Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

11(2) [Special Section]: 1-6 (e143).

REPORT

In vitro fertilizations with cryopreserved sperm of Rhinella

marina (Anura: Bufonidae) in Ecuador

^elen Proano and 2 0scar D. Perez

Escuela de Ciencias Biologicas, Pontificia Universidad Catolica del Ecuador, Avenida 12 de Octubre 1076y Roca, Apartado 17-01-2184, Quito,

ECUADOR

Abstract. —Considering worldwide amphibian population decline, sperm cryopreservation should be a priority

for conservation of species in areas of high biodiversity, such as the Neotropics. In this study, we present

the results of two cryopreservation experiments involving Rhinella marina sperm. Freezing was performed

in a -80 °C freezer and dimethyl sulfoxide (DMSO) was used as cryo protective agent. In the first experiment,

the effects of 5%, 10%, and 16% DMSO were evaluated in sperm lysis and fertilization capacity. Samples were

incubated for 10 minutes at 4 °C before freezing. For thawing, two procedures were tested: 21 °C thawing to be

used immediately and 4 °C thawing, to be used two hours later in in vitro fertilizations. The best treatment was

10% DMSO plus thawing at 4 °C, that achieved 20% successful fertilizations. In the second experiment, two

solutions were tested: 10% DMSO with and without HEPES. Freezing and post-thawing in vitro fertilizations were

performed after a two hour incubation period at 4 °C. A considerable improvement in fertilization percentages

was obtained in this experiment, with a 75% for DMSO alone, and a 70% for DMSO + HEPES. These results

provide good perspectives for future implementation of sperm cryopreservation in Neotropical institutions for

local threatened species.

Keywords. Dimethyl sulfoxide, fertilization percentages, Neotropics, sperm cryopreservation, in vitro fertilization,

Assisted Reproductive Technologies, toad

Citation: Proano B and Perez OD. 2017. In vitro fertilizations with cryopreserved sperm of Rhinella marina (Anura: Bufonidae) in Ecuador. Amphibian

& Reptile Conservation 11(2) [Special Section]: 1-6 (e143).

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-

reptiie-conservation.org>.

Received: 08 October 2016; Accepted: 19 May 2017; Published: 7 August 2017

Introduction

The extinction crisis faced by amphibians can be con¬

sidered as dramatic as that of the Triassic or Cretaceous

periods with 31% of species threatened (Kouba et al.

2013). Captive breeding programs (CBP) have been

established to ameliorate current amphibian population

declines, especially for those species which are faced

with poorly understood threats and are rapidly disappear¬

ing (Bishop et al. 2012).The aim of dedicated CBP is to

maintain ex situ populations of target species with high

genetic diversity for research and future reintroduction.

Assisted reproductive technologies (ART) can be imple¬

mented by CBP’s when reproduction in captivity is dif¬

ficult to achieve (Clulow et al. 2014). ART research for

amphibians has specialized in gamete collection through

hormonal induction, in vitro fertilization (IVF), and

sperm cryopreservation in several anuran and some cau¬

date species (Bishop et al. 2012). This last technique is

very useful because it allows the maintenance of high

genetic diversity with a minimum amount of space and

resources (Clulow et al. 2014).

Sperm cryopreservation for amphibians still lags

behind that of other vertebrate classes (Clulow et al.

2014), though, there are various publications with Pipi-

dae (Sargent and Mohun 2005), Bufonidae (Browne et al.

1998; Beesley et al. 1998), Ranidae (Beesley et al. 1998;

Mansour et al. 2010; Mugnano et al. 1998), Eleuthero-

dactyliade (Michael and Jones 2004), Hylidae and Myo-

batrachidae (Browne et al. 2002) family members. In

these studies, testicular sperm is cooled by liquid nitrogen

(LN2) quenched in a cooling chamber or by immersion

in ethanol/dry ice slurry, and cooling rates determined by

a thermocouple. The most commonly reported cryopro-

Correspondence. 1 belen. olmos90@gmail. com (corresponding author) ; 2 odperez@puce. edu.ec

Amphib. Reptile Conserv.

August 2017 | Volume 11 | Number 2 | e143

Proano and Perez

Fig. 1 . Rhinella marina embryo at 31 Gosner stage from in vitro fertilization with cryopreserved sperm.

tective agents (CPA) are dimethyl sulfoxide (DMSO) and

glycerol at 5%, 10%, 15%, or 20% v/v diluted in saline

or sucrose solutions and high temperatures are employed

to achieve a fast thawing. However, the effectiveness of

the CPA varies according the species and the cryopreser-

vation protocol.

The standardization of a cry ©preservation protocol for

a species allows its inclusion into genome resource banks

(Clulow et al. 2014). Therefore, there is a need to stan¬

dardize gamete cryopreservation protocols for neotrop¬

ical species because they comprise approximately 49%

of the world’s amphibian species and 60% of all threat¬

ened species (Bolanos et al. 2008). Moreover, sperm

cryopreservation for conservation purposes in this region

has focused mainly on fish (Viveiros and Godinho 2009;

Carolsfeld et al. 2003) and mammal (Adams et al. 2009)

species. To the authors’ knowledge, there are only two

research papers describing sperm cryopreservation for

anuran neotropical species: one published by Michael

and Jones (2004) on Eleutherodactylus coqui, and the

other by Della Togna (2015) on Atelopns zeteki.

Here we present two experiments conducted with Rhi¬

nella marina sperm. This species is abundant in Ecuador

and belongs to the Bufonidae family, which encompasses

53% of the threatened species in the Neotropics (Bolanos

et al. 2008). Samples were frozen in a -80 °C freezer in

plastic racks and DMSO was used as CPA in both experi¬

ments. In the first experiment, DMSO was tested at three

different concentrations and with two thawing regiments.

The second experiment examined the effects of HEPES

buffer incorporation into the isotonic solution. HEPES

was used in the isotonic solution of our experiments

because it is an effective protector of sperm functionality

after short term storage in mammals (Will et al. 2011),

and it improved sperm motility after 48 h storage in pre¬

vious trials (unpublished data). Glycerol, the other com¬

mon CPA, was not used in these experiments, because, at

a 10% concentration, it had lower fertilization percent¬

ages (13.43 ± 7.42%) than DMSO 10 % (38.50 ± 6.29%)

in a previous experiment under similar experimental pro¬

cedures (unpublished data).

Materials and Methods

General animal and sperm collection

Rhinella marina male and female adults were col¬

lected in Jama, Manabf Province, Ecuador (00° 11.160’S

080°17.547’W) during the rainy seasons between late

December and late March of 2013 and 2015. Six males

and four females were collected in the first field trip, and

six males and two females in the second one. In both

cases, individuals were transported to Pontificia Univer-

sidad Catolica del Ecuador (PUCE) in Quito, Pichincha

Province, Ecuador, and maintained for two weeks in 56.6

L plastic boxes, provided with two water containers and

fed crickets twice a week in accordance with Barnett et

al. 2001.

For surgical removal of the testicles, individuals

were anaesthetized with a 0.5% w/v solution of MS-222

(Sigma-Aldrich E10521-10G), pH 7, for 15-20 minutes

(Wright 2001). A half testicle was used in every freezing

treatment, thus whole or half testicle was left in the ani¬

mal to obtain a control sperm suspension (fresh sperm)

when IVF was performed. After testicle removal, ani¬

mals were sutured with Vycril 3-0, and were placed in

individual aquaria for recovery.

The testicles were held on ice in suspension buffer

(SB: 104.4 mM NaCl, 2 mM KC1, 6.1 mM Na,HP0 4 , 1

mM KH,P0 4 , pH 7.4; Beesley et al. 1998) with HEPES

(Gibco 15630-080) at a final concentration of 2.5 mM.

The testes for each treatment were bisected and weighed

to the nearest 0.03 g. Each half was placed in a 1.5 ml

microfuge tube with the corresponding experimental

Amphib. Reptile Conserv.

August 2017 | Volume 11 | Number 2 | e143

In vitro fertilizations with cryopreserved sperm of Rhinella marina

solution. In all cases, except for the DMSO treatment

in experiment two, DMSO was diluted to experimen¬

tal concentrations in SB with HEPES 2.5 mM. Mac¬

eration of testicles was performed with Novo Surgical

0250-22 scissors. The tubes were centrifuged briefly,

and the supernatant was placed in another 1.5 ml tube.

The resulting sperm suspension was distributed, in dif¬

ferent volumes in each experiment, in 600 pi microfuge

tubes, and placed in plastic racks for freezing in a -80

°C freezer. The sperm concentration was determined by

duplicate counts with an improved Neubauer chamber.

For control sperm solutions in both experiments, the

remaining testicle in each animal was removed after

euthanasia by administration of the same 0.5% MS-222

solution, but for one and a half hours, and the heart was

removed to ensure death (Wright 2001). Testicles were

macerated in 1.5 ml microfuge tubes containing SB with

HEPES, after a brief centrifugation, supernatant was

placed in other 1.5 ml tube and held at 4 °C until use.

Experiment one (El, n = 6 males). The half testicle

was macerated in two ml of any of the following solu¬

tions: SB + HEPES, 5%, 10%, or 15% DMSO. DMSO

sperm solutions were divided in 250 pi aliquots to be fro¬

zen. Samples were maintained 10 minutes at 4 °C and

one hour at -20 °C before being placed in a -80 °C freezer.

One week later, sperm samples were left in their respec¬

tive plastic racks until ice melted at room temperature

(RT, 21 °C) or at 4 °C. For IVF, sperm samples thawed at

RT were used immediately, while sperm samples thawed

at 4 °C were used after two hours at 4 °C. Embryos that

reached gastrula stage (Gosner’s 11 stage) were recorded

and a gastrula rate was calculated per petri dish. Sperm

counts were made only for RT treatments.

Experiment two (E2, n = 6 males). Half testicle was

macerated in 500 pi of SB + HEPES; 10% DMSO; or

10% DMSO + 2.5 mM HEPES. DMSO suspensions were

divided into 100 pi aliquots and placed in a plastic rack

to be held at 4 °C for two hours before freezing at -80

°C for three days. Thawing procedure at 4 °C from El

was employed. Embryos at second cleavage (Gosner’s 4

stage) were recorded and maintained until tail bud stage

(Gosner’s 17 stage), cleavage and tail bud rates were cal¬

culated per petri dish.

In vitro fertilization

For both experiments, ovulation in females was induced

by injection of fresh pituitary homogenate from one

female of the same species. Twelve hours after hormone

administration, females were euthanized as previously

described for males. Two females were induced to ovula¬

tion in El, eggs from one female were used for RT thaw¬

ing treatment and eggs from the other one, for 4 °C thaw¬

ing treatment. Eggs from only one female were used for

all treatments in E2. Eggs were removed from the ovi¬

duct and placed in a petri dish for fertilization. Experi¬

ment one (El) used lOOpl of sperm solution for 208 ±

Amphib. Reptile Conserv. 3

20 eggs, while experiment two (E2) used 50 pi of sperm

for 116 ± 18 eggs per petri dish. Sperm suspension was

pipetted directly from the fresh or thawed sample onto

the eggs without any previous CPA wash or dilution.

Around two minutes later, the eggs were covered with

six ml of filtered tap water, and after 10 minutes, 20 ml of

water were added. Embryos were reared to tail bud stage

(Gosner’s 17 stage) in 10 cm Petri dishes filled with fil¬

tered tap water that was changed daily.

Statistics

Two factor ANOVA and Wilcoxon test were performed

for El and E2, respectively, using SPSS 20. Gastrula rate

data of El were analyzed by CPA and thawing procedure

factors. Cleavage rates within each DMSO treatment of

E2 were analyzed by a Wilcoxon test because data size

was lower than 30 samples, a = 0.05 for both analyses.

Results and Discussion

In both experiments, IVF’s with cryopreserved sperm

resulted in embryo development that reached tail bud

stage, although different embryo survival rates were

achieved in each experiment. DMSO 10% + HEPES 2.5

mM treatment was present in both experiments and had

20% embryos in E1, and 54% in E2. These slower embryo

rates in E1 could be due to the freezing procedure, which

may allowed melting and recrystallization when moving

samples from 4 °C to -20 °C and from -20 °C to -80 °C

freezers. Besides, it is important to take into consider¬

ation factors such as the different sperm concentration,

the frozen volume and the pre-freezing DMSO incuba¬

tion period in E2.

DMSO 10% with 4 °C thawing regiment was the best

treatment for El (Table 1), and though it was not signifi¬

cantly different from the other DMSO concentrations, it

was used in E2 with some modifications. First, assuming

a high tolerance of R. marina sperm, samples were incu¬

bated with DMSO 10% not only after thawing, but before

freezing for two h at 4 °C, resulting in high embryo rates,

close to control treatment (Table 2). This could indicate

that sperm cells needed this amount of time before freez¬

ing to allow DMSO to enter the cells and protect them

from cryoinjury, and before IVF to restore all their func¬

tionality after thawing osmotic stress (Hammerstedt et al.

1990).

Sperm concentration and frozen volume were also

modified. A half testicle in two ml of solution in El

resulted in 1.07; 1.25; and 0.99 x 10 7 sperm/ml for

DMSO 5 %, 10 %, and 15 %, respectively. Half a tes¬

ticle in 500 pi in E2 resulted in 3.41 and 3.23 x 10 7

sperm/ml for DMSO 10 % and DMSO 10 % + HEPES,

respectively. Frozen volume in El and E2 were 250 pi

and 100 pi, respectively. A smaller volume with higher

sperm concentration might reduce the volume of water

in the extracellular space, making less probable for ice

August 2017 | Volume 11 | Number 2 | e143

Proano and Perez

Table 1. Gastrula and abnormal embryo rates from El (n = 6

males).

Treatment

Gastrula rate

(M ± SD %) Subgroups*

Abnormal embryo rate

(M ± SD %)

Control

91.28 ±7.58

DMSO 5% - RT

03.26 ±4.00

DMSO 5% - 4C

19.48 ±21.73

10.99 ±2.98

DMSO 10% - RT

10.73 ± 13.00

DMSO 10% - 4C

23.17 ±27.13

10.43 ±4.64

DMSO 15% - RT

02.44 ±3.13

DMSO 15%-4C

07.90 ± 8.96

18.52 ± 10.76

M = mean, SD = standard deviation, RT = Room temperature thawing, 4C = 4

°C thawing.

♦Subgroups by DMSO factor (p < 0.001, df = 15, F = 93.97) from two factor

ANOVA.

to form during the time that the system reaches equilib¬

rium at -80 °C. A reduction in ice nucleation avoids intra¬

cellular ice formation, and sperm lesions by ice crystals

or hyperosmotic stress during freezing and/or thawing

(Rubinsky 2003), thus contributing to protect sperm fer¬

tilizing capacity in E2. Spenn lysis can be inferred by the

decreased post thawing sperm concentration in E2 (Table

2), but percentage of viable sperm cannot be determined

because of the absence of membrane integrity or motil¬

ity evaluation.

Experiment one (Table 1) showed significant differ¬

ences in gastrula rates by CPA factor only between con¬

trol and all DMSO treatments (p < 0.001, df = 15, F =

93.97). There were significant differences in gastrula

rates for thawing factor, with 4 °C thawing better than

RT (p < 0.001, df = 15, F = 20.94). No interaction was

found between CPA and thawing factors. Gastrula rates

for DMSO concentrations at 4 °C were 19%, 23%, and

7% for DMSO 5%, 10%, and 15%, respectively. While

gastrula rates for RT thawing were 3%, 10%, and 2% for

DMSO 5%, 10%, and 15%, respectively (Table 1).

It is interesting that a slow thawing at 4°C had a higher

gastrula rate than RT thawing considering that fast thaw¬

ing is recommended to avoid recrystallization or osmotic

injuries due to a prolonged exposure to the hyposmotic

medium generated during melting (Rubinsky 2003) thus,

anuran cryopreservation protocols use thawing tempera¬

tures of 21 °C and 30 °C (Browne et al. 1998; Sargent

and Mohun 2005). Besides, a prolonged CPA exposure

can be considered toxic (Fuller 2004), but in this case,

samples used two h later gave higher gastrula rates than

samples used immediately. Moreover, tail bud stage was

reached by embryos of all DMSO treatments. These gas¬

trula rates could indicate a high tolerance of R. marina

sperm to prolonged DMSO exposure, as seen for other

species like Rana temporaria which had been exposed

to DMSO for 60 minutes with no detrimental effects on

viability or motility (Mansour et al. 2010). Whether it

was the temperature or the incubation time that led to

higher gastrula rates reached by 4 °C thawing remains to

be clarified.

