threatened to

Endangered.

Key words. Endangered species, Hemiphractidae, IUCN, molecular phylogenetics, molecular taxonomy, Venezuela

Citation: Kok PJR, Russo VG, Ratz S, Aubret R 2016. On the distribution and conservation of two “Lost World” tepui summit endemic frogs, Stefania

ginesi Rivero, 1968 and S. satelles Seharis, Ayarzaguena, and Gorzula, 1997. Amphibian & Reptile Conservation 10(1): 5-12 (ell5).

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: 08 March 2016; Accepted: 29 March 2016; Published: 12 April 2016

Introduction

The frog genus Stefania (Hemiphractidae) is endemic

to an iconic South American biogeographical region

named “Pantepui” (Mayr and Phelps 1967; McDiarmid

and Donnelly 2005) (Fig. 1). Pantepui, often referred to

as the “Lost World” because of Arthur Conan Doyle’s

famous novel (1912), lies in the western Guiana Shield.

The region harbors numerous isolated Precambrian

sandstone tabletop mountains more formally known as

“tepuis” (Fig. 2). Although Pantepui was initially re¬

stricted to tepui slopes and summits above 1,500 m el¬

evation (Mayr and Phelps 1967; Rull and Nogue 2007),

Steyermark (1982), followed by Kok et al. (2012) and

Kok (2013a), expanded the original definition of Pan¬

tepui to include the intervening Pantepui lowlands (200-

400 m asl) and uplands (400-ca. 1,200 m asl) in order

to better reflect the biogeography and biotic interactions

in the area (Kok 2013a). The genus Stefania currently

includes 19 species, 15 of which are restricted to tepui

slopes or summits (Duellman 2015; Frost 2015). Stefa¬

nia species are direct-developers (eggs and juveniles car¬

ried on the back of the mother) and occupy various types

of habitats from lowland rainforest to tepui bogs (Kok

2013a; Schmid et al. 2013; Duellman 2015). The genus

Stefania was erected by Rivero (1968) to accommodate

Cryptobatrachus evansi and a few related new species all

morphologically divergent from other Cryptobatrachus.

Shortly later, Rivero (1970) recognized two species-

groups within Stefania : the evansi group including spe¬

cies having the head longer than broad and found in the

lowlands and uplands of Pantepui, and the goini group

including species having the head broader than long and

found in the highlands of Pantepui. Kok et al. (2012),

followed by Castroviejo et al. (2015), showed that, based

on molecular data, these groups are actually not recip¬

rocally monophyletic. A complete molecular phyloge¬

netic analysis of the genus Stefania is still lacking, and

Correspondence. Email: ^Philippe.Kok@vub.ac.be (Corresponding author); 4 valerio.giovanni.russo@gmail.com ;

5 Sebastian. Ratz@vnb. ac. be; 6 faubret@gmail. com

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e115

Kok et al.

Fig. 1. Left: Map of Pantepui and its location within South America (inset); the thick blue line indicates the Rio Caroni. Right: Map

of the area under study showing localities mentioned in the text (yellow dots represent known localities of occurrence of Stefania

safeties, white dots represent known localities of occurrence of Stefania ginesi). Numbers indicate sampled localities and Roman

numerals indicate unsampled localities, as follows: (1) Aprada-tepui. Venezuela; (2) Murisipan-tepui, Venezuela; (3) Upuigma-

tepui, Venezuela; (4) Angasima-tepui, Venezuela; (5) Abakapa-tepui, Venezuela; (6) Chimanta-tepui, Venezuela; (7) Amuri-tepui,

Venezuela; (i) Kamarkawarai-tepui, Venezuela; (ii) Murei-tepui, Venezuela; (iii) Churi-tepui, Venezuela; (iv) Akopan-tepui, Ven¬

ezuela.

relationships between many species or populations are

unknown. Likewise, the exact distribution of some tepui

summit species is uncertain (e.g., Gorzula and Senaris

1999). Among these, two tepui summit endemic Stefania

species are known from several isolated tepui summits:

Stefania ginesi Rivero, 1968, which is reported from six

tepuis in the Chimanta massif (Chimanta-tepui, Amuri-

tepui, Abakapa-tepui, Churi-tepui, Akopan-tepui, and

Murei-tepui; Senaris et al. 1997; Gorzula and Senaris

1999; Barrio-Amoros and Fuentes 2012; Fig. 1), and Ste¬

fania satelles Senaris, Ayarzagiiena, and Gorzula, 1997,

which has a highly disjunct distribution, being reported

from Aprada-tepui (in the Aprada Massif), Angasima-

tepui, and Upuigma-tepui (two southern outliers of the

Chimanta massif), and from Murisipan-tepui and Ka¬

markawarai-tepui (in the Los Testigos Massif, north of

the Chimanta massif) (Senaris et al. 1997; Gorzula and

Senaris 1999; Fig. 1). Stefania ginesi is listed as Least

Concern (LC) by the International Union for Conserva¬

tion of Nature (IUCN) (Stuart et al. 2008) and S. satelles

is listed as Near Threatened (NT) (Stuart et al. 2008).

However, preliminary data suggest that their respec¬

tive distributions could be more restricted than initially

thought because more than two species could be involved

under these names (the authors, unpublished; see also Se¬

naris et al. 2014 regarding the distribution of S. ginesi).

Herein we used molecular phylogenetics to investigate

the relationships among three populations of S. ginesi and

four populations of S. satelles. We also aim at providing

a more precise distribution of these two taxa in order to

refine their conservation status. Indeed, tepui ecosystems

are reported as particularly sensitive to global warming

(Nogue et al. 2009), and tepui summit organisms might

be seriously threatened by habitat loss due to upward

displacement (Rull and Vegas-Vilarrubia 2006; see also

below). Likewise, climate envelope distribution models

of tepui ecosystems based on future scenarios show that

potential distributions become drastically smaller under

global warming (Rodder et al. 2010). Species restricted

to tepui summits are thus clearly at risk of extinction, and

there is an urgent need to evaluate their exact taxonomic

status and precise distribution.

Materials and Methods

Tissue sampling and molecular data

We combined available GenBank sequences of Stefania

ginesi and S. satelles for fragments of the mitochondrial

16SrRNA gene (16S) and the protein-coding mitochon¬

drial gene NADH hydrogenase subunit 1 (NDl) with 40

novel DNA sequences of Stefania ginesi and S. satelles :

nine of fragments of 16S, five of NDl, 13 of the nuclear

recombination activating gene 1 (RAG1), and 13 of the

nuclear CXC chemokine receptor type 4 gene (CXCR4).

We combined this dataset with DNA sequences of four

additional members of the genus Stefania from out¬

side the studied area (three species from east of the Rio

Caroni: S. scalae, an upland species, S. riveroi and S.

schuberti, two highland species; and one highland spe-

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e115

“Lost World” tepui summit endemic frogs, Stefania ginesi and S. satelles

Fig. 2. Typical Pantepui landscape. Photograph taken on 8 th June 2012 from the summit of Upuigma-tepui, showing Angasima-tepui

on the left and Akopan-tepui and Amuri-tepui on the right. Note stretches of savannah mainly caused by anthropogenic fires. Photo

PJRK.

cies from west of the Rio Caroni: S. riae ; in total 16 novel

sequences), and with Fritziana ohausi , member of the

clade sister to Stefania (Castroviejo et al. 2015), which

was selected as outgroup (see Table 1). Novel sequences

have been catalogued in GenBank under the accession

numbers KU958582-958637.

Total genomic DNA was extracted and purified using

the Qiagen DNeasy® Tissue Kit following manufactur¬

er’s instructions. Fragments of 16S (ca. 550 base pairs

[bp]), of ND1 (ca. 650 bp), and of RAG1 (ca. 550 bp)

and CXCR4 (ca. 625 bp) were amplified and sequenced

using the primers listed in Kok et al. (2012) and Biju and

Bossuyt (2003) under previously described PCR condi¬

tions (Biju and Bossuyt 2003; Roelants et al. 2007; Van

Bocxlaer et al. 2010). PCR products were checked on

a 1% agarose gel and were sent to BaseClear (Leiden,

The Netherlands) for purification and sequencing. Chro¬

matograms were read using CodonCode Aligner 5.0.2

Table 1. List of Stefania taxa and outgroup used in this study, with localities and GenBank accession numbers. Sequences newly

generated are in boldface. IRSNB = Institut Royal des Sciences Naturelles de Belgique, Belgium; MZUSP = Museu de Zoologia,

Universidade de Sao Paulo, Brazil.

Voucher

16S

ND1

RAG1

CXCR4

Genus

Species

Locality

Country

Coordinates

Elevation (m)

IRSNB 16724

JQ742191

JQ742362

KU958600

KU958619

Stefania

scalae

Salto El Danto

Venezuela

N 5°57’52’

’ W 61°23’31”

1208

Uncatalogued

JQ742172

JQ742343

KU958601

KU958620

Stefania

riae

Sarisarinama-tepui

Venezuela

N 4°41’W 64°13’

ca. 1100

IRSNB 15703

JQ742177

JQ742348

KU958602

KU958621

Stefania

riveroi

Yuruanl-tepui

Venezuela

N 5°18’50’

’ W60°51’50”

2303

IRSNB15716

JQ742178

JQ742349

KU958603

KU958622

Stefania

riveroi

Yuruani-tepui

Venezuela

N 5°18’50’

’ W 60°51’50”

2303

IRSNB 16725

JQ742173

JQ742344

KU958604

KU958623

Stefania

“ ginesi ”

Abakapa-tepui

Venezuela

N 5°11’23’

’W 62° 17’52”

2137

IRSNB 16726

JQ742174

JQ742345

KU958605

KU958624

“ginesi ”

Abakapa-tepui

Venezuela

N5 o lr07 ,

’ W 62°17’21”

2209

IRSNB 15839

JQ742175

JQ742346

KU958606

KU958625

Stefania

“satelles ”

Angasima-tepui

Venezuela

N 5°02’36’

’ W 62°04’51”

2122

IRSNB 15844

JQ742176

JQ742347

KU958607

KU958626

Stefania

“satelles ”

Angasima-tepui

Venezuela

N 5°02’36’

’ W 62°04’51”

2122

IRSNB 16727

KU958582

KU958593

KU958608

KU958627

Stefania

“satelles ”

Upuigma-tepui

Venezuela

N 5°05’ 10’

’ W 61°57’32”

2134

IRSNB 16728

KU958583

—

KU958609

KU958628

Stefania

satelles

Aprada-tepui

Venezuela

N 5°24’39’

’ W 62°27’00”

2551

IRSNB 16729

KU958584

—

KU958610

KU958629

Stefania

satelles

Aprada-tepui

Venezuela

N 5°24’43’

’ W 62°27’03”

2576

IRSNB 16730

KU958585

KU958594

KU958611

KU958630

Stefania

“ ginesi ”

Amuri-tepui

Venezuela

N 5°08’34’

’ W 62°07’08”

2215

IRSNB16731

KU958586

KU958595

KU958612

KU958631

Stefania

“ ginesi ”

Amuri-tepui

Venezuela

N 5°08’35’

’ W 62°07’08”

2213

IRSNB 16732

KU958587

KU958596

KU958613

KU958632

Stefania

schuberti

Auyan-tepui

Venezuela

N 5°45’56’

’ W 62°32’25”

2279

IRSNB 16733

KU958588

KU958597

KU958614

KU958633

Stefania

schuberti

Auyan-tepui

Venezuela

N 5°45’56’

’ W 62°32’25”

2279

IRSNB 16734

KU958589

KU958598

KU958615

KU958634

Stefania

“satelles ”

Murisipan-tepui

Venezuela

N 5°52’03’

’ W 62°04’30”

2419

IRSNB 16735

KU958590

KU958599

KU958616

KU958635

Stefania

“satelles ”

Murisipan-tepui

Venezuela

N 5°52’03’

’ W 62°04’30”

2419

IRSNB 16736

KU958591

—

KU958617

KU958636

Stefania

ginesi

Chimanta-tepui

Venezuela

N 5°19’12’

’ W 62°12’07”

2180

IRSNB 16737

KU958592

—

KU958618

KU958637

Stefania

ginesi

Chimanta-tepui

Venezuela

N 5°19’12’

’ W 62°12’07”

2180

MZUSP 139225

JN157635

KC844945

KC844991

—

Fritziana

ohausi

n/a

Brazil

n/a

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e115

Kok et al.

0.93

Stefania ginesi Abakapa

IRSNB16726

IRSNB16736

Stefania ginesi Chimanta

IRSNB16737

IRSNB16728

Stefania satelles Aprada

IRSNB16729

0.57

Stefania satelles Angasima

IRSNB 15844

— Stefania satelles Upuigma

1RSNB16727

Stefania ginesi Amuri

•IRSNB16731

IRSNB16734

Stefania satelles Murisipan

IRSNB16735

Eye color Tepui summit

incataiogued Stefania riae

Stefania schuberti

IRSNB16733

■ irsnbi 6724 Stefania scalae

Stefania riveroi

1 IRSNB15716

•

size

ca. 28 km 2

•

ca. 95 km 2

•

ca. 4.4 km 2

•

ca. 2 km 2

•

ca. 0.7 km 2

•

ca. 37 km 2

•

ca. 0.5 km 2

Fig. 3. Phylogenetic relationships as recovered in the MrBayes analysis (concatenated dataset, 2359 bp), outgroup not shown.

Values at each node represent Bayesian posterior probabilities; asterisks indicate values > 95%. Stefania ginesi sensu stricto, and

S. satelles sensu stricto are highlighted in red. Relation between eye color and tepui summit surface is indicated on the right side of

the figure. Photos PJRK.

(http://www.codoncode.com/index.htm) and a consensus

sequence was assembled from the forward and reverse

primer sequences. MAFFT version 7 (http://maflft.cbrc.

jp/alignment/server/) was used to perform preliminary

alignment using G-INS-i and default parameters. Mi¬

nor alignment corrections were made using MacClade

4.08 (Maddison and Maddison 2005). Protein-coding

sequences were translated into amino-acid sequences to

check for unexpected stop codons. Alignment-ambiguous

regions of 16S were excluded from subsequent analyses.

Molecular phylogenetic analyses

The combined 16S + ND1 + RAG1 + CXCR4 dataset

(totalling 2,359 bp after exclusion) was subjected to phy¬

logenetic inference using Bayesian analyses. Optimal

partitioning schemes were estimated with PartitionFinder

vl. 1.1 (Lanfear et al. 2012) using the “greedy” algorithm,

the “mrbayes” set of models, and the Bayesian Informa¬

tion Criterion (BIC) to compare the fit of different mod¬

els. Bayesian posterior probabilities (PP) were used to

estimate clade credibility in MrBayes 3.2.2 (Ronquist et

al. 2012) on the CIPRES Science Gateway V 3.3 (https://

www.phylo.org/, Miller et al. 2010). The Bayesian analy¬

ses implemented the best substitution models inferred by

PartitionFinder vl. 1.1 partitioned over the different gene

fragments, flat Dirichlet priors for base frequencies and

substitution rate matrices and uniform priors for among-

site rate parameters. Four parallel Markov chain Monte

Carlo (MCMC) runs of four incrementally heated (tem¬

perature parameter = 0.2) chains were performed, with a

length of 20,000,000 generations, a sampling frequency

of 1 per 1,000 generations, and a burn-in correspond¬

ing to the first 1,000,000 generations. Convergence of

the parallel runs was confirmed by split frequency SDs

(<0.01) and potential scale reduction factors (~1.0) for

all model parameters, as reported by MrBayes. All analy¬

ses were checked for convergence by plotting the log-

likelihood values against generation time for each run,

using Tracer 1.5 (Rambaut and Drummond 2009). Effec¬

tive sample sizes (ESS) largely over 200 were obtained

for every parameter. Results were visualized and edited

inFigTree 1.4.1 (Rambaut 2014).

Results

Stefania ginesi and S. satelles as currently recognized

are recovered non-reciprocally monophyletic (Fig. 3).

Our molecular phylogeny also reveals the occurrence of

five candidate species (sensu Padial et al. 2010) that have

been misidentified for more than a decade as S. ginesi

(two candidate species) or S. satelles (three candidate

species) (e.g., Senaris et al. 1997; Gorzula and Senaris

1999). Preliminary morphological analyses (in progress)

indicate a few, sometimes subtle, morphological charac¬

ters allowing discrimination among these candidate spe-

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e115

“Lost World” tepui summit endemic frogs, Stefania ginesi and S. satelles

cies and S. ginesi and S. satelles. Our combined results

indicate that S. ginesi sensu stricto is likely restricted to

its type locality, Chimanta-tepui, as we suspect that pop¬

ulations from other tepuis in the Chimanta Massif that

were not sampled in this study will prove to be distinct as

well. As for Stefania satelles, the species is restricted to

its type locality, Aprada-tepui.

Discussion and conservation recommendations

We assumed that misidentifications were likely due

to a rather conserved external morphology (e.g., head

broader than long, skin strongly granular, absence of

prominent cranial crests) of all tepui summit species pre¬

viously identified as Stefania ginesi or S. satelles. This

conserved morphology appears to be symplesiomorphic,

and probably the result of an allopatric non-adaptive ra¬

diation (lineage diversification with minimal ecological

diversification, see Rundell and Price 2009). It is, how¬

ever, intriguing that two slightly divergent phenotypes (a

“satelles phenotype” with brown eyes and a “ ginesi phe¬

notype” with blue eyes) evolved independently in each

subclade (see Fig. 3). Interestingly, selection towards one

of these two phenotypes seems closely associated with

the size of the summit surface on which the species occur

(see Fig. 3). The “ ginesi phenotype” is found on large

tepui summits (surface > 25 km 2 ) in the central Chimanta

Massif, whereas the “ satelles phenotype” is found on

much smaller tepui summits (surface < 5 km 2 ) in the pe¬

riphery of the core Chimanta Massif. Disentangling this

phenomenon and the nature of the ecological constraints

possibly involved and their influence on phenotypic tra¬

jectories is beyond the scope of this paper and will be

treated in a separate study.

Most importantly, our results have direct implica¬

tions on the conservation status of the populations un¬

der study. A complete taxonomic revision of the genus

is in progress, but meanwhile we wish to emphasize the

restricted distributions of all the populations previously

known as Stefania ginesi or S. satelles. Our results argue

for the upgrading of the conservation status of S. gine¬

si from LC to Endangered (EN), and that of S. satelles

from NT to EN, based on the same argument recently

developed for other species restricted to the summit of

one or two tepuis, e.g., Pristimantis imthurni and P. jam-

escameroni (Kok 2013b), or P. aureoventris (IUCN SSC

Amphibian Specialist Group 2014), thus in accordance

with criteria B1 a-b (iii) and B2 a-b (iii) of the IUCN

Red List of Threatened Species (IUCN 2014). We indeed

argue that (1) extents of occurrence of S. ginesi and S.

satelles are much less than 5,000 km 2 (less than 100 km 2

and five km 2 , respectively); (2) areas of occupancy of S.

ginesi and S. satelles are much less than 500 km 2 (less

than 100 km 2 and five km 2 , respectively); (3) there is an

inferred and projected decline in the quality of habitat

due to the effects of global warming upon tepui ecosys¬

tems, with an expected 2-4 °C increase in temperature

in the region through the next century (IPCC 2007). As

stressed by Nogue et al. (2009) and Rodder et al. (2010),

this rise in temperature will likely cause a decrease in

habitat suitability for tepui biota. In addition, numerous

anthropogenic fires in the region (Means 1995; Rull et al.

2013, 2016), coupled with a global rise of temperature,

may cause an up to 10% decrease in precipitation (IPCC

2007) instigating an increase in fire range and intensity

(Rull et al. 2013, 2016); and (4) the altitudinal range of

Stefania ginesi and S. satelles allows no vertical migra¬

tion in order to avoid these threats. As mentioned by Rull

and Vegas-Vilarrubia (2006), the inability to migrate to

compensate for the climate change is a key threat to tepui

summit biota.

There is an urgent need to gain a greater understand¬

ing of species boundaries and distributions in Pantepui,

especially in Venezuela where the threats are the highest

due to ongoing uncontrolled anthropogenic fires (Rull

et al. 2013, 2016). However, it is assumed that an even

greater threat to Pantepui biota is global climate change.

Local actions (such as stopping fires), even if necessary,

might only have a limited impact on the long-term sur¬

vival of Pantepui organisms. Conservation awareness is

critically important in the area, notably due to the inac¬

cessibility of tepui ecosystems where an out of sight, out

of mind effect may have taken place.

This study adds to the many studies now available

demonstrating that estimates of amphibian diversity

based on morphology alone are often misleading. Molec¬

ular data have indeed been shown to be of great help in

detecting cryptic species (e.g., Hebert et al. 2004; Vences

et al. 2005; Fouquet et al. 2007; Burns et al. 2008; Fou-

quet et al. 2016). Unfortunately, while everyone seems to

agree that gaining a greater understanding of the world

biodiversity is needed in order to prioritize biodiversity

conservation (e.g., Wilson 2016), the task turns more and

more often into a bureaucratic obstacle course, if not an

impossible mission for scientists working with molecular

data.

Acknowledgments. —PJRK’s work is supported by

a postdoctoral fellowship from the Fonds voor Weten-

schappelijk Onderzoek Vlaanderen (FW012A7614N).

Many thanks are due to C.L. Barrio-Amoros (Doc Frog

Expeditions, Costa Rica) and C. Brewer-Carias (Caracas,

Venezuela) for the loan of tissue samples. C. Brewer-

Carias also provided invaluable advice and help with lo¬

gistics in Venezuela.

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Kok et al.

Philippe J.R. Kok is a Belgian evolutionary biologist and herpetologist. He obtained his Ph.D. in biology at the

Leiden University (The Netherlands) in 2013. He is currently postdoctoral researcher at the Vrije Universiteit

Brussel, Belgium, where he teaches Field Herpetology to the second year Master students. His interests are

eclectic, the main ones being the evolution, systematics, taxonomy, biogeography, and conservation of amphib¬

ians and reptiles in the Neotropics, more specifically from the Guiana Shield. His work now primarily focuses

on vertebrate evolution in the Pantepui region.

Valerio G. Russo is an Italian herpetologist and naturalist mainly interested in Neotropical and Mediterranean

biodiversity. He obtained his Master’s degree in biology in 2015 at the Vrije Universiteit Brussel (VUB), Bel¬

gium, with a thesis on the systematics of the frog genus Stefania. He is now collaborating as an independent

researcher with the Biology Department of the VUB.

Sebastian Ratz has a Bachelor’s degree in biology from the University of Tubingen, Germany. He currently

works on his Master thesis (phylogeography of the genus Oreophrynella) at the Vrije Universiteit Brussel, Bel¬

gium. His main interests focus on the diversity and evolution of Neotropical amphibians.

Fabien Aubret is a French evolutionary biologist and herpetologist. He completed his Doctoral and Post¬

doctoral studies between 2001 and 2008 in Australia (University of Western Australia and University of Syd¬

ney). Since 2009, he has been working as a full time researcher for the CNRS (National Centre for Scientific

Research) at the Station of Theoretical and Experimental Ecology (SETE, Moulis, France). Fabien’s research

is mostly empirical, with an experimental backbone, and involves a variety of snake and lizard models. His

research is pluri-disciplinary and involves eco-physiology, phenotypic plasticity, climate change, thermoregula¬

tion, and reproductive biology.

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e115

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [Special Section]: 13-16 (e117).

SHORT COMMUNICATION

New records of the Critically Endangered frog

Pristimantis pardalinus (Craugastoridae) in the eastern

Andean slopes of central Peru

12 Rudolf von May

1 Department of Ecology’ and Evolutionary Biology), University> of Michigan, 2051 Ruthven Museums Building, 1109 Geddes Avenue., Ann Arbor,

Michigan 48109, USA 2 Museum of Vertebrate Zoology>, University> of California, Berkeley, 3101 Valley Life Sciences Bldg., Berkeley, California

94720, USA

Key words. Andes-Amazon, bromeliad, cloud forest, endemic, phytotelmata, Red List

Citation: von May R. 2016. New records of the Critically Endangered frog Pristimantis pardalinus (Craugastoridae) in the eastern Andean slopes of

central Peru. Amphibian & Reptile Conservation 10(1) [Special Section]: 13-16 (el 17).

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: 25 November 2015; Accepted: 12 April 2016; Published: 20 May 2016

The eastern slopes of the Andes exhibit high levels of

amphibian diversity and endemism coupled with diverse

ecosystems and steep elevational gradients (Catenazzi

and von May 2014). In central Peru, high-elevation eco¬

systems such as the Andean grassland, montane scru¬

bland, and the upper cloud forest have experienced high

levels of habitat loss and degradation, potentially affect¬

ing many amphibian species (Lehr and von May 2004;

von May et al. 2008). Conservation of key areas along

these Andean slopes is a priority because the type locali¬

ties of many amphibian species described a long time

ago (e.g., Boulenger 1912), or more recently (e.g., Lehr

et al. 2006), remain unprotected. Equally important is to

resurvey these sites to determine if species that have not

been seen in decades are still there (e.g., Lehr and von

May 2004; Lehr 2007), to assess their current popula¬

tion status and identify threats to their survival. This is

especially critical for endemic and range-restricted spe¬

cies, many of which are vulnerable to local threats such

as habitat loss and disease.

One example of such endemic and range-restricted

species is Pristimantis pardalinus , a terrestrial breeding

frog known from a single locality in central Peru (Lehr

et al. 2006). A recent assessment focusing on the extinc¬

tion risk of 39 potentially threatened amphibian species

in Peru (Jarvis et al. 2015) determined that P. pardali¬

nus , which was previously categorized as Data Deficient

Correspondence. Email: r~vonmay@gmail.com

(DD) according to the International Union for Conserva¬

tion of Nature Red List (IUCN 2012a), should be cat¬

egorized as Critically Endangered (CR). Given that the

species was known from a single locality, had a small

Extent of Occurrence (EOO <100 km 2 ), and faced on¬

going threats (e.g., agricultural expansion, overgrazing,

and human settlement), the status of P. pardalinus was

“up-listed” from DD to CR Blab(iii) (IUCN 2014).

Though the change in the conservation status of this spe¬

cies, which resulted from the application of the IUCN

Red List Categories and Criteria (IUCN 2012b), was an

important step, Jarvis et al. (2015) emphasized that addi¬

tional field assessments are needed in order to understand

the geographic distribution, population size, and threats

affecting this and many other species.

In this report, I provide new distributional data for

P. pardalinus based on field observations and the col¬

lection of voucher specimens. I used the morphologi¬

cal diagnoses provided by Lehr et al. (2006) to identify

specimens and took measurements to the nearest 0.1 mm

with calipers under a stereomicroscope. Specimens were

deposited in the Herpetological Collection of the Museo

de Historia Natural, Universidad Nacional Mayor de San

Marcos, Lima, Peru (MUSM) and in the Herpetological

Collection of the Museum of Vertebrate Zoology, Uni¬

versity of California, Berkeley, California, USA (MVZ).

On 14 March 2014, two field assistants and I surveyed

five sites located 10-15 km E-SE from Huasahuasi, the

Amphib. Reptile Conserv.

May 2016 | Volume 10 | Number 1 | e117

von May

Fig. 1. Map showing the currently known distribution of Pristimantis pardalinus. The yellow triangle indicates the location of the

type locality and the red stars indicate the location of new records reported in this study. The inset shows the location of the study

area in Peru (red box).

type locality of P. pardalinus (Fig. 1). We focused our

search on hillsides located next to the Carretera Central

road, Palca District, Tarma Province (Fig. 2). The hab¬

itat at the selected sites was a mix of scrubland domi¬

nated by terrestrial bromeliads and Peruvian feather

grass; two sites also had small patches of cloud forest

vegetation. Altogether, we inspected approximately 150

terrestrial bromeliads between 9:00 h and 16:00 h, and

found seven individuals of P. pardalinus at two sites. All

individuals were found inside bromeliads of the genus

Tillandsia. These bromeliads are commonly distributed

along various sections of the road connecting Palca and

San Ramon, as well as the road connecting the Carretera

Central and Huasahuasi (Fig. 2). One individual (MUSM

33278) was collected from the first site (11°19’15.78”S,

75 o 33 , 07.81 ,, W) at 2,702 m elevation and six indi¬

viduals (MUSM 33279-33281; MVZ 272273-272275)

were collected from the second site (11°17 , 17.77”S,

75°33’47.77”W) at 2,591 m elevation. Morphometric

data for all specimens are shown in Table 1. We surveyed

three additional sites along the Carretera Central (section

connecting Palca and San Ramon) and one site along the

road connecting the Carretera Central and Huasahuasi

(the type locality), but did not find additional individuals

of P. pardalinus (Fig. 1).

This report represents an extension of >10 km of the

known geographic range of P. pardalinus , based on spec¬

imens collected 10.56 km and 12.96 km, respectively,

from the type locality. Furthermore, I note that the el¬

evation given in the species description, 2,640 m, was in

error. A recent inspection of satellite images provided by

Google Earth and Fallingrain, a Global Gazeteer, indi¬

cate that the holotype and paratopotypes of P. pardalinus

were actually collected at ca. 2,800 m a.s.l. Therefore,

the currently known elevational distribution of P. parda¬

linus ranges from 2,591 to 2,800 m a.s.l. Given that the

three known localities of P. pardalinus are situated out¬

side protected areas, the long-term conservation of this

species will depend on the type of land use at these lo¬

calities. This is especially relevant considering that large

areas of potentially suitable habitat have already been

converted to cultivated land (Huasahuasi is one of the

main potato production centers in Peru). Thus, P. parda¬

linus should be considered a species of special concern

(von May et al. 2008) and the protection of the remaining

habitats in the region should be included in future ini¬

tiatives directed by the Servicio Nacional Forestal y de

Fauna Silvestre (SEFOR), Peru’s Wildlife Service.

Acknowledgments. —I thank Jesus H. Cordova San¬

ta Gadea, Director of the Herpetological Collection of

the Museo de Historia Natural, Universidad Nacional

Mayor de San Marcos, Lima, Peru and Carol Spencer,

Curator of the Herpetological Collection of the Mu-

Amphib. Reptile Conserv.

May 2016 | Volume 10 | Number 1 | e117

New records of the frog Pristimantis pardalinus

Fig. 2. Local collaborator Elmer Mapelli surveying bromeliads on rocky outcrop along the Carretera Central, Palca District, Tarma

Province, 2,591 m elevation (A). Six individuals of P. pardalinus , including MVZ 272273 (B), were found at this site.

seum of Vertebrate Zoology, University of California,

Berkeley, California, USA, for providing access to the

herpetological collections at each institution. I thank El¬

mer Mapelli and Patricio Valverde for assistance in the

field. Research and collecting permits were issued by the

Ministry of Agriculture in Peru (Resolucion Directoral

N° 120-2012-AG-DGFFS-DGEFFS y Resolucion Direc¬

toral N° 064-2013-AG-DGFFS-DGEFFS). I thank Jes¬

sica Deichmann and an anonymous reviewer for provid¬

ing helpful comments on the manuscript. Fieldwork in

central Peru was supported by grants from the National

Science Foundation (Postdoctoral Research Fellowship

in Biology, DBI-1103087) and the National Geographic

Society Committee for Research and Exploration (Grant

#9191-12).

Table 1. Measurements (in mm) of individuals of Pristimantis pardalinus found in this study. Individual collection number and sex

noted for each individual. SVL = snout-vent length, TL = tibia length, FL = foot length, HL = head length, HW = head width, ED

= eye diameter, TY = tympanum diameter, IOD = interorbital distance, EW = upper eyelid width, IND = mternarial distance, E-N

= eye-nostril distance.