In E2, cleavage rates (Table 2) were 97%, 75%, and

70% for Control, DMSO 10%, and DMSO 10% + H,

respectively. Wilcoxon test found no significant differ¬

ences between Control and DMSO 10% (z — *-1.78, p

= 0.075), nor between DMSO 10% and DMSO 10% +

H (z = -0.52, p = 0.6); but there were significant differ¬

ences between Control and DMSO 10% + H (z = -2.20,

p = 0.028). There was an embryo reduction from second

cleavage to tail bud stage in all treatments to 82%, 60%,

and 54% tail bud embryos for Control, DMSO 10% and

DMSO 10% + H, respectively (Table 2).

Since there were only three ovulating females used in

this study, maternal effects could have influenced fertil¬

ization rates, so egg condition was revised before IVF.

As expected from collection in the same locality during

rainy season, only stage VI eggs were found in the ovi¬

ducts of all females, indicating that they were in a similar

reproductive status and the capability of eggs to be fer¬

tilized (Rastogi et al. 2011). Oogenetic stage VI is deter¬

minant for embryonic development because well differ¬

entiated animal and vegetal poles, a maximum size, and

a postvitellogenetic condition indicate that oocytes are

ready for ovulation (Dumont 1972). Ovulation in these

females resulted in high gastrula and cleavage rates in

control treatments from El (91%) and E2 (97%), both

reaching tailbud stage.

Embryo developmental period in cryopreserved sperm

treatments from El and E2 did not differ with the control

treatments; all embryos developed in seven days from

fertilization to tail bud stage. However, some abnormali¬

ties in tail bud stage were found in all treatments from

El, 4 °C thawing with DMSO 5%, 10%, and 15 % had

11%, 10%, and 18% abnormal embryos (Table 1). There

is a 15% embryo reduction from second cleavage to tail

bud stages in all treatments from E2. Apparently, it is

not unexpected in natural frog populations to exhibit 2%

abnormal embryos. Possible causes might be environ-

Table 2. Sperm concentration, cleavage and tail bud rates in control, DMSO 10%, and DMSO 10% + HEPES 2.5 mM treatments

from E2 (w = 6 males).

(M ± SD x 10 7 sperm/ml)

Cleavage rate

(M ± SD %)

Subgroups*

Tail bud rate

(M ± SD %)

Control

2.50 ± 1.26

97.38 ± 01.84

82.74 ±8.12

DMSO 10%

3.41 ±2.38

1.78 ± 1.42

75.67 ±25.22

a, b

59.99 ±23.21

DMSO 10%+ H

3.23 ±2.06

1.28 ±0.93

70.35 ± 19.74

54.46 ±21.14

PF = Pre-freezing sperm concentration, PT = Post-thawing sperm concentration, M = mean, SD = standard deviation, H = HEPES 2.5 mM. * Subgroups from Wilcoxon

test.

Amphib. Reptile Conserv.

August 2017 | Volume 11 | Number 2 | e143

In vitro fertilizations with cryopreserved sperm of Rhinella marina

mental factors, such as UV radiation, extremes in pH, or

thermal variations (Paskova et al. 2011). Higher percent¬

ages of abnormal embryos (60 %) can be possibly caused

by xenobiotics, which interfere with embryo mechanisms

for reactive oxygen species (ROS) regulation (Paskova et

al. 2011). Captivity rearing conditions could cause ROS

regulation to fail, with the consequential embryo abnor¬

malities and mortality seen in El and E2, respectively.

The presence of higher abnormal embryo percentages in

captivity should be considered when planning to perform

IVF for captive propagation.

We considered that HEPES could help to protect

sperm functionality being one of Good’s buffer qualities

maintaining adequate pH values in culture media and has

been used successfully in mammalian sperm cryopreser-

vation (Will et al. 2011). Moreover, it has been used in a

chemotaxis experiment with Xenopns laevis sperm (Al-

Anzi and Chandler 1998) and we found it to retain sperm

motility after a 48 h period at RT and 4 °C (unpublished

data). But no improvement in cleavage or tail bud rates

were found by the addition of this reactive to cryopreser-

vation solutions (Table 2). The effect of HEPES on the

cryopreservation of R. marina sperm remains unclear,

though, it seems to be unnecessary.

The reported embryo rates in the present study sug¬

gest that frozen volume, sperm concentration, and

DMSO incubation time can be key elements in improv¬

ing embryo rates from IVF with cryopreserved sperm.

Rhinella marina sperm seems to tolerate prolonged

DMSO exposures at 4 °C, with favorable effects on

sperm response to freezing and thawing. Nevertheless,

freezing rates and cell viability or motility tests should be

conducted to make possible stronger conclusions about

the present data. We hope that this report leads to in-

depth studies that can be applied to the conservation of

more Neotropical species using ART.

Acknowledgements. —We thank the volunteers of the

Laboratory of Developmental Biology from PUCE for

their assistance, particularly Gabriela Maldonado for her

help with embryo and sperm counts. Special thanks to

Natalie Calatayud for her useful comments and sugges¬

tions. This study was funded by PUCE grants in 2013

and 2015 to Oscar Perez.

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Belen Proano graduated in Biological Sciences from Pontificia Universidad Catolica

del Ecuador (PUCE) in 2013. As an associated researcher at PUCE for two years,

her investigations focused on reproductive biology and the application of Assisted

Reproductive Technologies in Ecuadorian anurans under captivity conditions. Currently,

she is working on personal projects away from the scientific environment, but with the

same interest in understanding the wonder of life.

Oscar Perez was born in Quito Ecuador. He obtained a doctoral degree in 2008

from Pontificia Universidad del Ecuador in collaboration with Duquesne University,

Pennsylvania, USA. His advisors were Dr. Richard Elinson and Dr. Eugenia del Pino. Dr.

Perez is interested in the evolutionary comparison of development and the reproductive

biology of Ecuadorian vertebrates. His current research focus is in finding new alternative

models in developmental biology using the great Ecuadorian mega-diversity country as

his playground. More particularly, his interest is in frog oogenesis—oocyte organization

can vary between species and these variations can modify the developing pathway of the

future embryo. Comparative methodologies are applied to find variations in oogenesis

patterns in order to understand how these variations can modify embryogenesis features.

These analyses employ a diversity of techniques such as histology, immunohistochemistry,

genetic cloning, and bioinformatics tools in order to identify genes of importance for

oogenesis and embryogenesis. All these efforts are focused towards shedding light on the

reproduction and preservation of Ecuadorian fauna and its unique development features.

Amphib. Reptile Conserv.

August 2017 | Volume 11 | Number 2 | e143

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

11(2) [General Section]: 1-16 (e141).

urn:lsid:zoobank.org:pub:31FA8B4B-718B-4440-AE19-9E1AC95524BD

Description of two new species similar to Anolis insignis

(Squamata: Iguanidae) and resurrection of Anolis

(Diaphoranolis) brooksi

Steven Poe and 2 Mason J. Ryan

13 Department ofBiology) and Museum of Southwestern Biology, University of New Mexico, Albuquerque, New Mexico 87131, USA

2 Arizona Game and Fish Department, 5000 W. Carefree Highway, Phoenix, AZ 85086, USA

Abstract. —The spectacular giant anole lizard Anolis insignis is widely distributed but infrequently collected

outside of northern Costa Rica. We recently collected several individuals similar to Anolis insignis from

localities in Panama and southern Costa Rica. These populations differ from type locality A. insignis in male

dewlap color and morphology. We associate one set of these populations with Anolis ( Diaphoranolis ) brooksi

Barbour from Darien, Panama, and describe two additional populations as new species.

Keywords. Central America, Costa Rica, lizard, Panama, Reptilia, taxonomy

Citation: Poe S and Ryan MJ. 2017. Description of two new species similar to Anolis insignis (Squamata: Iguanidae) and resurrection of Anolis

(. Diaphoranolis ) brooksi. Amphibian & Reptile Conservation 11(2) [General Section]: 1-16 (el41).

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 journ

reptile-conservation.org>.

Received: 04 July 2016; Accepted: 09 June 2017; Published: 16 July

Introduction

Costa Rica and Panama contain perhaps the most stud¬

ied herpetofauna of the Neotropics for ecology and sys-

tematics (Savage 2002; Donnelly et al. 2005). The early

works of Taylor (e.g., 1956) and then Savage (e.g.,

1975), along with the development of the Organization

for Tropical Studies (OTS) and the efforts of the Univer¬

sity of Costa Rica (UCR), have established Costa Rica

as a center of herpetological research. The Smithsonian

Tropical Research Institute (STRI) has been instrumental

in fostering herpetological work in Panama.

The Anolis lizards of Costa Rica and Panama are

well studied (Taylor 1956; Savage 2002), but new spe¬

cies continue to be discovered (e.g., Kohler 2011; Poe et

al. 2015). As of 28 February 2016 the Reptile Database

lists 42 species of Anolis from Costa Rica and 45 spe¬

cies from Panama. Relatively unexplored regions such

as the southern Cordillera de Talamanca in Costa Rica

and the Darien Region of eastern Panama are likely to

produce new discoveries, and detailed molecular studies

such as those undertaken in frogs (Crawford et al. 2010)

are likely to unearth cryptic diversity of Anolis.

We have conducted extensive fieldwork on Anolis in

Costa Rica and Panama since 2006. During this time, we

have collected numerous individuals of Anolis that might

Correspondence. 3 anolis@unm.edu

Amphib. Reptile Conserv.

title Amphibian & Reptile Conservation-, official journal website <amphibian-

317

standardly be assigned to the spectacular and rarely col¬

lected giant anole species A. insignis (Fig. 1). We have

noticed numerous differences between populations of

this species that are consistent within geographically dis¬

tinct populations. We now possess enough material to

confidently distinguish and recognize three species of

Anolis similar to A. insignis. Herein we resurrect a previ¬

ously synonymized name and describe two new species.

Materials and Methods

We adopt the evolutionary species concept (Simpson

1961; Wiley 1978) and operationalize this concept by

identifying species based on traits that are consistent

within hypothesized species but differ among species.

Measurements were made with digital calipers on

preserved specimens and are given in millimeters (mm),

usually to the nearest 0.1 mm. Specimens are referenced

from the Museum of Southwestern Biology (MSB), the

Museum of Comparative Zoology (MCZ), the Los Ange¬

les County Museum (LACM), the Museo de Vertebra-

dos, University of Panama (MVUP), and the University

of Costa Rica (UCR). Snout-vent length (SVL) was mea¬

sured from tip of snout to anterior margin of the cloaca.

Head length was measured from tip of snout to anterior

margin of the ear opening. Head width was measured at

July 2017 | Volume 11 | Number 2 | e141

Poe and Ryan

Fig. 1. Anolis insignis, male, Pocosol, Alajuela, Costa Rica.

the broadest part of the head, between the posterolateral

corners of the orbits. Femoral length was measured per¬

pendicularly from the longitudinal midline of the venter

to the knee, with limb bent at a 90° angle. Terminology

and characters for qualitative conditions and scale counts

follow standards established by Ernest Williams (e.g.,

Williams et al. 1995).

We tested for the objective identification of hypoth¬

esized groups (i.e., species) using the Multiresponse Per¬

mutation Procedure (MRPP; Mielke 1984) as described

by McCune and Grace (2002). Like the commonly-used

discriminant function analysis (DFA), MRPP is among

the class of techniques used to test for the distinctiveness

of a-priori hypothesized groups. We use this test rather

than DFA because we are not confident making distri¬

butional assumptions about our data and we suspect the

nonparametric nature of this approach will treat our small

sample sizes more conservatively. We hypothesized

groups based on male dewlap color pattern and geogra¬

phy (see below) and employed the following characters:

number of lamellae on 4 th toe (counted in the manner of

Williams et al. [1995]), number of postmental scales,

number of postrostral scales, number of scales across

the snout at the second canthals, number of supralabial

scales to the center of the eye, number of scales between

the supraorbital semicircles, number of scales from the

interparietal to the supraorbital semicircles, number of

loreal rows. As none of these traits are the basis for our

diagnoses (see below), this analysis provides a some¬

what independent check of our species inferences. We

used Euclidean distances of standardized data (i.e., mean

= 0, standard deviation =1) and present observed and

expected Delta (i.e., the test statistic), P- value based on

99 randomizations, and Chance Corrected Within Group

Agreement (i.e., effect size). Sexual dimorphism, if pres¬

ent, appeared to be less than interspecific dimorphism for

the studied traits. Therefore to increase our small sample

sizes we analyzed both sexes together. We demonstrate

this lack of clustering by sex in two ways. First, we per¬

formed the same MRPP analysis but grouped by sex. Sec¬

ond, we performed Principal Component Analysis (PCA)

of the above characters and present bivariate graphs of

the first two principal components labeled by sex and by

hypothesized species. Although PCA may not be appro¬

priate for statistical interpretations and tests given our

small sample sizes and high observation-to-variable ratio

(see below; although we note that similar PCA results

are obtained with subsamples of variables), we believe

this technique nevertheless to be useful for the limited

purpose of visualizing gross differences in clustering pat¬

terns by sex versus by species. Statistical analyses were

performed in Stata (2013) and Microsoft Excel.

The hypothesized new species were found to form

a well-supported clade with Anolis insignis , A. micro-

tus , and A. ginaelisae (Bayesian Posterior Probability

of 100%) by Poe et al. (2015), who included all known

Dactyloa-cl&dQ Anolis in their phylogenetic analysis.

Terminal taxaNSPE, NSP.F, NSPL in Poe et al.’s (2015)

Fig. 5 correspond to species described herein. In order to

more finely examine the interrelationships of the insig-

nis- like anoles, we added new morphological data to the

data matrices of Poe et al. (2015) and Poe et al. (2017),

and analyzed these data for A. insignis , A. microtus, A.

ginaelisae , the three additional species described here,

and two Dactyloa-c\adQ outgroups (A. frenatns , A. fra-

seri). We eliminated characters that did not vary in the

ingroup and added characters based on our examina¬

tion of specimens for the current study. The final matrix

includes 18 characters of morphology and 50 genes of

DNA sequence data. Additional details of data proper¬

ties and collection (i.e., gene names, data sources, par¬

titioning) are in Poe et al. (2017). Morphological char¬

acters were rescaled differently from Poe et al. (2017)

to account for new data and our restricted taxon sample.

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Two new species similar to Anolis insignis and resurrection of Anolis brooksi

Fig. 2. Dewlaps of A) Anolis brooksi , male, El Cope, Panama; B) A. brooksi , female, El Cope, Panama; C) A. savagei, male, Las

Cruces, Costa Rica; D) A. savagei, female, Las Cruces, Costa Rica; E ) A. kathydayae, male, Fortuna, Panama; F) A. kathydayae,

female, Fortuna, Panama.

Although this data matrix includes 24,897 characters, we

note that only the morphological dataset is informative

for the interrelationships of A. insignis and the other three

species discussed in depth in this paper, as only two of

the discussed species are scored for some DNA sequence

data. The included DNA data are useful for establishing

the monophyly of these forms with A. microtus and A.

ginaelisae and examining genetic divergences as they

relate to hypothesized species (see below). The phyloge¬

netic matrix analyzed for this paper is available electron¬

ically at: stevenpoe.net. The morphological characters

and data matrix are in Appendices 1 and 2 respectively.

We analyzed this matrix using a Bayesian phyloge¬

netic approach as implemented in MrBayes (Huelsen-

beck and Ronquist 2001) using the model parameters and

settings of Poe et al. (2017), except that a heating temper¬

ature of 0.01 was used and the analysis was carried out

for 2,000,000 generations. That is, we included separate

GTR + G models for each of 15 DNA partitions of the

50 genes (including partitions by codon position for the

best-sampled protein coding genes COI and ND2) with

partitions determined by Partitionfmder (Lanfear et al.

2012) and model-averaging across the entire GTR model

space for each gene partition (“nst=mixed” in MrBayes).

Morphological character evolution was modeled with the

“standard” MrBayes model. We checked for convergence

of parameter values by examining estimated sample sizes

in Tracer (Rambaut et al. 2014).

Results

Four very different male dewlap types are recognizable

(Figs. 1, 2) and correlate with geography. Male speci¬

mens from central and northern Costa Rica have orange-

Amphib. Reptile Conserv.

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Poe and Ryan

Fig. 3. Graph of principal components 1 and 2 for traits used in MRPP analysis of species of Anolis studied here, labeled by A) sex

and B) putative species.

red dewlaps; those from southwestern Costa Rica have

pale pink dewlaps with black streaks; those from the For-

tuna area in Panama have white dewlaps; and those from

eastern Panama (Santa Fe, El Cope, Cerro Azul) have

peach-tan dewlaps. We hypothesize that these differences

represent inter- rather than intraspecific variation for four

reasons. First, we observed at least three adult males

within each range, with no significant variation in male

dewlap color pattern at any locality or between locali¬

ties where a particular dewlap type was found. Second,

the degree of difference among these male dewlap color

patterns would be unprecedented as intraspecific varia¬

tion in Anolis. Third, each male dewlap-group is distin¬

guishable by additional traits (see below). Fourth, groups

identified by male dewlap color are different according to

MRPP. The MRPP analysis was significant (P = 0.01, 99

randomizations; Delta = 3.09, Deltanull = 3.85), reject¬

ing the null hypothesis of random assignment of indi¬

viduals to groups. The Chance Corrected Within Group

Agreement was 0.20, indicating 20% within group agree¬

ment above that expected by chance. The MRPP analy¬

sis was nonsignificant when individuals were grouped by

sex rather than by hypothesized species (P = 0.24, 99

randomizations; Delta = 3.75; Deltanull = 3.85), which

is compatible with our observation of a lack of sexual

dimorphism in these characters. Figure 3 shows that our

studied individuals do not cluster morphologically by sex

according to PCA of traits used in the MRPP. Based on

this evidence, we are comfortable pooling our samples

by sex within species for the MRPP analysis.

We associate the Costa Rican specimens examined

from near the city of San Jose with the nominate species

Anolis insignis Cope 1871 (Type locality: “San Jose”).

Our central Panama specimens from Cerro Azul, Panama

province, and El Cope, Code province may be an unrec¬

ognized lineage. Alternatively, on geographic and mor¬

phologic grounds they may be associated with Diapho-

ranolis brooksi Barbour (holotype MCZ 16297) from the

Darien of Panama—an individual previously determined

to be “an unquestioned juvenile of A. insignis ” (Savage

and Talbot 1978). As a preserved specimen, the A. (=

Amphib. Reptile Conserv.

Diaphoranolis) brooksi holotype specimen appears sim¬

ilar to juveniles we collected at El Cope, and we lack

adult dewlap photos and adult specimens for the Darien

population. We choose to assign our easternmost form

to A. brooksi pending future collection of A. insignis-

like anoles in Darien. The distinctive populations from

Fortuna, Chiriqui, Panama, and Las Cruces, Puntarenas,

Costa Rica, currently lack names.

Below we redescribe Anolis insignis from specimens

near the city of San Jose Costa Rica, and A. brooksi from

specimens from El Cope and Cerro Azul in Panama. We

describe two new species from Las Cruces, Costa Rica,

and Fortuna, Panama respectively. We describe variation

in A. insignis and A. brooksi and describe holotype speci¬

mens for the two new species. Comparisons among the

four species are summarized in Table 1. The results of

our phylogenetic analysis of these species are summa¬

rized in Fig. 4. We infer that the Markov Chain Monte

Carlo analysis was run long enough to sample parameters

in proportion to their true posterior probability distribu¬

tions based on low standard deviation of split frequencies

(0.011) and estimated sample sizes well above 200 for all

parameters, as recommended by Rambaut et al. (2014).

Systematics

Anolis insignis Cope 1871

(Figures 1,5)

Holotype

Lost (Savage and Talbot 1978); from “Costa Rica: Pro-

vincia de San Jose: near Ciudad San Jose; probably from

near La Palma,” according to Savage and Talbot (1978)

and Savage (1974).

Examined specimens

LACM 149495 collected by J. Hagnauer and N.J. Scott

in January 1975 (no day provided) and LACM 149496

collected by G. Hagnauer and W. Timmerman in April

1974 (no day provided) from Costa Rica, Alajuela, Vicin¬

ity of Bijagua (10.7333; -85.1; 425 m); LACM 149500

July 2017 | Volume 11 | Number 2 | e141

Two new species similar to Anolis insignis and resurrection of Anolis brooksi

Anolis species similar to A. insignis. Numbers on clades are

posterior probabilities.

collected by K. Timmerman 20 June 1984 and LACM

149497 collected by H. Hespenheide and E. Fisher (no

date provided) from Costa Rica, Puntarenas, Monteverde

(10.3; -84.816667; 1,455 m); LACM 149498 collected

by P. Siegfried (no date provided) from Costa Rica, Ala-

juela, Poco Sol (10.3667; -84.6167; 580 m).

Diagnosis

Anolis insignis and the three species described below are

the only Central American Anolis to combine large size

(> 120.0 mm SVL), smooth scales on the upper thigh,

and short limbs (Savage and Talbot 1978). Anolis insig¬

nis is diagnosed from the three species described below

by its orange-red male dewlap (Fig. 1; white, peach-tan,

and pink with dark streaks, respectively by species, in

the other forms). It further differs from the Southwest¬

ern Costa Rican form in its lack of a postorbital blotch

(present in the Southwestern Costa Rican form); from the

Fortuna form in its prominent postcloacal scales in males

(obscure in the Fortuna form); from A. brooksi in some

scale counts (Table 1; e.g., greater number of postros-

trals) and details of color pattern (Savage and Talbot

1978; e.g., absence of narrow black lines dorsally).

External description (in mm)

Snout-vent length (SVL) to 157.0 mm male, 140.0 mm

female; head length-SVL ratio 0.24-0.25, head width-

SVL ratio 0.14-0.16; ear height-SVL ratio 0.015-0.028;

femoral length-SVL ratio 0.24-0.25; tail length-SVL

ratio 1.9-2.1. Dorsal head scales mostly smooth, a few

with weak keels or rugosity apparently reflecting under¬

lying bone or ossification, pustules present in some spec¬

imens; frontal depression present, anterior half of snout

raised in two faint parallel rows; rostral overlaps mental

anteriorly; lateral edges of mental scales extend farther

posteriorly than rostral; 9-11 scales across snout between

second canthals; 2-3 scales between supraorbital semi¬

circles; 2-3 scales separating interparietal and supraor¬

bital semicircles; suboculars in contact with supralabials;

five loreal rows; no elongate superciliaries, first super¬

ciliary is smaller than first canthal; anterior row of small

scales following canthals along edge of orbit; circumna-

sal scale separated from rostral by one scale; interpari-

Table 1. Morphological traits of species similar to Anolis insignis. Measurements are in millimeters. Means are given with ranges

in parentheses. Measurement characters were scored only for adults.

Anolis insignis

n — 2 males, 3 females

A. brooksi

n = 3 males, 2 females

A. kathydayae

n — 2 males, 2 females

A. savagei

n = 1 male, 1 female

Snout to vent length male

154.5

152.7

142.3

141.1

(152.0-157.0)

(129.5-176.0)

(136.6-148.0)

(1411)

Snout to vent length female

139.0

134.0

136.1

(juvenile)

(138.0-140.0)

(134.0)

(136.1)

Head length male

37.4

36.0

36.4

33.0

(36.2-38.6)

(30.5-41.4)

(34.8-38)

(33.0)

Head length female

34.6

34.8

33.9

(33.6-35.3)

(34.8)

(33.9)

Head width male

21.6

21.4

21.6

21.0

(20.8-22.4)

(18.1-24.7)

(20.7-22.5)

(21.0)

Head width female

21.3

20.8

21.2

(20.4-22.6)

(20.8)

(21.2)

Ear height male

3.0

3.7

4.2

2.9

(2.3-3.6)

(3.4-4.1)

(3.9-4.5)

(2.9)

Ear height female

3.5

3.7

4.1

(2.9-3.9)

(3.7)

(4.1)

Amphib. Reptile Conserv.

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Poe and Ryan

Table 1 (continued). Morphological traits of species similar to Anolis insignis. Measurements are in millimeters. Means are given

with ranges in parentheses. Measurement characters were scored only for adults.

Anolis insignis

n — 2 males, 3 females

A. brooksi

n — 3 males, 2 females

A. kathydayae

n — 2 males, 2 females

A. savagei

n = 1 male, 1 female

Femoral length male

37.9

37.5

36.3

30.4

(36.7-39.1)

(31.6-43.4)

(34.2-38.5)

(30.4)

Femoral length female

34.5

33.4

32.7

(33.5-35.1)

(33.4)

(32.7)

4 th toe length male

25.2

21.4

22.4

19.9

(24.7-25.7)

(19.1-23.7)

(20-24.8)

(19.9)

4 th toe length female

23.4

21.5

20.2

(22.4-24.7)

(21.5)

(20.2)

Tail length

294.0

291.6

284.0

245.0

(287.0-310.0)

(240.0-355.0)

(275.0-292.0)

(245.0)

Number of dorsal scales in 5% SVL

9.5

11.6

9.0

8.0

(7-11)

(11.0-12.0)

(9.0)

(8.0)

Number of ventral scales in 5% SVL

9.5

8.5

10.0

8.0

(8.0-11.5)

(8-9)

(10.0)

(8.0)

Number of scales across snout at sec-

10.0

10.4

10.0

8.5

ond canthals

(9.0-11.0)

(10-11)

(9.0-11.0)

(8.0-9.0)

Number of scales between supraorbital

2.2

3.4

3.2

2.0

semicircles

(2.0-3.0)

(3.04.0)

(3.0-4.0)

(2.0)

Number of scales between interparietal

2.6

3.0

3.2

1.5

and supraorbital semicircles

(2.0-3.0)

(2.0-4.0)

(3.0-4.0)

(1.0-2.0)

Number of postrostral scales

7.8

6.8

5.7

6.5

(7.0-10.0)

(6.0-7.0)

(5.0-6.0)

(6.0-7.0)

Number of postmental scales

7.4

6.0

5.0

7.5

(6.0-9.0)

(5.0-7.0)

(4.0-5.0)

(7.0-8.0)

Number of scale rows separating

suboculars and supralabials

Number of supralabials from rostral to

8.2

8.0

7.2

7.0

center of eye

(8.0-9.0)

(7.0-9.0)

(7.0-8.0)

(7.0)

Number of lamellae under phalanges II

26.6

26.4

25.5

26.7

& III of 4 th toe

(25.0-27.0)

(25.5-27.5)

(23.5-27.0)

(25.0-28.5)

Number of loreal rows

5.0

5.4

6.0

4.5

(5.0)

(5.0-6.0)

(6.0)

(4.0-5.0)

Posterolateral extent of mental

<= rostral

>= rostral

<=rostral

<rostral

etal length-SVL ratio 0.014-0.017; 8-9 supralabials to

center of eye; 6-9 postmentals; 7-10 postrostrals; scales

in supraocular disc only slightly differing in size; mental

partially divided posteriorly, extending posterolaterally

equal to or shorter than rostral, with straight posterior

border; 0-2 keeled enlarged sublabials.

Dewlap reaches well posterior to axillae in males

and females; dewlap scales in rows of multiple scales in

both sexes; no axillary pocket; pair of distinct, abruptly

enlarged postcloacal scales in males; dorsal scales

smooth; zero enlarged middorsal rows, 7-11 longitudi¬

nal rows in 5% of SVL; ventral scales in transverse rows,

smooth, 8-12 scales in 5% of SVL; supradigitals multi-

carinate; toepads expanded; 25-27 lamellae under third

and fourth phalanges of fourth toe; thigh scales smooth

Amphib. Reptile Conserv. 6

dorsally and ventrally, unicarinate anteriorly, multicari-

nate at knee; tail with a double row of middorsal scales.

Distribution and habitat

We have no experience with Anolis insignis in life. Sav¬

age (2002) reports that this is an uncommon canopy spe¬

cies that inhabits undisturbed forests.

With our recognition of multiple species within what

was previously considered Anolis insignis , we restrict the

range of A. insignis sensu stricto to the Cordillera Tilaran

and Cordillera Central of Costa Rica. We currently con¬

sider the range of A. insignis to encompass localities for

A. insignis-WkQ anoles collected in Northern and Central

Costa Rica. Assuming this range, the known elevation of

A. insignis is 425 m (Bijagua, CRE 3715, UCR 8783) to

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Two new species similar to Anolis insignis and resurrection of Anolis brooksi

Fig. 5. Dorsal headscales of X) Anolis kathydayae , MSB 96613;

B) A. brooksi, MSB 75647 C) A. savagei, MSB 96616; D) A

insignis LACM 149500.

1,500 m (La Palma, Holotype).

Anolis brooksi Barbour 1923

(Figures 2, 5-7)

Fig. 6. Adult male individuals of A) Anolis brooksi, El

Cope, Panama; B) A. savagei, Las Cruces, Costa Rica; C) A.

kathydayae, Fortuna, Panama.

collected by Thomas Barbour and Winthrop Brooks, in

April, 1922.

Examined specimens

Parque Nacional G.D. Omar Torrijos H., Code Prov¬

ince, Panama; 8.668, -80.593, 775 m: MSB 79924, MSB

79922, MSB 79923, MSB 75647, MSB 79925. Speci¬

mens examined but not scored for quantitative analysis:

Cerro Azul, Panama, Panama: MVUP 2007. Mt. Sapo,

Darien, Panama: MCZ 16297 (holotype).

Holotype Diagnosis

MCZ 16297 Diaphoranolis brooksi , juvenile female, Anolis insignis, A. brooksi, and the two species described

from Mt. Sapo, Darien, Panama, 2,500 feet elevation; below are the only Central American Anolis to combine

Amphib. Reptile Conserv.

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Poe and Ryan

Fig. 7. Dewlaps of males of Anolis brooksi from A) Cerro Azul, Panama (MVTJP 2007); B) Santa Fe, Panama

(not collected).

large size (> 120.0 mm SVL), smooth scales on the upper

thigh, and short limbs (Savage and Talbot 1978). Ano¬

lis brooksi is diagnosed from the three other insignis-

like anole species discussed here by its peach-tan male

dewlap (Fig. 2; orange-red in A. ins ignis; white, pale

pink with dark streaks, respectively by species, in the

other two forms). It further differs from the Southwest¬

ern Costa Rican form in its lack of a postorbital blotch

(present in the Southwestern Costa Rican form) and its

female dewlap color pattern (white or brown with dark

streaks; pale pink with dark streaks in the Southwestern

Costa Rica form); from the Fortuna fonn in its prominent

postcloacal scales in males (obscure in the Fortuna form)

and its female dewlap color pattern (white or brown with

dark streaks; patternless white in the Fortuna fonn); from

A. insignis in some scale counts (Table 1; e.g., fewer

postrostrals) and details of color pattern (Savage and Tal¬

bot 1978; e.g., presences of narrow black lines dorsally).

Description (measurements in mm)

Snout-vent length to 176.0 mm male, 134.0 mm female;

head length-SVL ratio 0.24-0.26, head width-SVL ratio

0.14-0.16; ear height-SVL ratio 0.023-0.028; femoral

length-SVL ratio 0.24-0.25; tail length-SVL ratio 1.9-

2.1. Dorsal head scales mostly smooth; frontal depres¬

sion present, anterior half of snout raised in two faint

parallel rows; rostral overlaps mental anteriorly; lateral

edges of mental extend farther posteriorly than rostral;

10-11 scales across snout between second canthals; 3-4

scales between supraorbital semicircles; 2-4 scales sepa¬

rating interparietal and supraorbital semicircles; suboc¬

ulars in contact with supralabials; 5-6 loreal rows; no

elongate superciliaries, first superciliary is approxi¬

mately equal in size to first canthal; row of small scales

following canthals along edge of orbit; circumnasal scale

separated from rostral by 1-2 scales; interparietal length-

SVL ratio 0.014-0.015 (or absent); 7-9 supralabials to

center of eye; 5-7 postmentals; 6-7 postrostrals; some

enlarged scales present in supraocular disc (or all scales

approximately equal), decreasing gradually in size; men¬

tal partially divided posteriorly, extending posterolater-

ally beyond rostral, with posterior border straight or in

convex or concave arc; 1-2 keeled enlarged sublabials.

Dewlap reaches well posterior to axillae in males and

females; dewlap scales in rows of multiple scales in both

sexes; no axillary pocket; distinct, abruptly enlarged

postcloacal scales present in males; dorsal scales smooth;

zero enlarged middorsal rows, 11-12 longitudinal rows

in 5% of SVL; pair of middorsal scale rows raised in larg¬

est specimen; nuchal crest present with slightly enlarged

triangular middorsal scales; ventral scales in transverse

rows, smooth, 8-9 scales in 5% of SVL; supradigitals

multicarinate; toepads expanded; 25-28 lamellae under

third and fourth phalanges of fourth toe; thigh scales

smooth dorsally and ventrally, unicarinate anteriorly and

multicarinate at knee; tail with a double row of middor¬

sal scales.

Color pattern in life

Adult males from El Cope (MSB 75647) and Cerro Azul

(MVUP 2007) appeared mainly tan dorsally, with diffuse

banding of white, black, green, peach, and dark brown.