Character

MUSM

33278

male

MUSM

33279

male

MUSM

33280

juvenile

MUSM

33281

male

MVZ

272273

female

MVZ

272274

male

MVZ

272275

juvenile

SVL

25.00

24.60

20.82

24.84

30.03

25.28

20.69

12.61

12.49

10.21

11.96

16.99

12.17

10.29

10.53

10.88

8.68

10.79

14.97

10.67

8.13

9.28

8.33

7.02

8.58

10.80

8.53

6.84

9.15

9.30

7.44

9.29

11.49

8.89

7.45

3.13

3.20

2.24

3.45

3.50

3.63

2.43

1.27

1.24

0.97

1.45

1.61

1.71

0.97

IOD

3.36

2.92

2.62

3.34

4.36

3.26

2.66

2.05

2.22

1.94

2.20

2.50

2.31

1.93

IND

2.04

2.05

1.56

2.04

2.41

2.11

1.36

E-N

2.82

2.64

2.09

2.80

3.64

2.70

2.16

Amphib. Reptile Conserv.

May 2016 | Volume 10 | Number 1 | e117

von May

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AmphibiaWeb. 2015. AmphibiaWeb: Information on

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British Museum. Annals Magazine Natural History

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T136132A43291649.en [Accessed: 25 November

2015],

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ters 1874 (Amphibia, Anura, Leptodactyidae). Zoo-

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dae) from the eastern Andes of central Peru with com¬

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tological Monographs 20 : 105-128.

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pleura Boulenger, 1912 (Amphibia: Anura: Hylidae).

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von May R, Catenazzi A, Angulo A, Brown JL, Carrillo

J, Chavez G, Cordova JH, Curo A, Delgado A, En-

ciso MA, Gutierrez R, Lehr E, Martinez JL, Medina-

Muller M, Miranda A, Neira DR, Ochoa JA, Quiroz

AJ, Rodriguez DA, Rodriguez LO, Salas AW, Seimon

T, Seimon A, Siu-Ting K, Suarez J, Torres C, Twomey

E. 2008. Current state of conservation knowledge on

threatened amphibian species in Pern. Tropical Con¬

servation Science 1: 376-396.

Rudolf von May is a postdoctoral research fellow at the Department of Ecology and Evolutionary Biology at

the University of Michigan. His current research seeks to understand how amphibian and reptile communities

are stmctured across habitats and elevations, taking into account the phylogenetic relatedness among species

present in those communities.

Amphib. Reptile Conserv.

May 2016 | Volume 10 | Number 1 | e117

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [Special Section]: 17-20 (e118).

SHORT COMMUNICATION

New distributional records of the Amazon River Frog

Lithobates palmipes (Spix, 1824) in Peru

^oy Santa-Cruz, 2 J. Amanda Delgado C., 3 Cinthya Y. Salas, and 4 Rudolf von May

U3 Museo de Historia Natural, Universidad Nacional de San Agustin de Arequipa, PERU 2 Museo de Historia Natural, Universidad Nacional de

San Antonio Abad del Cusco, Cusco, PERU 4 Department of Ecology> and Evolutionary Biology, University of Michigan, 2051 Ruthven Museums

Building, 1109 Geddes Avenue., Ann Arbor, Michigan 48109, USA

Key words. Amazonia, lowland rainforest, Rana, Ranidae, true frogs

Citation: Santa-Cruz R, Delgado C JA, Salas CY, von May R. 2016. New distributional records of the Amazon River Frog Lithobates palmipes (Spix,

1824) in Peru. Amphibian & Reptile Conservation 10(1) [Special Section]: 17-20 (el 18).

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: 25 November 2015; Accepted: 12 April 2016; Published: 30 May 2016

The Amazon River Frog Lithobates palmipes (Spix,

1824) is an aquatic-breeding species that inhabits various

types of rainforest throughout the lowlands of northern

South America, including both the Amazon and Orinoco

basins, part of the Guyana Shield, the Atlantic Forest, the

Cerrado and neighboring areas in Brazil (Hillis and De

Sa 1988; Acosta-Galvis 1999; Canedo and Bilate 2005;

Oliveira et al. 2010; Ferreira and Faria 2011; Ramalho

et al. 2011; Santos and Vaz-Silva 2012; Rodrigues et al.

2013; Frost 2015). According to Hillis and De Sa (1988),

this species belongs to the complex Rana palm ipes. Frost

et al. (2006) placed this and other closely related species

in the genus Lithobates , a name originally proposed

by Fitzinger in 1843. Differences in the recommended

species name vary according to different classification

criteria (e.g., Rana palmipes vs. Lithobates palmipes ),

and were thoroughly discussed by Hillis (2007).

However, this species may contain cryptic species (Hillis

and Wilcox 2005). In this report, we use the binomen

Lithobates palmipes because it is still widely accepted,

though we recognize that an equally valid alternative

would be to treat Lithobates as a subgenus of Rana in

order to preserve a long-standing taxonomy (Hillis and

Wilcox 2005; AmphibiaWeb 2015).

Previous studies documenting the distribution of

L. palmipes in South America (e.g., Hillis and De Sa

1988; Canedo and Bilate 2005; Rodrigues et al. 2013)

provided records from Loreto Region, northern Peru (a

Region in Peru is equivalent to a federal state; it was

formerly known as Departamento), but its distribution

along the Peruvian Amazon remains poorly known. It is

notable that L. palmipes had not been detected in other

well-studied lowland sites in Peru, such as Panguana

Biological Station (Schltiter et al. 2004), Cuzco

Amazonico (Duellman 2005), Los Amigos Biological

Station (von May et al. 2009, 2010), and Cocha Cashu

Biological Station in Manu National Park (Catenazzi et

al. 2013), despite intensive surveys conducted at those

sites. In this report, we provide new distributional data

for L. palmipes in Peru and update the map of its known

distribution in South America. We used the morphological

diagnoses provided by Hillis and De Sa (1988) to identify

specimens and took measurements to the nearest 0.1 mm

with calipers under a stereomicroscope.

Our report is based on field observations and the

collection of voucher specimens from two localities in

southern Peru, and an additional observation (with a

photographic voucher) from northern Peru (Fig. 1). On

09 April 2009, a juvenile individual of L. palmipes was

collected at Lechemayo, Carabaya Province, Puno Region

(13°15’7.39”S, 70°20 , 18.44”0, 390 m elevation). This

specimen was deposited in the Herpetological Collection

oftheMuseo de Historia Natural, Universidad Nacional de

San Antonio Abad del Cusco, Peru, with voucher number

Correspondence. Email: ' chara53@hotmail.com ; 2 amanditadc@gmail.com\ 3 cinthyasalas84@hotmail.com;

A rv onmay@umich.edu (corresponding author).

Amphib. Reptile Conserv.

May 2016 | Volume 10 | Number 1 | e118

Santa-Cruz et al.

Fig. 1. Known distribution of Lithobates palmipes in South America and location of new records in Peru. White circles represent

literature data and red circles indicate the new records in San Martin (http://www.inaturalist.org/observations/2384262), Madre de

Dios (MUSA-3722, MUSA-3723) and Puno (MHNC-7864) regions.

MHNC-7864 (snout-vent length 57.07 mm). On 28 May

2011, two individuals of L. palmipes were collected at the

Reserva Comunal Amarakaeri, Manu Province, Madre

de Dios Region (12°46’20.26”S, 70°56 , 44.56”O, 367

m elevation). Both specimens were found on the ground

at a slow-moving stream dissecting a middle floodplain

forest scattered with bamboo. These specimens were

deposited in the Herpetological Collection of the Museo

de Historia Natural (MUSA), Universidad Nacional de

San Agustln de Arequipa, Peru, with voucher numbers

MUSA-3722 and MUSA-3723 (snout-vent length

119.30 mm and 118.10 mm, respectively; see Table 1 for

additional morphometric data). The third locality record

is supported by a field observation made by Alessandro

Catenazzi on 15 July 2002 at Callanayacu, at the border

of the Cordillera Azul National Park, San Martin Region,

320 m (photographic voucher: http://www.inaturalist.org/

observations/2384262). In addition to the new records

reported here, we updated the known distribution of L.

palmipes in Bolivia using georeferenced data published

by Reichle (2007).

This report represents an extension of >175 km of the

known geographic range L. palmipes in southwestern

Amazonia. Furthermore, it is worth noting that two

other species of Lithobates have been recorded in Peru:

L. bwana and L. catesbeianus (Catenazzi and von

May 2014). One of these, the American Bullfrog, L.

catesbeianus , is an exotic species that has invaded various

South American ecosystems and its presence in northern

Peru was confirmed recently (Cossios 2010). As such, this

exotic species could pose a threat to many native aquatic-

breeding frogs including L. palmipes. Given that both L.

palmipes and L. catesbeianus may inhabit similar types

of water bodies such as slow-moving streams, seasonal

ponds, swamps, and flooded forests (Duellman 1978;

La Marca et al. 2010), continuous field assessments in

areas where these species have been sighted is a priority

(Catenazzi and von May 2014).

Acknowledgments. —We thank Evaristo Lopez Teje¬

da, Director of the Museo de Historia Natural, Universi¬

dad Nacional de San Agustln de Arequipa (MUSA), and

Juan Carlos Chaparro Auza, Curator of Herpetology at

the Museo de Historia Natural, Universidad Nacional

de San Antonio Abad del Cusco (MHNC), for provid¬

ing access to the herpetological collections. Research

and collecting permits were issued by the Ministry of

Agriculture (Resolucion Directoral N° 0398-2010-AG-

DGFFS-DGEFFS) and the Ministry of the Environment

(Resolucion Jefatural de la Reserva Comunal Amara¬

kaeri N° 001 -2010-SERNANP-RCA). We thank Ales¬

sandro Catenazzi for sharing one locality record reported

in this paper. We thank Jessica Deichmann and an anony¬

mous reviewer for providing helpful comments on our

manuscript. RvM thanks the National Science Founda¬

tion for a Postdoctoral Research Fellowship in Biology

(DBI-1103087).

Amphib. Reptile Conserv.

May 2016 | Volume 10 | Number 1 | e118

New distributional records of Lithobates palmipes in Peru

Fig. 2. Individuals of Lithobates palmipes recorded in this study. (A) Adult, female (MUSA-3722) from Reserva Comunal Amara-

kaeri, Manu Province, Madre de Dios Region, Peru. (B) Juvenile MHNC-7864 from Lechemayo, Carabaya Province, Puno Region,

Peru. Photographs by Roy Santa Cruz (A) and Amanda Delgado (B).

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SVL

114.2

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E-N

11.41

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structure as a function of forest type in Amazonian

Peru. Journal of Tropical Ecology 26(5): 509-519.

Roy Santa Cruz is an amphibian and reptile researcher at the Herpetological Collection of the Museum of

Natural History, Universidad Nacional de San Agustin de Arequipa, Peru (MUSA). His current research inter¬

ests include taxonomy and ecology of amphibian and reptiles in Peru.

J. Amanda Delgado C. is a Peruvian Biologist associated with the Natural History Museum of Universidad

Nacional San Antonio Abad del Cusco (MHNC). Her research interests include taxonomy and diversity of

amphibians and reptiles. She is also involved in the creation and management of new Regional Conservation

Areas in southeastern Peru.

Cinthya Y. Salas is a Peruvian biologist and a researcher at the Herpetological Collection of the Museum of

Natural History of the Universidad Nacional de San Agustin de Arequipa, Peru (MUSA). Her research interests

include ecology and conservation of amphibians and reptiles.

Rudolf von May is a postdoctoral research fellow at the Department of Ecology and Evolutionary Biology at

the University of Michigan. His current research seeks to understand how amphibian and reptile communities

are stmctured across habitats and elevations, taking into account the phylogenetic relatedness among species

present in those communities.

Amphib. Reptile Conserv.

May 2016 | Volume 10 | Number 1 | e118

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [Special Section]: 21-33 (e121).

A new species of Andean microteiid lizard

(Gymnophthalmidae: Cercosaurinae: Pholidobolus)

from Peru, with comments on P. vertebralis

^ablo J. Venegas, 12 Lourdes Y. Echevarria, 3 Simon E. Lobos, 4 Pedro M. Sales Nunes,

and 5 0mar Torres-Carvajal

1 Division de Herpetologia-Centro de Ornitologia y Biodiversidad (CORBIDI), Santa Rita N°105 36 Of. 202, Urb. Huertos de San Antonio, Surco,

Lima, PERU 2 Laboratorio de Sistemcitica de Vertebrados, Pontificia Universidade Catolica do Rio Grande do Sul, Porto Alegre, RS, BRAZIL

35 Museo de Zoologia, Escuela de Biologia, Pontificia Universidad Catolica del Ecuador, Avenida 12 de Octubre 1076 y Roca, Apartado 17-01-

2184, Quito, ECUADOR 4 Universidade Federal de Pernambuco, Centro de Biociencias, Departamento de Zoologia, Av. Professor Moraes Rego,

s/n. Cidade Universitaria CEP 50670-901, Recife, PE, BRAZIL

Abstract. —Based on morphological and molecular evidence, herein is reported the discovery of

a new species of Pholidobolus from the Andes of northwestern Peru. The new species is known

from the montane forests of Cajamarca and Lambayeque departments, at elevations of 1,800-

2,300 m. It differs from other species of Pholidobolus in lacking prefrontal scales and having both

strongly keeled dorsal scales and a diagonal white bar in the rictal region. Additionally, it is shown

that records of P. vertebralis from Peru are based on misidentified specimens. The southernmost

distribution records of P. vertebralis are from northwestern Ecuador. Also, an updated identification

key for species of Pholidobolus is provided.

Key words. Andes, hemipenial morphology, lizards, Pholidobolus vertebralis , systematics

Citation: Venegas PJ, Echevarria LY, Lobos SE, Nunes PMS, and Torres-Carvajal O. 2016. A new species of Andean microteiid lizard (Gymnoph¬

thalmidae: Cercosaurinae: Pholidobolus) from Peru, with comments on R vertebralis. Amphibian & Reptile Conservation 10(1) [Special Section]:

21-33 (e121).

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: 06 November 2015; Accepted: 04 May 2016; Published: 30 July 2016

Introduction

Lizards in the New World family Gymnophthalmidae

Merrem 1820 are small, with elongate bodies and

relatively short limbs, which are reduced in various

degrees in some species and nearly absent in others

(Pianka and Vitt 2003). Gymnophthalmidae comprises

47 taxa traditionally ranked as genera with 253 species

(Uetz and Hosek 2016). The diversity of gymnophthalmid

lizards is high in both the Amazonian rainforests and the

Andes (Presch 1980). Some genera like Euspondylus,

Gelanesaurus , Macropholidiis , Pholidobolus , Petracola ,

Proctoporus , and Riama are restricted to the Andes

and reach high elevations. For example, Proctoporus

bolivianus can be found at 4,080 m in Peru (Duellman

1979).

Species of Pholidobolus occur between 1,800 and

4,000 m along the northern Andes from northern Peru

in the Huancabamba Depression to extreme southern

Colombia (Torres-Carvajal and Mafla-Endara 2013).

Montanucci (1973) defined Pholidobolus using

morphological characters and recognized five species:

P. affinis (Peters 1863), P. annectens (Parker 1930), P.

macbrydei Montanucci 1973, P. montium (Peters 1863),

and P. prefrontalis Montanucci 1973. Twenty-three

years later Reeder (1996) described P. huancabambae.

However, recent taxonomic changes have been proposed

based on molecular phylogenetic evidence. Two species

of Pholidobolus , P. annectens , and P. huancabambae ,

were allocated in its sister clade, Macropholidus (Torres-

Carvajal and Mafla-Endara 2013). More recently,

“ Cercosaura ” dicra (Uzzell, 1973) and “C.” vertebralis

Correspondence. Emails: x sancarranca@yahoo.es (Corresponding author); Hourdese.20@gmail.com', Hobossimon@gmail.com ;

4 pedro. mmes@gmail. com ; 5 omartorcar@gmail. com

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e121

Venegas et al.

O’Shaughnessy 1879 were found to be members of

Pholidobolus (Torres-Carvajal et al. 2015), increasing

the number of species in this genus to seven, including

the recently described P. hillisi (Torres-Carvajal et al.

2014).

Morphologically, members of Macropholidus and

Pholidobolus can be distinguished from each other

by the presence of a single palpebral disk in the lower

eyelid in Macropholidus (divided in Pholidobolus ), and

the lack of a lateral fold in Macropholidus (present in

Pholidobolus). Nonetheless, the phylogenetic position

of P. anomalus Muller 1923, a geographically disjunct

species from southern Peru, is still uncertain (Montanucci

1973; Reeder 1996; Torres-Carvajal and Mafla-Endara

2013).

Herein, based on morphological and previously

published molecular evidence (Torres-Carvajal et al. 2015

and 2016), we report the discovery of a new species of

Pholidobolus collected in different field trips to montane

forests in the Andes of northwestern Peru. This discovery

increases the number of species of Pholidobolus to eight.

Materials and Methods

All type specimens of the new species described in this

paper were deposited in the herpetological collection

of Centro de Omitologia y Biodiversidad (CORBIDI),

Lima, Peru. Specimens used for comparisons are housed

at Museo de Zoologia, Pontificia Universidad Catolica

de Ecuador, Quito (QCAZ) (Appendix I). The following

measurements were taken with digital calipers and

recorded to the nearest 0.1 mm, except for tail length

(TL), which was taken with a ruler and recorded to the

nearest millimeter: head length (HL), head width (HW),

shank length (ShL), axilla-groin distance (AGD), and

snout-vent length (SVL). Sex was determined either

by dissection or by noting the presence of everted

hemipenes. We follow the terminology of Reeder (1996)

for the description of the holotype and scale counts, and

Montanucci (1973) for the diagnosis. Morphological

data from other species of Pholidobolus were taken from

the literature (Montanucci 1973; Reeder 1996; Torres-

Carvajal et al. 2014).

The left hemipenis of the holotype (CORBIDI 12734)

was prepared following the procedures described by

Manzani and Abe (1988), modified by Pesantes (1994)

and Zaher (1999). The retractor muscle was manually

separated and the everted organ filled with stained

petroleum jelly. The organs were immersed in an alcoholic

solution of Alizarin Red for 24 hours in order to stain

eventual calcified structures (e.g., spines or spicules),

in an adaptation proposed by Nunes et al. (2012) on the

procedures described by Uzzell (1973) and Harvey and

Embert (2008). The terminology of hemipenial structures

follows previous literature (Dowling and Savage 1960;

Savage 1997; Myers and Donnelly 2001, 2008; Nunes

etal. 2012).

Results

Systematics: The taxonomic conclusions of this study

are based on the observation of morphological features

and color pattern, as well as on previously inferred phy¬

logenetic relationships based on molecular data (Torres-

Carvajal et al. 2015). We consider this information as

species delimitation criteria following a general lineage

or unified species concept (de Queiroz 1998, 2007).

Pholidobolus ulisesi sp. nov.

urn:lsid:zoobank.org:act:283DAECE-3FD5-496D-963B-A4E8E4DC8CA7

Figs. 1-3.

Cercosaura vertebralis —Doan and Cusi 2014 (part):

1,195-1,200.

Pholidobolus sp.—Torres-Carvajal et al. 2015: 286.

Pholidobolus sp.—Torres-Carvajal et al. 2016: 70 (Fig.

2 ).

Holotype: CORBIDI 12734, an adult male from

Bosque de Huamantanga (5°39 , 48.09” S, 78°56 , 35.8”

W), at 2,211 m elevation, Huabal district, Jaen province,

Cajamarca department, Peru, collected on 7 March 2013

by PJ. Venegas.

Paratypes (17): CORBIDI 12740-46 juveniles, COR¬

BIDI 12735-36,12739 adult males, CORBIDI 12737-38

adult females, all collected with the holotype; CORBIDI

00871-73, an adult female, an adult male and a juvenile,

respectively, from El Chaupe (5°14'8.16” S, 79°5'56.58”

W), at 2,016 m elevation, Namballe district, San Ignacio

province, Cajamarca department, Peru, collected by M.

Dobiey on 24 August 2008; CORBIDI 14889, an adult

female, and CORBIDI 14896, a juvenile, from San Feli¬

pe de Jaen (5°45’ 10.854” S, 79°14’ 19.881” W), at 2,641

m elevation, Jaen province, Cajamarca department, Peru

collected by K. Garcia on 26 September 2014.

Photo voucher specimen: Canaris (6°03 26.18 S,

79° 16 00.35 "W), at 2,318 m elevation, Ferrenafe prov¬

ince, Lambayeque department, Peru, captured and re¬

leased by PJ. Venegas on 25 May 2007 (Fig. 3D).

Diagnosis: Pholidobolus affinis , P. dicrus (Fig. 4A), P.

hillisi (Fig. 4B), P. prefrontalis , and P. vertebralis (Fig.

4C) differ from the new species in having prefrontal

scales. Pholidobolus montium and P. macbrydei have stri¬

ated and quadrangular dorsal scales (strongly keeled and

hexagonal in P. ulisesi), and lack the conspicuous narrow,

pale brown, vertebral stripe present in P. ulisesi. In addi¬

tion, the new species has fewer dorsal scales (28-31, x

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July 2016 | Volume 10 | Number 1 | e121

A new species of Andean microteiid lizard

1213 A

CORBIDI

Fig. 1. Holotype of Pholidobolus ulisesi sp. nov. (CORBIDI 12734; male, SVL = 45.5 mm) in dorsal (top) and ventral (bottom)

views. Photographs by OTC.

= 29.75) than P. affinis (45-55), P. montium (35-50), P.

prefrontalis (37-46), and P. macbrydei (31-43).

Characterization: (1) Two or three supraoculars, an-

teriormost larger than others; (2) prefrontals absent; (3)

femoral pores absent in both sexes; (4) two to six opaque

lower eyelid scales; (5) scales on dorsal surface of neck

striated, becoming strongly keeled between forelimbs

and tail; (6) two or three rows of lateral granules at mid¬

body; (7) lateral body fold present; (8) usually two rows

of keeled ventrolateral scales on each side; (9) dorsum

dark brown with a distinct pale brown middorsal stripe,

slender at midbody, becoming grayish brown towards

the tail; (10) labial stripe white becoming cream or pale

brown along ventrolateral region; (11) sides of body dark

brown; (12) cream stripe along forearm; (13) a distinct

diagonal white bar with dark brown edges on each side of

the mandible, extending from sixth infralabial to proxi¬

mal pregular; (14) orange spots on sides of body, usually

above forelimb and the base of tail in adult males.

Description of holotype: Adult male (CORBIDI

12734; Fig. 1-3A); SVL 45.5 mm; TL 104 mm; dorsal

and lateral head scales juxtaposed, finely wrinkled; ros¬

tral hexagonal, 2.03 times as wide as high; frontonasal

quadrangular, wider than long, longer than frontal, later¬

ally in contact with nasal, loreal, and first superciliary;

prefrontals absent; frontal pentagonal, longer than wide,

slightly wider anteriorly, in contact with frontonasal and

supraocular I on each side; frontoparietals hexagonal,

longer than wide, with medial suture, each in contact

laterally with supraoculars I and II; interparietal roughly

heptagonal, its lateral borders parallel to each other; pa-

rietals slightly smaller than interparietal, pentagonal and

positioned anterolaterally to interparietal, each in contact

anteriorly with supraocular II and dorsalmost postocu¬

lar; postparietals three, medial scale smaller than laterals;

supralabials seven, fourth longest and below the center

of eye; infralabials five, fourth below the center of eye;

temporals enlarged, irregularly pentagonal or hexagonal,

juxtaposed, finely wrinkled; two finely wrinkled supra-

temporals, dorsal conspicuously larger than ventral one;

nasal divided, irregularly tetragonal, longer than wide, in

contact with rostral anteriorly, first and second supralabi¬

als ventrally, frontonasal dorsally, loreal posterodorsally

and frenocular posteroventrally; nostril on ventral aspect

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e121

Venegas et al.

Fig. 2. Head of the holotype of Pholidobolus ulisesi sp. nov.

(CORB1DI 12734) in dorsal (A), ventral (B), and lateral (C)

views. Photographs by OTC.

of nasal, directed lateroposteriorly, piercing nasal suture;

loreal rectangular; frenocular enlarged, in contact with

nasal, separating loreal from supralabials; supraoculars

two, with the first being the largest; four elongate super-

ciliaries, first one enlarged, in contact with loreal; palpe¬

bral disk divided into two pigmented scales; suboculars

three, elongated and similar in size; three postoculars,

ventral one smaller than the others; ear opening verti¬

cally oval, without denticulate margins; tympanum re¬

cessed into a shallow auditory meatus; mental semicir¬

cular, wider than long; postmental pentagonal, slightly

wider than long, followed posteriorly by three pairs of

genials, the anterior two in contact medially and the pos¬

terior one separated by postgenials; all genials in contact

with infralabials; gulars imbricate, smooth, widened in

two longitudinal rows; gular fold incomplete; posterior

row of gulars (collar) with two enlarged scales medially,

larger than the anterior gulars.

Scales on nape similar in size to dorsals, except for

the anteriormost that are widened; scales on sides of neck

small and granular; dorsal scales elongated, imbricate,

arranged in transverse rows; dorsal scales on nape stri¬

ated, becoming progressively keeled from forelimbs to

tail; number of dorsal scales between occipital and poste¬

rior margin of hind limbs 30; dorsal scale rows in a trans¬

verse line at midbody 19; dorsals separated from ven-

trals by two longitudinal rows of large keeled scales on

each side; longitudinal fold between fore and hind limbs

present; ventrals smooth, wider than long, arranged in

21 transverse rows between collar fold and preanals; six

ventral scales in a transverse row at midbody; subcaudals

smooth; limbs overlap when adpressed against body;

axillary region composed of granular scales; scales on

dorsal surface of forelimb striated, imbricate; scales on

ventral surface of forearm small and imbricate, those on

ventral surface of arm granular; two thick, smooth thenar

scales; supradigitals (left/right) 3/3 on finger I, 6/6 on II,

8/8 on III, 9/9 on IV, 6/6 on V; supradigitals 3/3 on toe

I, 6/6 on II, 10/9 on III, 12/11 on IV, 8/8 on V; subdigital

lamellae of forelimb single, 6/6 on finger I, 11/12 on II,

15/16 on III, 16/16 on IV, 9/8 on V; subdigital lamellae

on toes I and II single, on toe III paired on the middle,

on toe IV paired except for a few ones, on toe V paired

at the base; number of subdigital lamellae (pairs when

applicable) 6/6 on toe I, 10/11 on II, 16/17 on III, 21/21

on IV, 12/12 on V; groin region with small keeled, imbri¬

cate scales; scales on dorsal surface of hind limbs keeled

and imbricate; scales on ventral surface of hind limbs

smooth; scales on posterior surface of thighs granular

and on shanks striated and imbricate; femoral pores ab¬

sent; preanal pores absent; cloacal plate paired, bordered

by two scales anteriorly, smaller than cloacal scales.

Additional measurements (mm) and proportions of

the holotype: HL 9.91; HW 6.95; ShL 3.9; AGD 25.6;

TL/SVL 2.05; HL/SVL 0.21; HW/SVL 0.15; ShL/SVL

0.08; AGD/SVL 0.56.

Coloration in preservative (Figs. 1 and 2): Dor¬

sum dark brown with a grayish brown vertebral stripe

that is four scales broad at midbody, and extends from

occiput onto tail; vertebral stripe wide anteriorly becom¬

ing slightly slender at midbody; dorsal surface of head

brown, sides of head and body dark brown; two bright

cream spots on each side above insertion of forelimbs;

light stripe extending ventrolaterally from lips to inser¬

tion of hind limbs, white on lips and grayish brown along

the body; a distinct diagonal white bar with dark edges

on each side of the mandible, extending from the sixth

infralabial onto the proximal pregular; dorsal surface of

limbs dark brown with a cream stripe along the arms;

gular region pale gray, chest and venter dark gray; ventral

surface of tail dark gray.

Coloration of holotype in life (Fig. 3A): Similar to

that in preservative, but the bright cream spots on each

side above forelimbs are replaced by two black ocelli

with red centers, and the sides of the base of the tail have

scattered red flecks. The iris is light brown.

July 2016 | Volume 10 | Number 1 | e121

Amphib. Reptile Conserv.

A new species of Andean microteiid lizard

Table 1. Squamation characters of Pholidobolus ulisesi. Range, followed by mean ± standard deviation, is given for quantitative

characters (if applicable). ^Includes adults of both sexes and 10 juvenile specimens of undetermined sex.

Pholidobolus ulisesi

Characters

Males

n = 5

Females

n = 4

All specimens*

n = 19

Dorsal scales between occipital and posterior margin of

hind limb

28-31

29.6 ± 1.14

29-32

30.6 ± 1.14

28-32

30.05 ± 1.13

Dorsal scale rows in a transverse line at midbody

19-22

20.4 ± 1.14

18-21

19.6 ± 1.34

17-22

20.05 ± 1.43

Ventral scales between collar fold and preanals

20-21

20.75 ±0.5

20-23

20.8 ± 1.3

20-23

21.06 ±0.87

Ventral scale rows in a transverse line at midbody

6-8

6.8 ± 1.1

6-8

6.56 ±0.92

Subdigital lamellae on Finger IV

15-16

15.6 ±0.55

15-17

15.8 ±0.84

11-18

15.05 ± 1.65

Subdigital lamellae on Toe IV

20-21

20.4 ±0.55

18-22

20.6 ± 1.52

15-22

19.32 ± 1.95

Maximum SVL

45.52

57.46

TL/SVL

1.92-2.28

2.12 ± 0.18

(n = 3)

1.83-2.17

2.05 ±0.19

(» = 3)

1.83-2.28

2.05 ±0.18

(n = 7)

Fig. 3. Four individuals of Pholidobolus ulisesi sp. nov. in life. (A) holotype (CORBIDI 12734); (B) adult female (CORBIDI

12737); (C) juvenile (CORBIDI 12744); (D) adult male from Canaris (photo voucher). Photographs by PJV.

Variation: Variation in measurements and scutellation

of Pholidobolus ulisesi is presented in Table 1. Usually

two supraoculars, 2/3 (left/right) in specimen CORBI¬

DI 12742; superciliaries usually four, 3/4 in CORBIDI

12749, 6/5 in CORBIDI 00873, and 5/5 in CORBIDI

00872; little intrusive scales present on each side, in the

posterior angle of frontonasal in three specimens (COR¬

BIDI 12735, 12741, 12744); usually seven supralabials,

7/6 in CORBIDI 00871, 12738 and 6/6 in CORBIDI

12742—43; infralabials usually six, 5/5 in CORBIDI

12738, 12740, 12742, 6/5 in CORBIDI 00873, 12744

and 5/6 in CORBIDI 12735, 12743. Rows of ventrolat¬

eral keeled scales vary from two rows in nine specimens

(56% of the type series), one row on each side in three

specimens (CORBIDI 00872, 12741, and 12745), three

rows on each side in one specimen (CORBIDI 12739),

and absent in two adult specimens (CORBIDI 00871

and CORBIDI 00873). Usually two scales on posterior

cloacal plate, only two specimens (CORBIDI 12737-38)

have three scales, and two other specimens (CORBIDI

00871 and 00873) have four scales.

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e121

Venegas et al.

Fig. 4. Four species of Pho/idobolus. (A) adult female of P.

diems (QCAZ 5304); (B) adult male of P. hillisi (QCAZ 4999);

of P. sp. from La Granja (CORBIDI 1678). Photographs by:

(A) and (B) Santiago R. Ron, (C) OTC, and (D) PJV

Males can be distinguished from females by having

the contacted margins of rostral and mental distinctly

dark brown or black (indistinct or not contrasting in fe¬

males), and by the presence of red or orange spots above

the insertion of forelimbs and on the sides of the base

of tail (absent in females; Fig. 3B). Females are longer

(maximum SVL 57.4 mm, n = 4) than males (maximum

SVL 45.5 mm, n = 5). Juvenile CORBIDI 12743 (Fig.

3C) differs from adults in having a fragmented dirty

cream stripe along the flanks above the ventrolateral

stripe.

Hemipenial morphology: The left hemipenis of the

holotype of Pholidobolus ulisesi (Fig. 5) was everted

during preservation and prepared posteriorly. The organ

extends along approximately eight millimeters in length.

The lobes of the organ are partially everted and the hemi¬

penis is fully expanded. The hemipenial body is roughly

conical in shape, with the basis distinctly thinner than the

rest of the organ, and bears two small lobes with apical

folds in the apex. The sulcus spermaticus is central in

position, originating at the base of the organ, and pro¬

ceeding in a straight line towards the lobes. The sulcus

is broader in the region of the lobular crotch, where it

is divided by a small fleshy fold; its branches lie on the

medial region of the lobes, and end in their tips among

folds. The sulcate face of the hemipenial body presents

two nude areas parallel to the sulcus spermaticus that run

along the entire hemipenial body.

The lateral and asulcate faces of the hemipenis are or¬

namented with a series of roughly equidistant flounces

with calcareous spinules. Twenty-three rows of flounc¬

es extend along the body of the organ. There are four

proximal rows restricted to a central position on the basal

asulcate face of the hemipenis, all of them are roughly

chevron-shaped. The four proximal flounces on the sides

are diagonally positioned; the third to fifth flounces are

separated from a complementary flounce positioned on

the asulcate face and oriented in an inverse diagonal. The

subsequent flounces towards the lobes cross the sides of

the organ from the sulcate to the asulcate face, forming

chevrons with vertices in the central region of each side

pointing towards the basis of the organ. These chevron¬

shaped rows become reduced in size progressively to¬

wards the hemipenial apex. Similar to the description

of the hemipenis of Cercosanra vertebralis by Uzzell

(1973), the five distalmost lateral flounces of the hemipe¬

nis have an enlarged tooth in the vertex of the chevrons.

The lateral flounces are separated in two groups by

a nude area in the central asulcate face that increases in

size in the apical region, becoming Y-shaped. The region

between the asulcate and lateral sides are marked by a

conspicuous unevenness forming a distinctive bulge,

which is also present in other species of the Macropholi-

dus + Pholidobolus clade {Macropholidus annectens,

M. huancabambae, M. ruthveni, Pholidobolus affinis , P.

hillisi , P. maebrydei, P. montium , P. prefrontal is, P. verte¬

bralis r; Nunes, 2011; Torres-Carvajal et al. 2014).

The hemipenis of the holotype of P. ulisesi described

herein (Fig. 5) is broadly congruent with the illustrated

by Doan and Cusi (2014) for a specimen of P. ulisesi ,

considered by them as P. vertebralis (see “Discussion”

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e121

A new species of Andean microteiid lizard

hereafter). Although Doan and Cusi (2014) reported a

reduced count of flounces ornamenting the organ (14

versus 23 in the holotype of P. ulisesi ), their Fig. 5B

clearly shows at least 18 visible flounces ornamenting

the hemipenis sides, plus other flounces not countable

due the positioning of the organ and the lack of focus

in some areas of the hemipenis photograph. Similar to

the hemipenis of P. ulisesi described by Doan and Cusi

(2014), but contrasting with the hemipenis of P. verte-

bralis illustrated by Hemandez-Ruz and Bernal-Gonza-

lez (2011) for a specimen from Narino, Colombia, the

hemipenis of the holotype of P. ulisesi presents the four

flounces in basal position at the asulcate face separated

from the other flounces ornamenting the hemipenis later¬

ally. In the drawing presented by Hernandez-Ruz (2005)

for Cercosaura ampuedai (synonym of P vertebralis ac¬

cording to Doan and Cusi [2014]) such flounces are not

visible, probably due the distally misplaced tie made dur¬

ing hemipenial preparation.

Distribution and natural history observations:

Pholidobolus ulisesi is known from five localities at ele¬

vations of 1,900-2,300 m in Cajamarca and Lambayeque

departments, northern Peru (Fig. 6). All recorded locali¬

ties lie within the Huancabamba depression, a region

where the relatively low altitude of the Andean moun¬

tains causes fragmentation of montane habitats, and the

northern extreme of the Central Andes at Cordillera Oc¬

cidental in northern Peru. According to the terrestrial

ecorregions of the world by Olson et al. (2001), P. ulisesi

occurs within Eastern Cordillera real montane forest and

Maranon dry forest.

Pholidobolus ulisesi was found during the day in sun¬

ny and cloudy conditions in secondary montane forest, in

the edges of primary montane forest and recently opened

areas for cattle ranching, as well as in small plantations

of bean and coffee. In the open cattle-ranching areas, P.

ulisesi was found moving on fallen trees or hiding un¬

der trunks; in secondary montane forest, the lizards were

found foraging within herbaceous vegetation and run¬

ning through the patches of grass. They were especially

abundant in coffee and bean plantations, where they were

observed running through the herbaceous vegetation and

hiding in leaf litter. Sympatric squamate reptiles collect¬

ed with P. ulisesi were Chironius monticola and Dipsas

peruana at El Chaupe and Huamantanga, and Chironius

monticola , Epictia teaguei , Erythrolamprus taeniurus ,

Micrurus peruvianus, Stenocercus arndti , S. huanca-

bambae , and S. stigmosus at Quebrada La Iraca.

Etymology: The specific epithet “ ulisesi ” is a noun in

the genitive case and a patronym for Ulises Gamonal

Guevara, for his significant contribution to the archaeol¬

ogy of Cajamarca in northwestern Peru. One of his major

contributions is the discovery of the >6,000-year-old Fa-

ical cave paintings in San Ignacio, declared as Cultural

Patrimony of the Nation.

Remarks: In a molecular phylogeny of Cercosaura and

related taxa, Torres-Carvajal et al. (2015) showed, with

high support, that Pholidobolus ulisesi {Pholidobolus

sp. in their paper) and P. hillisi are sister species. To¬

gether they form a clade sister to all other species of

Pholidobolus. In addition, these authors found that both

“ Cercosaura ” vertebralis and “ Cercosaura ” dicra were

nested within Pholidobolus , and were therefore referred

to this genus (Torres-Carvajal et al. 2015). An identical

topology can be observed in a recent molecular phylog¬

eny of the clade Cercosaurinae by Torres-Carvajal et al.

(2016). Therefore, we adopt this taxonomic change in the

discussion below.

Fig. 5. Left hemipenis of Pholidobolus ulisesi sp. nov. (CORBIDI 12734 - holotype) in sulcate (left), lateral (middle), and asulcate

(right) views. Photographs by PMSN.

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Venegas et al.

ECUADOR

Guayaquil

PERU

80°0'0"W

5°0'0"S'

0W-

Altitude

0-250

250 - 500

500- 1000

1000 - 1500

1500 - 2000

2000 - 2500

2500 - 3000

3000 - 3500

Chachapoyas

□ 3500 - 4000

_1 4000 - 4500

1 4500+

100 150 200

■ —i— Kilometers

- 0 ° 0 ' 0 "

-5°0'0"S

80°0'0"W

Fig. 6. Distribution of Pholidobolus in Ecuador and Peru (circles): P affinis (green); P diems (black); P. hillisi (purple); P. maebry-

dei (blue); P. montium (gray); P. prefrontalis (brown); P. ulisesi sp. nov. (red); P. vertebralis (orange); and P. sp. (pink). Localities

for P ulisesi are: (1) Bosque de Huamantanga (type locality); (2) El Chaupe; (3) Estacion Biologica Chichilapa in the Santuario

Nacional Tabaconas Namballe, taken from Doan and Cusi (2014); (4) San Felipe de Jaen; (5) Canaris; (6) Quebrada La Iraca (near

La Granja village); and (7) Quebrada Checos (near La Granja village) taken from Doan and Cusi (2014).

Discussion

Pholidobolus vertebralis has been repeatedly reported

for Peru based on misidentified specimens. Uzzell (1973)

reported one specimen (LACM 58811) of this species (as

Prionodactylus vertebralis ) from Piura, 11 miles E of

Canchaque, on the western slope of the Huancabamba

Mountains. He noted, however, that this specimen was

different morphologically from other specimens of P.

vertebralis. Doan and Cusi (2014) confirmed this speci¬

men as P. vertebralis even though they also noted im¬

portant morphological differences with other specimens,

such as the absence of prefrontal scales, an undivided

palpebral disk, and the absence of a light vertebral stripe.

After reviewing several specimens of C. vertebralis from

Ecuador (n = 22; see Appendix 1), we found that all have

prefrontal scales, a divided palpebral disk, and a light

vertebral stripe (“ vertebralis ” refers to that stripe). Based

on photographs of specimen LACM 58811, as well as its

examination by staff of the herpetological collection at

the Natural History Museum of Los Angeles County, we

were able to identify it as Macropholidus huancabam-

bae Reeder 1996. Besides the differences between this

specimen and other specimens of P. vertebralis noted by

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July 2016 | Volume 10 | Number 1 | e121

A new species of Andean microteiid lizard

Key to species of Pholidobolus

la. Loreal scale usually present and frequently in contact with supralabials; dorsals striated; conspicuous light verte¬

bral stripe absent.2

1 b. Loreal scale present, not in contact with supralabials; dorsals keeled; conspicuous light vertebral stripe present. 5

2a. Prefrontal scales present.3

2b. Prefrontal scales absent. 4

3a. Ocelli on flanks present, supraoculars three. P. affinis

3b. Ocelli on flanks absent, supraoculars two. P. prefrontalis

4a. Sexual dimorphism strong, with males having distinctly broader heads and colorful flanks (red stripes and white

flecks). P. macbrydei*

4b. Sexual dimorphism not very marked, with males having slightly broader heads and inconspicuously colored flanks

(different tones of brown stripes). P. montium

5a. Prefrontal scales absent. 6

5b. Prefrontal scales present.7

6a. Diagonal white bar along rictal region, extending from the posteriormost infralabial to the proximal pregular...

. P. ulisesi

6b. Diagonal white bar in the rictal region absent. P. sp.

7a. Vertebral stripe bifurcates anteriorly at midbody. P. diems

7b. Vertebral stripe straight, not bifurcated.8

8a. Diagonal white bar in the rictal region, extending from the proximal pregular to the forelimb. P. hillisi

8b. Diagonal white bar in the rictal region absent. P. vertebralis

* We observed some specimens of Pholidobolus macbrydei with small loreal scales, not contacting supralabials, as well as specimens

lacking a loreal scale.

Uzzell (1973) and Doan and Cusi (2014), the dorsal and

flank scales are similar in size, whereas in P. vertebralis

flank scales are noticeably smaller than dorsals.

Doan and Cusi (2014) also reported two new localities

for Pholidobolus vertebralis in Peru based on mis identi¬

fied specimens of P. ulisesi and an undescribed species

of Pholidobolus. These localities lie in the Cajamarca

department, one in the Tabaconas Namballe Natural

Sanctuary (P. ulisesi ) and the other in Quebrada Che-

cos, approximately one km away from La Granja vil¬

lage (P. sp.) (see Fig. 6). Although P. ulisesi is similar to

P. vertebralis (Fig. 4C) in having a dark brown dorsum

with a conspicuous narrow middorsal pale stripe, and a

white labial stripe that extends posteriorly as a cream or

pale brown stripe along the ventrolateral region, it dif¬

fers from P. vertebralis (character states in parenthesis)

in lacking prefrontal scales (prefrontals present), and in

having a diagonal white bar in the rictal region (rictal bar

absent); ocelli above forelimbs and along the sides of the

base of tail (ocelli also present along the flanks); a cream

stripe along the forearm (stripe absent, one or two ocelli

along the foreami); a gray venter in adults of both sexes

in preservative (creamy gray with dark gray reticulations

or dark gray with pale marks); middorsal stripe between

3-4 scales wide at midbody (only two scales wide); and

slender hemipenial body (robust). In addition, P. ulisesi

is smaller than P. vertebralis, with a maximum SVL of

45.5 mm in males ( n = 5) and 57.4 mm in females, n =

4 (males 58.9 mm, n = 5, and females 68.4 mm, n = 5).

The specimens of Pholidobolus sp. from Quebrada Che-

cos reported by Doan and Cusi (2014), and a specimen

examined by us from Quebrada la Iraca, both localities

approximately two km apart, can be easily distinguished

from P. vertebralis by lacking prefrontal scales, and from

P. ulisesi by lacking the rictal diagonal white bar and a

white stripe on the forearm. We acknowledge that the

differences in color pattern between P. ulisesi and P. sp.

might only represent interpopulational variation within P.

ulisesi, which should be addressed with the examination

of further specimens, as well as phylogenetic analyses of

molecular data.

In conclusion, there are no voucher specimens of

Pholidobolus vertebralis from Peru, and its presence in

this country has been based on misidentified specimens

of Macropholidiis huancabambae, P. ulisesi, and an un¬

described species of Pholidobolus. Furthermore, we also

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July 2016 | Volume 10 | Number 1 | e121

Venegas et al.

examined the single specimen of P. vertebralis reported

by Uzzell (1973) from southwestern Ecuador (AMNH

18312) and conclude that it represents another unde¬

scribed species of PhoUdobolus. Thus, the southernmost

records of P. vertebralis are from northwestern Ecuador

around its type locality (Intag, Imbabura province). Fi¬

nally, as noted by Uzzell (1973), the few records of P.

vertebralis east of the Andes in Ecuador are most likely

based on erroneous locality data, as has been noted for

other species of amphibians and reptiles from the same

localities (e.g., Uzzell 1973).

Acknowledgments. —We thank C. Raxworthy and D.

Kizirian (AMNH) for the loan of specimens; G. Pauly

and N. Camacho (LACM) for providing photographs

and data of specimen LACM 58811. P.J. Venegas is in¬

debted to Nature and Culture International (NCI) and

with J. Puicon-Carrillo, ex-coordinator of Programa De-

sarrollo Rural Sostenible, for funding the field surveys

in Cajamarca and promote the regional conservation of

natural areas. Also, we are grateful for A. Garcia for his

logistic support and company in the field surveys at Caja¬

marca. Specimens were collected with the following per¬

mits: 110-2007-INRENA-IFFS-DCB, N°08C/C-2008-

INRENA-IANP, 118-2007-INRENA-IFFS-DCB, and

0581 -2011 -AG-DGFFS-DGEFFS. PMSN is grateful to

Fundagao de Amparo a Pesquisa do Estado de Sao Paulo

(FAPESP) (Grant # 2012/00492-8) and to Fundagao de

Amparo a Ciencia e Tecnologia do Estado de Pernambu¬

co (FACEPE) for financial support. OTC and SEL were

financially supported by Secretaria de Educacion Superi¬

or, Ciencia, Tecnologia e Innovacion (SENESCYT) and

Pontificia Universidad Catolica del Ecuador.

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Pablo J. Venegas graduated in Veterinary Medicine from Universidad Nacional Pedro Ruiz Gallo, Lambayeque,

Peru, in 2005. He is currently curator of the herpetological collection of Centro de Ornitologia y Biodiversidad

(CORB1DI). His current research interest is focused on the diversity and conservation of the Neotropical her-

petofauna, with emphasis in Pem and Ecuador. He worked as a researcher of the Museo de Zoologia (QCAZ),

Pontiflcia Universidad Catolica del Ecuador, in Quito between 2014 and 2015. So far he has published more than

50 scientific papers on taxonomy and systematics of amphibians and reptiles.

Lourdes Y. Echevarria graduated in Biological Sciences from Universidad Agraria La Molina, Lima, Peru, in

2014. As a student, she collaborated in the order and management of the herpetological collection of Centro de

Ornitologia y Biodiversidad, Lima, developing a great interest in reptiles, especially lizards. Lor her undergradu¬

ate thesis, Lourdes worked on the “Review of the current taxonomic status of Petracola ventrimaculatus (Cerco-

saurim: Gymnophthalmidae) using morphological and ecological evidence.” She worked as a researcher of the

Museo de Zoologia (QCAZ), Pontiflcia Universidad Catolica del Ecuador, in Quito on 2015. Currently, she is a

postgraduate student at Pontiflcia Universidade Catolica do Rio Grande do Sul (PUCRS) in Porto Alegre, Brazil

where she is working on Hemiphractidae phylogenetics and biogeography. She is also working on more articles

about lizard’s systematics.

Simon E. Lobos graduated in Biological Sciences from Pontiflcia Universidad Catolica del Ecuador (PUCE)

in 2013. As a student, he joined the Museo de Zoologia QCAZ, Pontiflcia Universidad Catolica del Ecuador in

Quito, where he developed a great interest in reptiles. He has been studying the systematics of gymnophthalmid

lizards for the last four years. For his undergraduate thesis, Simon worked on the “Molecular systematics of liz¬

ard Alopoglossus (Autarchoglossa: Gymnophthalmidae) in Ecuador.” This manuscript is the third lizard species

description coauthored by Simon. He is also coauthor of other recent papers on lizard systematics.

Pedro M. Sales Nunes graduated in Biological Sciences from Universidade de Sao Paulo (USP) in 2003, and

in 2006 received a Master’s degree in Zoology from the same institution under the supervision of Dr. Hussam

Zaher. In 2011 he received a Ph.D. degree from the same institution with the thesis entitled “Hemipenial Mor¬

phology of the Microteiid Lizards ( Squamata: Gymnophthalmidae )” under the supervision of Dr. Miguel Trefaut

Rodrigues. Between 2012-2014 he was a postdoctoral fellow at the USP, Sao Paulo, Brazil, also working under

the supervision of Dr. Miguel Trefaut Rodrigues. He is currently Curator of the Herpetological Collection at

the Universidade Federal de Pernambuco (UFPE), Recife, Brazil, and an Adjunct Professor at the Department

of Zoology in the same institution. His production is focused on taxonomy and systematics of South American

squamate reptiles.

Omar Torres-Carvajal graduated in Biological Sciences from Pontiflcia Universidad Catolica del Ecuador

(PUCE) in 1998, and in 2001 received a Master’s degree in Ecology and Evolutionary Biology from the Univer¬

sity of Kansas under the supervision of Dr. Linda Tmeb. In 2005 he received a Ph.D. degree from the same in¬

stitution with the thesis entitled “Phylogenetic Systematics of South American Lizards of the Genus Stenocercus

(Squamata: Iguania).” Between 2006-2008 he was a postdoctoral fellow at the Smithsonian Institution, National

Museum of Natural History, Washington DC, USA, working under the supervision of Dr. Kevin de Queiroz. He

is currently Curator of Reptiles at the Zoology Museum QCAZ of PUCE and an Full Professor at the Department

of Biology in the same institution. He has published more than 45 scientific papers on taxonomy, systematics,

and biogeography of South American reptiles, with emphasis on lizards. He is mainly interested in the theory and

practice of phylogenetic systematics, particularly as they relate to the evolutionary biology of lizards.

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e121

Venegas et al.

Appendix 1

Additional specimens examined

Macropholidus huancabambae. —PERU: Piura: 11 miles E of Canchaque, on the western slope of the Huancabamba Mountains, LACM 58811.

Pholidobolus affinis — ECUADOR: Provincia Chimborazo: Colta, 1°41’56”S, 78°46’25”W, 3,215 m, QCAZ 9899-01; Sicalpa, 1°42’18”S,

78°46’32”W, 3,212 m, QCAZ 11887. Provincia Cotopaxi: Cutuchi river, San Miguel de Salcedo, 1°2’9”S, 78°35’53”W, 2,640 m, QCAZ 9641.

Provincia Tungurahua: six km N Mocha to 400 m Panamerican Highway, 1°22’ 1”S, 78°39’ 16”W, 3,205 m, QCAZ 9895-97; Ambato surroundings,

1°14’59,8”S, 78°37’33”W, QCAZ 9340-73, 9375-9443; Chamisa on road to Guadalupe, 1°21’44”S, 78°30’39”W, 2,348 m, QCAZ 7266; Cotalo

on path to Mucubi Community, 1°25’46”S, 78°31’3”W, 2,626 m, QCAZ 9839; Patate, 1°18’42”S, 78°30’36”W, 2,199 m, QCAZ 9847-50; Poatug

Hamlet, Aya Samana, 1°16’58”S, 78°29’29”W, 2,573 m, QCAZ 10005, 10008, 10011-13, 10016, 10018; Poatug Hamlet, Terremoto, 1°16’23”S,

78°29’29”W, 2,547 m QCAZ 9997-10000, 10002-10004; San Miguehto on path to Pillaro, 1°13’12”S, 78°31’31”W, 2,689 m, QCAZ 9844; San

Miguelito on path to Teran, 1°12’58”S, 78°31’42”W, 2,741 m, QCAZ 9843.

Pholidobolus dicrus .—ECUADOR: Provincia Morona Santiago: Guarumales, 2°34’ 0.0006” S, -78° 30’ 0” W, 1,700 m, QCAZ 5292, 5304. Provin¬

cia Tungurahua: Rio Blanco, Via Banos-Puyo, 1° 23’ 55.6434”S, 78° 20’ 24”W, 1,600 m, QCAZ 6936, no locality data QCAZ 8015.

Pholidobolus hillisi .—ECUADOR: Provincia Zamora-Chinchipe: near San Francisco Research Station on Loja-Zamora road, 3°57’57”S,

79°4’45”W, WGS84, 1,840 m, QCAZ 4998-99, 5000; San Francisco Research Station, 3°58’14”S, 79°4’41”W, 1,840 m, QCAZ 6840, 6842, 6844.

Pholidobolus macbrydei. —ECUADOR: Provincia Azuay: 10 km S Cutchil, 3°8’2”S, 78°48’47”W, 2,900 m, QCAZ 823-24; 1.2 km E Osoran-

cho, 2°46’8”S, 78°38’10”W, 2,390 m, QCAZ 826; 6.2 km S Cutchil, 3°6’32”S, 78°48’4”W, 2,800 m, QCAZ 827; 20 km NE Cuenca, 2°51’0”S,

78°51’14”W, QCAZ 1359; seven km Sigsig, 2°59’56”S, 78°48’25”W, 2,890 m, QCAZ 1537; 6 km S Ona, 3°29’49”S, 79°9’47”W, QCAZ 3658;

20 km Cuenca-El Cajas, 2°46’39”S, 79°10’12”W, 3,508 m, QCAZ 9932-34, 9936-38, 10020; Cochapamba, 2°47’50”S, 79°24’56”W, 3,548 m,

QCAZ 10133-35; Cochapata, 3°25’47”S, 79°3’35”W, 3,074 m, QCAZ 12605-07; Cuenca, Cuenca-Azoguez Panamerican Highway 2°53’43”S,

78°57’30”W, 2.486 m, QCAZ 6985; El Cajas National Park, path to Patul Community, 2°44’28”S, 79°14’5”W, 4,092 m, QCAZ 8010-11; El Cajas

National Park, Patul river, 2°41’37”S, 79°13’56”W, 3,610 m, QCAZ 8893; El Cajas National Park, Zhurcay river, 3°2’30”S, 79°12’56”W, 3,766

m, QCAZ 8900-01; El Cajas National Park, 2°42’21”S, 79°13’32”W, 3,600 m, QCAZ 8946; El Capo, 2°46’43”S, 79°14’43”W, 4,100 m, QCAZ

4997; Giron, San Gregorio Community, Quinsacocha paramo, 3°6’22”S, 79°13’4”W, 3,242 m, QCAZ 8510-11; Giron, San Gregorio Community,

Quinsacocha paramo, 3°2’30”S, 79°12’56”W, 3,766 m, QCAZ 8894-99, 8902-05, 8907; Giron, San Gregorio Community, Quinsacocha paramo,

3°2’30”S, 79°12’57”W, 3,766 m, QCAZ 8906; Guablid, 2°46’30”S, 78°41’51”W, 2,453 m, QCAZ 9913-17, 9919-20, 9940-41; Gualaceo-Limon

road, 2°56’53”S, 78°42’43” W, 3,110 m, QCAZ 819-22; Gualaceo-Limon road, 8.1 km O Azuay-Morona Santiago border, 2°57’50”S, 78°42’7”W,

3,140 m, QCAZ 825; Gualaceo, 2°52’56”S, 78°46’31”W, 2,298 m, QCAZ 9606; Gualaceo-Plan de Milagro road, 2°54’35”S, 78°44’4”W, 2,624

m, QCAZ 10875; Las Tres Cruces, 2°46’30”S, 79°14”53”W, QCAZ 4136; Maylas, Gualaceo-Macas road, 2°58’25”S, 78°41’41”W, 3,100 m,

QCAZ 7269; Mazan Protected Forest, 2°52’29”S, 79°7’26”W, 2,700 m, QCAZ 1296-97; Mazan Protected Forest, 2°52’31”S, 79°7’45”W, 3,189

m, QCAZ 8008, 8013; Ona-La Paz road, 3°22’42”S, 79°11’20”W, 2,969 m, QCAZ 6031; Patacocha hill, 3°7’16”S, 79°3’54”W, 3,340 m, QCAZ

6144; Pucara, Tres Chorreras, 3°12’49”S, 79°28’3”W, QCAZ 11038; Quinoas river, 3°5’14”S, 79°16’40”W, 3,200 m, QCAZ 1564-66; San Anto¬

nio, 2°51’40”S, 79°22’43”W, 2,943 m, QCAZ 9668; San Vicente-Cruz path, 2°47’43”S, 78°42’53”W, 3,044 m, QCAZ 11416-17, 11420; Sigsig,

3°7’46”S, 78°48’14”W, 2,969 m, QCAZ 5605-08; Sigsig road, 3°3’17”S, 78°47’19”W, 2,574 m, QCAZ 9605; Tarqui, 3°0’57”S, 79°2’40”W,

2,627 m, QCAZ 8512. Provincia Canar: Canar, 2°33’39”S, 78°55’51”W, QCAZ 9947; Culebnllas, 2°25’35”S, 78°52’12”W, 4,000 m, QCAZ

1349; Guallicanga ravine, 2°25’56”S, 78°54’8”W, 3,960 m, QCAZ 10048-49; Guallicanga river, 2°28’24”S, 78°58’22”W, 3,048 m, QCAZ

10051-52; Ingapirca, 2°32’43”S, 78°52’28”W, 3,400 m, QCAZ 1551; Juncal, 2°28’24”S, 78°58’22”W, 3,048 m, QCAZ 10050; Mazar Protected

Forest, 2°32’48”S, 78°41’54”W, QCAZ 7376-84, 7883; Mazar Reserve, La Libertad, 2°32’45”S, 78°41’46”W, 2,842 m, QCAZ 10970-72. Pro¬

vincia Chimborazo: Alao, 10 km Huamboya, 1°52’22”S, 78°29’51”W, 3,200 m, QCAZ 1567-68; Atillo Grande, Magdalena lake, 2°11’15”S,

78°30’25”W, 3,556 m, QCAZ 9214; Atillo Grande, Frutatian lake, 2°12’57”S, 78°30’5”W, 3,700 m, QCAZ 9216-18; Culebrillas, Sangay National

Park, 1°57’39”S, 78°25’55”W, 3,345 m, QCAZ 9612; Pungala, Eten Community, Timbo, 1°55’45”S, 78°32’14”W, 3,408 m, QCAZ 9616-21;

Pungala, Melan Community, 1°52’30”S, 78°32’52”W, 3,564 m, QCAZ 9626-29, 9631; Ozogoche, 2°22’7”S, 78°41’20”W, 4,040 m, QCAZ 6006-

07; Shulata, 2°20’22”S, 78°50’36”W, 3,228 m, QCAZ 5597-98;. Provincia El Oro: Guanazan, 3°26’24”S, 79°29’13”W, 2,638 m, QCAZ 7891,

7894. Provincia Loja: 17.1 km S Saraguro, 3°43’45”S, 79°15’53”W, 3,150 m, QCAZ 828; 26 km N Loja, Huashapamba Native Forest, 3°39’30”S,

79°16’20”W, 2,894 m, QCAZ 8651; Cordillera of Lagumllas, Jimbura, 4°49’1”S, 79°21’43”W, 3,600 m, QCAZ 3785; Cordillera of Lagumllas,

Jimbura, 4°37’42”S, 79°27’49”W, 3,450 m, QCAZ 6145-47; Fierro Urco, 3°42’38”S, 79°18’ 18”W, 3,439 m, QCAZ 6949-50; Gurudel, 3°39’22”S,

79°9’47”W, 3,100 m, QCAZ 5078-79; Jimbura, Jimbura lake, 4°42’32”S, 79°26’48”W, 3,036 m, QCAZ 6945-48; Jimbura, path to Jimbura lake,

4°42’34”S, 79°26’8”W, 3,348 m, QCAZ 10054-62; Military antemia, Saraguro, 3°40’46”S, 79°14’16”W, 3,190 m, QCAZ 3673-75, 9632; San

Lucas, 3°43’55”S, 79°15’38”W, 2,470 m, QCAZ 2861; Saraguro, 3°37’13”S, 79°14’9”W, 3,100 m, QCAZ 3606, 3754; Urdaneta, 3°36’6”S,

79°12’31”W, QCAZ 2019. Provincia Tungurahua: Poatug Hamlet, El Corral, 1°16’21”S, 78°28’5”W, 3,468 m, QCAZ 8047, 9995-96. Provincia

Zamora Chinchipe: Loja-Podocarpus National Park road, 3°59’44”S, 79°8’28”W, 2,776 m, QCAZ 10870-71; Valladolid, Podocarpus National Park,

4°29’3”S, 79°8’56”W, 1,800 m, QCAZ 3743.