The limbs and digits were banded with narrow double

lines of black or dark green. The tail was patterned with

distinct black and greenish bands. The dewlap was solid

peach-tan. An adult female (MSB 79925) appeared simi¬

lar to the males but possessed scant green dorsally, with

a white dewlap with prominent dark streaking. A dark

shoulder blotch is evident in individuals in some of our

photos of adults, but not in others. The iris is red. The

throat is fight and the tongue appeared peach in an El

Cope specimen but yellow in the specimen from Cerro

Azul. Males from Cerro Azul and Santa Fe had dew¬

laps similar to the El Cope specimen, but slightly paler

(Fig. 7). An uncollected specimen from Isla Escudo de

Veraguas, Bocas del Toro, that we tentatively allocate to

this species had a dewlap similar to those figured here

but with a brighter, slightly orange-yellow tint. An adult

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e141

Two new species similar to Anolis insignis and resurrection of Anolis brooksi

Sources: Esri, USGS, NOAA

Legend

Species

^ A. savage/'

^ A. brooksi

| A. kathydayae

A A. insignis

Kilometers

100

150

200

Fig. 8. Map of Panama and Costa Rica, showing localities for specimens referenced in text. Type localities are in red. Black

symbols are specimens examined (type locality specimens also were examined for all species). Gray symbols represent unexamined

specimens or photographic evidence discussed in text. Each point may represent multiple individuals (see text).

female dewlap figured by Lotzkat et al. (2013) was light

brown with dark streaks.

Distribution and habitat

We collected Anolis brooksi in El Cope and Cerro Azul

sleeping at night on saplings and tree branches from three

to five meters above the ground. Specimens were col¬

lected in dense secondary forest (El Cope) and in dis¬

turbed habitat (Cerro Azul). Photographic evidence of

male dewlap color pattern indicates the species is present

at Santa Fe, Veraguas (see below) and, potentially, Isla

de Escudo, Bocas del Toro (pers. obs.). Thus, A. brooksi

appears to occur from sea level to 970 m from Darien

north to Bocas del Toro.

Anolis savagei, new species

(Figures 2, 5, 6)

urn:lsid:zoobank.org:act: 1F0F7528-F3D6-43B3-993D-E7AEBCB5A39C

Holotype

MSB 96616, adult male, collected at Las Cruces, Puntar-

enas, Costa Rica; 8.78242, -82.95886,1,127 m; collected

by Steven Poe, Eric Schaad, Ian Latella, and Mason Ryan

on 20-23 March 2009.

Paratypes

UCR20635 (not scored; POE 2671); LACM 149499 col¬

lected by R.W. McDiarmid on 21 Aug 1971 from Costa

Rica, Puntarenas, San Vito de Java, OTS Las Cruces Bio¬

logical Station (8.816667; -82.966667; 1,100 m).

Diagnosis

Anolis insignis, A. brooksi, A. savagei , and the species

described below are the only Central American Ano¬

lis to combine large size (> 120.0 mm SVL), smooth

scales on the upper thigh, and short limbs (Savage and

Talbot 1978). Anolis savagei is distinguished from A.

insignis, A. brooksi , and the form described below by its

male dewlap color pattern of pale pink with dark streaks

(orange-red in A. insignis ; peach-tan in A. brooksi ; white

in the form described below; Figs. 1, 2) and presence of

a prominent postorbital blotch (absent in A. insignis, A.

brooksi, and the form described below).

Etymology

This name is a patronym to honor Dr. Jay M. Savage for

his contributions to Neotropical herpetology, especially

his seminal works, mentorship, and leadership in tropi¬

cal biology and conservation in Costa Rica. Dr. Savage

helped found the Organization of Tropical Studies (OTS)

and the type locality of this species is the Las Cruces

OTS field station.

Description of holotype

Snout-vent length 141.0 mm; head length-SVL ratio

0.23, head width-SVL ratio 0.15; ear height-SVL ratio

0.021; femoral length-SVL ratio 0.22; tail length-SVL

ratio 1.74. Dorsal head scales smooth, some rugose; fron¬

tal depression present, dorsum with weak parallel rows

evident anteriorly; rostral overlaps mental anteriorly;

eight scales across snout between second canthals; two

scales between supraorbital semicircles; one scale sepa¬

rating interparietal and supraorbital semicircles; subocu¬

lars in contact with supralabials; five loreal rows; zero

elongate superciliaries, first large scale posterior to can¬

thals is slightly smaller than first canthal; row of slightly

enlarged scales along anterior aspect of dorsolateral edge

of orbit; circumnasal scale separated from rostral by one

scale; interparietal length-SVL ratio 0.021; seven supra¬

labials to center of eye; seven postmentals; six postros-

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e141

Poe and Ryan

Fig. 9. Box plots showing variation between Anolis insignis (i), A. brooksi (b), A. kathydayae (k), and A. savagei (s). Traits are

number of scales between interparietal and supraorbital semicircles (ip), number of expanded lamellae on fourth toe (lm), number

of loreal rows (lr), number of postmental scales (pm), number of postrostral scales (pr), number of scales across the snout between

the second canthals (sc), number of scales between the supraorbital semicircles (so), number of supralabial scales from rostral to

center of eye (si), snout to vent length (sv), head length relative to sv (hi), femoral length relative to sv (fl), tail length relative to

sv (ta), toe length relative to sv (to), ear height relative to sv (eh), number of longitudinal dorsal scales in 5% of sv (d5), number of

longitudinal ventral scales in 5% of sv (v5).

trals; some enlarged scales present in supraocular disc,

decreasing gradually in size; mental partially divided

posteriorly, with posterior border in concave arc; lateral

edges of rostral extend farther posteriorly than mental;

two enlarged smooth sublabials; more posterior lateral

throat scales are keeled.

Dewlap reaches well posterior to axillae in males

and females; dewlap scales in rows of multiple scales

in both sexes; pair of distinct, abruptly enlarged post-

cloacal scales present; dorsal scales smooth, with no

enlarged middorsal rows, 12 longitudinal rows in 5% of

SVL; nuchal crest present with slightly enlarged middor¬

sal scales; ventral scales in transverse rows, smooth, 11

scales in 5% of SVL; supradigitals multicarinate; toepads

expanded; 28-29 lamellae under third and fourth phalan¬

ges of fourth toe; tail with a double row of middorsal

scales; thigh scales smooth dorsally and ventrally, mostly

smooth anteriorly with a few weakly unicarinate scales.

Color pattern in life

Color patterns of a male (MSB 96616) and female (UCR

20635) specimen were very similar. Dorsal color was

generally brown, with alternating tan and dark brown

irregular bands, the dark bands with some lighter blotch¬

ing within them. Photographic evidence (R. Stanley, I.

Latella; pers. comms.) indicates some individuals pos¬

sess green and pale peach-orange dorsally in addition to

brown. The dewlap in both sexes was pale pink with black

horizontal streaks. No shoulder blotch was observed, but

a prominent postorbital blotch was present in all adult

specimens examined (n = 5).

Distribution and habitat

We found Anolis savagei at night sleeping 5-6 m up on

narrow tree branches along trails in the closed canopy

secondary forest of Las Cruces Biological Station. More

work is needed on the ecology of this species. Specimens

examined for this paper are from the Cordillera de Tala-

manca in southwestern Costa Rica at 1,127 m. Two indi¬

viduals photographed from the western edge of Chirripo

National Park at 1,590 m (R. Stanley, pers. comm.) appar¬

ently are A. savagei based on the presence of a promi¬

nent postorbital blotch in each, and the darkly streaked

dewlap of the individual for which the dewlap is partially

visible. We have not examined the A. insignis -like speci¬

mens reported from near sea-level by Savage and Tal¬

bot (1978; Ballena, BM 1909.7.10.20; Rincon de Osa,

UCR 4387), but these are likely to be A. savagei based on

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July 2017 | Volume 11 | Number 2 | e141

Two new species similar to Anolis insignis and resurrection of Anolis brooksi

those authors’ emphasis of a postorbital blotch in these

specimens. Given these localities, A. savagei occurs on

the Pacific slope of the Cordillera de Talamanca from sea

level to at least 1,590 m, from Chirripo National Park

south to Las Cruces (Fig. 8).

Anolis kathydayae, new species

(Figs. 2, 5, 6)

urn:lsid:zoobank.org:act:31E4F176-EAll-4172-A0El-A9DE3AE65287

Holotype

MSB 96614 adult male from Panama, Chiriqui, trail

from paved road near Chiriqui/Bocas del Toro province

boundary at Fortuna pass; 8.78533, -82.21434, 1,178 m;

collected by Steven Poe and Julian Davis on 13 March

2013.

Paratypes

MVUP 2128, juvenile from Panama, Bocas del Toro,

side of Fortuna pass road, just north of Chiriqui/Bocas

del Toro boundary; 8.78008, -82.20584, 1,038 m; col¬

lected by Steven Poe and Julian Davis on 13 March 2013.

MSB 96612, same locality as holotype, collected by Ste¬

ven Poe and Caleb Hickman, December 2003. MSB

79921, MSB 96613, same locality as holotype, collected

by Steven Poe, Erik Hulebak, and Heather Maclnnes on

28 July 2005.

Diagnosis

Anolis insignis , A. brooksi , A. savagei , and A. kathy¬

dayae are the only Central American Anolis to combine

large size (> 120.0 mm SVL), smooth scales on the upper

thigh, and short limbs (Savage and Talbot 1978). Anolis

kathydayae is distinguished from these species by male

dewlap color pattern (white with light green or dull blue

tint in male A. kathydayae ; orange-red in male A. insig¬

nis', pale pink with dark streaks in A. savagei ; peach-tan

in A. brooksi ; Figs. 1, 2). It is further distinguished from

A. savagei and A. brooksi by female dewlap color pat¬

tern (solid white with greenish tint in A. kathydayae ;

white or brown with dark streaks in A. brooksi ; pale pink

with dark streaks in A. savagei ; unknown in A. insignis).

At least in our samples, A. kathydayae is further dis¬

tinguished from A. insignis by several scale characters

(Table 1; e.g., fewer postmentals, 4-5 versus 6-9 in A.

insignis ). Additionally, the two male A. kathydayae we

have examined display obscure, weakly enlarged post-

cloacal scales, whereas all male individuals of the other

insignis-like anoles we have examined display large, dis¬

tinct postcloacal scales.

Etymology

The name is a matronym to honor Kathy Day and the

Miller Institute for Basic Research in Science. Kathy

has contributed greatly to the professional and personal

development of scientists and the advancement of basic

science through her position running the Miller Institute.

Description of holotype

Snout-vent length 148.0 mm; head length-SVL ratio 0.26,

head width-SVL ratio 0.15; ear height-SVL ratio 0.030;

femoral length-SVL ratio 0.26; tail length-SVL ratio 2.0.

Dorsal head scales mostly smooth, some with weak keels

or wrinkling reflecting underlying bone or ossification;

frontal depression present, dorsum with weak parallel

rows evident anteriorly; rostral overlaps mental anteri¬

orly; 10 scales across snout between second canthals;

four scales between supraorbital semicircles; subocu¬

lars in contact with supralabials; zero elongate supercili¬

ary scales; first scale posterior to canthals is smaller than

first canthal; six loreal rows; circumnasal scale separated

from rostral by one scale; interparietal length-SVL ratio

0.018; seven supralabials to center of eye; six postmen¬

tals; six postrostrals; some enlarged scales present in

supraocular disc, decreasing gradually in size, bordered

medially by a partial row of small scales; mental partially

divided posteriorly, extending posterolaterally approxi¬

mately even with rostral, with posterior border in con¬

cave arc; one-two enlarged keeled sublabials.

Dewlap reaches well posterior to axillae in males

and females; dewlap scales in rows of multiple scales in

both sexes; no axillary pocket; postcloacal scales slightly

enlarged; dorsal scales smooth, pair of middorsal scale

rows slightly raised, nine longitudinal rows in 5% of

SVL; nuchal crest present with pair of slightly enlarged

triangular middorsal scale rows; ventral scales in trans¬

verse rows, smooth, 10 scales in 5% of SVL; supradigi-

tals multicarinate; toepads expanded, 27 lamellae under

third and fourth phalanges of fourth toe; tail with a double

row of middorsal scales; thigh scales smooth to weakly

keeled dorsally and ventrally, unicarinate anteriorly, mul-

ticarinate at knee.

Color pattern in life

An adult male (MSB 96614) had a tan body with discrete

dark green broad bands speckled with light tan. The ante¬

rior body to posterior head had a bluish-green wash. Dor¬

sal head scales were greenish-tan, outlined with darker

brown. A very faint blotch was present above the shoul¬

der. The iris was brown and the tongue was dark yel¬

low. The limbs and digits were greenish-tan, with darker

green bands. The tail was banded with sharply alternat¬

ing black and tan bands. The dewlap was white, with a

yellowish-green tint. Another adult male (MSB 96613)

was patterned similarly but mostly lacked green—the

anterior bluish-green wash was absent, and the bands

were dark brown to black with no greenish tint. The dew¬

lap of this individual was white, with faint blueish tint.

One adult female (MSB 79921) appeared dark greenish

with diffuse banding of white, darker green, and brown.

The dewlap appeared very pale yellow-green. A juvenile

female (SVL 87.0 mm; MSB 96612) appeared nearly

completely pale green, with faint white lateral bands and

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e141

Poe and Ryan

some darker green reticulations on the body and darker

green bands on the limbs and digits, and white blotches

dorsally on the head. This individual had a pale green¬

ish-yellow dewlap with some dark green reticulations. A

near-hatchling (MSB 96615) had a cream dewlap with

prominent black streaks.

Distribution and habitat

We found adults of Anolis kathydayae sleeping horizon¬

tally on narrow branches along a trail in secondary forest

three to five meters above the ground, and juveniles at

roadside habitat four to five meters above the ground on

twigs. Elevational range of these two sites is 1,038-1,178

m. Currently known distribution for A. kathydayae is the

Fortuna pass area of Panama.

Discussion

The four insignis- like Anolis species discussed here are

distinct in male dewlap color (Figs. 1, 2), which usually

varies little within species of Anolis , and in additional

morphological traits (Diagnoses; Table 1; Fig. 9). Below

we discuss the status of each species relative to previous

discussions on these forms and our own views of the dis¬

tinctiveness and importance of diagnostic traits for these

species, especially in light of our small sample sizes.

We also discuss some limited molecular data bearing on

these forms.

Savage and Talbot (1978) originally drew attention

to differences between Northern Costa Rican (i.e., Ano¬

lis insignis ), southern Costa Rican (i.e., A. savagei), and

Panamanian (i.e., A. brooksi, A. kathydayae ) “A. insig¬

nis” The postocular blotch of southern Costa Rican

forms discussed by these authors appears to be an auta-

pomorphic diagnostic trait for A. savagei. Including pho¬

tos, preserved specimens, and reports from Savage and

Talbot (1978), we are aware of eight specimens that are

assignable to A. savagei based on male dewlap color

of the population and locality. All eight of these speci¬

mens possess a postocular blotch, and all A. insignis, A.

brooksi , and A. kathydayae examined by us (including

photos, n = 18) lack a postocular blotch. Additionally, A.

savagei is quite distinct in overall morphology (Table 1;

Diagnoses; Fig. 9).

Anolis kathydayae is striking in its possession of

pale, patternless dewlaps in males and females (Fig. 2).

Although a few species of Anolis display intraspecific

variation in male dewlap color pattern, such variation

nearly always occurs within populations (e.g .,A. gemmo-

sus around Mindo, Ecuador; A. valencienni in northern

Jamaica) or at hybrid zones (e.g., distichus- group forms;

Glor and FaPort 2012). Thus we note the relative invari¬

ance of the distinctive male dewlap of A. brooksi across

El Cope in Code (Fig. 2), Santa Fe in Veraguas (Fig. 7),

Cerro Azul in Panama (Fig. 7), and possibly Isla Escudo

de Verguas in Bocas del Toro (pers. obs.; see above) as

evidence for the species status of this form relative to

Amphib. Reptile Conserv.

the other forms discussed here. We note the constancy

of the distinctive streaked dewlap of A. savagei between

Fas Cruces and Chirripo (a distance of -100 km), and

the presence of an orange-red male dewlap of A. insignis

over at least three localities in northern Costa Rica (Poco

Sol, Fa Fortuna, Monteverde; photographic evidence).

We know of no intermediate forms between these dew¬

lap types, although some minor variation occurs within

each of them. Thus we view the presence of the unusual

male and female dewlaps of A. kathydayae as strong evi¬

dence for the species status of this form, in addition to

the molecular evidence presented below and the external

morphological patterns shown in Table 1 and Fig. 9.

We observed three of the four species of insignis-likQ

anoles to differ consistently in female dewlap color (Fig.

2). Female Anolis brooksi have a white or brown dewlap

with black streaks, female A. savagei have a pale pink

dewlap with dark streaks, and female A. kathydayae have

a pale, patternless dewlap (we have not seen a confirmed

female dewlap of true A. insignis). We note that there is

considerable ontogenetic variation in this trait, with all

examined juvenile females in life (A. kathydayae, A.

brooksi) possessing some dark streaking on the dewlap.