Pholidobolus montium .—ECUADOR: Provincia Cotopaxi: two km S Chugchilan on road to Quilotoa, 0°48’24”S, 78°56’11”W, 2,917 m, QCAZ

8056-58; Latacunga, 0°52’27”S, 78°38’26”W, 2,857 m, QCAZ 873-74, 1411-12, 9642; Mulalo, 0°46’35”S, 78°34’40”W, 3,030 m, QCAZ 9639;

San Juan de Pasto Calle, 0°45’4”S, 78°38’51”W, 1,956 m, QCAZ 8053-54; South Illimza, 0°39’43”S, 78°42’40”W, 3,400 m, QCAZ 858-59,

1454. Provincia Imbabura: Atuntaqui, 0°19’59”N, 78°12’50”W, QCAZ 855; Cotacahi, Peribuela, Cuicocha Lake, Cotacachi-Cayapas Reserve,

0°17’34”N, 78°21’5”W, 3,082 m, QCAZ 9683, 9685-86; 0°23’4”N, 78°15’25”W, 2,900 m, QCAZ 6137, 6139; Cotacachi-Cayapas Reserve, Jose

Maria Yerovi Islets, 0°18’20”N, 78°21’41”W, 3,093 m, QCAZ 10959-60; El Juncal, 0°26’6”N, 77°57’58”W, QCAZ 6451. Provincia Pichincha: 16

km W Chillogallo, Quito-Chiriboga road, 0°17’46”S, 78°39’30”W, 3,100 m, QCAZ 797; five km E Pifo-Papallacta road, 0°15’3”S, 78°17’58”W,

2,800 m, QCAZ 1107-08; Alambi, 0°1’59”S, 78°34’26”W, 2,727-3,800 m, QCAZ 9691; Alangasi, 0°18’24”S, 78°24’40”W, QCAZ 1453, 1469;

Amaguana, Hacienda San Ignacio, 0°22’22”S, 78°30’14”W, QCAZ 1463-64, 5275; Calacali, Simon Bolivar Street, uphill through secondary road,

0°1’1”N, 78°30’49”W, 3,001 m, QCAZ 11674, 11676, 11678-79; Calacali Stadium, 0°0’0,3”S, 78°30’38”W, 2,833 m, QCAZ 11682; Carretas,

0°6’25”S, 78°26’46”W, QCAZ 875; Chillogallo, 0°16’48”S, 78°33’25”W, QCAZ 840-43; Cumbaya, LaPrimavera, 0°12’6”S, 78°25’40”W, QCAZ

7248; Guayllabamba, 0°3’23”S, 78°20’26”W, QCAZ 7905; Inga, 5.5 km SE La Merced, 0°17’51”S, 78°20’52”W, 2,798 m, QCAZ 5278; Lloa,

0°14’52”S, 78°34’33”W, QCAZ 4109; Lloa Stadium, 0°14’39”S, 78°35’12”W, 3,059 m, QCAZ 11661; Loreto, road to Mohnuco, Central Stadium,

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A new species of Andean microteiid lizard

Appendix 1 (continued)

Additional specimens examined

0°23’4”S, 78°24’30”W, 2,844 m, QCAZ 11663; Machachi, 0°29’50”S, 78°32’25”W, QCAZ 844-48,1374-77,1462; Machachi, The Tesalia Springs

Company S.A. surroundings, 0°30’27”S, 78°33’57”W, 2,900 m, QCAZ 1465-67, 830-31, 833, 860-61, 1459-61; Nono, 0°4’42”S, 78°34’24”W,

2,843 m, QCAZ 11653-55; Nono School, 0°4’4”S, 78°34’35”W, 2,754 m, QCAZ 11656-58; Pasochoa, 0°26’24”S, 78 o 30’15”W, 2,850 m, QCAZ

1451-52; Pomasqui, 0°3’3”S, 78°27’21”W, QCAZ 862-68; Pululahua Volcano, 0 o 2’34”N, 78°30’15”W, QCAZ 1450, 1520; Quito, Bellavista,

0°11’21”S, 78°28’35”W, QCAZ 1099; Quito, Chillogallo, 0°16’26”S, 78°33’23”W, QCAZ 8967; Quito, Itchimbla, 0°13’21”S, 78°29’56”W,

QCAZ 834, 1455-58, 1643, 2843; Quito, Garden of the Pontificia Umversidad Catolica del Ecuador (PUCE), 0°12’33”S, 78°29’28”W, 2,800 m,

QCAZ 856-57, 7032, 1295, 2853; Quito, Parque Metropolitano, 0°10’35”S, 78°27’40”W, QCAZ 4051; Quito, Umversidad Central del Ecuador,

0°11’59”S, 78°30’19”W, 2,800 m, QCAZ 3727; Rio Guajalito Protected Forest, 0°13’44”S, 78°48’22”W, QCAZ 1338-39; San Antonio de Pich-

incha, 0°0’33”S, 78°26’45”W, QCAZ 580-81, 790-92, 849, 1119-20, 1368, 1393, 2220, 2223, 2653; Tababela, International Airport, 0°6’21”S,

78°21’4”W, QCAZ 8046, 9044, 10064, 10974-76; Quito, Tumbaco, 0°12’34”S, 78°24’2”W, QCAZ 1113-14; Uyumbicho, 0°22’59”S, 78°31’6”W,

QCAZ 870.

Pholidobolus prefrontalis. —ECUADOR: Provincia Azuay: Sigsig, 3°7’46”S, 78°48’14”W, 2,480 m, QCAZ 1553; Provincia Canar: Canar,

2°33’29”S, 78°56’4”W, QCAZ 1410; Provincia Chimborazo: Alausi, 2°11’54”S, 78°50’42”W, 2,359 m, QCAZ 9907-9911; Tixan, 2°9’22”S,

78°48’3”W, 2,908 m, QCAZ 9951-54; Tixan, 2°9’22”S, 78°48’3”W, 2,908 m, QCAZ 9951-54.

Pholidobolus vertebralis—ECU ADOR. Provincia Carchi: Chilma Bajo, 0°51 ’53.83” N, 78°2’59.26”, W, 2,071 m, QCAZ 5057, 8671-8673, 8678,

8679, 8717, 8724, 0°51’50.31” N, 78 o 2’50.05” W, 2,022 m, QCAZ 8684-8689. Provincia Pichincha: Mindo, 0°3’2.41” S, 78°46’18.77” W, 1,700

m, QCAZ 2911, 2912, 2915, 0°4’40.98”S, 78°43’55.02”W, 1,601 m, QCAZ 7528; Cooperativa El Porvemr, El Cedral 0°6’50.40” N, 78°34’11.75”

W, 2,297 m, QCAZ 5081, 5082; Santa Lucia deNanegal, 0°6’48.70”N, 78°36’48.60”W, 1,742 m, QCAZ 10667,0°7’8.51”N, 78°35’58.70”W, 1,900

m, QCAZ 10750. LOCALITY IN ERROR: ECUADOR: Provincia Pastaza: Mera, AMNH 60586-97.

Pholidobolus sp.—ECUADOR: Provincia El Oro: El Chiral, 1,350 m, AMNH 18312.

Pholidobolus sp.—PERU: Cajamarca: Provincia de Chota: Quebrada La Iraca (near to La Granja village), 6°22’09.9”S, 79°08’04.61”W, 2,213 m,

CORBIDI 1679.

In accordance with the International Code of Zoological Nomenclature new rules and regulations (ICZN 2012), we have deposited this paper

in publicly accessible institutional libraries. The new species described herein has been registered in ZooBank (Polaszek 2005a, b), the of¬

ficial online registration system for the ICZN. The ZooBank publication LSID (Life Science Identifier) for the new species described here

can be viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/.” The LSID for this publication

is: um:lsid:zoobank.org:pub:CAB026AC-BlCE-4F43-B0C3-AB908645159F.

Separate print-only edition of paper(s) (reprint) are available upon request as a print-on-demand service. Please inquire by sending a request

to: Amphibian & Reptile Consenation , amphibian-reptile-conservation.org, arc.publisher@gmail.com.

Amphibian & Reptile Consen’ation is a Content Partner with the Encyclopedia of Life (EOL), http:///www.eol.org/ and submits information

about new species to the EOL freely.

Digital archiving of this paper are found at the following institutions: ZenScientist, http://www.zenscientist.com/index.php/filedrawer; Ernst

Mayr Library, Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts (USA), http://library.mcz.harvard.edu/

ernst_mayr/Ejournals/ARCns.

The most complete journal archiving and journal information is found at the official ARC journal website, amphibian-reptile-conservation,

org. In addition, complete journal paper archiving is found at: ZenScientist, http://www.zenscientist.com/index.php/filedrawer.

Citations

ICZN. 2012. Amendment of Articles 8,9,10,21 and 78 of the International Code of Zoological Nomenclature to expand and refine methods

of publication. Zootaxa 3450: 1-7.

Polaszek A et al. 2005a. Commentary: A universal register for animal names. Nature 437: All.

Polaszek A et al. 2005b. ZooBank: The open-access register for zoological taxonomy: Technical Discussion Paper. Bulletin of Zoological

Nomenclature 62(4): 210-220.

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e121

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [Special Section]: 34-39 (el30).

SHORT COMMUNICATION

New records, range extension and call description for the

stream-breeding frog Hyloscirtus lascinius (Rivero, 1970)

in Venezuela

^Fernando J. M. Rojas-Runjaic, 3 Edwin E. Infante-Rivero, 4 Cesar L. Barrio-Amoros

1 Lciboraldrio de Sistematica de Vertebrados, Pontijicici Universidade Catolica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681, Porto Alegre,

RS 90619-900, BRAZIL 2 Museo de Historici Natural La Salle. Apartado Postal 1930, Caracas 1010-A, VENEZUELA 3 Laboratorio de Ictiologia,

Centro MBUCV, Instituto de Zoologiay Ecologia Tropical, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, VENEZUELA

4 Doc Frog Expeditions, Uvita, Puntarenas, COSTA RICA.

Abstract .—The stream-breeding frog Hyloscirtus lascinius is known from a few localities

on the Eastern versant of the Cordillera Oriental of Colombia and Tama massif in Colombia

and Venezuela. It has also been reported from Cordillera de Merida in Venezuela but without

precise localities or voucher specimens. Herein we report H. lascinius from the Sierra de Perija

in Venezuela, and provide four locality records in Cordillera de Merida. The record of Perija

extends the known species’ distribution ca» 213 km NW from the northernmost locality previously

recorded. We also describe the advertisement call of this species for the first time, and provide

some notes on its natural history.

Keywords. Amphibia, Anura, Biogeography, Bioacustics, Sierra de Perija, Andes

Citation: Rojas-Runjaic FJM, Infante-Rivero EE, and Barrio-Amoros CL 2016. New records, range extension and call description for the stream¬

breeding frog Hyloscirtus lascinius (Rivero, 1970) in Venezuela. Amphibian & Reptile Conservation 10(1) [Special Section]: 34-39 (el 30).

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: 27 April 2016; Accepted: 07 November 2016; Published: 19 December 2016

The hylid genus Hyloscirtus Peters, 1882 was resurrected

by Faivovich et al. (2005) to accommodate all the stream¬

breeding frogs previously included in the Hyla armata ,

H. bogotensis, and H. larinopygion species groups. This

genus was supported by 56 molecular synapomorphies

and by having wide dermal fringes on fingers and toes (the

only putative morphological synapomorphy). Recently,

Duellman et al. (2016) described Colomascirtus for the

clade formed by the H. armatus and H. larinopygion

species groups, restricting Hyloscirtus to the

H. bogotensis species group.

Hyloscirtus as currently defined (Duellman et al.

2016), contains 17 species (Guayasamin et al. 2015) and

is distributed from Costa Rica and Panama in Central

America, through the Andes of Colombia, Venezuela,

Ecuador, Peru, and Bolivia in South America (Frost

2016). In Venezuela, only three species of Hyloscirtus are

currently known: H. jahni (Rivero, 1961), H. lascinius

(Rivero, 1970), and H. platydactylus (Boulenger, 1905).

Hyloscirtus lascinius was originally described by

Rivero (1970) as a member of Hyla Laurenti, 1768

(as Hyla lascinia). Its relationships with other stream¬

breeding frogs were earlier presumed by Rivero (1970),

Go in (in Rivero 1970) and Duellman (1972), but only

after two decades was this recognized as part of the Hyla

bogotensis group (Ruiz-Carranza and Ardila-Robayo

1991; Duellman et al. 1997). Faivovich et al. (2005)

transferred it to Hyloscirtus based only on morphological

evidence. However, molecular corroboration of his

taxonomic position was presented more recently by

Wiens et al. (2010), Pyron and Wiens (2011), Faivovich

et al. (2013), Almendariz et al. (2014), Guayasamin et al.

(2015), and Duellman et al. (2016).

Hyloscirtus lascinius is distributed on the eastern

versant of the Andean Cordillera Oriental and Tama massif

in Colombia at Norte de Santander department, between

1,730-1,960 m asl (Ruiz-Carranza et al. 1996; Bemal

and Lynch 2008; Sanchez 2010), and in the Venezuelan

Correspondence. Emails: l2 rojasnmjaic@yahoo.com (Corresponding author); 3 edwinfante@gmail.com ; 4 cesarlba@yahoo.com

Amphib. Reptile Conserv.

34 December 2016 1 Volume 10 I Number 1 I el30

Rojas-Runjaic et. al

Fig. 1. Hyloscirtus lascinius from Venezuela. (A) Campamento

Guacharaca, Sierra de Perija, Zulia state; (B) Near La Macana,

Merida state; (C) Quebrada La Rana, Merida state. Photos:

F.J.M. Rojas-Runjaic (A) and C.L. Barrio-Amords (B and C).

portion of Tama, Tachira state, between 1,250-1,700

m asl (Rivero 1970; Mijares-Urrutia 1992). La Marca

et al. (2004) and Barrio-Amoros (2004) mentioned its

presence on the western versant of Cordillera de Merida,

in Merida state, but without referring any precise locality

or voucher specimens.

Herein, we report the first records of Hyloscirtus

lascinius for Zulia state, northwestern Venezuela, and

formally documented its presence in several localities of

Merida state, based on museum specimens deposited at

Museo de Historia Natural La Salle, Caracas, Venezuela

(MHNLS), and uncollected specimens photographed

by CLBA. Also we describe for the first time the call of

this species. Call description is based on a 73 s digital

recording of the advertisement call of a single male

at Quebrada La Rana, in Santa Cruz de Mora, Merida

state, Venezuela, on 13 August 2006. Air temperature

was 17 °C. Calls were analyzed using Raven Pro 1.3

(Bioacoustics Research Program 2008).

Specimens from Zulia state (Fig. 1A) were found

during a survey of the inventory of amphibian and reptiles

of the Venezuelan side of Sierra de Perija, conducted by

Museo de Historia Natural La Salle (MHNLS). They

are five adult males (MHNLS 19163-19165, 19237—

19238; SVL: 41.6-44.0 mm) collected at Campamento

Guacharaca, Cano Tetari Kopejoacha, Rio Negro upper

basin, Machiques de Perija municipality, Sierra de Perija

(10°04 , 22”N, 72°51 , 16 ,, W, 1,661 m; Fig. 2) on 21-27

May 2009. The creek where the specimens were collected

(Fig. 3A) was surrounded by a primary ombrofilous

submontane/montane evergreen forest (Huber and

Alarcon 1988), with abundant ferns, Heliconiaceae,

Araceae, and Cyclanthaceae plants. All specimens were

found between 20:00-22:00 h, calling from branches of

76°W

68°W

66°W

12°N

10°N

8°N

6=N

Fig. 2. Distribution of Hyloscirtus lascinius in Venezuela and Colombia. 1: Campamento Guacharaca, Sierra de Perija, Zulia state,

Venezuela. 2: San Luis, Merida state, Venezuela. 3: Road Santa Cruz de Mora-La Macana, Merida state, Venezuela. 4: Quebrada

Ovalles, Merida state, Venezuela. 5: Quebrada De La Rana, Merida state, Venezuela. Yellow triangle: Tabor, Tama massif,

Tachira state, Venezuela (type locality); White pentagon: Chinacota, Norte de Santander department, Colombia (Sanchez 2010);

The record of headwaters of Rio Tachira, Norte de Santander, Colombia (Ruiz-Carranza et al. 1996) and additional localities

between Delicias and Tabor (Rivero 1970) are included in the yellow triangle that indicates the type locality.

Amphib. Reptile Conserv.

35 December 2016 1 Volume 10 I Number 1 I el30

Distribution and call description of Hyloscirtus lascinius

Fig. 3. Habitat of Hyloscirtus lascinius at Campamento

Guacharaca, Sierra de Perija, Zulia state (A). Males calling

from a branch; (B) and from a rocky wall; (C) at the edge of

the creek in Campamento Guacharaca. Photos: F.J.M. Rojas-

Runjaic.

bushes (Fig. 3B) or rocky walls (Fig. 3C) on the sides of

the creek and between 50-300 cm above ground.

When captured the frogs released a strong citrus smell.

At this locality Hyloscirtus lascinius was sympatric

with Hyloscirtus sp., Cryptobatrachus remotus Infante-

Rivero, Rojas-Runjaic and Barrio-Amoros, 2009,

Centrolene daidaleum (Ruiz-Carranza and Lynch, 1991),

Centrolene notostictum Ruiz-Carranza and Lynch,

1991, Hyalinobatrachium pallidum (Rivero, 1985),

Pristimantis rivasi Barrio-Amoros, Rojas-Runjaic, and

Barros, 2010, and Pristimantis sp.

This new record of Hyloscirtus lascinius for the

Venezuelan Sierra de Perija in Zulia state (Fig. 2) extend

the species’ distribution by ca. 276 km north (straight-

line) from Chinacota, Norte de Santander, Colombia,

the northeasternmost locality previously documented

(Sanchez 2010), and ca. 213 km NW (straight-line) from

San Luis, Merida state, Venezuela, the northernmost

locality known in the Cordillera de Merida (locality

record also in this this work).

Four more localities are reported herein (Fig. 2),

all from Cordillera de Merida, to confirm previous

statements (Barrio-Amoros 2004; La Marca et al.,

2004). These are: 1) San Luis, La Azulita Andres Bello

municipality, Merida state (08 o 41’27”N, 71°29’44”W;

ca. 1,614 m; CLBA personal observation); 2) creek on

the road Santa Cruz de Mora-La Macana, Pinto Salinas

municipality, Merida state (08°23’13”N, 71°37’35”W,

1,130 m; photographic record; Fig. IB); 3) Quebrada

Ovalles, above La Macana, Pinto Salinas municipality,

Merida state (08°22’56”N, 71°35’51”W, 1,478 m;

MHNLS 17913); and 4) Quebrada de la Rana, Pinto

Salinas municipality, Merida state (08°22’N, 71°24’W,

ca. 1,200 m; photographic record; Fig. 1C). In San

Luis, Hyloscirtus lascinius is sympatric with Scinax

manriquei Barrio-Amoros, Orellana and Chacon-Ortiz,

2004, Dendropsophus aff. minutus (Peters, 1872) and

Espadarana andina (Rivero, 1968). At the creek between

Santa Cruz de Mora and La Macana with Flectonotus

pygmaeus (Boettger, 1893), and Tachiramantis

lentiginosus (Rivero, 1984); and at Quebrada de la Rana

with Hyalinobatrachium pallidum , and Pristimantis cf.

vanadise (La Marca, 1984).

Based on all localities previously documented (Rivero

1970; Mijares-Urrutia 1992; Ruiz-Carranza et al. 1996;

Bemal and Lynch 2008; Sanchez 2010) and the referred

in this note, the species’ altitudinal range is extended to

0.2

0.4

0.6

1.4

0.8 1 1.2

Time (s)

Fig. 4. Oscillogram (A) and spectrogram (B) of the advertisement call of Hyloscirtus lascinius.

1.6

1.8

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36 December 2016 1 Volume 10 I Number 1 I el30

Rojas-Runjaic et. al

ca. 1,130-1,960 m.

The advertisement call of Hyloscirtus lascinius is a

single tonal note, emitted as single and sporadically

(6 of 22 notes), or in groups of two (one group; 2 of 22

notes), three (two groups; 6 of 22 notes) or four notes

(two groups; 8 of 22 notes) (Fig. 4). These notes (calls)

sound as metallic whistles. Note duration is 91-164 ms

(123 ± 16.2; n = 19), with a note interval (only among

notes emitted in groups) of 700-1,075 ms (888.1 ±

121.9; n = 10). Fundamental frequency is at 1.077-1.227

kHz (1.160 ± 0.040; n = 17) and the dominant frequency

(=peak frequency) is at 2.132-2.369 kHz (2.330 ± 0.074;

n = 19), with modulation frequency throughout the calls.

The Sierra de Perija, and extension of the Andean

Cordillera Oriental and natural border between

northeastern Colombia and northwestern Venezuela,

remains poorly explored and its anuran fauna is still

not well known. However, the finding of Hyloscirtus

lascinius in this mountain system, as well other amphibian

discoveries documented in the last decade (Infante-

Rivero et al. 2006a, b, 2009; Castroviejo-Fisher et al.

2007; Barrio-Amoros et al. 2008, 2010; Rojas-Runjaic

et al. 2010, 2011, 2012) show that this region harbors a

diverse amphibian fauna closely related to the amphibian

faunas of the Andean Cordillera Oriental and Cordillera

de Merida. We predict that new expeditions to Perija will

result in the discovery of numerous additional species.

These future findings will improve the knowledge of

the amphibian diversity of the Sierra de Perija, and its

biogeographical affinities with neighboring bioregions.

Hyloscirtus lascinius was classified as Least Concern

(LC) in the IUCN Red List of Threatened Species

because, although its extent of occurrence was estimated

less than 5,000 km 2 , it is considered an adaptable species,

and does not appear to be in decline (La Marca et al.

2004). At the western versant of the Cordillera de Merida,

the localities where we documented this species are

currently being modified for agricultural purposes, and

the habitat is declining in extent and quality, by which

these populations may be being affected. However,

the new locality in the Venezuelan Sierra de Perija is

within Parque Nacional Sierra de Perija and significantly

increase its species’ distribution, indicating that it has an

extent of occurrence much wider than previously known

and that, at least the populations of Perija are apparently

protected. Thus, we consider that the conservation status

of LC is adequate for H. lascninius.

Acknowledgements.— We thank Julio Cesar Madrid,

Johnny Romero, Andres Orellana, Carlos Gottberg, and

Erik Arrieta (f) for field assistance. To Amelia Diaz de

Pascual and Moises Escalona for the data provided on the

CVULA specimens. To Ygrein Roos for the assistance

in the verification of the identity of some specimens of

MHNLS. Also to Juan Manuel Guayasamin and J. Celsa

Senaris for their comments on an early version of the

manuscript. FRR acknowledge the funding provided

by Banco Federal through the project FED-MHNLS-09

“Inventario de las especies de anfibios y reptiles de

la vertiente venezolana de la Sierra de Perija, estado

Zulia,” under the mark of “Ley Organica de Ciencia,

Tecnologia e Innovacion.” Currently, FRR is supported

by a Ph.D. scholarship from Conselho Nacional de

Desenvolvimento Cientifico e Tecnologico (CNPq).

Permit for collecting (#4750: period 2008-2009) was

issued to FRR by the Venezuelan Ministerio del Poder

Popular para el Ambiente. The expedition to the Rio

Negro basin in the Parque Nacional Sierra de Perija,

was benefited with permit of the Instituto Nacional de

Parques (PAA-215-2008) also issued to FRR.

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new species of Hyalinobatrachium (Centrolenidae:

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of the Hyla bogotensis group. Occassional Papers

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Kansas , 11: 1-31.

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Hyla armata and Hylapulchella groups in the Andes

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of Hyla. Scientific Papers of the Natural History

Museum, The University of Kansas 3: 1-41.

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Faivovich J, Haddad CFB, Garcia PCA, Frost DR,

Campbell JA, Wheeler WC. 2005. Systematic review

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species of Myersiohyla (Anura: Hylidae) from Cerro

de la Neblina, Venezuela, with comments on other

species of the genus. American Museum Novitates

3792: 1-63.

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J, Bustamante L, Pyron RA, Penafiel N, Morochz

C, Hutter CR. 2015. Molecular phylogeny of stream

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CL. 2006b. Geographic Distribution: Anura:

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Cabello P. 2010. Amphibia, Anura, Centrolenidae,

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CL. 2011. A new frog of the genus Aromobates

(Anura, Dendrobatidae) from Sierra de Perija,

Venezuela. Zootaxa 2919: 37-50.

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2012. New records and distribution extensions of

centrolenid frogs for Venezuela. Check List 8(4):

819-825.

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especie de Hyla del grupo bogotensis (Amphibia:

Anura: Hylidae) de la Cordillera Oriental de

Colombia. Caldasia 16(78): 337-342.

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1996. Lista actualizada de la fauna de Amphibia

de Colombia. Revista de la Academia Colombians

de Ciencias Exactas, Fisicas y Naturales 20(77):

365-415.

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synapomorphies for species groups of Hyloscirtus

Peters, 1882 (Anura: Hylidae: Cophomantini).

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Pyron RA, Wiens, JJ. 2011. A large-scale phylogeny of

Amphibia including over 2800 species, and a revised

classification of extant frogs, salamanders, and

caecilians. Molecular Phylogenetics and Evolution

61(2011): 543-583.

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Amphib. Reptile Conserv.

38 December 2016 1 Volume 10 I Number 1 I el30

Rojas-Runjaic et. al

Fernando J. M. Rojas-Runjaic is a researcher at the Museo de Historia Natural La Salle in

Caracas, Venezuela, and curator of the amphibian, reptile, and arachnid collections. Fernando

has a B.S. in Biology from the Universidad del Zulia (Venezuela) and a Master’s degree

in Biodiversity and Conservation in Tropical Areas from the Universidad Internacional

Menendez Pelayo (Spain). Currently he is enrolled in the Ph.D. program in Zoology at

the Pontificia Universidade Catolica do Rio Grande do Sul (Brazil). His research interests

are broad and include diversity, phylogenetic systematics, taxonomy, biogeography, and

conservation of amphibians, reptiles and scorpions.

Edwin E. Infante-Rivero is a professor and researcher at the laboratory of Ichthyology at the

Institute of Zoology and Tropical Ecology in the Universidad Central de Venezuela (UCV).

He has a B.S. in Biology from the Universidad del Zulia, and a Master’s degree in Zoology

from UCV. Currently is a Ph.D student in the Zoology program at UCV. His research interests

are broad on vertebrates (fishes, amphibian, reptiles, birds), and focus on topics such as

systematics, taxonomy, biogeography, and conservation.

Cesar L. Barrio-Amoros is an anthropologist who has worked with herpetofauna in Spain,

Venezuela, and Costa Rica. His research interests include the biogeography and systematics

of amphibians and reptiles, with emphasis on Dendrobatoidea and Terrarana from Venezuela,

especially in the Guiana Shield. Now residing in Costa Rica, he is a free-lance investigator

and photographer. Cesar has authored or co-authored more than 200 papers, including the

description of 50 new species of amphibians and reptiles.

Amphib. Reptile Conserv.

39 December 2016 1 Volume 10 I Number 1 I el30

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [General Section]: 1-6 (e112).

SHORT COMMUNICATION

Monitoring a population of Cruziohyla craspedopus

(Funkhouser, 1957) using an artificial breeding habitat

v Clare Turrell, 'Brian Crnobrna, and 2 Marnie Smith-Bessen

1 Asociacion Fauna Forever, Avenida Aeropuerto km 1, Puerto Maldonado, PERU

2 Division of Tropical Environments and Societies, James Cook University, Townsville, Queensland 4811 AUSTRALIA

Abstract .—We report the detection of Cruziohyla craspedopus in Madre de Dios, Peru via use of

an artificial breeding habitat: 1) giving us crucial information about the population, 2) contributing

to the population by providing habitat, and 3) emphasizing the value of this method in detecting

elusive species.

Key words. Oviposition, primary forest, tree hole breeding, phytotelm breeding, canopy, frog, Peru, rare

Citation: Turrell C, Crnobrna B, Smith-Bessen M. 2016. Monitoring a population of Cruziohyla craspedopus (Funkhouser, 1957) using an artificial

breeding habitat. Amphibian & Reptile Conservation 10(1) [General Section]: 1-6 (el 12).

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: 09 February 2015; Accepted: 29 June 2015; Published: 31 January 2016

For the more elusive and endangered anuran species

in South America it can be difficult to get accurate

representations of abundance and population size.

Implementing different sampling techniques, including

those focusing on aquatic larval stages, may bring

population levels into clearer focus. Tadpoles are often

restricted to well-defined water bodies where they can

be easily caught for identification. Reproduction may

depend on the availability and quality of these water

bodies. The sparse literature addressing Amazonian

tadpole identification is underdeveloped and often

conflicting. Proper identification therefore represents

a major hurdle in this type of investigation. We aimed

to address problems with tadpole identification while

measuring the effect of breeding habitat location via use

of artificial breeding habitats (ABHabs) (Gascon 1994).

Adult amphibians with aquatic larvae often occupy

drastically different environments from their tadpoles

(Werner and Gilliam 1984). Cruziohyla craspedopus

(Funkhouser, 1957) inhabit the high xeric environments

of forest canopy, but are known to descend to the forest

floor to breed (Rodriguez and Duellman 1994). Ranging

from northern Ecuador to southern Peru (Faivovich et al.

2005), C. craspedopus has since been found throughout

the Brazilian Amazon (Lima et al. 2003; Meneghelli

et al. 2011; Venancio et al. 2014). While classified as

Least Concern by the IUCN (Angulo et al. 2004), it is

elusive and existing data does not adequately represent

the population. In a comprehensive article, Hoogmoed

and Cadle 1991 established the reproductive mode of

C. craspedopus , with important updates by Block et

al. (2003) and Rodrigues et al. (2011): phytotelm “tree

hole” breeding involving logs, hollows, or depressions at

ground level (pitfall buckets).

Resetarits and Wilbur (1989) mentioned the

following factors influencing breeding habitat choice

and oviposition site: species present at the pond,

vegetation structure, pond age, temperature, and degree

of permanence. Following Pearman (1993), who found

varied responses to ABHabs across species in terms

of area and depth, we used a variety of sizes, ranging

from 0.5 L to as much as 400 L, to account for differing

preferences. This was the approach of our pilot study at

the Las Piedras Biodiversity Station (LPS), Tambopata

Province, Madre De Dios Department, Peru (UTM 19 L

442465 8667117, 265 m asl, see Crnobrna et al., in prep,

for site description). We placed five clusters of five plastic

containers evenly across the primary terra firma forest

landscape (Fig. 1). Clearing overhanging vegetation was

often required, so we built makeshift oviposition sites

Correspondence. Email : 1 CTurrell 1 @sheffteld.ac. nk, 2 tripamirgus@gmail. cony 3 marnie.smithbessen@my.jcu.edu.an

Amphib. Reptile Conserv.

January 2016 | Volume 10 | Number 1 | e112

Turrell et al.

441000 442000 443000

Fig. 1. Site map for ABHab points at LPS: dashed line is ap¬

proximate separation of terra firma and flood plain forest.

from sticks and situated them over the larger ABHabs.

We used tadpole descriptions by both Duellman (2005)

and Hero (1990) to aid us in identification. Where the

literature did not provide, or where the descriptions were

not adequate, we kept individuals until metamorphosis

and/or preserved specimens in 10% formalin (CORBIDI

field # FF2222).

Our most successful ABHab (unique code P-2.1) was

a blue cylindrical kiddie pool with a 25 cm plastic rim

surrounding a 150 cm diameter plastic sheet. We left

this particular ABHab in the “driest” forest available as

expressed by its vicinity to water: all known permanent

and ephemeral water sources were more than 250 m

away. We drew water from a palm swamp 360 m away

until the pool was approximately one half full (200 L), but

subsequent rain filled it to its maximum capacity (more

than 400 L). After set up on 28 August 2013 successive

checks during the end of the dry season returned no

observations of anuran breeding or tadpoles (Table 1).

We left the site unattended between November 2013

and January 2014, during which time rain intensity was

highest (160 cm, SENAMHI2014). On 26 January 2014

we found approximately 20 Cruziohyla craspedopus

tadpoles. Phyllomedusa tomopterna and Dendropsophus

sp. tadpoles were simultaneously using the ABHab. This

was the first positive check of P-2.1. In addition to the

tadpoles found, there were also multiple aquatic insect

species, and the remaining jelly of two empty egg masses

present on our makeshift overhanging oviposition sites

(Fig. 2A). While we cannot rule out P. tomopterna as the

source, repetition of this protocol at other sites returned

Phyllomedusa species. We could not, however, observe

such persistent, low-viscosity masses at any other site (n

= 25)—implying the original egg masses seen at P-2.1

were indeed laid by C. craspedopus.