Our observations of adult female dewlap color pattern

suggest some taxonomic utility to this character in this

case, but these differences may not be evident in larger

sample sizes.

The Northern Costa Rican form (i.e., Anolis insignis)

and the widespread Panama form (i.e., A. brooksi) share

similar dorsal color patterns and their male dewlaps are

most similar among the species discussed here (Figs. 1,

2). There remains much work to be done on the system-

atics of these forms. The geographic patterns among the

insignis-likQ Anolis , including two similar geographi¬

cally intervening species (i ,e.,A. savagei, A. kathydayae ;

Fig. 8), suggests that conspecificity of A. brooksi and A.

insignis is unlikely. Still, this is a hypothesis that begs

continued investigation, as is the potential presence of

multiple species within A. insignis and A. brooksi. In par¬

ticular, we have little confidence that the populations that

we are calling A. brooksi are actually conspecific with

topotypical A. brooksi, for which we have examined only

a single preserved juvenile specimen (i.e., the holotype).

We elect to use this name because juveniles of the tan-

dewlap form (i.e., A. brooksi as we are recognizing it)

are indistinguishable from the holotype of A. brooksi,

and the range of the tan dewlap form approaches the A.

brooksi type locality to the east. To give the tan-dewlap

form a new name rather than assume its conspecificity

with A. brooksi seems unconservative under these cir¬

cumstances.

The low sample sizes of our analyses (Table 1; sup¬

plemented by photographic evidence and observations in

Savage and Talbot [1978] and Fotzkat et al. [2013]) are

unfortunate but currently unavoidable. The insignis-likQ

Anolis apparently are difficult to find, or possibly rare.

Fotzkat et al. (2013) included just two collected individ-

July 2017 | Volume 11 | Number 2 | e141

Two new species similar to Anolis insignis and resurrection of Anolis brooksi

uals of insignis- like anoles in their summary of the giant

anoles of Panama. Savage and Talbot (1978) studied all

specimens of insignis-hko anoles collected before 1978,

a total of 24 individuals. Vertnet lists just 28 records for

A. insignis as of 08 August 2016, after decades of inten¬

sive herpetological field work in Costa Rica and Pan¬

ama since Savage and Talbot (1978). Our new sample of

eleven collected specimens, plus additional photographic

vouchers, warrants a new treatment of these forms and

supports recognition of multiple species. However, we

recognize that the strength of our inferences is tempered

by our necessarily limited sampling. We have little doubt

that the taxonomic picture we have painted for these

forms, while pragmatic and warranted given the evidence

in front of us, is incomplete.

Some DNA sequence data has been generated for

Anolis brooksi and A. kathydayae under the name A.

insignis , but no molecular data exists for A. savagei

and true A. insignis. Castaneda and de Queiroz (2011)

included data from COI, ND2, and RAG1 genes for

an “A. insignis ” sample from Fortuna Reserve, i.e.,

near the type locality of A. kathydayae. Alfoldi et al.

(2011) included data for several genes for a sample

of A. “insignis ” from Cerro Azul, Panama Province

(POE 2154 in their appendix; now MVUP 2007). This

individual clearly is assignable to A. brooksi (Fig. 7).

Lotzkat et al. (2013) collected 16S data for an adult and

juvenile female specimen of “A. insignis ” from Santa

Fe, Veraguas, and Willie Mazu, Comarca Ngobe-Bugle

in Panama, respectively. Accurate identification of these

specimens is not straightforward because our diagnoses

are based mainly on adult male specimens and the

species in question generally overlap in scalation (Table

1). However, the adult female specimen of Lotzkat et al.

(2013), from Santa Fe, is referable to A. brooksi based

on female dewlap color pattern (Lotzkat et al. 2013:

Fig. 14C) and locality; a subadult male photographed

from Santa Fe (Fig. 7) clearly is A. brooksi. The juvenile

specimen (SMF 91477) may be A. kathydayae or A.

brooksi. The locality of this specimen is proximal to the

type and other known locality of A. kathydayae but at a

lower elevation on the Caribbean slope. This proximity

to the A. kathydayae type locality suggests A. kathydayae

as the most likely identification for this population, but

reported 16S distances suggest this sample represents

A. brooksi. The uncorrected 16S distance between the

Lotzkat et al. (2013) samples is just 0.004—a 16S distance

corroborated by comparison of the Willie Mazu sequence

with our Santa Fe sample (MVUP 2007). Perhaps this

specimen is A. kathydayae and 16S is evolving slowly in

one or both of A. kathydayae and A. brooksi, or perhaps

the specimen is A. brooksi and this species approaches A.

kathydayae on the Caribbean slope.

An alternative interpretation of the 16S result is con-

specificity of the Fortuna and Santa Fe populations (i.e.,

of Anolis brooksi and A. kathydayae as we have recog¬

nized them here), with the differences between these

populations noted herein attributed to intraspecific varia¬

tion. This interpretation seems unlikely given the consis¬

tent morphological differences between these forms (Fig.

2; Table 1; Fig. 9) and new information on mitochon¬

drial DNA distances for these populations. We sequenced

the mitochondrial ND2 gene of the Santa Fe tissue (data

included here in the phylogenetic analysis) as part of a

larger project (Poe et al. 2017) and found an uncorrected

(“p”) distance of 12.5% between the Castaneda et al.

(2011) “A. insignis ” sample (i.e., A. kathydayae) and the

Santa Fe sample (i.e., A. brooksi). This distance is simi¬

lar to pairwise species distances among many distinctive

species of Anolis (e.g., the A. microtns-A. brooksi [Santa

Fe] ND2 distance is 9.5%). Thus, information from the

ND2 gene corroborates our morphological inference of

separate species status for Fortuna (A. kathydayae) and

eastern (A. brooksi) populations of anoles similar to A.

insignis.

The phylogenetic analysis was unable to robustly

resolve the relationships of the new forms (Fig. 4). The

well-supported clades in the estimated tree—i.e., the

ingroup and the sister relationship of Anolis microtus

and A. ginaelisae —were well-established previous to

this work (Savage and Talbot 1978; Castaneda and de

Queiroz 2011; Lotzkat et al. 2013; Poe et al. 2015). The

poor support for the interrelationships of the four spe¬

cies discussed here indicates that external morphologi¬

cal data alone is inadequate to resolve them. Clearly,

additional phylogenetic work using DNA sequences is

needed on the insignis- like Anolis. Fresh sampling of

known coastal versions of these species in Caribbean

Panama and Pacific Costa Rica (Fig. 8; see localities in

Savage and Talbot [1978]) and incorporation of material

from the type localities of A. insignis, A. savagei and A.

brooksi would be especially informative, for questions of

species boundaries as well as phylogeny.

Acknowledgements. —We thank Eric Flores (Fig.

7B), Rick Stanley, Tom Kennedy (Fig. 6A), Ian Latella

(Fig. 2D), and Victor Acosta (Fig. 1) for providing pho¬

tos. Thanks to Julie Ray and Roberto Ibanez for facilitat¬

ing field work in Panama. Collecting and export permits

were provided by the Autoridad Nacional del Ambiente

de Panama in Panama and the Ministereo del Ambiente

y Energia in Costa Rica. Thanks to Eric Schaad, Erik

Hulebak, Heather Maclnnes, Julian Davis, Ian Latella,

and the UNM herpetology class for help in the field. We

thank the Los Angeles County Museum (Greg Pauly,

Nefti Camacho) for loan of specimens, and the Museum

of Comparative Zoology (Jim Hanken, Jonathan Losos,

Jose Rosado, Joe Martinez) for allowing examination of

specimens.

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Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e141

Two new species similar to Anolis insignis and resurrection of Anolis brooksi

Appendix 1

Morphological characters for phylogenetic analysis.

1. Maximum snout to vent length (SVL; mm; ordered). 0: < 120; 1: 120-129; 2: 130-139; 3: 140-149; 4: 150-159 5: >159.

2. Femoral length/SVL (ordered). 0: < 0.230; 1: 0.230-0.239; 2: 0.230-0.239; 3: 0.240-0.249; 4: 0.25-0.259; 5: >0.259.

3. Ear height/SVL (ordered). 0: < .017; 1: 0.17-0.019; 2:0.020-0.022; 3: 0.023-0.025; 4: 0.026-0.028; 5: >0.28.

4. Toe length/SVL (ordered). 0: < 0.16; 1: 0.16; 2:0.17; 3: 0.18; 4: 0.19; 5: >0.19.

5. Tail length/SVL (ordered). 0: < 1.75; 1: 1.75-1.84; 2: 1.85-1.94; 3: 1.95-2.04; 4: 2.05-2.14; 5: >2.14.

6. Mean number of longitudinal ventral scales in 5% of SVL (ordered). 0: < 8; 1: 8-8.4; 2: 8.5-8.9; 3: 9-9.4; 4: 9.5-9.9; 5: >9.9.

7. Mean number of longitudinal dorsal scales in 5% of SVL (ordered). 0: < 8.5; 1: 8.5-8.9; 2: 9-9.4; 3: 9.5-9.9; 4: 10-10.4; 5: >10.5.

8. Mean number of expanded lamellae on toe IV (ordered). 0: <23; 1: 23; 2: 24; 3: 25; 4: 26; 5: >26.

9. Mean number of scales across the snout at the second canthals (ordered). 0: < 7; 1: 7-7.9; 2: 8-8.9; 3: 9-9.9; 4: 10-10.9; 5:>11.

10. Mean number of scales between supraorbital semicircles (ordered). 0: 0: < 2; 1: 2; 2: 2.5; 3:3; 4: 3.5; 5:>3.5.

11. Elongate superciliary scale (longer than first canthal; frequency-coded). 0: absent; 5: present.

12. Mental (frequency coded). 0: extends along mouth posteriorly past rostral; 5: rostral extends posteriorly past mental.

13. Mean number of postmental scales (ordered). 0: < 6; 1: 6-6.4; 2: 6.5-6.9; 3: 7-7.4; 4: 7.5-7.9; 5: >7.9.

14. Number of postxiphisternal incriptional ribs (Etheridge 1959; Savage and Talbot 1978; frequency coded). 0:4; 5:5.

15. Number of supralabial scales from rostral to center of eye (ordered). 0: < 6.5; 1: 6.5-6.9; 2: 7.0-7.4; 3: 7.5-7.9; 4: 8.0-8.4; 5: >8.4.

16. Scales on upper surface of thigh (Savage and Talbot 1978; frequency coded). 0: smooth; 5: keeled.

17. Scales in supraocular disc (Savage and Talbot 1978; ordered). 0: small, approximately equal in size; 5: mix of large and granu¬

lar scales.

18. Male dewlap color (unordered). 0: pink; 1: white; 2: orange-red; 3: tan-peach; 4: pale pink with black streaks; 5: yellow.

Appendix 2

Coding for morphological characters in phylogenetic analysis.

A.fraseri

A. frenatus

A. ginaelisae

A. microtus

A. insignis

A. brooksi

A. kathydayae

A. savagei

0 13 15

3 5 4 5 4

0 4 0 3 5

1 2 0 3 4

4 4 3 2 3

5 4 4 0 3

3 4 5 0 3

3 0 2 0 0

1 5

5 4

2 0

0 0

4 3

2 5

5 2

5 5

0 2

3 5

0 1

0 0

5 4

4 4

5 2

1 (23) 0 1

5 5 0 5

0 0 0 0

10 0 1

10 4 3

3 0 0 1

3 0 4 0

10 5 4

0 5

5 3

5 2

5 4

5 2

5 0

5 5

0 5

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e141

Poe and Ryan

Steven Poe is Associate Professor in the Department of Biology and Associate Curator in the Division

of Amphibians and Reptiles of the Museum of Southwestern Biology at the University of New Mexico,

USA. His research focuses on taxonomy, phylogenetics, and comparative ecology and evolution,

especially of Anolis lizards. He has collected over 250 species of Anolis in 15 countries.

Mason J. Ryan is a snake conservation biologist at Arizona Game and Fish Department and Research

Associate at the University of New Mexico Museum of Southwestern Biology, USA. His research

focuses on tropical and desert amphibians and reptiles with an emphasis on disease, climate change,

conservation, and community ecology.

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e141

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

11(2) [General Section]: 17-32 (e142).

Stakeholder contributions to conservation of threatened

Northern Pine Snakes (Pituophis melanoleucus, Daudin,

1803) in the New Jersey Pine Barrens as a case study

Joanna Burger, 2 Michael Gochfeld, 3 Robert T. Zappalorti, 4 Emile DeVito, 5 Christian Jeitner,

6 Taryn Pittfield, 3 *David Schneider, and 3t Matt McCort

1 Division of Life Sciences, 604 Allison Road, Piscataway, New Jersey 08854, USA Environmental and Community Medicine, Robert Wood Johnson

Medical School, Piscataway, New Jersey 08854, USA 3 Herpetological Associates, Inc. 405 Magnolia Road, Pemberton, New Jersey 08068, USA

4 New Jersey Conservation Foundation, 170 Longview Road, Far Hills, New Jersey 08068, USA

Abstract .—The successful management and protection of endangered or threatened species generally falls

to state agencies. This paper suggests that while governmental agencies provide the legal, regulatory, and

management framework for snake conservation, it is often the universities, conservation organizations,

consultants, and concerned citizens that conduct the research needed for conservation efforts. Identification

of all the relevant stakeholders and their contributions is important for determining how to manage the threats

and enhance population viability. Managing the efforts of volunteers is hampered by the need to protect the

locations of sensitive nesting and hibernation habitat, while encouraging protection of the species overall. In

this paper we provide a template of the stakeholder categories that are often involved in research, management,

and conservation, and describe the types of agencies, organizations and people within each category and their

major contributions, using research with Pine Snakes (Pituophis melanoleucus). This suite of stakeholders

has been successfully involved with Pine Snake research for over 30 years, and helped with examining

key environmental and habitat needs. The contributions are synergistic and additive, lending continuity of

stakeholder involvement. We also suggest several stakeholder involvement actions that can be useful to a

range of conservationists.

Keywords. Environmental management, management framework, public participation, sensitive species, reptiles

Citation: Burger J, Gochfeld M, Zappalorti RT, DeVito E, Jeitner C, Pittfield T, Schneider D, McCort M. 2017. Stakeholder contributions to conservation

of threatened Northern Pine Snakes ( Pituophis melanoleucus, Daudin, 1803) in the New Jersey Pine Barrens as a case study. Amphibian & Reptile

Conservation 11(2) [General Section]: 17-32 (e142).

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: 10 February 2017; Accepted: 26 May 2017; Published: 20 July 2017

Introduction

Initially, decision-making and managing environmental

resources was a top-down approach, where the involve¬

ment of the public in research and conservation was

largely one way, with governmental agencies provid¬

ing information to the public. This evolved into two-way

communication where agencies also asked the public for

their input, perceptions, and concerns. The importance

of stakeholders and communities in environmental man¬

agement was initially acknowledged in the Environmen¬

tal Protection Agency’s risk assessment paradigm, which

included the public in the problem formulation phase

(USEPA 1992, 1998). Several subsequent authors rec¬

ognized the importance of a multi-stakeholder frame¬

work for environmental management, where a range

of stakeholders was involved in goal-setting for a proj¬

ect (Pittinger et al. 1998). The Presidential/Congressio¬

nal Committee on Risk Assessment and Risk Manage¬

ment (PCCRARM 1997) acknowledged that the National

Research Council’s (NRC 1983, 1996) risk assessment

paradigm required the addition of stakeholders and

risk management to the process. Public participation or

involvement is usually monitored as the success of the

process, or the success of the project (Chess and Purcell

1999), but not the success of stakeholder inclusion.

The realization of the importance of stakeholders in

decision-making was empowering, and has led directly to

the involvement of stakeholders in every phase of mon¬

itoring, assessment, research, and conservation (Bon-

Correspondence. x burger@biolog}>.rutgers.edu 2 mg930@eohsi.rutgers.edu i RZappalort@aol.com 4 emile@njconsen’ation.org

5 jeitner@biology. rutgers. edu 6 pittfield@biology>. rutgers. edu *dsclmeider@herpetologicalassociates. com f mmccort@herpetologicalassociates. com

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e142

Burger et al.

Fig. 1. Northern Pine Snake (Pituophis melanoleucus ) hissing when first encountered in the New Jersey Pine Barrens.

ney et al. 2009; Glowinski and Moore 2014). Partly the

stakeholder participation derived from analysis of eco¬

system services and governance (Paavola and Hubacek

2013). Three major advances followed: 1) stakeholder

was defined as all interested and affected parties, includ¬

ing governmental agencies, non-governmental orga¬

nizations, the private sector, and the general public, 2)

stakeholders could identify environmental issues and for¬

mulate the questions requiring answers, and 3) a wide

range of stakeholders could be involved in all phases of

designing and implementing an environmental manage¬

ment project. Although the last is an ideal approach, it is

seldom achieved in practice. Stakeholders may be par¬

ticularly important to predicting or deducing unintended

consequences of management. Yet, with decreasing fed¬

eral, state, and local personnel, and decreasing and lim¬

ited funding, involving a wide range of stakeholders in

projects to help conduct studies and participate in envi¬

ronmental management and conservation is an ideal

method of accomplishing more with less, while gaining

public support. Citizen science projects, and commu¬

nity participatory research, are becoming more common

and more powerful (Bonney et al. 2009; Dickinson et al.