On 26 January, we found live tadpoles in two Gosner

stages: stage 28 and stage 45 (Gosner 1960). At stage

28, the Cruziohyla craspedopus tadpole (Fig. 2B) had a

total length of 32 mm, a body length of 13 mm, width of

six mm and height of two mm; the interorbital distance

was five mm. The dorsum and tail were black. In some

individuals there was a slight yellow tint to the tail, and

in later stages small yellow specks on the dorsum. The

venter was a translucent purple, then white at very late

stages. The tail was pointed with a ventral fin equal in size

to the caudal musculature, and a slightly smaller dorsal

fin originating on the body. The greatest body width was

halfway down the body, and largest body height was

where the caudal musculature and the body meet. The

nostrils were a quarter of the distance from the snout to

the eyes. The eyes were yellow, situated dorsolaterally,

and visible from below. The spiracle was ventrolateral

and the oral disc bore a single row of marginal papillae

ventrally and laterally. The labial tooth row formula

(LTRF) observed was 2/2. However, both Duellman

(2005) and Hoogmoed and Cadle (1991) recorded the

LTRF to be 2/3, underscoring the difficulties in tadpole

identification.

We continued to monitor the site throughout 2014,

and in March another Gosner stage was observed (27).

Throughout April and May Cruziohyla craspedopus

tadpoles were present in the ABHab, presumably from

at least one more breeding event, although specimens

were not retrieved and “staged.” However, we saw no

diminishment in their numbers during this time, and no

newly metamorphosed or adult individuals were seen.

Delayed metamorphosis, to 100 days, could explain

Fig. 2. A) ABHab P-2.1 with empty egg mass; B) Tadpole of Cruziohyla craspedopus (preserved).

Amphib. Reptile Conserv.

January 2016 | Volume 10 | Number 1 | e112

Monitoring a population of Cruziohyla craspedopus

Table 1 . Timeline: P-2.1 ABHab

at Las Piedras Biodiversity Station

# coexistent size

classes of

C. craspedopus

larva

Gosner

stages

observed

Date

Activity

August 2013

Fauna Forever ABHab project starts at

Piedras Sation

28 August 2013

P-2.1 set up

n/a

September 2013

P-2.1 checked weekly

n/a

October 2013

final negative check, Fauna Forever

relocates to other field sites

n/a

December 2013

heavy rains begin, ABHabs at max.

volume

n/a

26 January 2014

First positive check, upwards of 20

tadpoles of 2 species, 2 emptied egg

masses

28, 45

30 January 2014

Most advanced stage tadpole collected

from P-2.1 metamorphoses in captivity

March 2014

positive check

May 2014

Fauna Forever relocates to Piedras

Station, begins to monitor P-2.1 nightly

not

collected

June 2014

Fauna Forever AHB project shuts down,

maintenance and checking of ABHabs ceases

these observations, but all documented metamorphosis

has been less than 100 days (Hoogmoed and Cadle

1991), making it a distant possibility in a typically water-

stressed species.

At P-2.1 we observed one breeding pair of Cruziohyla

craspedopus actively breeding in December and January.

Two details indicate that the number of individuals

involved was greater than the minimum two: 1) Multiple

egg masses as well as two distinct developmental and

size classes persisted at once, indicating staggered,

non-synchronous laying/hatching—a feat more easily

achieved by more than one female (Yeager and Gibbons

2013); 2) In past observations of C. craspedopus each

breeding habitat was attended by multiple individuals

including multiple males (Block et al. 2003; Hoogmoed

and Cadle 1991), which tended to congregate in typical

amphibian style. Considering that smaller, earlier stage

individuals were present in the ABHab into May of 2014,

the evidence suggests that at least three independent

breeding events took place involving many productive

females. We collected one individual in Gosner stage

45 on 26 January 2014, and it metamorphosed in our

captivity within four days (Fig. 3). Due to logistical

constraints we were unable to monitor the site during

this time, but according to the developmental scheme

demonstrated, a subset of 10-20 individuals would

have metamorphosed in late January. Asynchronous

metamorphosis remains a possibility, but undoubtedly

one well-placed artificial habitat contributed to the local

population of C. craspedopus. In such an understudied

species there is no way to know how much population

growth this represents, i.e., the individuals surviving

past metamorphosis weighed against naturally occurring

habitats’ size, hydroperiod, and predation.

Lamentably, even after repeated visits to P-2.1

at night, these tadpole observations represent the

only evidence of a breeding Cruziohyla craspedopus

population at LPS, regardless its productivity. Extensive

effort to find amphibians at LPS included, but was not

limited to, three years of coordinated surveys utilizing

100 m transects, call surveys, long opportunistic walks,

and time constrained searches of breeding habitats

(Cmobrna et al., in prep.). Despite this fact, only one

adult individual of C. craspedopus has ever been seen

at LPS, which was outside of herpetological fieldwork (a

solo night hike of devoted enthusiast Paul Rosolie, pers.

comm.). The well-documented difficulty in detecting the

species, in Madre De Dios and elsewhere, is reflected in

Duellman (2005), where almost 20 years of searching

found no C. craspedopus until a suitable breeding habitat

was discovered in 2000 (Block et al. 2003). Our results

indicate that while artificial habitats contribute to local

Amphib. Reptile Conserv.

January 2016 | Volume 10 | Number 1 | e112

Turrell et al.

Fig. 3. Newly metamorphosed juvenile Cruziohyla craspedo-

pus.

populations of C. craspedopus, they also appear to be

the only way to detect populations within logistical time

constraints and with any degree of certainty. The dual

advantage of this approach could be widely beneficial

to future surveys attempting to find this charismatic and

photogenic species.

It bears repeating that no natural habitat has been

encountered at LPS, which implies that the majority

of Cruziohyla craspedopus phytotelm breeding pools

are themselves in the canopy (Cmobrna and Turrell,

pers. obs.), and therefore can only be surveyed via

canopy access—a costly, training intensive, and at

times dangerous method. Similarly costly in training is

recognizing the advertisement call, which could confirm

the species on event of finding one near ground level. Call

surveys for C. craspedopus would need to be extremely

sensitive in picking up the soft and sporadic call (see

Read and Ron 2011), which would often be relegated to

anecdotal evidence.

The success of this ABHab was consistent with

Marsh et al. (1999) that degree of pond isolation was

more important than habitat quality. Although we spaced

ABHab points evenly throughout the forest, they only

numbered five and most were associated with available

aquatic habitats that the ABHabs would share species

with. P-2.1, however, was the only pool in unbroken terra

firma with no direct influence from any known water

body. We expected little to no amphibian occupancy on

the assumption that the pool was not in the vicinity of

any breeding sites visible from the ground. Yet even still

the pool harboured both common and elusive species.

This brings focus to the link between larval and adult

environments, which when broken can contribute to the

loss of amphibian diversity in imperilled ecoregions

(Becker et al. 2007). Although Cruziohyla craspedopus

populations are likely more abundant than indicated by

published records (Faivovich et al. 2005; Lima et al.

2003; Meneghelli et al. 2011; Venancio et al. 2014),

they are still subject to the specificity of their breeding

habitats—their availability limiting population growth.

It is worth noting that detection of C. craspedopus in

this way confirms its presence in primary forest, and one

could argue that this species can be used as a primary

forest indicator (Gardner et al. 2007; von May et al.

2010). Other “tree hole” breeders have been implicated

in deforestation and fragmentation studies (Zimmerman

and Bierregaard 1986; Ernst and Rodel 2008) because of

their perceived reliance on features of primary forests:

phytotelm structures of large trees. Future studies

should take into account oviposition parameters of C.

craspedopus in the event that the species’ absence in

ideally placed artificial habitats (Tocher et al. 1997;

Gagliardi 2008) and/or known naturally occurring

habitats does in fact indicate degraded forests.

Acknowledgments. —The Fauna Forever ABHab

project was the fruition of Chris Kirkby’s idea. Justin

Touchon generously donated the kiddie pools used. Erik

Wild and Matthew Tibbie reviewed early manuscripts.

Volunteer research assistants directly involved included

Chris Penker (Germany), Sarah Hrnyck (USA), Katrin

Luder (Switzerland), Tom Heather (New Zealand), and

Harry Turner (England). Special thanks to Andrew and

Amy-Jane Peabody. The Las Piedras Biodiversity Station

contributes to conservation efforts in Madre De Dios, and

the people directly responsible are JJ Durand, Paul Roso-

lie, and Mohsin Kazmi.

Literature Cited

Angulo A, Coloma LA, Ron S, Hoogmoed M, Castro F,

Rueda JV, Cisneros-Heredia D, Icochea J. 2014. Cru¬

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ened Species. Available: http://www.iucnredlist.org/

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Turrell et al.

Clare Turrell graduated from the School of Environmental Sciences at the University of East Anglia in 2013.

Ecology fieldwork in Kenya as part of this course inspired her to follow this passion and in 2014, Clare became a

herpetology intern for Fauna Forever.

Brian Crnobrna (sir-no-bur-na) graduated from the School of Environmental Studies (SES), otherwise known as

The Zoo School at the Minnesota Zoo, in 2001. Some other things might have helped him become the herpetofauna

coordinator for Fauna Forever starting in 2009, like serving as student reseach assistant in Peru and Namibia and

volunteering as a field biologist in Honduras, but SES was the most important. He’s currently being groomed for

grad school at Arizona State.

Marnie Smith-Bessen is a postgraduate student in the College of Marine and Environmental Sciences at James

Cook University. Her primary area of interest is the population ecology and conservation of herpetofauna in the

tropics.

Amphib. Reptile Conserv.

January 2016 | Volume 10 | Number 1 | e112

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [General Section]: 7-19 (e114).

Epidemiological surveillance and amphibian assemblage

status at the Estacion Experimental de San Lorenzo, Sierra

Nevada de Santa Marta, Colombia

v Luis Alberto Rueda-Solano, 2 Sandra V. Flechas, 'Maria Galvis-Aparicio, Andres A. Rocha-Usuga,

3 Edgar Javier Rincon Baron, 4 Borish Cuadrado-Pena, and 5 Rebeca Franke-Ante

1 Facultad de Ciencias Bdsicas, Universidad del Magdalena, Santa Marta, COLOMBIA 2 Departaniento de Ciencias Biologicas, Universidad de los

Andes, Bogota, COLOMBIA 2 Institute de Biologla, Universidad de Antioquia, Medellin, COLOMBIA 4 Barque Nacional Natural Sierra Nevada de

Santa Marta, COLOMBIA 5 Parques Nacionales Naturales de Colombia, Territorial Caribe, Santa Marta, COLOMBIA

Abstract. —Amphibian population declines and extinctions have occurred in conserved sites or

protected areas far from anthropogenic activities as a result of emerging infectious diseases such

as chytridiomycosis. Regular epidemiological surveillance, monitoring of key species, and the

implementation of biosecurity protocols are fundamental actions for the in-situ conservation of

amphibian fauna. Since 2008 biosecurity protocols have been implemented for all personnel that

enter the Estacion Experimental de San Lorenzo, a partly mountainous protected and conserved area

of the Sierra Nevada de Santa Marta with a high diversity of endemic and endangered amphibians.

Semiannual disease screenings of amphibians were carried out, as well as an amphibian inventory

and a survey of species of the genus Atelopus. To-date no mass mortality events have been reported

and Bd has not been detected. Nevertheless, some individuals of Ikakogi tayrona and Pristimantis

megalops showed symptoms of disease, the latter of which included individuals affected with

skin tumors. Deformities in individuals of Atelopus were also observed. The implementation of

epidemiological surveillance, monitoring of key amphibian species, and biosecurity protocols

are important strategies for the conservation management of the endemic amphibians within the

protected area of the Sierra Nevada of Santa Marta.

Key words. Anura, Atelopus , Pristimantis , tumors, chytridiomycosis, disease screening, mortality events, health, dis¬

ease, Batrachochytrium dendrobatidis , Bd

Citation: Rueda-Solano LA, Flechas SV, Galvis-Aparicio M, Rocha-Usuga AA, Rincon-Baron EJ,Cuadrado-Pena B, Franke-Ante R. 2016. Epide¬

miological surveillance and amphibian assemblage status at the Estacion Experimental de San Lorenzo, Sierra Nevada de Santa Marta, Colombia.

Amphibian & Reptile Conservation 10(1) [General Section]: 7-19 (el 14).

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 & Reptile Conservation ; official journal website <amphibian-

reptile-conservation.org>.

Received: 02 March 2015; Accepted: 09 June 2015; Published: 31 March 2016

Academic Editor and Translation: Mayra Oyervides, The University of Texas-Pan American, UNITED STATES OF AMERICA

Introduction

During the last decades amphibian population declines

and extinctions have been observed around the world,

causing concern from academic, scientific, and

governmental entities (Gascon et al. 2007; Mendelson

et al. 2006; Stuart et al. 2008). Close to 41% of

amphibian species worldwide are categorized under

some level of threat (Baillie et al. 2010; IUCN 2014)

and additionally about a quarter of amphibian species

are classified as Data Deficient (DD, IUCN 2014), which

makes it more difficult to determine the actual status

of populations. Colombia harbors approximately 215

threatened amphibian species, which represent slightly

more than a fourth of its entire amphibian fauna (Acosta-

Galvis 2014), making Colombia the country with the

Correspondence. Email: * biologohiisrueda@gmail.com (Corresponding author)

Amphib. Reptile Conserv.

March 2016 | Volume 10 | Number 1 | e114

Rueda-Solano et al.

greatest number of threatened amphibian species in the

world (IUCN 2014). Nevertheless, some species have

experienced serious declines while others remain stable.

In addition entire lineages have been affected almost to

the brink of extinction, as reported for the species of the

genus Atelopus (La Marca et al. 2005; Lotters 2007),

which in Colombia 76% (33 of 43) of the species are

categorized as Endangered and Critically Endangered

(IUCN 2014; Acosta-Galvis 2014).

Although habitat destruction continues to be the

main cause of population declines and extinctions

worldwide (IUCN 2014), it is puzzling that many species

have disappeared in well conserved, remote areas such

as primary forests in protected refuges (Crawford et

al. 2010; Crump et al. 1992; Lips et al. 2003; Pounds

et al. 2006). We now have a better understanding of

the pathogenic microscopic fungus Batrachochytrium

dendrobatidis ( Bd\ Longcore et al. 1999), which causes

the disease known as chytridiomycosis, one of the

leading factors behind mass mortality events previously

considered enigmatic. This fungus interferes with

the process of osmoregulation and affects electrolyte

balance, which often leads to the death of susceptible

individuals (Voyles et al. 2009). Furthermore, Bd seems

to inhibit the immune response of its hosts (Fites et al.

2013) which may present symptoms such as lethargy,

abnormal postures, and hyperemia (Berger et al. 2000;

Daszaketal. 1999).

In Colombia, there are about 565 protected areas

(RUNAP 2011), of these, 58 are administered by

the Parques Nacionales Naturales de Colombia, and

constitute approximately 11% of the continental territory

(PNNC 2015). These areas safeguard the country’s

biodiversity and represent a refuge for amphibian

assemblages, including endemic species or species with

narrow distributions, which may be susceptible to the loss

and fragmentation of their habitat. However, these areas

are still vulnerable to the threat imposed by Bd and other

emerging diseases. For example, it is known that for the

Parque Nacional Natural Gorgona, Bd has been present

for at least eight years (Flechas et al. 2012), however there

is no evidence of declines. Additionally, amphibians are

vulnerable to diseases produced by aquatic pathogens,

due to their dependency on aquatic environments. (Bosch

2003). For this reason authorities and administrators

of protected areas in Colombia consider emerging

diseases (especially chytridiomycosis) a challenge to the

protection of threatened amphibians.

The implementation of recurrent epidemiological

surveillance, monitoring of key amphibians species, and

biosecurity protocols, become fundamental to the in situ

conservation of amphibian assemblages in protected ar¬

eas. This way, early alerts are generated and can be used

to implement and carry out the best management prac¬

tices in a timely manner, thus reducing Bd (or disease)

outbreaks and transmission. This paper shows the imple¬

mentation of a pilot program (the first program of its kind

in the country) of these actions (surveillance, monitoring,

and protocols for amphibian species implemented for

disease control) set in a protected area, the Parque Na¬

cional Natural Sierra Nevada de Santa Marta, considered

one of the principle centers for amphibian endemism in

Colombia (Lynch et. al. 1997).

Materials and Methods

Study Area

The Sierra Nevada de Santa Marta (SNSM) was declared

a reserve of the Biosphere in 1979 by United Nations Ed¬

ucational Scientific and Cultural Organization (UNES¬

CO). Situated within three departments of the Colombian

Caribbean (Fig. 1, 2A.); 383,000 hectares belong to the

protected area (PNNC 2015). This area, comprises mul-

74M00"W

74°5‘0 , *W

74 e 0‘0'W

73 <, 55'0 , W

Fig. 1. Map of the Serrania de San Lorenzo, Sierra Nevada de Santa Marta, Colombia. Red square area highlights the Estacion

Experimental de San Lorenzo to 2,200 meters.

Amphib. Reptile Conserv.

March 2016 | Volume 10 | Number 1 | e114

Epidemiological surveillance and amphibian assemblage status

Fig. 2. Serrama de San Lorenzo (B) Querbrada San Lorenzo

(A) Sierra Nevada de Santa Marta, Colombia. Photographs by

Luis Alberto Rueda Solano.

tiple ecosystems, including dry and wet tropical forests,

sub-Andean and Andean forests, moors, and zones with

perpetual snow cover (PNNC 2015; ProSierra 2015).

With approximately 17 species of amphibians, 12 of rep¬

tiles, 14 of birds and one mammal, all of them endemic to

the area (PNNC 2015; ProSierra 2015), it is considered

one of the greatest centers of endemism in the country

and one of the irreplaceable protected areas of the world

(Le Saout et al. 2013; Lynch et al. 1997).

The study site is the Estacion Experimental de San

Lorenzo (11° 6’ 41.61” N 74° 3’ 17.13” W), located in

the Serrama de San Lorenzo, on the northwestern slope

of the SNSM, department of Magdalena, Colombia (Fig.

2A) at 2,200 m and comprising an area of400 ha. The sur¬

rounding vegetation is comprised of well conserved par¬

tially mountainous primary and secondary Andean for¬

est, which include tropical and subtropical rainforests of

the isomesothermic jungles (14 °C to 24 °C) (Hernandez-

Camacho and Sanchez-Paez 1992). The site’s native flora

includes woody species of Gustavia speciosa, Sloanea

sp. and some palm species of the genus Geonoma sp. and

Chamaedorea sp. (Cleef and Rangel 1984; Rangel and

Garzon 1995). Nevertheless, some hectares of non-native

vegetation are present. Coniferous forests with species

of Pinits patida and Cnpressns Insitanica introduced in

the early 90s, may influence the amphibian assemblage

that inhabit this area (Camero and Chamorro 1999). This

sector presents one rainy season, between April through

November, with a dry period between December through

March (Tamaris-Turizo et al. 2007). The mean annual

temperature is 12.8 °C, the mean annual precipitation is

2,446 mm and the relative humidity oscillates between

73-98% (Tamaris-Turizo and Lopez-Salgado 2006).

Epidemiological Surveillance

Since 2008, population census have been performed

employing the visual encounter survey (VES) meth¬

od (Rodda et al. 2001; Rueda et al. 2006; Heyer et al.

1994) in determining numbers of animals with clinical

signs of diseases, present deformities and/or individuals

found dead in the surrounding areas of the Estacion Ex¬

perimental San Lorenzo. Epidemiological surveillance

has been carried out through programmed visits every

six months by researchers and biology students from

the Universidad de Magdalena and through scheduled

or nonscheduled visits by staff of the Parque Nacional

Natural SNSM. We looked for anurans presenting leth¬

argy, macroscopic lesions, abnormal postures, hyper¬

emia, ulcers or the presence of fungi or other corporal

anomaly, such as deformities. Data was collected for all

individuals presenting clinical signs of a potential Bd in¬

fection. Swab samples from individuals were obtained

following the protocol described by Hyatt et al. (2007).

Frogs were captured using fresh disposable nitrile gloves

and held individually in bags until the sample was taken.

Each animal was swabbed by running a cotton swab ten

times over the ventral surface, the inner thigh area, and

the plantar surface for a total of 50 strokes. Cotton swabs

were kept dry and then stored at -20 °C until processing

of future laboratory analysis.

One single specimen of Pristimantis megalops with

clinical disease signs was analyzed using histological

methods. The samples of the skin areas affected with le¬

sions were fixed in FAA (Formalin alcohol-acetic) for

24^18 hr at 6 °C. Subsequently, dehydrated in graded se¬

ries of alcohols (30,40, 50, 60, 70, 80, 90, 95, and 100%)

and two cleared steps in xylene for two hours, then em¬

bedded in Paraplast Plus (Me Cormick®) for 12 hr at 55

°C (Luna 1968; Suvarna et al. 2012). Transverse and lon¬

gitudinal sections were obtained with a rotary microtome

Leica ® model (RM2125) set to 4-5pm thick. These were

stained with hematoxylin-eosin for general descriptions

and were previously stained using the Van Gieson tech¬

nique with alcian blue and Gill III hematoxylin (changes

made by the authors) to demonstrate collagen in the con¬

nective tissue. The sections were examined under a light

microscope Nikon Eclipse Ni-U® equipped with differ¬

ential interference contrast (CDI). The photographs were

obtained with DS-Fi2® Nikon digital camera using the

NIS Elements of Nikon software version 3.07. The image

processing was performed with Image-Pro Analyzer 6.3

program (Media Cybernetics). These analysis were car¬

ried out in the biology laboratory of the Universidad de

March 2016 | Volume 10 | Number 1 | e114

Amphib. Reptile Conserv.

Rueda-Solano et al.

Antioquia (Medellin, Colombia) and photographs taken

in the Laboratory of Biotechnology of the Sede de Inves¬

tigation Universitaria (SIU-UdeA).

Furthermore, a single specimen of Ikakogi tayrona

with clinical disease signs was analyzed using conven¬

tional Polymerase Chain Reaction (PCR) to determine

Bd presence. DNA was extracted from swabs using Ge-

neReleaser® (Bioventures Inc., Carlsbad, California,

USA). We used the primers developed by Annis et al.

(2004) to amplify the ITS1-ITS2 region specifically in

B. dendrobatidis: Bd\ a (5 ’ -CAGTGTGCCATATGT-

CACG-3’) and Bd2a (S’-CATGGTTCATATCTGTC-

CAG-3’). Amplifications were performed in an MJ Re¬

search Peltier Thermal Cycler (PTC-200), as follows: an

initial two minute denaturation at 95 °C followed by 35

cycles of DNA amplification (i.e., 45 sec at 95 °C, 45 sec

at 55 °C, and one min at 72 °C). A final extension at 72

°C for 10 min completed the amplifications. Each reac¬

tion consisted of 0.5 pL of each primer (1 M), 3.0 pL of

doubly distilled DNA-free water, 6 pL of GoTaq® Green

Master Mix (IX; Promega), and 2 pL of the DNA extract.

The amplified fragments were separated by electrophore¬

sis through 1% agarose gels. These analysis were carried

out in the genetic laboratory of the Universidad de los

Andes (Bogota, Colombia).

Biosecurity Protocols

To date the presence of Bd has not been reported at Ser-

rania de San Lorenzo. To prevent and reduce the risk of

transmission of Bd , since 2008 park administrators from

SNSM have been implementing a biosecurity proto¬

col for all foreign and national personnel that enter the

Estacion Experimental de San Lorenzo. The protocol

consists of disinfection of field equipment (boots, nets,

measuring devices) used as much by researchers as by

tourists who visit the protected area (Phillott et al. 2010).

Nevertheless, as a preventive measure in situ , field

boots are washed with approximately 200 mL of a com¬

mercial sodium hypochlorite solution diluted in three li¬

ters of water.

Amphibian Assemblage

To describe the amphibian assemblage from the San Lo¬

renzo sector, an inventory was carried out during seven

surveys throughout the months of October and Novem¬

ber of 2008, March of 2009, April and October of 2013,

and April and November of 2014. No surveys were made

in the years 2010-2012. Each field trip had a duration

of four days, and the study site was surveyed for eight

hours daily, employing the VES method in diurnal peri¬

ods (9:00-12:00 and 15:00-17:00 hrs) and nocturnally

(18:00-21:00 hrs). During the surveys, for each individ¬

ual data associated with the habitat and time of day of

the observation were registered. The number of research¬

ers varied among surveys (from two to seven), and thus

each survey had different effort levels (between 16 and

80 hours x person). To determine the relative abundance

(RA) of different species in the sector, a classification of

Table 1. Epidemiological surveillance from the year 2008 to 2014 for each of the reported amphibians within the sector of the ex¬

perimental station of San Lorenzo, 2,200 m altitude. Sierra Nevada of Santa Marta, Northern Colombia. Very rare (VR); Rare (R);

Common (C); Abundant (A); Very Abundant (VA).

Species

Microhabitat

Habit

N° diseased

individuals

Disease

type

N° de¬

formed

individuals

N°dead

individuals

Year of

reported

sick indi¬

vidual

Atelopus laetissimus

128

Terrestrial/

Shrubs

Nocturnal

Cutaneous

ulcers

2013

Atelopus nahumae

Terrestrial/

Shrubs

Diurnal/

Nocturnal

—

2013

Ikakogi tayrona

Shrubs

Nocturnal

Undeter¬

mined

2008

Pristimantis delicatus

Shrubs

Nocturnal

—

Pristimantis carmelitae

Terrestrial

Nocturnal

—

Pristimantis cristinae

Shrubs

Nocturnal

—

Pristimantis insignitus

Terrestrial

Nocturnal

—

Pristimantis megalops

477

Terrestrial

Diurnal/

Nocturnal

Fibropap-

illoma

(tumors)

2008;

2013;

2014

Pristimantis ruthveni

Rare

Shrubs

Nocturnal

—

Pristimantis sanctaemartae

211

Shrubs

Nocturnal

—

Pristimantis tayrona

Phytotelmata

Nocturnal

—

Pristimantis sp. nov. 1

Shrubs

Nocturnal

—

Pristimantis sp. nov. 2

Terrestrial/

Shrubs

Nocturnal

—

Bolitoglossa savagei

Shrubs/Phyto-

telmata

Nocturnal

—

Amphib. Reptile Conserv.

March 2016 | Volume 10 | Number 1 | e114

Epidemiological surveillance and amphibian assemblage status

Fig. 3. Healthy individuals of Ate/opus laetissimus (A) (Bufonidae); Atelopus nahumae (B) (Bufonidae); Pristimantis megalops (C)

(Craugastoridae); and Ikakogi tayrona (D) (Centrolenidae). Photographs by Luis Alberto Rueda Solano.

very rare, rare, common, abundant and very abundant,

according to the of individuals (ind.) recorded during all

the surveys was established. Species were very rare, if it

was observed equal to or less than nine ind.; rare, if it was

observed between 10-20 ind.; common, if it was observed

between 21-30 ind.; abundant if it was observed between

31-50 ind.; and very abundant, if it was observed over

50 ind. in the total of all surveys. For identification of the

species belonging to the genus Pristimantis we employed

the synopsis of Lynch and Carranza (1985) and for the

remaining species we used the information provided by

the American Museum of Natural History from its online

reference Amphibian Species of the World (Frost 2014).

Monitoring of Atelopus laetissimus and

Atelopus nahumae

Due to the importance represented by the species of At-

elopus, for being one of the most affected genus for Bd

(La Marca et al. 2005; Lotters 2007), we monitored both

species reported at this locality, A. laetissimus and A. na-

humae (Ruiz-Carranza et al. 1994). These species were

monitored during the same months of the amphibian in¬

ventories of the sector. Nevertheless, these surveys were

done during two additional days. Employing a low lying

transect of 50 m in length and five m wide over the stream

known as “La Quebrada San Lorenzo” located about 500

m north of the Estacion Experimental de San Lorenzo

at 2,100 m (Fig. 2B). Surveys were done twice daily for

individuals of Atelopus , in the morning (9:00-12:00 hrs)

and at night (18:00-00:00 hrs). For individuals of Atelo¬

pus, SVL, weight, and sex have been systematically re¬

corded since 2013. Each individual was handled with a

new pair of gloves as part of the biosecurity protocols to

prevent the transmission of Bd.

RESULTS

Epidemiological Surveillance

To date no mass mortality events have been reported, or

individuals with field evidence of Bd infections in the as¬

semblage of the 15 endemic species of amphibians at the

San Lorenzo sector, corresponding to 1,375 ind. mostly

healthy individuals of Bufonidae, Craugastoridae, Cen¬

trolenidae, and Plethodontidae (Table 1, Fig. 3). How¬

ever, 13 sick individuals of the species Ikakogi tayrona ,

Atelopus laetissimus , A. nahumae , and Pristimantis meg-

alops were recorded (Table 1, Fig. 4).

The sick individual of Ikakogi tayrona presented

symptoms similar to those of chytridiomycosis, such as

lethargy, pale skin, and hyperemia of the ventral skin

(Fig. 4A, B). Nevertheless, the conventional PCR labora¬

tory analysis yielded negative results for Bd. Pristiman¬

tis megalops , presented the greatest number of sick in¬

dividuals (Table 1), they were affected by white colored

tumors, bulgy and with a hard consistency (Fig. 4C, D).

Healthy skin of P. megalops is smooth and dark-brown in

color (Fig. 4, 5), while the area associated with the tumor

was hyperplastic with white coloration (Fig. 5A). The

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Rueda-Solano et al.

Fig. 4. Sick individuals of Ikakogi tayrona (A, B same individual); Pristimantis megalops (C, D), and malformation in Atelopus

nahumae (E) and Atelopus laetissimus (F) found in epidemiological surveillance 2008-2014 in La Estacion Experimental de San

Lorenzo (2,200 meters), Sierra Nevada de Santa Marta, North of Colombia. Photographs by Luis Alberto Rueda Solano (A, B, D),

Cesar Molina (C); Andres Rocha Usuga (E, F).

epidermis of the areas that are not associated with tumors

is thin with few cell layers and is delimited by a layer of

strongly pigmented skin cells (Fig. 5B). The dermis is

thick and has large collagen layers that are joined closely

to the underlying striated muscle through connective tis¬

sue (Fig. 5B). Neoplasia histologically corresponds to

a skin fibropapilloma or fibroma, formed mainly of fi¬

broblasts and marked anisocariosis characterized to be

heavy vascularization (Fig. 5C, D). The fibropapilloma is

white in color because the pigmented layer of the dermis

present in healthy skin disappears completely (Fig. 5E,

F). Also, dermal collagen layers are less compact, unor¬

ganized, and not associated with the muscle due to the

profuse growth of fibroblasts in the tumor (Fig. 5F). The

epidermis associated with neoplasia can present areas of

few cell layers similar to healthy skin or otherwise be

hyperplastic (Fig. 5F). Despite these individuals present¬

ing cutaneous disease, they did not present symptoms of

lethargy or malnutrition. No other similar characteristics

pertaining to these tumors were observed in other species

of the assemblage.

Regarding Atelopus laetissimus only one individual

presented a cutaneous ulcer on the abdomen, and two in¬

dividual of A. laetissimus and A. nahumae were found

with malformations of the extremities and face (Fig 4

E, F). Skin swab samples were collected for future Bd

analysis for Atelopus species. In 2013 we collected 11

samples for A. laetissimus and one sample for A. nahu¬

mae and in 2014, 13 samples for A. laetissimus and four

for A. nahumae were collected. These samples were de¬

posited dry and cool (-20 °C) at the biology laboratory of

the Universidad de Magdalena.

Amphibian Assemblage

All species for which epidemiologic surveillance was

carried out at the sector of San Forenzo are endemic to

the SNSM. The majority of individuals (61.5%) belong

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March 2016 | Volume 10 | Number 1 | e114

Epidemiological surveillance and amphibian assemblage status

Fig. 5. Healthy skin and fibropapilloma in Pristimantis megalops (A). Cross section of the healthy skin of Pristimantis megalops

(B): The epidermis is thin, can be seen pigmented layer and layers of collagen in the dermis. Cross section of Fibropapilloma (C).