2010). Citizen science is a method of integrating public

outreach and scientific data collection locally and region¬

ally (Cooper et al. 2007). An important aspect of citizen

science is to gather natural history information that might

otherwise go unnoticed (Dickinson et al. 2010). Stake¬

holder involvement, whether identified as citizen science

or participatory research offers opportunities (Conrad

and Hilchey 2011), particularly for conducting long-term

studies and monitoring for sustained conservation efforts

(see Lawrence 2006).

In this paper we describe the risks faced by Pine

Snakes {Pituophis melanoleucus ) as a case study to iden¬

tify the types of stakeholders that can be involved in

snake research and conservation (Fig. 1). We also give

examples of each type, and provide descriptions of the

different types of contributions that stakeholders can

make that lead to understanding the biology and conser¬

vation needs of snakes. Assessing stakeholder participa¬

tion can lead to increases in the wise use of professionals

and volunteers, but can also provide examples of oppor¬

tunities to engage people and use personnel, and provide

models of participation for others engaged in manage¬

ment of natural resources. This is a recently developed,

often overlooked approach that can increase the person¬

nel and provide logistic support needed to conduct long¬

term research. The threats in urban areas are partly off¬

set by the potential for many volunteers. This approach

has the added advantage of increasing public awareness,

knowledge, and appreciation for snakes in general. The

popular jargon for volunteers is citizen scientists (Cooper

et al. 2007; Dickinson et al. 2010), but using a range of

stakeholders involves more than just volunteers. Includ¬

ing stakeholders in management is particularly impor¬

tant, given the global decline of reptiles in general (Gib¬

bons et al. 2000).

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e142

Stakeholder contributions to conservation of threatened Northern Pine Snakes

Fig. 2. Female Northern Pine Snakes dig their own nests in the

New Jersey Pine Barrens, although in the southern part of their

range they do not do so. They bend their neck such that the

head forms a scoop capable of bringing sand out the entrance

(Fig 2a). While digging their body is hidden below ground, and

the dump pile of sand is visible (and serves to attract poachers;

Fig 2b).

Background on Pine Snakes: Northern Pine Snakes are

large constrictors that reach the northern limit of their

range in the New Jersey Pine Barrens. They are among

the top-level predators in the region and can grow to

almost two meters long (Conant and Collins et al. 1998;

Powell et al. 2016; Burger and Zappalorti, unpub. data).

This species is declining in many parts of its range, and

is not common anywhere. The declines of the species to

the south, and its threatened status in New Jersey, make

it imperative to understand the factors impacting popu¬

lation levels. The New Jersey population of Northern

Pine Snakes is isolated from other populations living to

the south by several hundred km (Burger and Zappalorti

2011a, 2016; Powell et al. 2016).

Fig. 3. Typical nesting area of Northern Pine Snakes in New

Jersey. They require relatively open areas where there is

complete sun penetration to the ground to provide sufficient

warmth to the incubating eggs (Burger 1989a, 1991a; Burger

and Zappalorti 2011a).

Fig. 4. Female Pine Snakes sometimes remain in their nests for

several days after egg-laying is complete, perhaps protecting

their clutch from being disrupted by other females that lay in

the same nest.

Pine Snakes in the New Jersey Pine Barrens are the

only North American snake that excavates their own nest

in open-canopy sandy areas, and show high fidelity to

these exact nest sites (Burger and Zappalorti 1991, Fig.

2). Open sandy areas with appropriate ground vegetation

to provide structure to support excavation, while main¬

taining sun penetration to the ground, are rare in the Pine

Barrens. Usually several females nest in the same open

clearing (Fig. 3), and sometimes several females lay eggs

in the same nest (Burger and Zappalorti 1991, 1992). The

nest tunnel can be more than two meters long. Clutches

can be distinguished because females exude a substance

that binds the eggs together. Excavation of nests can

take several days, and digging females usually rest dur¬

ing the hottest part of the day in the shade of pine trees.

Once part of the tunnel is excavated, females sometimes

remain in the tunnel during the heat of the day, and con¬

tinue to do so for a few days after a clutch is laid (Fig.

4). Nesting females and their nests are vulnerable to off¬

road vehicles (ORVs), poachers, and predators, as are

hatchlings (Burger 2006, 2007, Burger et al. 1992, 2007;

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e142

Burger et al.

Fig. 5. Pine Snakes hibernate in communal hibernacula that can contain up to 30 or more Pine Snakes (Burger et al. 1988; Burger

and Zappalorti 2011a, b, 2015,2016). Fig. 5a shows the depth hibernation chambers are below ground, a snake in a natural chamber

(Fig 5b) and in cement blocks from an old septic chamber (Fig. 5c, Pine Snake on right, Black Racer on left).

Burger and Zappalorti 2016). Northern Pine Snakes from

the New Jersey Pine Barrens are highly prized by col¬

lectors because of their vibrant black and white pattern.

Hatchlings emerge in the late summer or early fall,

and find their way to hibernacula by following adult

scent trails (Burger 1989a, 1990), or they hibernate in

old stump holes or other places. Adults have relatively

large territories, and radio-tracked snakes can be found

as far as 3-4 km away from hibernation and nesting areas

(Burger and Zappalorti 2011a, Zappalorti et al. 2014,

2015).

Snakes spend the winter in communal hibernacula

that they modify from old mammal burrows and old

stumps, digging long tunnels out into virgin sand, and

overwintering in chambers (Burger et al. 1988; Burger

and Zappalorti 2011a, 2015, 2016). The snakes usually

hibernate a meter or more below the ground in chambers

the size of their coiled body (Fig. 5). Traditional hiber¬

nacula are used for many years, and several we study

have been active for 30 + years. If a hibernaculum is

entered by mammalian predators, it may be abandoned

for several years, but snakes eventually return to use it

(Burger and Zappalorti 2011a). Both sexes show philopa-

try to hibernation sites, but females are more philopatric

than males (Burger and Zappalorti 2015). Once we have

Amphib. Reptile Conserv.

dug up a hibernacula, we rebuilt it with an appropriate

chamber and entranceway made of cement blocks that

prevent mammalian predators from entering. Our mark¬

ing and recapture methods have not adversely affected

the behavior or survival of the snakes (Burger and Zap¬

palorti 2011b).

Northern Pine Snakes are vulnerable to the usual

threats of insufficient food supplies, predators, inclement

weather, and finding hibernation sites (this is especially

true for hatchlings), but they also face human distur¬

bance, wanton killing, mortality on roads, and poaching.

They are vulnerable due to habitat loss and fragmen¬

tation, and human activities that lead to local extirpa¬

tions (Golden et al. 2009; Burger and Zappalorti 2011a;

2016). It is for this reason that the involvement of a full

range of stakeholders (including the public) is necessary

and important to the conservation of this large snake.

Involvement of stakeholders is an important aspect of the

Pinelands National Reserve management (New Jersey

Pinelands Commission 2009).

Materials and Methods

The objectives of this series of studies of Pine Snakes,

which has spanned over 40 years, are to 1) examine the

July 2017 | Volume 11 | Number 2 | e142

Stakeholder contributions to conservation of threatened Northern Pine Snakes

breeding and hibernation biology of Pine Snakes, 2)

understand the threats faced by Pine Snakes, and gather

information helping to preserve them, 3) understand the

possible role of contaminants, 4) conserve Pine Snake

populations in their preferred habitats, and 5) educate the

public about the importance and role of Pine Snakes in

the Pine Barrens ecosystem. Over the last 30 years as

it became clear that people, organizations and agencies

wanted to contribute, and to take part in a research and

conservation efforts to conserve Pine Snakes. Our intent

is to describe the various contributions of different orga¬

nizations and people to serve as an example for other

short or long-term studies with reptiles, whether threat¬

ened or not. All procedures were completed under appro¬

priate state permits and a Rutgers University protocol

approval (E86-017).

Results

Types of stakeholders: Understanding the biology of

species, and collecting data for management and con¬

servation traditionally fell to governmental agencies

and universities. However, many different categories of

stakeholders now participate and fund species conserva¬

tion and management. Table 1 lists the categories that are

relevant for Northern Pine Snakes, and that have partic¬

ipated in Pine Snake research and conservation activi¬

ties to a greater or lesser degree. A general description

of each stakeholder type follows, and may be useful for

other species of conservation concern (Table 1). This rep¬

resents a suite of stakeholders that may be involved in

many different types of environmental studies.

Stakeholder contributions to Pine Snakes conserva¬

tion: Within each stakeholder type there are different

organizations, groups, and individuals that contribute

to research and conservation of Pine Snakes in the New

Jersey Pine Barrens. Some stakeholder groups contrib¬

ute positively, without any negative effects, while oth¬

ers can have both positive and negative effects on Pine

Snakes (usually not the same people). Tables 2 and 3 list

the threat types, and the roles of stakeholders’ in conser¬

vation and research in the New Jersey Pine Barrens. The

references in Table 3 generally relate to Northern Pine

Snakes in the New Jersey Pine Barrens (or from other

regions), and not to other congeners. Much of the infor¬

mation available for Pine Snake life history and behavior

comes from either university studies, or those funded by

state agencies or industry, or a combination thereof, with

the help of volunteers (Fig. 6).

Discussion

Stakeholder involvement: Federal and state agen¬

cies (resource and regulatory) are usually thought of as

determining the status and trends of animals, protect¬

ing and conserving them, regulating or permitting their

use, and conducting research that leads to conservation

and management. With limited and sometimes declin¬

ing resources, agencies must set priorities, and different

agencies may have conflicting priorities (i.e., promot¬

ing multiple use vs protecting resources). While State

involvement has been valuable for Pine Snake conser¬

vation, there are other groups that play critical roles

in research and conservation. These roles are essential

Table 1. Types of stakeholders that can participate in research and conservation. Not all species, populations, or communities will

have this full range of stakeholders.

Type _

Independent Scientist

(university ; museum, other)

Natural Resource Agency

Management Agency

Regulator)> Agency>

Conservation Organization

Other Non-governmental

Agency

Environmental Justice

Community

Public

Consultant

Industry

Developer

Definition

Scientist engaged in designing and implementing research projects, leading to public talks, publication and

dissemination of results, and in some cases, to regulations or adaptive management.

State, federal, or local agency responsible for managing a biological resource (a species, population,

community, natural area, preserve, or ecosystem)

State, federal, or local agency responsible for managing a resource other than biological one (e.g., water

authorities)

State, federal, or local agency responsible for developing and enforcing regulations that pertain to a species,

population, community, or ecosystem (e.g., park, refuge), as well as media resources (e.g., water).

Non-governmental agency (NGO) with a conservation mission to protect species, populations, communities,

or ecosystems, including endangered and threatened species. Can be national, state, or local.

Any other NGO with a vested interest in the species, population, community, or ecosystem, either directly or

indirectly.

Any identifiable environmental justice community that is interested or affected by the resource; usually

involves low income or minority communities.

The general public, not otherwise engaged in any of the above categories, that is interested and affected by the

existence of a wildlife resource and the opportunity to experience it.

Business specifically set up with expertise to address environmental questions posed by governments, industry,

or developers.

Local or regional industry that overlaps in some way with a resource, through land, air, or water, or directly

with a species or community.

Entity that develops or changes the local or regional land use, usually for residential or commercial activities.

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e142

Burger et al.

Fig. 6. Volunteers of all ages are involved in our Pine Snake research, and the handling and measuring of snakes contributes to their

education, and results in their providing information about conservation to their families, friends, classmates, and others. Following

hibernation studies, the children (and adults) put the snakes back into their hibernation chambers.

because the NJDEP, Endangered and Nongame Species

Program has insufficient resources to gather data on all

the threatened and endangered species in the state. The

trend of decreasing resources may continue.

Engaging the members of conservation organiza¬

tions and the public in research activities has the added

advantage in that they often become committed to con¬

tinued work, to spreading conservation information, and

to specifically protecting Pine Snakes (and other snakes).

For many naturalists and conservationists, working with

state and university scientists provides a unique and rare

opportunity to work with endangered or threatened spe¬

cies, which is both rewarding and thrilling, while con¬

tributing to essential conservation knowledge. Allowing

children, especially teenagers, to participate results in

disseminating information and enthusiasm to their class¬

mates and friends (Fig. 6). It also increases their aware¬

ness of the importance of Pine Snakes and preserving

their environments.

The inclusion of stakeholders that participate in data

collection can result in connecting people to information

about the species around them (Lawrence 2006), as well

as increasing and expanding scientific literacy (Bonney

et al. 2009). These are valuable goals, particularly for

snakes, which often are feared (and therefore killed or

discouraged from urban areas). Partnerships among dif¬

ferent agencies and conservation organizations can lead to

both improved conservation of species, and to increased

collaboration among entities that will benefit future con¬

servation efforts (Bidwell and Ryan 2006). Stakeholder

involvement can have the added benefit of demonstrating

the adverse effects of some species (Young et al. 2013),

such as raccoons, that have increased because of human

provision of food in urban environments, especially on

sensitive, threatened Pine Snakes. More case studies on

stakeholder involvement in species conservation in urban

areas could lead to some general principles of involve¬

ment. For example, people living along canals could

monitor and track water snake numbers or their nest suc¬

cess, or people living near parks could track the num¬

ber or habitat use of local snakes. Others in the public

could record the location and date of turtle nests, of local

species, or place protective cages over nests to prevent

predation. In all cases, volunteers should coordinate with

scientists and local agencies (Fig. 7).

Problems with involving stakeholders in conserva¬

tion of a threatened species: There are several issues in

involving many different stakeholders: 1) Protection of

sensitive areas for Pine Snakes, 2) Protecting information

about sensitive locations, 3) Conflicts among and within

stakeholder groups, and 4) Securing help for field work

when needed. In addition, illegal activities threaten the

Pine Snake populations. Each will be discussed below.

The locations of sensitive areas for Pine Snakes need

to be protected because they can be exposed to snake

collectors that poach eggs, gravid females, and all Pine

Snakes they encounter. With 6-digit GPS locations avail¬

able on cell phones, this has become critical. Participants

must be aware of the need to protect location data. In

some years we have lost 40 % of our Pine Snake nests to

poachers; the average was 29 %/year (Burger et al. 1992;

Burger and Zappalorti 2011a). This is in addition to losses

to natural predators such as foxes, raccoons, and skunks.

It is imperative that everyone actively helping with Pine

Snake work and conservation be aware of the potential,

and avoid intentional or inadvertent disclosure of the

location of nesting and hibernating snakes. This includes

cautioning volunteers to avoid putting any information

on social media that could indicate such locations, and

warning them to turn off the GPS on their cameras and

cell phones. People readily agree with this, but often are

not aware of the problem. We are combating poaching

by removing clutches before poachers have a chance to

collect them. We hatch the eggs in the laboratory, and

replace the hatchlings in their original nests after they

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July 2017 | Volume 11 | Number 2 | e142

Stakeholder contributions to conservation of threatened Northern Pine Snakes

Table 2. Main threats faced by Pine Snakes in the New Jersey Pine Barrens and Opportunities for Stakeholder Involvement. These

are not exhaustive, but provide examples of major threats or risks to the snakes.

Threat Type

Major Threat

Opportunity for Stakeholder Involvement

Habitat Loss

Development

Mainly NJDEP, Pinelands Commission, Public pressure on agencies. Public can protect snakes, leave

habitat where possible on their properties.

Forestry practices

Mainly NJDEP (Parks and Forests), Pinelands Commission, Public pressure on agencies, conservation

organizations work to affect optimization for different sensitive species. Scientists of all stakeholder

groups develop information on Pine Snake habitat needs to lobby Parks and Forests; public lobby for

Pine Snakes. Conservation organizations and other publics can lobby for restrictions of off-road vehicles

to reduce mortality.

Infrastructure

development

NJ Department of Transportation (DOT). NJDEP (Endangered Species and Nongame Project) influence

DO T and work to build under-highway passages. NJDEP collect information on road-killed Pine Snakes

to identify sensitive regions. Public can report Pine Snakes dead on the roads with their locations to the

NJDEP database.

Fire

Natural fires originally set back succession, providing open areas for Pine Snakes to nest and hibernate.

Management of fires prevents the natural creation of open areas. State agencies (in collaboration

with Pinelands Commission) can manage controlled burns (or forest cutting) to create open areas;

conservationists and the public can lobby for creation of open areas, and can volunteer for such

management actions.