Detail of fibroblasts forming fibropapilloma (D): the tissue is highly vascularized. Detail of the epidermis and collagen layers

covering fibropapilloma (E-F). Histochemical staining in Fibropapilloma (F): The layers of collagen in the dermis can be seen in

magenta color, fb: fibroblasts; col: layers of collagen; de: dermis; ep: epidermis; gla: dermal glands; hs: healthy skin; lp: pigmented

layer; sm: striated muscle tissue; tu: tumor or fibropapilloma; vt: vascular tissue. Photographs by Edgar Javier Rincon Baron.

to the genus Pristimantis, with ten species (of 15 in total);

dominating in abundance, two of these are in the process

of being described (Table 1). Pristimantis megalops and

P. sanctaemartae were the most abundant. In this sector

both genera of endemic and monotypic species Ikakogi

tayrona and Geobatrachus walkeri were recorded. Only

one species of salamander was observed, Bolitoglossa

savagei with a moderate abundance (Table 1). Similarly,

both described species of Atelopus for this sector were

reported, A. laetissimus are more abundant; conversely

individuals of A. nahumae were rarely observed (Table

1 ).

Monitoring of Atelopus laetissimus and

Atelopus nahumae

In relation to the monitoring of the Atelopus species at

the San Lorenzo stream, 128 records of A. laetissimus

were obtained over the course of all the surveys, with

an annual mean of 18 ind. (n = 7 surveys SD ± 5.46). In

2008 a mean of 19.5 ind. (n = 2 surveys; SD±2.12) (Fig.

6), in 2009 only one survey was done in which nine ind.

were recorded (Fig. 6). From the year 2010 through 2012

no monitoring was done at the San Lorenzo stream. In

2013 a mean of 16 ind. (n = 2 surveys SD ± 1.41) of A.

laetissimus, making this the year with the fewest number

of records in comparison to other years (Fig. 6). Finally,

in the year 2014 the greatest mean was recorded with 24

ind. {n m2 surveys; SD ± 2.82) (Fig. 6). Although the

sequence of years is incomplete, the tendency in the past

two years has been a slight increase in the number of ob¬

servations of A. laetissimus individuals (Fig. 6).

Around 90% of the records of A. laetissimus observed

have been males, principally in the night hours perched

on leaves. Males had a snout-vent-length (SVL) and av¬

erage weight of 4.05 cm (n = 80 SD ± 0.46) and 4.87 g

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March 2016 | Volume 10 | Number 1 | e114

Rueda-Solano et al.

(,n = 80 SD ± 1.65) respectively. Only 11 females were

observed, with SVL and weight averages of 5.0 cm {n =

11 SD ± 0.46) and 6.5 g (n = 11 SD ± 1.23) respectively.

For A. nahumae , 10 individuals were recorded, two male

individuals in the year 2008, one female in the year 2013

and three males and four females in 2014. Due to the

scarce observations it was not possible to discern a popu¬

lation trend.

Discussion

Epidemiological surveillance in the amphibian assem¬

blage of the San Lorenzo sector showed a low number

of individuals with symptoms of disease and no dead in¬

dividuals were found over the years 2008, 2009, 2013,

and 2014. This contrasts with other localities in protected

areas and nonprotected areas of the American continent

in countries such as Canada (Greer et al. 2005), Unit¬

ed States (Green et al. 2002), Costa Rica (Crump et al.

1992; Lips and Papendick 2003), Panama (Crawford et

al. 2010; Lips et al. 2006; Lips 2003), Venezuela (Bo-

naccorso, Guayasamin, Mendez, and Speare 2003), and

Ecuador (Bustamante et al. 2005; Merino-Viteri et al.

2005), where mass mortalities and population and am¬

phibian assemblage collapses have been reported due to

emergent diseases.

Only one mass mortality event has been reported in

Colombia in the year 1997 in Serrania de los Paraguas

between the departments of Choco and Valle del Cauca

(Lynch and Grant 1998), nevertheless anecdotal infor¬

mation shows the absence of some amphibian species

in other localities which were previously more diverse

and abundant, for example the Parque Nacional Natural

Chingaza (A. Amezquita and C. Navas, pers. comm.).

This would not be the case for the amphibian assem¬

blage of the protected area of the San Lorenzo sector,

given that all the species reported have historical type

localities from this mountain range (Lynch and Carranza

1985) including the species of Atelopus (Ruiz-Carranza

et al. 1994) which would be vulnerable to declines due to

chytridiomycosis (Lips et al. 2003). Additionally, we did

not find evidence that any of these species are currently

experiencing population declines. On the contrary, in this

study new species were reported increasing the diversity

of endemic amphibians for this locality and for the bio¬

geographic region. Although, some anuran species still

inhabit this mountain range, we could not survey them

occurring in remote locations with restricted access; this

is the case of Atelopus arsyecue , A. walkeri , and A. car-

rikeri (Rueda-Solano 2008). Therefore, our conclusions

only focus on the populations of the protected area of the

Serrania de San Lorenzo, for other localities we do not

know the conservation status of amphibian assemblages.

Although biosecurity protocols are an important aspect

in the conservation of endemic amphibians, this study

does not formally test or validate their decontamination

protocols in this protected area.

In the special case of sick individuals found through¬

out the epidemiologic surveillance, the signs of illness

of the individual of Ikakogi tayrona were very similar to

those presented by individuals affected by chytridiomy¬

cosis (Daszak et al. 1999). Nevertheless, with negative

Bd results the possibility exists that the signs detected in

the field may be misinterpreted as typical of chytridio¬

mycosis, when in reality are part of the symptomatology

of other diseases which have not been previously diag¬

nosed. However, our results are limited mainly to the de¬

tection of signs of chytridiomycosis in the field, it is al-

26 -

8 -1-1-1-r

2008 2009 2013 2014

Fig. 6 . Monitoring of Atelopus laetissimus through the years in the Quebrada San Lorenzo Serrania de San Lorenzo, SNSM. Circle =

average number obtained from individuals in each year (w = 2 samples for the years 2008, 2013 and 2014) (w = 1 sample for 2009);

Error Bars = maximum and minimum individual in each year. Dotted line = trend in the number of individuals over time.

Amphib. Reptile Conserv.

March 2016 | Volume 10 | Number 1 | e114

Epidemiological surveillance and amphibian assemblage status

ways recommended to corroborate the field observations

with laboratory analysis for accurate detection of Bd.

In respect to the sick specimens of Pristimantis meg¬

alops , we are unaware of any reference regarding the

symptoms presented by these individuals in Colombia,

however similar neoplastic diseases are documented in

others amphibian species (Green and Harshbarger 2001;

Khudoley and Mizgireuv 1980). The origin of these spon¬

taneous neoplasms are still unclear and often limited to

specific species or populations (Stacy and Parker 2004),

as a matter of fact; P. megalops was the only species in

the assemblage that presented this cutaneous anomaly.

Similarly, we are unaware if these tumors may be lethal

for these individuals, above all because the individuals

reported to have these tumors did not appear to have af¬

fected locomotion or nutritional state. It is necessary to

conduct more studies about the tumor effects on these in¬

dividuals, at the physiological, behavioral, and ecologi¬

cal level. Additionally, it should be corroborated whether

this disease is unique to P. megalops or it can be found

among populations of amphibians at different localities.

Historically the populations of Atelopus laetissimns

and A. nahumae found at the Serrania de San Lorenzo

have been abundant (Carvajalino-Fernandez et al. 2008;

Granda-Rodriguez et al. 2008,2012; Ruiz-Carranza et al.

1994). Nevertheless, at our study site, A. laetissimus is

predominantly abundant, while A. nahumae is ecologi¬

cally rare, it may be due to our surveys coincide with

the upper extreme of its altitudinal distribution. This is

corroborated at lower localities (approx. 1,500 m) where

this species is predominantly abundant and A. laetissimus

is ecologically rare (Carvajalino-Fernandez et al. 2013;

Rueda-Solano, pers. observ.). In relation to the abun¬

dance of A. laetissimus at the San Lorenzo stream, we

do not have sufficient data to estimate a population trend,

nevertheless, in the last two years of monitoring (2013

and 2014) we have found a similar abundance, which

may suggest a stable population for this species in the

sector of the Sierra Nevada de Santa Marta. This conclu¬

sion coincides with previous studies which show relative

abundances of A. laetissimus and A. nahumae (Carvajali¬

no-Fernandez et al. 2008; Granda-Rodriguez et al 2008,

2012). At present A. laetissimus and probably A. nahumae

have stable and abundant populations, this strongly con¬

trast with the problematic declines and extinctions of the

entire Atelopus clade in the neotropics principally those

which inhabit high elevations (La Marca et al. 2005; Lot-

ters 2007). Nevertheless, we are unaware of the historical

population trend for these species, especially during the

years where reported declines were in Colombia (Lynch

and Grant 1998), where Bd may have caused declines in

these species, however it was not documented and may

currently coexist with Bd, as occurs for other species of

Atelopus (Flechas et al. 2012; Tarin et al. 2014), or in

other amphibian communities where there is a higher

prevalence of Bd and no evidence it affects the natural

populations (Guayasamin et al. 2014). We suggest ret¬

rospective studies including historical demography and

Bd diagnosis using museum specimens (Rodriguez et al.

2014; Cheng etal. 2011).

Similarly, the majority of records for Atelopus la¬

etissimus correspond to male individuals found active

during nocturnal surveys, perched on leaves of bushes

adjacent to streams. These observations coincide with

historical data, in similar proportions (Granda-Rodriguez

et al. 2008). In respect to females of these species, we

can only deduce they possess different home ranges than

males and encounters occur during reproductive seasons.

Our results provide strong evidence about the nocturnal

habits of the males of this species, which would employ

leaves to perch and forage from (Rueda-Solano, pers. ob¬

serv.). These observations are inconsistent with almost

all the species of the genus Atelopus, which are diurnal

(Lotters 1996), with one exception previously described

for Atelopus nocturnus (Bravo-Valencia and Rivera-Cor-

rea 2011).

Conclusion

The implementation of epidemiological surveillance,

monitoring of key amphibian species, and biosecurity

protocols at the San Lorenzo area have been constituted

as important strategies for the conservation management

of the endemic amphibians within the protected area of

the Sierra Nevada of Santa Marta. It is expected that

these actions be sustained and replicated at other pro¬

tected areas in Colombia and the world, with amphib¬

ian assemblages susceptible to Bd. At a minimum they

should serve as a baseline in establishing amphibian con¬

servation methods and best management practices for

in-situ programs. In like manner these actions should be

complemented with the utilization of laboratory methods

for the detection of Bd and other diseases.

Acknowledgments. —This work was the result of

joint efforts of several years among researchers, students

of the Universidad de Magdalena and administrators of

the Parques Nacionales Naturales de Colombia Author¬

ity. We are grateful to the Conservation Leadership Pro¬

gram for the financial support to the Atelopus Project Co¬

lombia and to the administrators of Parques Nacionales

Naturales of Colombia Territorial Caribe for their logis¬

tic support in our field trips. Gratitude is extended to the

ProAves foundation for allowing amphibian monitoring

work at the Reserve El Dorado and to the students of

Herpetology from the Universidad de Magdalena from

the years 2013 and 2014 for their valuable assistance

in the field. Thanks to Katiuska Rueda Solano and Pi¬

lar Ximena Lizarazo Medina for their help in this work

and Eudes Alfonso Vaca Blanco, as the person in charge

of overseeing the sector, maintaining the process of dis¬

infection in the park. Lastly but not least, we thank the

administration of the Parque Nacional Natural Sierra Ne¬

vada de Santa Marta for continuing their interest in epi-

Amphib. Reptile Conserv.

March 2016 | Volume 10 | Number 1 | e114

Rueda-Solano et al.

demiological surveillance and the timely understanding

of environmental factors and anthropogenic ones which

may influence wildlife conservation.

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Luis Alberto Rueda-Solano is a young biologist specializing in herpetology. His interests include ecology,

ecophysiology, behavior, and conservation of amphibians and reptiles. Luis graduated from Magdalena Uni¬

versity (Santa Marta, Colombia), and holds a Masters degree in biological sciences from Andes University (Bo¬

gota, Colombia). Currently, he works at Magdalena University where he is developing various herpetological

projects that promote knowledge and conservation of Colombian Caribbean herpetofauna.

Sandra Victoria Flechas is a graduate student at the Biological Sciences Department at Universidad de los

Andes in Bogota (Colombia). Her research focus in describing the current distribution of the fungal pathogen

(Batrachohytrium dendrobatidis, Bd) in Colombia and understanding factors that explain the presence of Bd

in the country. In addition, she studies the effect of the pathogen in populations of harlequin frogs of the genus

Atelopus. She is interested in the role of bacterial symbionts as a mechanism to help amphibians to counteract

the devastating effects of Bd.

Amphib. Reptile Conserv.

March 2016 | Volume 10 | Number 1 | e114

Epidemiological surveillance and amphibian assemblage status

Maria Alejandra Galvis Aparicio is a student of biology at the University of Magdalena and belongs to

Group Herpetologico. Currently, she is a fellow of the Conservation Leadership Programme (Atelopus Project

Colombia) and a fellow of the Colombian Association of Herpetology. Her research interests include microbiol¬

ogy and pathogy (especially, emerging diseases) that affect amphibians and reptiles, particularly the endemic

anurans of the Sierra Nevada de Santa Marta. She works on projects related to epidemiological monitoring of

Batrachochytrium dendrobatidis and other pathogens of frogs in the Serrania de San Lorenzo, Sierra Nevada

de Santa Marta.

Andres A. Rocha Usuga is a young student of biology at the University of Magdalena (Santa Marta, Colom¬

bia) with a special emphasis in herpetology. His research interests includes the behavioral ecology of frogs,

focusing on the reproductive biology of frogs and their conservation. He has participated in several research

projects that have served as a hub for the conservation of endangered species. He is currently studying as an

undergraduate thesis on the reproductive biology of Atelopus species.

Edgar Javier Rincon Baron holds a Ph.D. in biological sciences and is a professor at the University of An-

tioquia in the subjects of botany, plant anatomy, and microtechnology. Edgar manages and advises research

with applications in scanning electron microscopy, transmission, and confocal microscopy. He directs several

undergraduate and graduate work studies related to the processing and interpretation of section histology es¬

pecially from plants and animals. He specializes in biology plant breeding and the taxonomy of Monilophyta

and Lycophyta.

Borish Cuadrado-Pena is a biologist (University of Magdalena), with M.Sc. in Environmental Science (em¬

phasis Hydroclimatology). Borish has worked in PNN Sierra Nevada de Santa Marta since 2006 to 2014 coor¬

dinating activities such as ecological restoration, sustainable systems for conservation, research, monitoring,

and management of wildlife.

Rebeca Franke-Ante is a biologist (emphasis zoology), with a M.Sc. in biology (emphasis marine biology),

specializing in the territorial NPSC Caribbean, with support in protected areas of the Colombian Caribbean.

Rebeca’s topics of interest include monitoring, research, wildlife management, climate change, and connectiv¬

ity with restoration interests in wetlands, migration, infectious diseases, conservation, diversity, fish, and birds..

Amphib. Reptile Conserv.

March 2016 | Volume 10 | Number 1 | e114

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [General Section]: 20-27 (e116).

PERSPECTIVES

Tadpoles, froglets, and conservation:

A discussion of basic principles of rearing and release

procedures

^Joseph R. Mendelson III and 3 Ronald Altig

1 Department of Herpetology’, Zoo Atlanta, 800 Cherokee Avenue SE, Atlanta, Georgia 30312, USA 2 School of Biology’, Georgia Institute of

Technology’, 301 Ferst Drive, Atlanta, Georgia 30332, USA 3 Department of Biological Sciences, Mississippi State University, Mississippi State,

Mississippi 39762, USA

Abstract .—We outline component features of the captive environment and the natural world that

should be considered when designing a program for head-starting and releasing amphibians,

presumably as part of a conservation project. The main points indicate the importance of accounting

for features of the basic biology of amphibian larvae, the biology of the focal species, and highlight

the types of error risks based on generalities, human convenience, and logistical limitations.

Similarly, we urge consideration and evaluation of the quality of the metamorphs that are produced

over the sheer quantity produced and released. While most of the examples are taken from pond¬

breeding species, the general principles are relevant, and details may be modified to fit amphibian

species with larvae in other habitats.

Key words. Amphibians, conservation, larvae, reintroductions, translocations, head-start, captive breeding

Citation: Mendelson III JR, Altig R. 2016. Tadpoles, froglets, and conservation: A discussion of basic principles of rearing and release procedures.

Amphibian & Reptile Conservation 10(1): 20-27 (el 16).

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: 08 November 2015; Accepted: 17 March 2016; Published: 22 April 2016

Captive rearing of endangered frogs is a large, expensive

(Mattioli et al. 2006), and time-consuming component

of conservation efforts. We discuss a number of factors

intended to improve the chances of successful rearing

and release of captive individuals (McGregor and Zip-

pel 2008). Central themes are 1) acceptance that captive-

reared animals differ in a number of ways from those

from natural populations (e.g., Griffiths and Pavajeau

2008; Gawor et al. 2012); 2) rearing protocols must ad¬

dress the specific biology of each species (e.g., Morrison

and Hero 2003); 3) avoidance or reduction of activities

that cater only to our conveniences; and 4) evaluation of

criteria to judge success based on metrics other than just

numbers released. We emphasize the need for long-term

monitoring of the success of the releases. Most sugges¬

tions center around testing, improving, and standardizing

species-specific procedures once those that produce in¬

dividuals of the highest probable fitness are verified, not

just the most individuals. Tadpole mortality varies across

experimental venues (Melvin and Houlahan 2012), so

survival and fitness likely vary according to husbandry

regime, release protocols, and even captive breeding it¬

self.

We argue that each taxon-specific system should di¬

rectly address several types of questions. Do artificial

environments and particularly the food sources used in

captive programs alter reared froglets relative to what

wild individuals experience (i.e., rapid acclimation to

captivity of Griffiths and Pavajeau 2008)? Do these al¬

terations adversely manifest themselves in the survival

and fitness of 1) the released animals; 2) the popula¬

tion in which the animals are released; or 3) the meta-

Correspondence. Email: l jmendelson@zooatlanta.org (Corresponding author); 3 r altig@biologyy.msstate.edu

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e116

Mendelson and Altig

populations with which they interact? The real question

is: do the introductions contribute in a beneficial way to

conservation, or is there the appearance of conservation

(through numbers) when actions actually degrade the fit¬

ness of the population (e.g., via artificial selection)? How

do the morphological, immunological (Venesky et al.

2012), and hematological (Davis and Maerz 2011; Davis

2012) conditions of reared froglets compare to those of

wild individuals? For example, Bums et al. (2009) found

that the first-generation of captive-bred and reared gup¬

pies had smaller brains than wild individuals, and Fraser

(2008) noted that re-introduced leopard frogs showed ab¬

normal behaviors in the wild. It must be recognized that

captive breeding, often with limited choices of mates and

usually with small numbers of individuals, constrains or

eliminates the possible beneficial effects of sexual se¬

lection (Williams and Hoffman 2009). Captive rearing

of larvae from the wild may avoid problems related to

sexual selection, but nonetheless impose selective pres¬

sures on the offspring. Because fisheries researchers have

faced these problems for years, they have a good per¬

spective based on the pitfalls of releasing captive-reared

individuals into the wild. Studies from fisheries science

have indicated that the quality or fitness of captive-reared

individuals are not necessarily equivalent to those of wild

individuals (e.g., Araki et al. 2007; Christie et al. 2012).

Amphibian conservationists would be advised to review

the references cited herein and create means of measur¬

ing and assessing the biological quality of wild and cap¬

tive tadpoles.

Within these contexts, we recognize the limitations of

funding, the expectations of administrators, and the ex¬

pertise of personnel sometimes can work against the suc¬

cess of a given program. Stakeholders and participants

in conservation programs may be pressured to, or evalu¬

ated on, the release of as many individuals as possible, as

soon as possible, with minimal cost. These realities can

lead to the implementation of inordinate or biologically

inappropriate actions that may not increase the chance

of the timely release of more viable individuals. For ex¬

ample, we have heard comments such as “ Ido not under¬

stand. Some of them took off swimming across the pond

and some of them got balled up in sand like they did not

know what was going on.” In fact, reared individuals may

not perform successfully at basic tasks like prey recogni¬

tion or seeking refuge, but they can be given a chance

to leam (i.e., pre-release training of Griffiths and Pava-

jeau 2008, or the “soft-release” concept as it is termed in

some conservation programs). Other statements that we

have overheard include “ That skinny tadpole likes to lie

on its side,” “I do not know why that tadpole swims in a

spiral, ” “These tadpoles constantly swim up and down

the wall of the aquarium” All of these cases describe

tadpoles that either are sick, stressed, or otherwise un¬

suitable for release, and examples of at least the first two

cases should certainly be culled to improve the collective

quality of the cohort (e.g., Nye and Cameron 2005).

We looked at two kinds of relevant information from

the natural world to get a relative idea of what is faced in

terms of the odds of survival in amphibian breeding. This

perspective is crucial for evaluating captive programs,

but seems to be poorly considered by practitioners. Some

stakeholders we have observed seem to evaluate pro¬

grammatic success by trying to maximize the number of

metamorphic individuals that are released. Survival rates

from egg to metamorphosis range from 0-20 % and are

commonly 1-5 % (Wells 2007: table 14.5, fig 14.9). Sur¬

vival from metamorphosis to first reproduction ranges

from 6-26% (Herreid and Kinney 1966; Licht 1974).

Greenberg and Tanner (2005) tracked the success of 23

breeding events of Scaphiopus holbrookii at eight sites in

Florida over nine years; five of these events were consid¬

ered successful by producing a minimum of 100 meta-

morphs likely derived from >107 eggs deposited on site.

Semlitsch et al. (1996) reported only one reproductive

event at one site that produced significant numbers of

metamorphs of S. holbrookii over 16 years. High levels

of mortality are typical of many amphibian reproduction

efforts, and efforts of husbandry to avoid such mortality

may not be desirable.

Survival and fitness are correlated with environmental

conditions and diet. Dietary requirements of metamorphs

may be easily underestimated. For example, a grand co¬

hort of 7,000 (1,000 each of seven species) metamorphs

can consume at least 2.3 x 10 6 insects the size of Dro¬

sophila in the first post-metamorphic month (JRM and

RA, unpubl. data). This number, about 2,250 g, will pro¬

duce about 930 g of frog tissue (RA, unpubl. data). A

frog needs about 20 cal/day/g body weight for mainte¬

nance at 20 °C (Mazur 1968). At 5796.6 cal/g dry weight

of fly tissue (Cummins and Wuycheck 1971), one can

calculate that a frog could consume about 2.2^1.2 times

the calories needed for maintenance during the first post-

metamorphic month. Also, it must be recognized that

specific conditions at one point in the rearing process can

influence the quality of an individual much later in on¬

togeny (e.g., Scott et al. 2007; Gervasi and Foufopoulos

2008; Gagliano and McCormick 2009; Uller et al. 2009;

Van Allen et al. 2010) or perhaps more importantly, in

subsequent generations (Frost et al. 2010). Because the

behavioral, immunological, morphological, and physi¬

ological qualities of reared individuals seldom are mea¬

sured or evaluated in amphibian programs, there is little

idea if techniques (e.g., diet) are producing individuals

of good quality. There are many factors that modify vari¬

ous qualities of metamorphs and postmetamorphs (e.g.,

Alford 1986; Blouin 1991, 1992; Gramapurohit et al.

2004; Relyea and Auld 2005, and many others). In light

of these data, the percentage of individuals produced are

likely quite a poor estimator of success after release, and

success should not be equated with the introduction of

the largest possible number of tadpoles or metamorphs

into the wild.

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Tadpoles, froglets, and conservation

The Natural World

We emphasize that not all aquatic larvae are adapted to

similar aquatic environments. For example, phytotelmata

are very different environments than are streams or ponds.

Yet, the default rearing vessel for amphibians in captive

programs is a stock, straight-walled aquarium. Focusing

on the natural pond, for this example, let us consider this

is an environment with a large surface area per volume

for gas exchange and waste dispersion, wind and temper¬

ature induce water movements, and populations of plants

and animals dispense with metabolites. The sloping

banks allow tadpoles to escape some predators and seek

water of the desired temperatures during development,

and those approaching metamorphosis can safely switch

from gas exchange across the gills to the lungs. During

their daily movements about the pond, tadpoles learn di¬

rections to proper areas involving temperature, food, and

refuge. The default enclosure with vertical walls and uni¬

form depth violates all of these natural conditions, and

the larvae have no exposure to differing microclimates,

a natural light cycle, or the myriad ecological conditions

that wild individuals encounter. Careful consideration of

the egg deposition sites chosen by breeding frogs, and

post-hatching behaviors of tadpoles should inform all

aspects of the rearing enclosure, its placement, and envi¬

ronment. We should consider whether it is even possible

to raise high-quality frogs indoors, or tropical frogs in a

typical Nearctic zoo?

With respect to the natural world, and returning to the

idea of pre-release training (Griffiths and Pavajeau 2008),

we suggest that survival after release would increase

appreciably if the simple tenets of Y-axis orientation

(Ferguson et al. 1965; Taylor and Ferguson 1969) were

implemented. This type of orientation allows individuals

to move about their habitat productively as they receive

input visually or via the pineal complex (i.e., nonvisual,

including the patterns of light polarization; Taylor and

Adler 1970). An accurate sense of time is involved, and

the biological clock must be reset each day by witness¬

ing sunrise in order to stay synchronized with changes

in day lengths. A fixed light-dark cycle in the laboratory

does not entrain the animals in any way because there is

no appropriate movement of the “sun” and no changes

in day length. Naive tadpoles released without training

may have a higher probability of being predated, and a

released metamorph (i.e., small size with poor locomo¬

tor skills, large surface-volume ratio and thus rapid water

loss, likely with small energy reserves, and no idea of the

locations of proper refuges) that makes one wrong direc¬

tional choice has a high probability of dying.

We advocate that the adoption of the research proto¬

col of Taylor and Ferguson (1969) into the release pro¬

cedures would surely improve the success of the project.

All that is needed to follow our pond-breeding example

is: construct a meshed, wire cage with a top and bottom

(about 100 x 50 x 30 cm placed with its long axis about

a third on land and two-thirds in the water, place tadpoles

or metamorphs in the water and include moist cover for

froglets, and wait for at least two days). Tadpoles and

froglets of stream-breeding species (Preininger et al.

2012) obviously would also benefit from this procedure

with some innovation of different meshed enclosures for

stream species. The point is that, by experiencing even

two sunrises, the animals will know the Y-axis, and when

they are released, the animals can be expected to have

a much better chance at survival because they are more

likely to make the appropriate decisions.

The Culture World

We comment on several related topics that we feel are

important; in all cases, stage refers to Gosner’s (1960)

table. The various forms of egg clutches (Altig and Mc-

Diarmid 2007) may be quite fragile, but individual eggs

are much more robust than one would assume. Clutches

can be pulled or cut apart without damaging the ova to

improve the surface area/volume of the groups. In the

case of pond breeding species, eggs should be placed in

water not much deeper than the groups of eggs. Different

protocols are advisable, of course, for species that breed

in substantially different environments (e.g, streams or

phytotelmata). The natural history of the species and the

specific conditions under which its larvae develop must

be considered and incorporated into the rearing proto¬

cols.

With respect to tadpoles, consider those of the Costa

Rican Leaf Frog Agalychnis lemur that occur in very

shallow, virtually non-moving swamps in nature; this

must be considered when rearing this species in the

lab. Many zoos and labs rely on stock aquaria, or simi¬

larly shaped tanks, that often are poor choices for rear¬

ing containers. They have a small surface area/volume,

and this is a problem exacerbated by the tendency to put

too many individuals in a given tank. If a caretaker la¬

ments the management of air stones and water changes,

then the system is incorrect by definition. Information

on management of water quality are reviewed in Poole

and Grow (2012) and Pessier and Mendelson (2010).

Tadpoles that swim up and down the glass are signal¬

ing that they are stressed by inappropriate temperatures,

oxygen concentrations, or lack of naturalistic gradients

of these crucial variables. Patterns of temperature varia¬

tion can unpredictably influence developmental rate and

morphology (Arrighi et al. 2013). Similarly, inappropri¬

ate quantities of food or refugia, waste buildup, or popu¬

lation density will also cause stress in tadpoles. As an

example of our concept, consider a hypothetical pond¬

breeding species. Shallow pans are not recommended.

They have a reasonable surface area/volume, but their

total volume is small and thus water chemistry is quickly

overwhelmed by food and feces, and catastrophic water

loss to evaporation is easy to miss. A plastic wading pool

or some similarly shaped, shallow enclosure is the best

April 2016 | Volume 10 | Number 1 | e116

Amphib. Reptile Conserv.

Mendelson and Altig

because of the large surface area/volume. Aeration is not

needed and water changes will only be necessary 1-2

times during the rearing of a batch of 300 tadpoles in a

143-cm diameter pool. Water depth in these pools does

not need to exceed 6-8 cm and usually 4 cm is sufficient

for most pond-breeding species; it is the surface area that

is important because deeper water does not increase the

usable space for more tadpoles. Flow-through systems

decrease the manual work involved but are probably the

worst at providing the animals the chance to acquire the

proper intestinal bacteria.

We contend that the fear of the chytrid fungus, other

pathogens, and caretakers’ zeal for cleanliness has stimu¬

lated keepers to create overly clean environments, and

this action deprives the tadpoles of acquiring intestinal

symbiosis (Pryor and Bjorndal 2005a,b). Coprophagy,

especially after the material has developed large popu¬

lations of microbes and protozoan, is a viable feeding

tactic of tadpoles (Steinwascher 1978). Careful manage¬

ment of diseases in captivity is based more on common-

sense husbandry protocols, rather than absolute steriliza¬

tion (Pessier and Mendelson 2010). Proper work-flow

regimes, dedicated equipment, and vigilant monitoring

of water quality are recommended over routine water-

changes (in the absence of measurements to validate

such) and bleaching of tanks and substrates.

Tadpoles and metamorphs have been fed many differ¬

ent foods (e.g., Modzelewski and Culley 1974; Claussen

and Layne 1983; Jorgensen 1989; Pryor 2003; Hailey et

al. 2007; Pramuk and Gagliardo 2008; and many oth¬

ers), but decisions and choices of foods do not appear

to be based on data derived specifically from develop¬

mental performances, size, or energetic reserves present

at metamorphosis. Amphibians sequester most of their

body fat in the fat bodies attached to the anterior ends

of the kidneys. Assessing the size of the fat body (i.e.,

dissection of a few specimens, or perhaps development

of techniques using sonography or electrical conductiv¬

ity; Walsberg 1988) of individuals raised on different di¬

ets would be a valuable endeavor. Part of the problem

is that caretakers are just starting to distinguish between

the consummatory and digestive diets of tadpole, with

the real distinction being between what tadpoles ingest

versus what they digest (Altig et al. 2007; Schiesari et al.

2009; Whiles et al. 2009). How oral structures influence

feeding performances (e.g., Venesky et al. 2010a,b) also

remains to be determined. The main point is that tadpoles

swallow large quantities of the products of primary pro¬

ductivity (e.g., plants or algae), but when one considers

periphyton, it is likely that these plants or algae may not

represent the primary energetic intake (Altig et al. 2007).

For example, one might consider adding a bit of clay

soil to the rearing containers (Hailey et al. 2007; Akers

et al. 2008). Adding supplements of natural foods, such

as wild-collected aquatic plants or algae, is a viable idea,

but one can inadvertently add vicious predators as well as

potential pathogens and parasites. Algae can be allowed

to develop in tanks, and there are protocols for clean¬

ing plant materials before introduction to tanks (Pessier

and Mendelson 2010); in either case, the important pe¬

riphyton will develop. Tetramin® fish foods (TetraWerke,

Melle, Germany), which contain considerable amounts

of animal-based material, and powdered rabbit pellets

have been used successfully by the authors, but there are

no data on actual developmental performances. There are

now many recipes for tadpole diets applied to a number

of different programs for ranids (Abrahamse and Hayes

2009) and other taxa. These kinds of feed promote rapid

production of microorganisms which likely serve as the

primary food source for tadpoles. The point to keep in

mind is that the knowledge-base for tadpole diets in the

wild and nutritional needs is poor and far from taxonomi-

cally complete. Basic research in the form of controlled

studies is necessary for virtually all species.

If the program is designed to release post-metamor-

phic individuals and rearing conditions have been suit¬

able, then the majority of tadpoles will reach metamor-

phic stages 41-42 (i.e., eruption of front legs) at nearly

the same time (Wells 2007). If a large proportion (e.g.,

75-90 %) of the tadpoles do not metamorphose over a

short period of time, then one may assume an excessive

population density or some other factor has impeded nor¬

mal growth. Metamorphs should be held until tail resorp¬

tion is complete because tailed individuals have reduced

locomotor abilities. If post-metamorphic frogs are to be

maintained in captivity, then abundant and diverse small

prey must be available. A single-species diet of fruit flies

alone does not match the diversity of nutrients available

to free-ranging froglets.

Release of Reared Individuals

After considering the release options outlined above, the

release of tadpoles and froglets should be coordinated

with when metamorphosis of the target species occurs at

the release locality. This detail will increase the chances

of there being proper weather conditions and sufficient

food available, and one might consider verifying the lo¬

cal prey base (Goldstein 2007). Iterative assessments, via

monitoring, and appropriate modifications of the release

environment may be required. In the real context of the

interactions of biotic and abiotic conditions, local popu¬

lations increase and decrease through time. Populations

in “good localities” (sources) persist for long periods,

and populations in sites of some unknown lesser condi¬

tions (sinks) appear and disappear abruptly on short time

scales. If a population did not succeed at the target site

under the natural conditions, one should question the log¬

ic of a restoration attempt at that site; at least we should

understand the reasons for its original failure. Also, the

concept of source-versus-sink populations presents a dif¬

ficult decision if one wishes to establish a population at

an entirely new site (e.g., Pellitteri-Rosa et al. 2008; Mc-

Murry et al. 2009; Ruiz et al. 2010). In any case, popu-

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e116

Tadpoles, froglets, and conservation

lations at sink-sites may sometimes represent important,

if ephemeral, connectivity across the meta-population

landscape. It is quite unlikely that any two adjacent pools

present the same conditions, and in such a case, one must

set aside human notions and conveniences, know the bi¬

ology of the target species very well, evaluate the new

site in detail, and diversify as much as is feasible with

the number of individuals available. Various factors that

change as a site undergoes succession can also change

the likelihood of a given site being a viable site for rein¬

troduction. Also, released individuals likely perform dif¬

ferently relative to other local taxa (Tingley et al. 2011).

Although there can be repercussions in doing so, one

might consider reducing the chance of predation. Snakes

can decimate tadpoles and froglets, but predatory fishes

usually are not a problem in ephemeral sites, streams, or

other types of sites where many frogs breed. Examples

of the types of things to be considered for pond sites in¬

clude comparisons of sites with no aquatic vegetation or

short, sparse vegetation; stands of tall vegetation (e.g.,

especially cattails), zones covered by water lilies, and

dense stands of emergent vegetation are not acceptable.

These vegetation structures provide excessive organic

debris that can reduce oxygen concentrations and exces¬

sive shade that inhibits proper periphyton growth. Dense

stands of filamentous algae and algal mats are not ac¬

ceptable because these populations reduce the oxygen

concentration and some of these organisms are toxic. All

manners of emergent, submergent, and floating vegeta¬

tion must be considered with direct respect to the antici¬

pated micro-habitat use of the released species.

One should also consider the qualities of adjacent

terrestrial areas. High densities of froglets can occur at

release sites, so additional refuges ought to be provided

if one suspects that refugia could possibly be a limited

resource. Artificial refugia made from PVC pipe (nar¬

row gauge; cap on the bottom and a T-cap at the top;

small drain hole about two cm from bottom to avoid fill¬

ing with water; painted black) placed upright in the local

environment will be used by post-metamorphic treefrogs

(RA, unpubl. data). Pushing a rod into soft soil at a low

angle and removing it leaves preliminary burrows for

toads and ranid frogs, and pieces of PVC pipe laid on the

ground and covered with soil to avoid overheating pro¬

vides similar burrows. At the same time, this technique

is subject to invasion by introduced fire ants (Solenopsis

invicta) in southeastern North America; the ants use the

tubes to help establish a mound and consume any frog¬

lets that may enter. Untreated wooden cover boards also

can be quite useful as retreats if there is enough local

moisture.

As an alternative to rearing tadpoles in pools in the

laboratory, and the various concerns raised here above,

it may be preferable to simply transfer eggs to the new

site. Breaking a clutch into smaller pieces to enhance

aeration would be prudent because the eggs likely are not

placed in the same manner (e.g., attached to a twig off

the bottom) as was done by the ovipositing frogs. Protec¬

tion from egg predators (e.g., mesh enclosures) would be

advisable. Egg transfers between already inhabited sites

can facilitate genetic connectivity between sites, if that

is what the management plan recommends. The program

should not reduce genetic diversity. Reintroduction to a

new or extirpated site may need multiple releases, not

only for establishment, but for genetic management (e.g.,

if only Fj’s from captive adults were released, and there

is no connectivity to other populations, there would be

inbreeding depression, genetic drift, etc.). Long-term ge¬

netic maintenance should be considered when deciding

where and how often animals are released.

Conclusions

The study of amphibian declines is difficult, and the

search for solutions is frustrating (e.g., Beilby et al.

2009). When release programs either succeed or fail, we

often are never sure of the reasons why in either case, and

volumes of anecdotal information are produced. Knowl¬

edge of the genetic diversity of the populations that are

released (e.g., Charmantier and Garant 2005) is crucial,

and throughout our rearing attempts, we must be certain

that caretakers are not perpetuating any initial problems

(e.g., Walker et al. 2008). We understand that some of the

points we have raised may violate restrictions of funds,

personnel, facilities, and time. But, we suggest that the

bar should be raised at every available chance. If imple¬

mented at the design-phase of a conservation project, our

recommendations require fewer resources than traditional

programs so long as the crucial component of long-term

post-release monitoring is equivalent. More field data on

the biology of the species involved are needed, and many

of the practical or financial limitations can be overcome

by rather minor changes in techniques based on better

knowledge of species biology. No protocol will ever ap¬

proach total success, especially when details of why the

targets met their demise in the first place. Some research¬

ers who have made multiyear releases of head-started

frogs at a site, but have not yet started routine monitoring

seem uninformed. Perhaps the biggest idea in this discus¬

sion is that it must be remembered that imposing non¬

natural conditions (Gawor et al. 2012) on tadpoles and

froglets by the seemingly simple act of culturing these

organisms (Denver and Middlemis-Maher 2010) should

underscore all aspects of the design and evaluation of a

conservation program. The quality of the released indi¬

viduals, the release protocol, and post-release monitor¬

ing are the most important factors to reconsider in any

amphibian reintroduction or relocation program.

Acknowledgments. —Ray Semlitsch and Lisa Wal¬

lace of Mississippi State University gratefully provided

pertinent information, and helpful comments on early

drafts were provided by Brian Kubicki, Robert Hill, Jen¬

nifer Pramuk, Michelle Christman, Steven Whitfield, and

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e116

Mendelson and Altig

Henry Mushinsky. Ron Gagliardo contributed substan¬

tially to the original manuscript.

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Joseph R. Mendelson III is Director of Research at Zoo Atlanta, Adjunct Professor at Georgia Tech, and

Scientific Advisor for Amphibian Ark. He has been studying Neotropical amphibians for about 25 years.

Ronald Altig is Professor Emeritus Mississippi State University after a 30-year career in teaching and

researching larval amphibians.

Amphib. Reptile Conserv.

April 2016 | Volume 10 | Number 1 | e116

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [General Section]: 28-33 (e119).

A new case of facultative paedomorphosis in Smooth Newts,

Lissotriton vulgaris (Caudata: Salamandridae), in Turkey

^ilara Kizil, llhan Bayryam Ismail, 2 Anthony Olivier, and ^Kerim Cigek

1 Section of Zoology, Department of Biology, Faculty of Science, Ege University, TR-35100, Bornova, Izmir, TURKEY 2 Tour du Valat, Centre de

recherche pour la conservation des zones humides mediterraneennes, Le Sambuc, 13 200 Arles, FRANCE

Abstract .—A report of the occurrence of a dimorphic population of Smooth Newts in Lake Sazli

(Izmir, Western Anatolia, Turkey). During fieldwork on March 2015, 29 aquatic individuals (seven

males, 22 females) were captured and of these individuals, five were paedomorphic (four males,

one female). The metamorphic (83%) and paedomorphic (17%) ratio of the population is skewed

to metamorphic. The mean snout-vent length (SVL) was 28.30 mm (range = 26.00-30.41), and total

length (TL) was 57.66 mm (53.35-61.40) in paedomorphs. The average exterior gill length was 4.12

mm (2.64-4.71). The SVL was 39.99 mm (range = 33.44-39.93), and TL was 69.06 mm (66.19-79.17) in

metamorphs. The possible reasons for the presence of facultative paedomorphosis in the population

are discussed, with the dimorphic paedomorph hypothesis supported.

Key words. Salamander, Izmir, Lake Sazli, Gediz Delta

Citation: Kizil D, ismail iB, Olivier A, and Qigek K. 2016. Anew case of facultative paedomorphosis in Smooth Newts, Lissotriton vulgaris (Caudata:

Salamandridae), in Turkey. Amphibian & Reptile Conservation 10(1) [General Section]: 28-33 (el 19).

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: 11 February 2016; Accepted: 18 March 2016; Published: 22 June 2016

Facultative paedomorphosis is an environmentally-

induced polymorphism that results in the coexistence of

sexually mature, gilled, and fully aquatic paedomorphic

individuals and transformed, metamorphic individuals

in the same population (Denoel et al. 2005a).

Paedomorphosis has been known for more than one

century and can be either obligate or facultative in 57

urodeles species (Denoel et al. 2005a). Facultative

paedomorphosis has been particularly reported in

numerous European newts (e.g., Kalezic et al. 1994;

Denoel 2005; Denoel et al. 2009a; Skorinov et al. 2009).

Lissotriton vulgaris has a high tendency to be

paedomorphic (Beebee and Griffiths 2000) and there

are many reports of the phenomenon throughout its

distribution range (e.g., Skorinov etal. 2009;Litvinchuket

al. 1996; Litvinchuk 2001; Denoel et al. 2009b; Covaciu-

Marcov et al. 2011; Stanescu et al. 2014). In Turkey,

there are four records of facultative paedomorphosis in L.

vulgaris from Thracian and Marmara and Aegean regions

of Turkey (Yilmaz 1983; £evik et al. 1997; ^igek and

Ayaz 2011; Bozkurt et al. 2015, Fig. 1).

On March 13, 2015, during an amphibian and reptile

monitoring survey in the Gediz Delta, paedomorphic

Smooth Newts were observed in a population sampled

at Lake Sazli, which is located in the northeastern part of

the Gediz Delta, approximately 15 km west of Menemen

(38.600149°N, 26.911006°E, at about sea level, Fig.

1). The subspecies Lissotriton vulgaris schmidtlerorum

(Raxworthy 1988) inhabits the Izmir region. The

lake surface area is nearly 30 ha, and is surrounded

by agricultural (corn, cotton, and wheat) areas and

Ouercus sp. dominated shrubs (Fig. 2a). Emergent

aquatic vegetation of the lake includes Common Reed

(Phragmites australis ), Reed Mace ( Typha sp.), Common

Spike Rush ( Eleocharis sp.), Tufted Sedge ( Carex sp.),

and Rushes ( Juncus sp.) (Gediz Delta Management Plan

2007). Climatic conditions in the study area (Izmir) are

mainly Mediterranean, with a mean annual temperature

Correspondence. E-mails: *kerim.cicek@hotmail.com (or) kerim.cicek@ege.edu.tr (+90) 2323112409; Fax: (+90) 232388103;

Postal Address: Section of Zoology, Department of Biology, Faculty of Science, Ege University, Bornova, Izmir, TR-35100, TURKEY

(Corresponding author).

Amphib. Reptile Conserv.

June 2016 | Volume 10 | Number 1 | e119

Kizil et al.

Fig. 1 . The map of Gediz Delta (Izmir, Turkey). 1: Kumkoy, Istanbul, 2: Kiiguk^ekmece, Istanbul, 3: Ihsaniye, Karasu, Sakarya,

4: Lake Suluklii, Manisa, 5: Lake Ikiz, Izmir, The star shows the record (Lake Sazli, Gediz Delta, Izmir), The dotted line shows

Ramsar protected area borders.

of 18 °C (8.9-28 °C) and annual rainfall of 690 mm

(20-1,430) (1950-2015, Turkish Meteorological Service

2016).

Sampling was conducted from the shore to one m

depth by two persons using 45 cm radius dip nets. 29

aquatic individuals (seven males, 22 females) were

captured, and of these individuals, five were found to

be paedomorphic (four males, one female, Fig. 2b, c).

The paedomorphs were captured 30-50 cm depth on

shore of lake. The males presented sexual characteristics,

including the distended cloacae, spots on the side, and

a well-developed dorsal crest. The paedomorphs were

easily discerned from the metamorphs by their gills. The

sex of the captured individuals was recorded; snout-vent

length [from the tip of the snout to the posterior of the

cloacal opening], total length, and exterior gill length

(mm) were measured with digital callipers to 0.01 mm

precision. Three paedomorphic individuals were brought

to the laboratory and kept in an aquarium (40 x 30 x 30

cm, in 25 cm natural spring water) for a brief period to

be photographed. The paedomorphs were then released

back to where they had been captured.

The mean snout-vent length (SVL) was 28.30 mm

(range = 26.00-30.41, SE = 0.76), and total length (TL)

was 57.66 mm (53.35-61.40, SE = 1.09) in paedomorphs.

The average exterior gill length (GL) was 4.12 mm

(2.64-4.71, SE = 0.38). The SVL was 39.99 mm (range =

33.44-39.93, SE = 0.81), and TL was 69.06 mm (66.19-

79.17, SE = 1.33) in metamorphs (Table 1).

According to these data, the metamorphic (83%) and

paedomorphic (17%) ratio of the population is skewed

to metamorphic. This species shares its habitat with four

other amphibians: The Balkan-Anatolian Crested Newt

(Triturus ivanbureschi), Levant Water Frog (Pelophylax

bedriagae ), Oriental Tree Frog (Hyla orientalis ), and

the Variable Toad ( Bufotes variabilis)\ five reptiles,

the European Pond Turtle (. Emys orbicularis). Western

Caspian Turtle ( Mauremys rivulata ), Grass Snake

{Natrix natrix ), Dice Snake ( Natrix tessellata ), and East-

Four-Lined Ratsnake ( Elaphe sauromates). Fifteen fish

species belonging to eight families are cited in the Gediz

Delta Management Plan (2007), including the introduced

exotic Western Mosquitofish ( Gambusia affinis).

Ba§oglu et al. (1994) stated that when L. vulgaris

larvae complete their metamorphosis, their TL can be 35

to 40 mm, adults can be 70 to 80 mm in TL in Western

Anatolia. In the western Anatolian population, the adult

snout-vent length ranged from 28.8 to 35.4 mm in males,

and 30.5 to 36.4 mm in females, while TL ranged from

54.6 to 65.9 mm in males, and 56.1 to 66.8 mm in females

(Olgun et al. 1999). The average SVL of L. vulgaris was

40.15 mm (34.7—43.7), 40.77 mm (35.3-44.6) in females

(Colleoni et al. 2014). Stanescu et al. (2014) reported that

the SVL of paedomorphs was 32.7 mm for females, 34.5

mm for males from the Danube Delta Biosphere Reserve

(Romania). They also indicated metamorphs were larger

than paedomorphs. Colleoni et al. (2014) reviewed sexual

dimorphism in newts and found female-biased sex size

dimorphism in the species. Bozkurt et al. (2015) found

paedomorphic L. v. koswigi from (Sakarya) northwestern

Turkey and they measured 31.60 mm for males and

30.06 mm for females. The authors claimed that the

size of paedomorphs is larger than metamorphs. In the

Montenegrin Smooth Newts paedomorphs may or may

Amphib. Reptile Conserv.

June 2016 | Volume 10 | Number 1 | e119

Facultative paedomorphosis in Lissotriton vulgaris in Turkey

Fig. 2. The general view of habitat (A) and a male paedomorphic Lissotriton vulgaris (B, C) from Lake Sazli (Izmir, Turkey). The

arrows show the cloaca (B) and the gills (C).

not be bigger than metamorphs in their developmental

pathway (i.e., metamorphosis versus paedomorphosis),

as well as their species and population (Denoel et al.

2009a).

The gill length (GL) of paedomorphs measured 2.58

to 7 mm in northwestern Turkey (Bozkurt et al., 2015),

5.88 mm (3.30-7.90) in Lake Stiluklti (western Turkey,

£igek, and Ayaz, 2011), 6.7 mm in Romania (Covaciu-

Marcov and Cicort-Lucaciu 2007), and 1.5 mm in

Ukraine (Litvinchuk 2001). Our data was within the

range of previous reports. GL varies among populations

and might be connected to the oxygen level or other water

quality parameters, but this remains to be demonstrated.

According to Semlitsch (1987), facultative

paedomorphosis is controlled by environmental and

genetic factors that allow individuals to cope with habitat

variation, take advantage of environmental heterogeneity

in the presence of open niches, and increase their fitness.

It can occur in a variety of habitats from deep oligotrophic

alpine lakes to small eutrophic temporary ponds, arid

areas and humid forests (Whiteman 1994; Denoel et

al. 2001). In addition, paedomorphs are encountered

in different regions and latitudes, independently of

environmental and habitat conditions (Whiteman 1994;

Denoel et al. 2001; Denoel et al. 2005a).

Three main hypotheses explain the appearance and

maintenance of facultative paedomorphosis: i) the best

of a bad lot, ii) the paedomorphic advantage, and iii) the

dimorphic paedomorph hypothesis (Whiteman 1994).

The paedomorph advantage hypothesis corresponds to the

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June 2016 | Volume 10 | Number 1 | e119

Kizil et al.

Table 1 . Summary statistics of paedomorphic and metamorphic Lissotriton vulgaris from Lake Sazli (Izmir, Turkey).

Paedomorphic Males

Paedomorphic Females

Mean

Range

SVL (mm)

28.30

26.91-30.41

0.75

TL (mm)

57.52

55.49-61.40

1.09

GL (mm)

4.02

2.64M.71

0.38

SVL (mm)

26.00

TL (mm)

53.35

GL (mm)

4.52

Metamorphic Males

Metamorphic Females

Mean

37.46

68.80

36.95

68.48

Range

35.53-39.93

66.19-73.48

—

33.44-38.37

66.62-79.17

—

1.08

1.54

0.84

0.89

basic model ofWilbur and Collins (1973). It predicts large,

fast-growing individuals in good growing habitats to be

paedomorphic, while individuals smaller than minimum

size for the paedomorphs completely metamorphose

to escape competition with larger paedomorphs. More

unlikely, the best of a bad lot model predicts the reverse

solution in poor habitats with low growth conditions.

The larger larvae metamorphose, while the smallest ones

keep a larval form and become reproductively mature.

The latter hypothesis, the dimorphic paedomorph,

suggests that the phenomenon results from the two

other hypotheses according to the local conditions

experienced by each individual (Whiteman 1994). The

metamorphic and paedomorphic ratio of populations can

exhibit variations across populations and species (Denoel

et al. 2001). The fluctuations in natural populations of

paedomorphic urodeles may be related to both natural

and anthropogenic factors (Denoel et al. 2005b). The

absence of predators and the abundance of food resources

are known to favor the delay of metamorphosis and the

appearance of paedomorphs (Denoel et al. 2001). At

Lake Suluklii (£i?ek and Ayaz 2011), the metamorph/

paedomorph rate changes from year to year with no

paedomorphs found in five samples in 2015 (K.£.,

pers. obs). The fluctuation in the population size of fish

could affect the metamorph/paedomorph rate (Denoel

et al. 2015). The Lake Sazli population could favor the

third hypothesis due to the observation that the size of

paedomorphs is lower than metamorphs and presence of

potential predators in the habitat.

Habitat is an essential key in the persistence of

facultative paedomorphosis in natural populations

of newts (Denoel 2005). Denoel and Ficetola (2014)

compared the likelihood of multiple potential

environmental determinants impacting facultative

paedomorphosis. They observe that paedomorphs

prefer deep ponds, with conditions favorable to aquatic

breathing (high oxygen content), with no fish and

surrounded by a suitable terrestrial habitat. Despite the

presence of predators, Lake Sazli has dense aquatic

vegetation, abundant food sources and is surrounded by

unsuitable terrestrial habitat. There is limited available

shelter to hide and save terrestrial forms. Particularly,

the presence of aquatic shelters has been shown to favor

the coexistence between newts and fish (Winandy et

al. 2015). This might have allowed the co-occurrence

of newts with fish in the studied population but more

surveys are needed to explore these patterns.

Although several studies have documented the

presence and the cause of facultative paedomorphosis in

Europe (Denoel et al. 2005a and reference herein), data

remain limited on Asian species and subspecies. Better

monitoring of facultative paedomorphosis in this part

of the world would help to explore hypotheses that may

provide a more comprehensive understanding of this

phenomenon.

Acknowledgments. —We would like to thank

Oguzkan Cumhuriyet and Omer Donduren for his help

during the field trips. Dr. Lisa Ernoul, Dr. Arnaud Bechet

(Tour du Valat), Charles La Via, and Dr. Mathieu Denoel

for improving the English, and their valuable comments

on the earlier version of this manuscript. This study is

part of a project supported by Doga Dernegi (http://www.

dogademegi.org/) and the Tour du Valat (www.tourduva¬

lat. org). We are grateful to these organizations and the

PACA Regional Council for their generous financial sup¬

port.

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Kizil et al.

Dilara Kizil graduated with a biological sciences degree from Ege University (Izmir, Turkey) in 2015. She

is studying for her Masters at Ege University, specializing in ecology. Dilara’s undergraduate thesis was the

herpetofauna of Gediz Delta. Currently her interests are the ecology of Turkish reptiles, focusing on turtles and

tortoises. She is working with Kerim £igek at Ege University and Anthony Olivier of Tour Du Valat Research

Center (France) on the ecology of amphibians and reptiles.

ilhan Bayryam ismail is an ecologist and naturalist interested in amphibians. He graduated with a biological

sciences degree from Ege University (Izmir, Turkey) in 2012, where he is earning his Master’s degree in zool¬

ogy with a thesis on the life history of the Levant Water Frog in Manisa, Turkey.

Anthony Olivier is a French biologist working at the Tour du Valat Institute. His research interests include

ecology and conservation of amphibians and reptiles.

Kerim Ci^ek j s 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 in 2005. Kerim’s Master thesis was on the food composition of Marsh

Frog (Rana ridibunda) in Central Anatolia. He completed his Ph.D. thesis at Ege University in 2009, with

the population dynamics of Uludag Frog (Rana macrocnemis). Kerim is currently working at Ege University,

Izmir, Turkey, has authored or co-authored over 70 peer-reviewed scientific publications, and is co-editor for

the journal Biharean Biologist.

Amphib. Reptile Conserv.

June 2016 | Volume 10 | Number 1 | e119

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

10(1) [General Section]: 34-45 (el20).

First population assessment of two cryptic Tiger Geckos

(Goniurosaurus ) from northern Vietnam:

Implications for conservation

^ai Ngoc Ngo, 2 ’ 3 Thomas Ziegler, 4 Truong Quang Nguyen, 4 Cuong The Pham, ^ao Thien Nguyen,

5>6 ’ 7 Minh Due Le, and 2 ’ 3 *Mona van Schingen

1 Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18 Hoang Ouoc Viet Road, Hanoi, VIETNAM 2 Cologne Zoo,

Riehler Strafe 173, 50735, Cologne, GERMANY 3 Department ofTerrestrial Ecology?, Institute of Zoology?, University? of Cologne, Ziilpicher Strafie

47b, 50674, Koln, GERMANY * Institute of Ecology? and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Ouoc Viet

Road, Hanoi, VIETNAM 5 Faculty > of Environmental Sciences, Hanoi University? of Science, Vietnam National University, 334 Nguyen Trai Road,

Hanoi, VIETNAM 6 Centre for Natural Resources and Environmental Studies, Hanoi National University, 19 Le Thanh Tong, Hanoi, VIETNAM

''Department of Herpetology?, American Museum of Natural History, Central Park West at 79th Street, New York, New York 10024, UNITED STATES

OF AMERICA

Abstract. —The Cat Ba Tiger Gecko Goniurosaurus catbaensis Ziegler, Nguyen, Schmitz, Stenke

and Rosier, 2008 is a recently discovered species endemic to Cat Ba Island, Hai Phong, Vietnam.

Morphologically, G. catbaensis resembles G. luii Grismer, Brian, Viets and Boyle, 1999, which was

originally described in 1999 from southern China and was recorded in 2006 also in northeastern

Vietnam. Both species inhabit remote limestone habitats, which suffer ongoing degradation and

fragmentation due to agricultural development and the expansion of touristic sites. Tiger Geckos

experience increasing interest in the international pet trade, which already resulted in local

population extirpation of G. luii due to unsustainable overexploitation for commercial use. However,

impacts of anthropogenic pressures on wild populations, distribution ranges, and population

sizes of Goniurosaurus species remain imperfectly studied. Herein we used a capture-recapture

method to provide preliminary population size estimation of the endemic island dwelling species,

G. catbaensis, in comparison to its cryptic continental relative, G. luii, in order to evaluate their

conservation status and assess the level of threats. Our study revealed relatively small population

sizes and provided evidence for the negative impact of humans on the two Goniurosaurus species.

Our research emphasizes the necessity to support the conservation of the species and their natural

habitats, especially on the Cat Ba Archipelago. We further provide a new provincial record of G. luii

in Vietnam from Lang Son Province and record for the first time evidence for the occurrence of G.

catbaensis on further offshore island in the Ha Long Archipelago.

Key words. Eublepharidae, distribution, population size, new record, endemism, conservation measures

Citation: Ngo HN, Ziegler T, Nguyen TQ, Pham CT, Nguyen TT, Le MD, van Schingen M. 2016. First population assessment of two cryptic Tiger Geck¬

os ( Goniurosaurus ) from northern Vietnam: Implications for conservation. Amphibian & Reptile Conservation 10(1) [General Section]: 34-45 (el 20).

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 <amphlblan-

reptlle-conservatlon.org>.

Received: 12 January 2016; Accepted: 16 June 2016; Published: 15 July 2016

Introduction

Tiger Geckos of the genus Goniurosaurus have a restrict¬

ed distribution range in Asia, from northern Vietnam over

southern China eastwards to the Ruykuyu Archipelago

of Japan. Currently, 17 species are recognized; most of

them are endemic to small areas (Grismer et al. 1994,

1999; Seufer et al. 2005; Yang and Chan 2015; Ziegler

et al. 2008). Tiger Geckos are popular in the internation¬

al pet trade and the species G. luii was reported being

extirpated at its type locality in southern China shortly

after its description (Stuart et al. 2006). Although Tiger

Geckos are considered to be threatened by extinction due

to overexploitation for the illegal trade and habitat de-

Correspondence. Email: mschinge@smail.uni-koeln.de (corresponding author).

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e120

Ngo et al.

kilometers

Fig. 1. Distribution of the cryptic species Goniurosaurus catbaensis (gray circles) and G. luii (black circles): Filled circles represent

study areas; dashed circle represents new record of G. luii ; questions marks indicate former localities or sites where the current pres¬

ence is unclear. Occurrence records were represented as big circles to prevent showing exact locality data.

struction, only the members of the G. kuroiwae group

are listed on the IUCN Red List of Threatened Species

(Ota 2010). Knowledge about the status of the remaining

Goniurosaurus populations is poor and for most species

are not yet available. This study aims to contribute to¬

wards a better understanding about the population status

of the two species, Goniurosaurus catbaensis and G. luii

in Vietnam. Both species are members of the G. luii spe¬

cies group, which contains eight known species, G. ara-

neus Grismer, Viets and Boyle 1999 from the northern

portion of Vietnam, G. bawangJingensis Grismer, Haitao,

Orlov, and Anajeva 2002 from Hainan Island, China, G.

luii Grismer, Viets and Boyle 1999 from northern Viet¬

nam and southeastern mainland of China, G. catbaensis

Ziegler, Nguyen, Schmitz, Stenke, and Rosier 2008 from

Cat Ba Island of Vietnam, G. huuliensis Orlov, Ryabov,

Nguyen, Nguyen, and Ho 2008 from northern Vietnam,

G. liboensis Wang, Yang, and Grismer 2013 from the bor¬

der region between Guangxi and Guizhou provinces, G.

kadoorieorum Yang and Chan 2015, and G. kwangsien-

sis Yang and Chan 2015 from Guangxi Province, China.

The members of this species group are morphologically

very similar and their phylogenetic relationships are only

partly resolved (Grismer et al. 1994, 1999; Seufer et al.

2005; Vu et al. 2006; Yang and Chan 2015; Ziegler et

al. 2008). Hence, we investigated one of the most poor¬

ly known species, G. catbaensis, which is an endemic

flagship species for Cat Ba Island in the Gulf of Tonkin,

northern Vietnam. This island belongs to one of the most

attractive tourist sites in Vietnam, but the impact of the

tourism on this ecosystem and its biodiversity is not yet

fully understood.

Population size estimations provide essential informa¬

tion for the classification of the threat level of a species

and are crucial for wildlife management and manage¬

ment of the long-term survival of populations and spe¬

cies (Reed et al. 2003; Traill et al. 2007). We therefore

conducted the first population assessment of G. cat¬

baensis , including population density, size, and structure,

and evaluation of human impacts on the population. In

comparison, we likewise studied its cryptic sibling spe¬

cies G. luii on the mainland of northern Vietnam, which

is also karst adapted, and occupies a similar ecological

niche (Grismer 1999; Ziegler et al. 2008). Goniurosau¬

rus luii indeed shows a wider distribution range, but its

natural history is still poorly known and data on its abun¬

dance in Vietnam is completely lacking to date (Grismer

1999; Yang and Chan 2015). By comparing populations

of two closely related species, one from an island with

the other in mainland ecosystems, we expected lower

population densities in the mainland, stronger human im¬

pacts at tourist sites, and finally aimed to gain insights for

improved conservation strategies for the Tiger Geckos in

the future.