Human

Disturbance

Off-road vehicles

Conservation organizations, scientists, and the public pressure state and local officials, including NJDEP

(ENSP [Endangered and Nongame Species Program], PF [Parks and Forests] ) and law enforcement to

manage off-road vehicles to reduce mortality on snakes and other wildlife, while providing for legitimate

off-road recreational activity at levels which do not threaten natural resources.

Poaching

NJDEP, law enforcement (both ENSP and PF) to monitor sensitive nesting and hibernation areas during

peak activity times (spring, early summer nesting season, fall). Conservation organizations and private

citizens to pressure government agencies and Pinelands Commission to enforce laws. Citizens can stop

poachers when they see them, and raise awareness among neighbors about poaching.

Predators

Natural predators

Scientists from all stakeholder categories need to monitor natural predation rates to determine if actions

by NJDEP are required. Public can report any incidences of predation on Pine Snakes to NJDEP database.

Enhanced natural

predators

Scientists from all stakeholder categories need to monitor whether there are increases in natural predators

that are due to availability of food; state agencies, Pinelands Commission, and others conduct educational

programs to explain the importance of not feeding animals, or leaving food available.

Human

commensals

NJDEP, Pinelands Commission and conservation organizations can educate the public about the threats

from dogs and other pets to natural ecosystems, including snakes. All stakeholders need to make the

effects of releasing pets into the wild known to the general public.

Prey Base

Population

variations

NJDEP (ENSP and PF) and Pinelands Commission can fund and encourage studies on variations in prey

populations, and the relationship to habitats and fragmentation. This infonnation could be used to address

habitat and development restrictions. To better provide prey for Pine Snakes, the public should not control

rodents on undeveloped property that they own.

Management

Needs

Lack of

enforcement

NJDEP, law enforcement to ensure that personnel are used effectively to maximize protection during peak

Pine Snake activity Periods. Conservation organizations and public to reinforce these needs. Public can

report any infractions.

Lack of key

infonnation

While NJDEP and Pinelands Commission require specific infonnation on habitat needs and threats that

pose a risk to populations, university scientists and other scientists have a responsibility to conduct studies

to address specific needs. Public volunteers can help in monitoring, assessments, and conservation studies

with time, money, and expertise. They can volunteer for research projects to allow long-term studies to

continue.

Lack of personnel

and money

Conservation organizations and the public to lobby government agencies to devote more personnel and

money to protection and conservation of Pine Snakes and other sensitive Pinelands Species. Industry

and developers can set aside some funding for necessary assessments and monitoring of projects and

mitigations to determine efficacy. Public can contribute to research and conservation projects.

Education about

Pine Snakes

All stakeholders can play a role in education, but public advocates (conservation organizations, Pinelands

Commission) can continue to include Pine Snake conservation as part of their educational programs.

All volunteers can educate their neighbors, friends, and family about the role of Pine Snakes and their

threatened status in the state.

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Burger et al.

Fig. 7. Volunteers contribute directly to conservation efforts by helping to remove trees that are obstructing sun penetration to nests

or hibernation sites (Fig. 7a), or taking data on snake behavior (Fig. 7b).

have shed (and we remain until they have emerged, dis¬

persed, and are no longer visible; Fig. 8).

The number of NJDEP conservation officers and

Park Police has declined, and numbers are inadequate to

effectively cover all the areas that need to be patrolled

for the range of species protected under their responsibil¬

ity. Although there are key seasons for Pine Snake activ¬

ity, some of the hotspots are not close together, making

it more difficult to patrol them and apprehend poachers.

Many of the nesting areas have been known for many

decades, and poachers regularly check them, including

putting out “sucker boards” for snakes to hide under

(where they can readily find them to poach).

Conflicts among stakeholder groups: There can be

conflicts among stakeholder groups, even among state

agencies, and these should be acknowledged (Young et

al. 2013). The Department of Environmental Protection

has a number of divisions that have different mandates

with respect to habitats and the animals within them. For

example, the Endangered and Nongame Species Program

(ENSP) is charged with protection of all animal species,

except for fish and game species. The Division of Parks

and Forestry (PF) is charged with managing the forests,

which can include cutting, special use permits, and other

activities. In some cases the activities conflict with the

protection of habitat for a species, such as Pine Snakes.

Pine Snakes require open areas for nesting and for hiber¬

nation sites (Burger and Zappalorti 1986, 2011a), but

these need to be close to suitable forest for foraging and

summer dens (Burger and Zappalorti 1988b, 1989). Cut¬

ting large swaths of forest removes effective habitat,

results in fragmentation, and churns up potential nesting

areas. Pine Snakes do not nest in sugar sand, nor in sand

with many dense roots, but prefer some roots from Hud-

sonia to stabilize the soil (Burger and Zappalorti 1986,

1988a). However, removal of small areas of trees can

open the canopy and be optimal for Pine Snakes (Burger

and Zappalorti 2011a), as well as for other snakes (Webb

et al. 2005).

The pressures within each agency can also differ. For

example with Pine Snakes, ENSP desires to keep off¬

road vehicles (ORVs) away from sensitive areas (nesting,

hibernation) to avoid habitat destruction, and direct mor¬

tality, and would keep ORVs out of the forest during peak

snake movement and activity periods (spring, nesting,

fall). By contrast ORV users petition Parks and Forests to

allow them to use ORVs in the forests at other times. Off

road vehicle users have strong lobbying groups. Agency

management is likely to listen to a vociferous group with

many members. However, ORVs churn up nesting areas,

killing eggs and hatchlings, and making habitat unus¬

able for nesting, and they also unintentionally run over

basking or moving snakes because large Pine Snakes are

cryptic and invisible to a motorbike moving through nar¬

row forest trails at excessive speeds (Burger et al. 2007).

Conclusions

Key contribution of stakeholders to conservation:

Including a variety of stakeholders who have a strong

interest in the conservation of a rare plant or wildlife spe¬

cies typically has a positive outcome. A good example

of stakeholder cooperation was the planning and writ¬

ing of a comprehensive management and recovery plan

for the Gopher Tortoise (Gopherus polyphemus), which

was subsequently listed as a state “threatened” species

(Florida Fish and Wildlife Conservation Commission

2012). Input from expert Gopher Tortoise stakeholders

provided their years of knowledge and experience which

was included in the recovery and management plan (Ash¬

ton and Ashton 2008). This case, however, did not have

as inclusive a group of stakeholders, including non-gov¬

ernmental agencies (NGOs) and the general public.

Our case study illustrates how a range of stakeholders

can aid in research and conservation of Pine Snakes in

a number of ways, and help ensure that long-term stud¬

ies provide the information needed for their continued

protection. The various stakeholders we cooperated with

have contributed markedly to conserving Pine Snake

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July 2017 | Volume 11 | Number 2 | e142

Stakeholder contributions to conservation of threatened Northern Pine Snakes

Table 3. Agencies and entities that directly contribute to research and conservation of Pine Snakes in New Jersey. The examples

given relate to Pine Snakes and are used to provide an indication of the ways stakeholders can participate, having a positive or

negative effect (+/-).

Type

Example

+/-

Contribution

Independent

Scientist

Rutgers University,

Other universities or

colleges, museums

Design, oversee, and implement research and conservation on Pine Snakes, leading to

publication in refereed literature and provision of information to the public. Train students,

both graduate and undergraduate, and organize volunteers to participate in research projects

(Burger etal. 1987, 1991; Burger 1989b, 1990, 1991a,b, 1998a,b, 2006; Burger and Gochfeld

1985; Rudolph et al. 2007; Miller et al. 2012.

Resource

Agency

NJ Department

of Environmental

Protection (NJDEP),

Endangered and

Nongame Species

Program

Responsible for listing species (endangered, threatened, species of special concern), and

gathering information where needed to protect the species and enhance populations, if needed.

Pine Snakes are listed as threatened in NJ, and the ENSP has had to respond to delisting calls

by developers (the state prevailed). Lead evaluations of the status of all nongame species,

and oversee and engage in research, including snakes (Burger and Zappalorti 1988a, b, 1989,

1992; Schwartz and Golden 2002; Golden and Jenkins 2003; Golden et al. 2009). NJDEP

also bans ORVs on public lands (NJDEP 2002).

NJDEP; Division of

Parks and Forests

Responsible for administering NJ state parks and forests. Bass River State Forest and Wharton

State Forest have been involved with actively preventing off-road vehicles on nesting and

hibernation sites, and habitat manipulation to improve nesting habitat (Burger et al. 2007;

Burger and Zappalorti 201 la, b).

NJ Natural Fieritage

Program

Lists and catalogues all sightings of endangered, threatened, and special concern species.

Information is useful to federal and state agencies, consultants, and others. Exact locations of

Pine Snakes are not disclosed generally to other that state or federal agencies.

Pinelands Commission

of the Pinelands

National Reserve

Responsible for administering the Pinelands National Reserve, including protecting habitat for

threatened and endangered species, such as the Pine Snake (NJPC 2009).

Other Agency

Ocean County

Department of

Emergency Services

Provide facilities and office space for snake research (Burger and Zappalorti 1988).

Regulatory

Agency

NJ Department

of Environmental

Conservation, Law

enforcement

Responsible for enforcing state endangered species laws. Pine Snakes are heavily poached by

snake collectors in some years (Burger and Zappalorti 2011a, b).

Conservation

Organization

New Jersey

Conservation

Foundation

Major mission is the protection and conservation of NJ’s species, populations, communities,

and ecosystems. Engage in independent and collaborative research with Pine Snakes, protection

of Pine Snakes on their properties, organizes volunteers to help with research projects. Provide

funding where possible. Mobilize interest in conservation measures and influence protective

laws and regulations. Provide expertise and volunteers to aid in conservation, such as placing

barriers to ORV traffic on nesting and hibernation sites (Burger et al. 2007).

Pineland Preservation

Alliance

Dedicated to upholding the tenets of the (NJ) Pinelands Preservation Act, and protecting the

plants and animals of the Pinelands; provides volunteers to assist in research and conservation

projects, especially protecting sensitive areas from illegal off-road vehicle use.

The Nature Conservancy

Work to conserve species and habitats; fund projects (Burger and Zappalorti 2015; Zappalorti

etal. 2015).

New Jersey Audubon

Provide volunteers to assist in research and conservation projects.

Other Non¬

governmental

agencies

Outdoor hiking clubs:

Burlington County

Naturalists, Batona Trail

Club

+/-

Report sightings of rare species, assist with filling in knowledge gaps in distribution for rare

species.

Environmental

Justice

Communities

Some retirement

communities

+/-

Some retirement communities are on low/fixed incomes; some retirees fear snakes, do not

protect them, and kill them on sight; dogs can become predators. The original residents of the

Pine Barrens (“Pineys”), who had small farms in the pines, protected Pine Snakes because

they eat rats and mice. They left places for them to nest at the edges of fields (Burger and

Zappalorti 2011a).

Public

Naturalists

Gather information, produce reports and books about animals or habitats (field guides; Conant

and Collins 1998; Boyd 1991).

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Burger et al.

Table 3 (continued). Agencies and entities that directly contribute to research and conservation of Pine Snakes in New Jersey.

The examples given relate to Pine Snakes and are used to provide an indication of the ways stakeholders can participate, having a

positive or negative effect (+/-).

Type

Example

+/-

Contribution

Conservationists,

hunters.

+/-

Volunteer to help with research projects, help build hibemacula and collect data on life history

characteristics. Help monitor populations (Gerald et al. 2006a, b). Hunters maintained hunting

lodges in the Pines, keeping open areas around their lodges which are used by Pine Snakes for

nesting and hibernation sites.

Buck Run Hunt Club,

Burrs Mill Hunt Club

Provide access and volunteers to help with research and conservation of Pine Snakes. Help

build hibemacula and provide information on nesting sites and timing of nesting. Maintain

open nesting areas for snakes (Burger and Zappalorti 1986, 1991; Zappalorti and Burger

1986; Burger et al. 1988).

Other recreationists

+/-

Hikers, photographers, and others that walk through the Pine Barrens forests or roads. Usually

protective of snakes, but may inadvertently kill or injure snakes. All foot and vehicular traffic

within the pines can kill or injure snakes, and carry invasive seeds, leading to habitat changes.

Retirement communities

+/-

Some retirees are protective of Pine Snakes, while others are afraid, and discourage, injure,

or kill them.

Traffic

There is significant mortality on paved roads, and on the sand roads that pass through the

forest. Some people aim their cars toward the snakes, deliberately killing them (Himes et al.

2002; Golden et al. 2009).

Off-road vehicle

enthusiasts

Some recreationists (ORVs) make trails in the pines or on nesting areas, disrupting nests and

killing snakes or destroying the underground nests (running over them; Burger et al. 2007).

Snake enthusiasts and

poachers

+/-

Snake enthusiasts help protect snakes and contribute time and money to snake research and

conservation. Poachers can be a problem (poaching of nests averaged 29%/year, but was as

high as 40%, Burger et al. 1992).

Consultants

Companies and

scientists

+/-

Professionals that bid for work from state agencies and industry to census, monitor, or study

species. Also conduct un-paid scientific studies. Contract work for the state always provides

useful information (Zappalorti and Burger 1986; Zappalorti et al. 2014, 2015).

Herpetological

Associates

Consulting firm dedicated to providing sound scientific information to agencies, conservation

organizations, and industry about amphibians and reptiles. Also conducts independent

herpetological research (Zappalorti and Burger 1986; Burger and Zappalorti 2011a).

Industry>

Varied

+/-

Provide funding for studies on their lands that they wish to develop; such funding results in

information on nesting, hibernation sites, movement, and activity ranges (Gerald et al. 2006a,

b).

Developers

General contractors

+/-

If in appropriate habitat, need to conduct an assessment of Pine Snake presence and abundance,

depending upon contractor can be positive or negative; can produce important information on

Pine Snakes (Zappalorti et al. 2015; Burger and Zappalorti 2011a), or can census at the wrong

times or with the wrong methods.

Builders Association

ofNJ

-/+

Challenged the threatened status of Pine Snakes; request delisting of rare species. Provide

funding for state-required threatened or endangered species studies on proposed development

site (Golden et al. 2009).

populations in New Jersey. They did so by volunteering

to aid with research and conservation projects, educating

the public about the role and importance of Pine Snakes

in the Pinelands ecosystem, aiding in enforcement of laws

and regulations, and providing funds for specific research

tasks. For example, volunteers helped our research by

searching for nest sites, and aiding with hibernation and

radio-tracking studies. They greatly aided conservation

efforts by cutting small groups of trees to provide open

nesting habitat, removing herbaceous cover to increase

the suitability of nesting areas, and adding logs to pro¬

vide hiding places for hatchlings (Fig. 7). We note in

passing that our project started before Pine Snakes were

listed as a threatened species by the State of New Jersey,

and it was our data (aided by stakeholders) that contrib¬

uted to their listing.

We suggest that other herpetological studies can be

greatly improved with the inclusion of stakeholders (Fig.

9). Each stakeholder group has the potential to contrib¬

ute in many ways. State and county governmental agen¬

cies should be encouraged to enact laws and regulations

to provide protection for herpetological communities, as

well as to provide surveillance and law enforcement. The

involvement of state agencies and NGOs has persuaded

landowners to allow researchers to conduct studies on

their land, and to consider easements or the purchase

of land to provide wildlife corridors in connecting criti¬

cal habitats. Land managers, either government agency,

NGO, or private interests have directly aided in targeted

conservation activities. In doing so they became aware

of partnerships in field conservation to improve habitat

(e.g., removal of vegetation or invasive species), prevent

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July 2017 | Volume 11 | Number 2 | e142

Stakeholder contributions to conservation of threatened Northern Pine Snakes

Figure 8. Several Pine Snake females often nest in the same

nest. Here we (R. Zappalorti and J. Burger) have removed four

clutches (note they are bound together, making it possible to

identify the eggs of three different females). Once females

lay eggs, they exude a liquid that binds the eggs together.

This partly prevents other females from disrupting the clutch

and accidentally removing them to the outside while they are

digging their own side chambers.

ORV entry (adding fencing, building berms, or other bar¬

riers), or educate the public about the importance of pro¬

tecting Pine Snakes within their ecosystems.

NGOs can disseminate information through newslet¬

ters and programs on conservation needs, solicit volun¬

teers from their organizations, and encourage contribu¬

tions of money, equipment and time. Indirectly NGOs

can advocate for state and local government to enact pro¬

tection measures (laws, regulations), and provide conser¬

vation officers. By their example, NGOs can demonstrate

the criticality of conservation for endangered or threat¬

ened species.

Many other organizations and individuals can also

directly contribute to conservation of reptiles. For exam¬

ple, companies can provide volunteers and educate their

employees about the importance of a range of species.