Materials and Methods

Study areas: Study sites were selected based on previ¬

ous surveys of the authors’ on Cat Ba Island, Hai Phong

City and in Ha Lang District, Cao Bang Province, north-

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First population assessment of two cryptic Tiger Geckos

Fig. 2. A: Macrohabitat and B: Microhabit of Goniurosaurus luii in Ha Lang District, Cao Bang Province, North Vietnam; speci¬

mens of G. luii from C: Cao Bang Province and D: Lang Son Province (new record). Photos Mona van Schingen, Marta Bernardes,

and Tao Thien Nguyen.

ern Vietnam (e.g., Lehmann, 2013; Ziegler et al. 2008, see

Fig. 1). Both areas comprise large limestone karst forma¬

tions with secondary evergreen forest and share zoogeo¬

graphic affinities (Fig. 2A, 3A). The limestone karst for¬

est provides diverse habitats for a unique flora and fauna,

and is recognized as arcs of biodiversity (Clements et

al. 2006). While habitats in Cao Bang Province—situ¬

ated at the border to China—lie outside protected areas,

Cat Ba Island was recognized as “Cat Ba Archipelago

Biosphere Reserve” (CBBR) by the UNESCO in 2004,

due to its significant ecosystem and biodiversity values

(CBBR Authority 2013). Besides the recent discovery

of G. catbaensis by Ziegler et al. (2008), Cat Ba Island

harbors 282 further species of terrestrial vertebrates, of

which 22 are listed in the Red Data Book of Vietnam. Cat

Ba is renowned for the endemic, Critically Endangered

Golden-headed Langur ( Trachypithecus poliocephalus).

Cat Ba Archipelago as well as the adjacent Ha Long

World Heritage Area account as the most popular tour¬

ist destination in Vietnam, annually attracting more than

one million tourists (CBBR Authority 2013), and thus is

facing several challenges from rapid tourism as well as

aquaculture development, and climate change. During

the present study, two sites on Cat Ba Island, which dis¬

tinctly differed in the number of tourists, were selected in

order to evaluate if the presence of tourists might impact

wild populations of Goniurosaurus (Fig. 1).

Field surveys: During a field survey in 2012 several

Goniurosaurus cf. luii specimens were observed in Lang

Son Province. One voucher specimen was collected and

deposited in the collection of the Institute of Ecology and

Biological Resources (IEBR), Hanoi (TD-LS2012.1).

The specimen was determined by comparison with fur¬

ther specimens of G. luii from Cao Bang Province (ML-

19, TAO-182, IEBR 3252, CB-2012.2, IEBR 3254,

and IEBR 3253) and based on data from the literature

(Grismer et al. 1999; Yang and Chan 2015). Exact local¬

ity data is not presented herein to prevent poaching (see

also Yan and Chan 2015). For assessment of the popu¬

lation status of G. catbaensis and G. luii , field surveys

were conducted between June and August 2014, May

2015 and during a short time in June 2015, which is the

non-hibernation season of Goniurosaurus (Grismer et al.

1999). Seven transects (1,100 to 4,200 m in length) along

limestone cliffs or caves were repeatedly surveyed in pe¬

riods of several days on Cat Ba Island and in Cao Bang

Province. Surveys took place after sunset between 7:30

and 11:30 pm, when lizards were found active or forag¬

ing. Captured animals were individually marked with a

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July 2016 | Volume 10 | Number 1 | e120

Ngo et al.

Fig. 3. A: Macrohabitat of Goniurosaurus catbaensis at the coast of Cat Ba Island; B: Limestone cliffs, the typical microhabitat

of G. catbaensis ; C: Adult male of G. catbaensis marked for population assessment; D: First evidence for the occurrence of G.

catbaensis within limestone cave of small offshore Island in Ha Long Bay archipelago. Photos Hai Ngo, Tao Thien Nguyen, and

Minh Le Pham.

permanent pen (Edding Eraser) and released on the same

spot after taking measurements (see Fig. 3C). This mark¬

ing technique has the advantage of being non invasive,

inexpensive, and enables the short-term identification of

animals, while markings last until the next shedding of

the animals.

Prism version 5.0 for Windows, GraphPad Software, La

Jolla California USA, www.graphpad.com. We further

compared densities of Goniurosaurus in areas, which are

frequently visited by tourists with areas where access is

limited in order to evaluate if tourism affects wild popu¬

lations.

Population analysis: To estimate population sizes,

we applied a “Capture-recapture Method” after Huang

et al. (2008) by using an “Invisibility Rate Index,” which

compensates for animals present but not detected dur¬

ing surveys. The method is described in more details in

Huang et al. (2008) and van Schingen et al. (2014). Esti¬

mated population sizes were only applied for the specific

surveyed sites, and did not encompass the entire popula¬

tions of the species. Since it is impossible to survey all

suitable habitats, density estimations in reference to the

transect line were used as relative abundances of respec¬

tive species. To assess the population structure, lizards

were categorized into three age classes based on snout-

vent length (SVL >105 mm = adult, SVL > 85 mm =

subadult, and SVL < 85 mm = juvenile), sexes, and in

case of females, into gravid and non-gravid specimens.

To test for differences in population structure between

the island species G. catbaensis and the continental G.

luii, a Chi 2 test with a = 0.05 was applied with GraphPad

Molecular analysis: In order to confirm the taxonomic

assignment of the newly collected specimens of Goniuro¬

saurus cf. luii from Lang Son and Cao Bang, a fragment

of the mitochondrial ribosomal gene, 16S, was amplified

using the primer pair 16Sar and 16Sbr (Palumbi et al.

1991) for four samples (TD-LS2012.1, TAO-182, ML-

19, IEBR-3254). Tissue samples were extracted using

DNeasy blood and tissue kit, Qiagen (California, USA).

Extracted DNA from the fresh tissue was amplified by

PCR mastermix (Fermentas, Canada). The PCR volume

consisted of 21 pi (10 pi of mastermix, five pi of water,

two pi of each primer at 10 pmol/pl, and two pi of DNA

or higher depending on the quantity of DNA in the final

extraction solution). PCR condition was: 95 °C for five

minutes to activate the taq; with 40 cycles at 95 °C for 30

s, 50 °C for 45 s, 72 °C for 60 s; and the final extension

at 72 °C for six minutes.

PCR products were subjected to electrophoresis

through a 1% agarose gel (UltraPure™, Invitrogen). Gels

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First population assessment of two cryptic Tiger Geckos

Table 1 . Totally observed specimens, densities and estimated population size of Goniurosaurus catbaensis and G. luii in 2014 and

2015.

2014

2015

June

July

August

May

Total Cat Ba Island

Species

G. catbaensis

Total observed

—

D[ind/km of transect]

—

1.3

1.0

1.3

Population size

—

Cat Ba National Park

Species

G. catbaensis

Total observed

—

D[ind/km of transect]

—

0.9

1.1

0.9

Population size

—

Viet Hai Commune

Species

G. catbaensis

Total observed

—

D[ind/km of transect]

—

1.7

0.8

1.5

Population size

—

Ha Lang District, Cao Bang

Province

Species

G. luii

Total observed

—

D[ind/km of transect]

0.8

—

Population size

—

were stained for 10 minutes in IX TBE buffer at two pg/

ml of ethidium-bromide, and visualized under UV light.

Successful amplifications were purified to eliminate PCR

components using GeneJET™ PCR Purification Kit

(Fermentas, Canada). Purified PCR products were sent

to Macrogen Inc. (Seoul, South Korea) for sequencing.

Sequences generated in this study were aligned with

one another using De Novo Assemble function in the

program Geneious v.7.1.8 (Kearse et al. 2012). They

were then compared with other sequences using the Ba¬

sic Local Alignment Search Tool (BLAST) in GenBank.

Results

New population record of G. luii : Four sequences

of 574 bps were obtained from the Goniurosaurus speci¬

men collected in Lang Son Province. The sequences are

almost identical to each other, except in two positions,

and 99% to 100% similar to those of Goniurosaurus

luii from GenBank, specifically the sequences with ac¬

cession numbers EU499390, EU499391, KC765083,

KM455054. The results confirmed that Goniurosaurus

samples collected in Lang Son Province and in Cao Bang

Province are conspecific with G. luii. Thus, our finding

represents the first record of G. luii and the second re¬

corded Goniurosaurus species from Lang Son Province,

northern Vietnam. Previously, only G. huuliensis was

known from Huu Lien Nature Reserve, Huu Lung district

in the South of Lang Son Province occurring at eleva¬

tions of about 370 m (Orlov et al. 2008). Goniurosau¬

rus luii was recorded from the north at similar elevations

of about 364 m above sea level. Based on our current

knowledge no sympatric occurrence of the two species

has been recorded so far, but exact distribution boundar¬

ies remain unknown. The microhabitats of G. luii in Lang

Son Province were densely vegetated limestone caves,

which are similar to those observed in Cao Bang Prov¬

ince (Fig. 2B). The Geckos had been found active during

night on cliffs or cave walls about 0.5-2.5 m above the

ground. Most interestingly, our morphological examina¬

tion of the newly recorded G. luii specimens from Lang

Son Province showed that no significant differences in di¬

agnostic characters compared with the recently described

G. kadoorieorum (see Table 1, Fig. 3D). But the newly

recorded specimens of G. luii from Lang Son Province

slightly differed from both G. luii and G. kadoorieorum

in having more nasal scales surrounding the naris (9 vs.

6-7 in G. kadoorieorum and 6-8 in G. luii) and more

ciliaria (59-60 vs. 47-54 in G. kadoorieorum and 50-56

in G. luii) (see Yang and Chan 2015).

Extended distribution range of G. catbaensis : In

this study G. catbaensis was recorded—besides already

known sites in Cat Ba National Park (NP) and Viet Hai

commune—on karst formations at the coastline of Cat

Ba Island (Fig. 3B). An adult female was found on rocks

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July 2016 | Volume 10 | Number 1 | e120

Ngo et al.

Table 2. Morphological characters of Goniurosaurus luii from Guangxi (type series; Grismer et al. 1999), Lang Son and Cao Bang

Province compared with G. kadoorieonmi (Yang and Chan 2015). Length given in mm.

G. luii (type series,

Guangxi, China, n=6)

G. luii (Lang Son)

(17=1)

G. luii (Cao Bang) (n=6)

G. kadoorieorum (n= 5)

SVL

—

112.13

78.93-121.21 (104.0±18.4)

68.9-118 (104.28±20.3)

TaL

—

62.42

54.95-65.67 (58.37±6.25)

59.8-69.5 (60.59±5.38)

—

53.41

36.58-60.1 (49.41 ±9.24)

30.5-55.6 (49.3±10.61)

—

28.92

21.74-30.68 (27.9±4.12)

18.3-30.5 (26.82±4.98)

—

20.46

14.99-21.8 (18.72±3.09)

12.5-20.4 (18.28±3.46)

—

12.85

7.89-14.95 (11.38±2.82)

8.3-13.3 (11.64±1.98)

—

12.24

9.13-12.87 (11.35±1.71)

7.6-12.6 (11.28±2.11)

—

11.58

8.35-12.36 (10.23±1.78)

6.5-10.9 (9.5±1.77)

SVL:TaL

—

1.79

1.35-2.34 (1.86±0.42)

1.15-1.83 (1.56±0.36)

SVL:HL

—

3.88

3.51-3.96 (3.71±0.17)

3.77-3.98 (3.88±0.08)

SVL: AG

—

2.09

2.07-2.17 (2.11±0.06)

2.05-2.26 (2.13±0.08)

HL:HW

—

1.41

1.39-1.66 (1.49±0.1)

1.39-1.5 (1.47±0.05)

SE:EE

—

1.06

1.03-1.23 (1.11±0.08)

1.14-1.25 (1.19±0.04)

SPL

9-12 (9.5±0.55)

10/9

10-12 (10.92±0.67)

10-11 (10.3±0.48)

IFL

9-11 (10±0.63)

10/10

8-11 (9.9±0.9)

—

9/9

6-8 (7.25±0.75)

6-7 (6.2±0.42)

—

3-1 (1.5±0.84)

PostIN

—

3-5 (4.0±0.89)

3-9 (5.2±2.49)

2-4 (3±0.89)

2-5 (3.83±0.98)

4-5 (4.8±0.45)

—

7-11 (8.67±1.37)

8-11 (9.6±1.52)

14-17 (15 8±1.17)

—

15-19 (16.7±1.16)

CIL

57-61 (59.5±1.87)

59/60

50-56 (53.83±1.75)

47-54 (51.7±2.58)

119-144 (134.5±12)

122

105-132 (118.5±11.47)

124-132 (129.2±3.11)

GST

9-14 (12.2±1.34)

11/12

9-12 (11.2±0.94)

11-13 (12±1.05)

33-34 (33.8±0.75)

32-35 (33.3±1.21)

30-34 (32.6±1.67)

DTR

—

19-23 (21.5±1.52)

22-24 (23.2±0.84)

LF1

—

9/10

9-10 (9.83±0.39)

10-11 (10.2±0.42)

LF4

—

19/20

19-20 (19.54±0.69)

17-19 (17.8±0.79)

LT1

—

11/9

9-10 (9.9±0.32)

10-11 (10.6±0.52)

LT4

21-24 (23.5±1.38)

24/24

22-25 (23.5±1.08)

21-24 (22.3±0.95)

PP (male)

23-29 (26±2.58)

—

17-24 (20.5±4.95) ( n=2 )

26-28 (26.75±0.96)

PP (female)

—

22 (pitted)

18-24 (20.0±3.5) (pitted, n= 3)

Absent

PAT

—

2/2

1-2 (1.75±0.45)

1-2 (1.4±0.52)

on the ground at an elevation of eight m above sea level.

This sighting is the first observation of G. catbaensis in

immediate proximity to the sea and provides an extended

distribution range from forested areas to completely open

areas close to the sea. In addition, first evidence for the

occurrence of G. catbaensis on a small island within the

Ha Long Bay is recorded based on a photo documenta¬

tion by a tourist (Fig. 3D). The specimen was observed

on the wall of a limestone cave on a very small offshore

island. Based on color pattern and discernible scalation,

the photographed specimen revealed to be G. catbaensis ,

although not all diagnostic characters for that species

could be confirmed due to the lack of a voucher speci¬

men.

Population status: During the present study, G. cat¬

baensis and G. luii were found along five and seven

transects, respectively. A total of 43 individuals of G.

catbaensis and 15 individuals of G. luii were captured.

Based on an estimated invisibility rate index of 0.6, the

G. catbaensis population on Cat Ba Island was estimated

to comprise 16, 24, and 16 individuals in May, July, and

August, respectively (Table 1). Furthermore, the encoun¬

ter rates of G. catbaensis were always higher in Viet Hai

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July 2016 | Volume 10 | Number 1 | e120

First population assessment of two cryptic Tiger Geckos

G. catbaensis

■ Male Subadult juvenile ■ Non - 6ra ' ,id f avid

Female Female

May

July

SVL [mm]

C '

Z5 ■

9 > ,

August

100

SVL [mm]

120

Fig. 4. A: Average population structure of Goniurosaurus catbaensis and a continental G. luii population from Cao Bang Province

(May-August vs. June, respectively); B: Frequency histogram of Snout-vent length of G. catbaensis for the months May, July, and

August.

Commune than in other sites near the headquarters of Cat

Ba NP, where more tourists frequented. In comparison,

the continental subpopulation of G. luii was estimated

to comprise about 21 individuals within the investigated

site (Table 1). Monthly mean densities of G. catbaensis

ranged between 1 and 1.3 individuals per km of surveyed

transect, while densities were generally higher in Viet Hai

Commune than in other sites within Cat Ba NP (Table 1;

Fig. 4). With regard to temporal variations, the highest

density of G. catbaensis was observed during the month

of August compared to May and July. In comparison, the

continental subpopulation of G. luii was estimated at the

density of 0.8 individuals per km/transect, slightly lower

than the density of G. catbaensis.

The investigated population of G. catbaensis on aver¬

age consisted mainly of adult males, followed by adult

females, subadults, and juveniles (39%, 33%, 18%, 10%,

respectively; see Fig. 4). In comparison, the population

structure of continental G. luii slightly differed, with

adult females constituting the major proportion of the

local subpopulation, followed by adult males, juveniles,

and subadults (65%, 14%, 14%, 7%, respectively, see

Fig. 4). Females were more abundant in populations of

G. luii than in those of G. catbaensis (Fig. 4). However,

population structures did not differ significantly between

the two cryptic species (Chi : = 5.2; df = 3 \p — 0.158).

Most of the observed adult females were gravid (33% in

Cao Bang vs. 54% on Cat Ba) between May and July. In

July, all five encountered females of G. catbaensis were

gravid, while no gravid females were observed in Au¬

gust. Frequency histograms of SVL showed a tendency

of a monthly shift in presence of age classes in G. cat¬

baensis (Chi 2 = 1.227, df = 6, p = 0.9755; Fig. 4). In¬

dividuals with SVL less than 90 mm were only found

from July onwards. Similarly, animals with SVL larger

than 120 mm were observed from July onward, while the

largest individuals (SVL up to 110 mm) were recorded in

August (Fig. 4).

Discussion

Distribution: While most of the Goniurosaurus species

are endemic and restricted to a small distribution range,

our new record of G. luii from Lang Son Province ex¬

tended the distribution of this species in northern Viet¬

nam. However, the distribution range of G. luii probably

overlaps with its cryptic relatives, i.e., G. araneus, G.

kadoorieorum , and G. kwangsiensis (Chen et al. 2014;

Yang and Chan 2015). Chen et al. (2014) indicated that

G. luii and G. araneus occur sympatrically in Guangxi

Province, southern China, being only divided by a riv¬

er system as a geographic boundary, which also is the

barrier for G. yingdeensis and G. indet. The recently

described G. kadoorieorum and G. kwangsiensis were

also discovered from Guangxi Province, although infor¬

mation about exact locality was not provided (Yang and

Chan 2015). Our morphological investigation of G. luii

from Vietnam revealed no distinct morphological differ¬

ences between G. luii and G. kadoorieorum. Thus, the

validity of the newly described G. kadoorieorum should

be verified by genetic analyses in the future. A similar

case of cryptic diversity within a small geographic range

is found in the G. kuroiwae complex, consisting of five

species, in the Ryukyu Archipelago, Japan (Chen et al.

2014). Definite overlaps in distribution ranges of differ¬

ent Goniurosaurus species have only been reported for

less closely related congeners, G. lichtenfelderi and G.

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July 2016 | Volume 10 | Number 1 | e120

Ngo et al.

luii thus far. Although G. luii is the second known species

of Goniurosaurus (after G. huuliensis) from Lang Son

Province, they are not sympatric inhabitants. While G.

luii was recorded from northern Lang Son, G. huuliensis

had been only recorded from Huu Lien Nature Reserve in

the southern part of the province. It can be assumed that

the two species are geographically separated, however

exact distribution boundaries have not yet been identi¬

fied.

At present, G. catbaensis is endemic to Cat Ba Archi¬

pelago and it is expected to be found on other offshore is¬

lands in Ha Long Bay. Besides, other reptile species such

as Pseudocalotes brevipes and Rhynchophis boulengeri

have been observed at the coastline of Cat Ba Island,

while R. boulengeri was even observed swimming in the

ocean (Nguyen et al. 2011). In addition, two specimens

of the Gekko palmatus complex were sighted on a small

offshore island in Cat Ba Archipelago in 2015. These

findings highlight the potential of Cat Ba Archipelago

and Ha Long Bay as a laboratory for future studies to

understand island biogeography of tropic lizards. These

studies are recommended to investigate species commu¬

nities, species relationships, and explore if similar spe-

ciation processes are underway on these small offshore

islands comparable to those reported in Anolis lizards on

Caribbean islands (e.g., Losos and Schluter 2000; Losos

and Thorpe 2004).

Population status: Species with restricted distribution

ranges are especially vulnerable to anthropogenic threats,

such as habitat loss or degradation, overexploitation, and

climatic changes (Hanski 1991; Reed et al. 2003; Traill et

al. 2007). The population size plays a crucial role in long

term survival of species, and a minimum viable size of

at least 3,000-7,000 individuals is required to maintain

a stable population over a longer time period (Reed et

al. 2003; Traill et al. 2007). Preliminary estimates of G.

catbaensis revealed a small population size that varied

monthly between 16 and 24 individuals. These values

only reflect the situation at the two known sites of the

species on the island and might not capture the popula¬

tion over the entire range of the taxon. We assume that

future surveys will probably uncover further occurrenc¬

es, which is supported by the recent sighting of potential

G. catbaensis on a small offshore island. However, G.

catbaensis is still relatively restricted in its distribution

and exclusively relies on the presence of limestone habi¬

tats in remote areas. Thus, the total population size of the

species is assumed to be relatively small, and not exceed¬

ing the size of a minimum viable population.

Accordingly, G. catbaensis had been found in a fairly

low density of 1.2 individuals per km, which only occurs

in the sites containing suitable habitats such as limestone

cliffs and caves. The habitats only cover a portion of the

Cat Ba Archipelago, since karst fomiations alone repre¬

sent only one of several ecosystems present on Cat Ba

Island with an area of about 170 km 2 (CBBR Authority,

2013). Our findings suggest that higher abundances of G.

catbaensis were generally found in remote sites, which

were less frequently visited by tourists. The results might

indicate a negative impact of tourism on the population

of G. catbaensis. As tourism is developing rapidly on the

island, wild populations and suitable habitats are likely to

steadily decrease in the future.

A comparative investigation of the continental G. luii

population in Cao Bang Province revealed similar size

estimations of about 21 individuals (vs. 16-24 individu¬

als of G. catbaensis on Cat Ba), based on the same num¬

ber of surveyed transects. The sites, where G. luii was

observed, were remote and far away from human settle¬

ments. This observation affirms our assumption that the

presence of humans negatively impacts the occurrence of

Goniurosaurus species.

Population structure: In both investigated species,

adult females represented the dominant group, which

might be an indication for a territorial or aggressive be¬

havior between males, which probably disperse more

than females (Vitt and Pianka 1994). The proportion of

males tends to be higher in the island population of C.

catbaensis compared with the continental population of

G. luii. This finding might be explained by the limited

dispersal ability on the island due to limited availability

of suitable habitats. Accordingly, the fact that relatively

higher numbers of gravid females were found on the is¬

land, compared with the continental population, might

have resulted from the respective higher density of males

in the population. Furthermore, gravid females in both

populations of G. catbaensis and G. luii were encoun¬

tered between May and July, confirming the observation

of Grismer et al. (1999) that July is the reproduction sea¬

son of G. luii.

Implications for Conservation

Tiger Geckos, in particular Goniurosaurus luii and G.

araneus, have been used for traditional medicine by lo¬

cal people and became very popular in the trade since the

1990s (Grismer et al. 1999; Chen et al. 2014; Yang and

Chan 2015; Ziegler et al. 2015). Grismer et al. (1999)

reported an exemplary case of one dealer exporting over

10,000 individuals of G. luii and G. araneus to the USA

for the pet trade. Already before its description in 1999,

G. luii had been overexploited for commercial use in

China, which presumably led to the extirpation from its

type locality in Pingxiang (Grismer et al. 1999; Stuart

et al. 2006). According to Yang and Chan (2015), local

villagers mentioned to have been paid by dealers for col¬

lecting large quantities of live Goniurosaurus, which is a

common scenario within the non-sustainable reptile trade

(e.g., Huang et al. 2008). A similar scenario might have

taken place simultaneously in Vietnam. As a result, even

extensive field surveys, e.g., Nguyen et al. (2009), Orlov

et al. (2008), Ziegler et al. (2008), and by our team in

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e120

First population assessment of two cryptic Tiger Geckos

2010 and 2014 in Cao Bang Province, failed to record

any specimen of G. araneus.

These findings emphasize how fast local populations

of range-restricted species can be extirpated due to over-

exploitation (e.g., Huang et al. 2008; van Schingen et al.

2015). The international demand for Goniurosanrus spe¬

cies among hobbyists still remains high. The long term

monitoring of local pet markets and internet sources

by Yang and Chan (2015) showed that almost all Gon-

iurosaurus species are subject to extensive pet trade.

Sometimes, the species fetch alarmingly high prices.

Observations by our team confirmed the regular trade in

respective species in international reptile fairs, e.g., in

Hamm and Dortmund, Germany, or on internet platforms

such as www.terraristik.com. The species are available

for sale from as low as 15 EUR up to several hundred

Euros per individual.

In many cases, the origin of the species and their le¬

gal export permits remain questionable. Among them, G.

catbaensis has been observed in European pet markets,

even though it was only described relatively recently.

Anthropogenic threats, such as poaching, habitat degra¬

dation, and introduced predators together with a small

distribution range of 1,600 km 2 imperiled the insular G.

kuroiwae species group, endemic to the Ryukyu Archi¬

pelago of Japan, leading to its inclusion in the IUCN Red

List as Endangered (Ota 2010). Yang and Chan (2015)

argued that most Goniurosaurus species from China and

Vietnam are similarly or even more threatened than the

Japanese species, since Japan is more advanced in spe¬

cies conservation management.

Our study suggests that the insular G. catbaensis is

very sensitive to the impacts of humans, and subject to

overexploitation to supply the international pet trade.

Besides illegal collection, habitat destruction for touris¬

tic purposes has dramatically increased the pressure on

the wild population of G. catbaensis. According to in¬

terviews with local villagers, several karst areas of Cat

Ba Island, comprising unique and important habitats

for the species, have recently been converted to a huge

tourist resort and further tourism development has been

planned. Such development would seriously threaten G.

catbaensis and the unique fauna and flora of the Cat Ba

Archipelago, which requires urgent conservation mea¬

sures to protect the species from imminent extinction.

Recommendations

Due to the restricted distribution range of G. catbaensis

and the rising anthropogenic threats to its natural popula¬

tions, we recommend to include this species in the IUCN

Red List. Since this study provided evidence for negative

impact of tourism on the presence of G. catbaensis , the

public access to core habitats of the species needs to be

restricted by local authorities. Based on our results, G.

catbaensis was found more frequently at some spots in

the vicinity of Viet Hai Village. The sites should there¬

Amphib. Reptile Conserv.

fore be considered a priority zone for the species con¬

servation. Future surveys will evaluate the relevance of

further sites as key habitats for conservation of G. cat¬

baensis. Furthermore, the Vietnamese authorities should

strictly control illegal collection of G. catbaensis as well

as other Goniurosaurus species. Currently, all Goniuro¬

saurus species are considered to be threatened by com¬

mercial use (Chen et al. 2014; Grismer et al. 1999; Yang

and Chan 2015; Ziegler et al. 2015) and the international

demand for Tiger Geckos still remains high. Because of

their restricted distribution ranges and low densities, all

Goniurosaurus species are especially vulnerable to un¬

sustainable harvest, which already caused the local ex¬

tinction of at least one species. As a first step to reduce

poaching and to control the international trade in Goniu¬

rosaurus species, we further recommend assessment of

trade status for all species of the genus Goniurosaurus

with a view to including them in the appendices of the

Convention of International Trade in Endangered species

(CITES).

Acknowledgments. —For supporting field work and

issuing relevant permits, we thank the authorities of

the Cat Ba National Park, Hai Phong City, Hai Phong

City, Minh Le Pham from the management department

of Ha Long Bay, and Forest Protection Department of

Cao Bang Province. We are very thankful to H.A. Thi,

M. Bernardes, and L. Barthel for their assistance in the

field, and to Jakob Hallermann (Hamburg), Vinh Quang

Luu (Hanoi), and Ulrich Schepp (Bonn) for commenting

on a first draft of the manuscript. We are grateful to T.

Pagel and C. Landsberg (Cologne Zoo), M. Bonkowski

(University of Cologne), C.X. Le and T.H. Tran (IEBR,

Hanoi), and M.T. Nguyen, L.V. Vu (VNMN, Hanoi)

for their support of research and conservation work in

Vietnam, and H.T. Ngo for laboratory assistance. Nature

conservation-based biodiversity research is mainly fund¬

ed by Cologne Zoo, the Institute of Ecology and Bio¬

logical Resources (IEBR), the Amphibian Conservation

Fund of German zoo associations (Verband der Zoolo-

gischen Garten, VdZ, e.V.) and private participants in the

German-speaking region as well as Stiftung Artenschutz,

the European Association of Zoos and Aquaria (EAZA),

the Nagao Natural Environment Foundation (Japan), the

World Association of Zoos and Aquariums (WAZA), the

Alexander von Humboldt Foundation (VIE 1143441)

and the University of Cologne. Research of T.T. Nguyen

is funded by the President of the Vietnam Academy of

Science and Technology (VAST). Cologne Zoo is part¬

ner of the World Association of Zoos and Aquariums

(WAZA): Conservation Project 07011 (Herpetodiversity

Research).

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Hai Ngoc Ngo is a young scientist who has been involved in several projects at the Institute of Ecology and

Biological Resources (IEBR) since 2013, and has been a researcher of the Vietnam National Museum of Nature

since 2014. He recently graduated with his M.S. in 2015 at Ha Noi University of Science. He has participated

in numerous held surveys to study herpetology in Vietnam and has much experience in held research and con¬

servation work. He is now focusing on ecology, phylogeny, and conservation of endemic endangered lizards

in Vietnam.

Thomas Ziegler has been the Curator of the Aquarium/Terrarium Department of the Cologne Zoo since 2003.

He is also the coordinator of the Cologne Zoo’s Biodiversity and Nature Conservation Projects in Vietnam and

Laos. Thomas studied biology at the University Bonn (Germany), and conducted his diploma and doctoral

thesis at the Zoological Research Museum Alexander Koenig in Bonn, focusing on zoological systematics and

amphibian and reptile diversity. As a zoo curator and project coordinator he tries to combine in situ and ex situ

approaches, viz., to link zoo biological aspects with diversity research and conservation in the Cologne Zoo, in

rescue stations and breeding facilities in Vietnam, and in Indochina’s last remaining forests. Beginning in Feb¬

ruary 2009 he has been an Associate Professor at the Zoological Institute of Cologne University. Since 1994,

Thomas has published 355 papers and books, mainly dealing with herpetodiversity. He was involved in the first

record of Goniurosaurus luii from Vietnam (Vu et al. 2006) and in the discovery of Goniurosaurus catbaensis

(Ziegler et al. 2008).

Truong Quang Nguyen is a researcher at the Institute of Ecology and Biological Resources (IEBR), Vietnam

Academy of Science and Technology (VAST), and is a member of the Biodiversity and Nature Conservation

project of the Cologne Zoo. He finished his Ph.D. in 2011 at the Zoological Research Museum Alexander

Koenig (ZFMK) and the University of Bonn, Germany (DAAD Fellow). From 2011 to 2014 he worked as

a postdoctoral student in the Zoological Institute at the University of Cologne. He conducted numerous held

surveys and is the co-author of seven books and more than 150 papers relevant to biodiversity research and

conservation in Southeast Asia. His research interests are systematics, ecology, and phylogeny of reptiles and

amphibians of Southeast Asia.

Cuong The Pham is a Ph.D. candidate and researcher at the Institute of Ecology and Biological Resources

(IEBR) Vietnam Academy of Science and Technology (VAST). He is member of the Cologne Zoo’s Biodi¬

versity and Nature conservation projects in Vietnam. He has published several papers mainly dealing with

Vietnam’s herpetodiversity. He is experienced in biodiversity and held research and has conducted numerous

held surveys in Vietnam.

Tao Thien Nguyen is the curator of herpetology and the current head of the Department of Nature Conserva¬

tion at the Vietnam National Museum of Nature (VNMN) of the Vietnam Academy of Science and Technology.

His research interests are in the taxonomy, evolutionary origin, and diversihcation of amphibians and reptiles,

as well the practical elucidation of the phylogeny of various amphibian and reptile groups. Tao obtained his

Ph.D. at the Kyoto University, Japan with a focus on the molecular and morphological systematics and distribu¬

tion pattern of various rhacophorid species. He has extensive experience in taxonomy and ecology of amphib¬

ians and reptiles throughout Vietnam. Since 2007, he has published more than 70 papers on herpetology.

Amphib. Reptile Conserv.

July 2016 | Volume 10 | Number 1 | e120