Awareness of the plight of reptiles might result in man¬

agers altering the timing of activities (e.g., reduction of

activity during critical nesting periods), and enhance¬

ment of vigilance throughout the year to avoid unnec¬

essary harm. Companies can also develop a culture of

ongoing contributions of research funds or volunteer

assistance with held research and conservation.

Individuals can volunteer to aid projects, provide

funding for projects, advocate at local, state and federal

levels to protect reptile communities, and provide local

information not necessarily known by others. Some peo¬

ple have historical knowledge of populations, nest and

hibernation sites used, and changes in predator (or prey)

abundance in a particular habitat. In one particular exam¬

ple, the site engineer at a hazardous material cleanup

site became aware of both gestating, state-endangered

female Timber Rattlesnakes (Crotalus horridus ) and

nesting Pine Snakes, and mentioned their presence to an

adjacent non-profit conservation landowner. An innova¬

tive approach to enhancing the rattlesnake gestation and

Pine Snake nesting sites was developed and implemented

as part of the hazardous material cleanup. A permit was

obtained for this new plan, and it was actually less expen¬

sive than the original remediation plan which would have

ruined the gestation and nesting areas with unnecessary

tree plantings.

In ah the above examples, individuals are key. People

working for governmental agencies, NGOs, businesses,

and other organizations, as well as volunteers, can all

contribute to advancing research and conservation of

reptiles.

Acknowledgements. —We thank the many agencies

and individuals who have helped study and preserve Pine

Snakes in the New Jersey Pine Barrens, especially Dave

Jenkins and Dave Golden of the Endangered and Non¬

game Species Program, the Division of Parks and For¬

estry of the New Jersey Department of Environmen¬

tal Protection, New Jersey Conservation Federation,

Nature Conservancy, Rutgers University, Drexel Uni¬

versity, Herpetological Associates staff members, and

other Burger graduate students, as well as Kris Schantz,

Cynthia Coritz, and Walter Bien. This research was per¬

formed under Rutgers University Protocol number E86-

017, and appropriate state permits. The views, opinions,

and data presented in this paper are the responsibility of

the authors, and not the funding agencies.

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Joanna Burger is a Distinguished Professor of Biology at Rutgers University, as well as a member of

the School of Public Health, Institute for Marine and Coastal Sciences, the Biodiversity Center, and

the Environmental and Occupational Health Sciences Institute. Dr. Burger received her B.S. in Biology

from the State University of New York at Albany, her M.S. in Zoology and Science Education from

Cornell University, her Ph.D. in Ecology and Behavioral Biology at the University of Minnesota in

Minneapolis, Minnesota, and an honorary Ph.D. from University of Alaska. She is an ecologist, human

ecologist, behavioral biologist, and ecotoxicologist who has worked with several species, including

Pine Snakes, lizards, turtles, and sea turtles for over 40 years in many parts of the world. Her primary

research has been in behavioral ecology, ecotoxicology, risk assessment, and biomonitoring. Additional

research involves public perceptions and attitudes, inclusion of stakeholders in solving environmental

problems, and the efficacy of conducting stakeholder-driven and stakeholder-collaborative research.

She has been a member of the Endangered and Nongame Species Council of NJ since the mid-1970s,

and has served on several National Academy of Sciences Boards and committees. She received the

Brewster Medal from the American Ornithologist’s Union, the Distinguished Achievement Award from

the Society of Risk Analysis and is a fellow in the American Association for the Advancement of

Science.

Amphib. Reptile Conserv.

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Stakeholder contributions to conservation of threatened Northern Pine Snakes

Michael Gochfeld, M.D., Ph.D., is an environmental toxicologist and physician who received

his Ph.D. in evolutionary biology from the City University of New York/American Museum

of Natural History Program, and an M.D. from Albert Einstein College of Medicine. He

teaches evidence-based medicine and toxicology and conducts research on population biology,

reproductive success and heavy metal contamination in birds. He has been involved with

the Pine Snake studies from the beginning. He is Emeritus Professor of Environmental and

Occupational Medicine in the Environmental and Occupational Health Sciences Institute at

Rutgers Robert Wood Johnson Medical School and Rutgers School of Public Health.

Robert T. Zappalorti is the principal herpetologist and CEO of Herpetological Associates,

Inc. (HA). He founded HA in 1977, and continues to specialize in conservation, management

and mitigation plans for threatened and endangered plants and wildlife. His firm also provides

environmental monitoring, habitat evaluations of adverse impacts from developmental

projects and conservation plans. Robert has conducted numerous herpetological surveys for

rare species under contract with utility companies, state, federal, and NGO clients that included

expert witness and testimony. Mr. Zappalorti has published over 45 peer reviewed papers and

book chapters and is a wildlife photographer. Many of his photographs have appeared in books

and magazines, including National Geographic. Robert is an international guest speaker at

numerous museums, zoos, and universities since 1964 to present. Between 1974 and 1977 he

served as Associate Curator of Herpetology and Education, at the Staten Island Zoological

Society. His responsibilities included lecturing, teaching, herpetological research, inventory of

zoo specimens, zoo exhibit planning, assist zoo veterinarian with animal care, public relations,

education programs, film-making, and wildlife photography. Between 1964 and 1974 he was

a Reptile Keeper at the Staten Island Zoological Society, and reported directly to the late Carl

F. Kauffeld, Director and Curator of Reptiles.

Emile D. DeVito has been the Manager of Science and Stewardship at the New Jersey

Conservation Foundation since 1989. He received a doctorate in Ecology in 1988 for research

on bird communities and vegetation landscapes in New Jersey’s Pine Barrens. Dr. DeVito

directs field research on NJCF preserves, partnering with faculty and graduate students at

nearby universities. He assists in developing and implementing management plans for

NJCF’s 25,000+ acres of holdings designed to protect and enhance biological diversity, and

has participated in recent Pine Snake studies. He is a trustee of the Pinelands Preservation

Alliance and the NJ Natural Lands Trust. He serves on the Endangered and Non-Game Species

Advisory Committee within the NJ Division of Fish and Wildlife, and the Highlands Coalition

Natural Resource Committee.

Christian Jeitner received his B.S. from Stockton University in 1998. He worked as a Marine

Fisheries Technician at Rutgers University Marine Field Station conducting fish assemblage

surveys. In 2001 he joined Joanna Burger’s research team at Rutgers University Department

of Cell Biology and Neuroscience as a Senior Laboratory Technician. He began studying eco-

toxicology and received his M.S. in 2009 researching heavy metal levels in Dolly Varden

from the Aleutian Islands in Alaska. Currently his research focuses on contaminants in fish

and birds, animal behavior, Pine Snake studies, and human and ecological risk at DOE sites.

Taryn Pittfield received her B.S in Ecology and Natural Resources and Marine Sciences

(2008) from Rutgers University. She then interned with the Smithsonian Environmental

Research Center in the Invertebrate Zoology Lab. Upon returning to New Jersey she has since

worked as a Senior Research Technician with Dr. Joanna Burger in the Behavioral Toxicology

lab at Rutgers University. She earned her M.S. in Wildlife Ecology and Conservation (2016) at

Rutgers; her thesis research focused on the effects of human recreation on emydid turtles in an

urban canal of New Jersey. Further research interests focus around the ecology of reptile and

avian species, their biology and inter-relationships with each other and humans, particularly

in urban areas.

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e142

Burger et al.

Matthew P. McCort received his B.S. in Environmental Studies from the Richard Stockton

College of New Jersey in 2000. He has been with Herpetological Associates, Inc., since 2000

working as a professional herpetologist and has specialized in the ecology of the reptiles and

amphibians of the northeastern United States. Matthew has assisted in research on and conducted

surveys for endangered, threatened, and rare wildlife species throughout the northeastern states

as well as in South Carolina, Georgia, Florida, and Aruba.

David W. Schneider received his Associate of Science degree in Biology from Burlington

County College in 1997 and a Bachelor of Science degree in Biology from Richard Stockton

College in 2000. David has been employed by Herpetological Associates, Inc., since 2000

and conducts surveys and manages various projects dealing with the study of endangered and

threatened reptiles and amphibians in the northeast and southeastern United States. David has

35 years of experience with New Jersey Pine Barrens herpetofauna and is an expert in the

ecology of this region.

Amphib. Reptile Conserv.

July 2017 | Volume 11 | Number 2 | e142

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

11(2) [General Section]: 33-43 (e145).

Reproductive biology of Tylototriton yangi (Urodela:

Salamandridae), with suggestions on its conservation

1j *Kai WANG, 2 Zhiyong YUAN, 3 Guanghui ZHONG, 4 Guangyu LI, and 5 Paul A. Verrell

'Sam Noble Oklahoma Museum of Natural History and Department of Biology>, University of Oklahoma, Norman, Oklahoma 73072, USA 2 College

of Forestry, Southwest Forestry University, Kunming, Yunnan, 650224, CHINA "Sichuan Academy of Forestry, Chengdu, Sichuan, 610081, CHINA

4 Tsinghua University, Beijing, 100084, CHINA 5 School of Biological Sciences, Washington State University, Pullman, Washington 99163, USA

Abstract. —Despite the long-term establishment and the species richness of the knobby newt genus Tylototriton,

taxonomy of its members remained controversial, and little is known about the reproductive biology of its

members, especially about their courtship behavior. Here we provide information on the reproductive biology

of the Tiannan Knobby Newt, T. yangi, including the pre-spermatophore-deposition courtship behavior both

in the field and in captivity, morphology of its eggs and larvae, and breeding habitat at the type locality. We

compare different aspects of the reproductive biology interspecifically within the T. verrucosus group, and

provide suggestions for future behavioral studies. In addition, with information about the reproductive biology

of the species, we offer recommendations for its conservation accordingly.

Keywords. Comparative ethology, courtship behavior, development, habitat, larvae morphology, sexual isolation

Citation: WANG K, YUAN Z, ZHONG G, LI G, Verrell PA. 2017. Reproductive biology of Tylototriton yangi (Urodela: Salamandridae), with suggestions

on its conservation. Amphibian & Reptile Conservation 11(2) [General Section]: 33-43 (el 45).

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-

reptiie-conservation.org>.

Received: 19 December 2016; Accepted: 10 May 2017; Published: 30 November 2017

Introduction

Although most biologists embrace the evolutionary spe¬

cies concept, wherein a species is defined as an indepen¬

dent evolutionary lineage, species delimitation can be

difficult in practice using standard morphological and

molecular approaches, especially for organisms with

conservative morphologies and complex evolutionary

histories (Sites and Marshall 2004; Marshall et al. 2006;

Barley et al. 2013). The knobby newts of the genus Tylo¬

totriton Anderson, 1871 represent a classic example of

such a challenging species-complex. Despite the estab¬

lishment of the genus Tylototriton for more than a cen¬

tury, the species boundary of its type species, Tylototri¬

ton verrucosus Anderson, 1871, remains controversial

to date, mostly due to the unsettled issue regarding its

type specimens (Nussbaum et al. 1995; Chanda et al.

2000; Nishikawa et al. 2013, 2014; Phimmachak et al.

2015). As a consequence, species boundaries and taxo¬

nomic validity of remaining members of the T. verruco¬

sus group remain unclear (Nishikawa et al. 2013, 2014;

Phimmachak et al. 2015).

In contrast to the traditional morphological approach,

ecological and ethological approaches, which exam-

Correspondence. * Correspondence: kai.wang-2@ou.edu

ine reproductive ecology and courtship behavior, may

provide additional evidence to delimit species bound¬

aries and reveal insights into the evolutionary histo¬

ries of organisms (Topfer-Hofmann et al. 2000; Rundle

and Nosil 2005; Marshall et al. 2006). In salamanders,

courtship behavior patterns and pheromones used during

courtship are known to be species-specific, and differ¬

ences in courtship behavior and courtship chemicals can

lead to sexual isolation among sympatric species, as well

as among conspecific but allopatric populations (Verrell

and Mabry 2003; Rissler and Apodaca 2007). Therefore,

assessing behavioral differences during courtship among

congeners of the genus Tylototriton may provide critical

insights on its complex systematics and taxonomy.

However, much information on the reproductive biol¬

ogy, including courtship behavior, is lacking for many

members of the genus Tylototriton , particularly species

that were recently described (Nishikawa et al. 2015; Her¬

nandez 2016). One such example is the Tiannan Knobby

Newt, Tylototriton yangi (Hou, Li, Lv, 2012). First

described by Hou et al. (2012) from the T. verrucosus

group, limited detailed information was known regard¬

ing its typical habitat and reproductive ecology since its

original description (Fei et al. 2012; Hernandez 2016).

Amphib. Reptile Conserv.

November 2017 | Volume 11 | Number 2 | e145

Wang et al.

PERM

PEP#}

PBP#2

PEPflf 7

PBP#G PBPlrt)

PUP o. pep^ p

PEP#10

PBPtll

PBP#J3PBP#12

f - •

PEP# 14

/ >

PBP«6

PBP#17

Vy ? % $ t *

Fig. 1. Location of the study site (the type locality of Tylototriton yangi) at Gejiu, Honghe Prefecture, Yunnan Province, PR China.

Numbered locations of potential breeding pools (abbreviated as PBP) are shown in yellow.

Understanding the reproductive biology of T. yangi in a

comparative framework will facilitate future studies to

investigate the evolution of reproductive biology of the

genus. Furthermore, since the known distribution range

of T. yangi overlaps greatly with that of major tin-mining

sites in China, it is imperative that we understand its hab¬

itat requirements and reproductive biology so that effec¬

tive conservation efforts can be developed and applied.

Here we provide detailed descriptions of the breeding

habitats, pre-spermatophore courtship behavior both in

the held and captivity, and morphology of eggs and lar¬

vae of the Tiannan Knobby Newt, T. yangi. In addition,

we compare our descriptions to those available for other

species in the T. verrucosus group, provide directions for

future behavioral and ecological studies of the species

group, and suggest conservation strategies.

Materials and Methods

Field observations

Field observations were conducted at the type locality of

T. yangi in mixed plantations near Gejiu, Honghe Pre¬

fecture, southern Yunnan Province, from May 16th to

May 18 th , and from May 27 th to May 28 th 2014 (Fig. 1).

Detailed locality information is not provided here to pre¬

vent potential poaching. Potential breeding pools (PBP)

were located and surveyed twice during each day (first

during the day, second from dusk until midnight). Plants

and other animals around and within the PBPs were col¬

lected and photographed. These samples were later iden¬

tified to species after fieldwork. Behavioral observations

and recordings were made at night when the newts were

active. Behavior patterns were recorded using a Nikon

D7000 digital camera.

Observations in captivity

Five males and five females of T. yangi were collected

from areas around Gejiu and Mengzi of Honghe Pre¬

fecture, Yunnan, China on May 28 th . Collecting permits

were obtained from Kunming Institute of Zoology, Chi¬

nese Academy of Sciences, and animal care followed the

Animal Welfare Protocol of Kunming Institute of Zool¬

ogy, Chinese Academy of Sciences. Sexes were sepa¬

rated and housed in same-sex groups in four 60 x 30 x

40 cm plastic containers with five cm of water and live

aquatic plants. Newts were fed live bloodworms and

were allowed to acclimate to the captive environment for

four days prior to the staging of heterosexual encounters.

For the heterosexual encounters, two trials, with two rep¬

lications each, were conducted at different water depth

to determine whether water depth influences courtship

behavior. For the first trial, two active males and one

of the largest females were placed in a circular plastic

container (diameter one m) filled with 15 cm of water

and observed at 1 a.m. on June 5 th and again on June 6 th .

All interactions among individuals were observed for 60

minutes, and courtship behavior patterns were recorded

using a Nikon D7000 digital camera. For the second trial,

the same animals were placed into the same plastic con¬

tainers with only five cm of water observated at 1 a.m.

Amphib. Reptile Conserv.

November 2017 | Volume 11 | Number 2 | e145

Reproductive biology of Tylototriton yangi

Fig. 2. Habitat in which Tylototriton yangi was found at the type locality of Gejiu. Examples of typical breeding pools are shown at

the right corner (from left to right, PBP#17 and PBP#12), and positions of other pools are indicated by white arrows. Photographs

by Kai WANG.

on both June 5 th and June 6 th . Pre-spermatophore deposi¬

tion courtship behavior patterns were recorded using the

same equipment as in the first trial. After the observation

sessions, all adults were released back to the wild.

Eggs and larval morphology

Embryos produced by females in captivity were main¬

tained until hatching. Larvae were fed with live blood¬

worms and housed in five plastic containers. Photographs

were taken at different developmental stages until larvae

completed metamorphosis. Juveniles were kept for one

week after metamorphosis and then released into the wild

at the type locality.

Results

Breeding habitat

The dominant habitat type was secondary mixed forest

with scattered water sources. Seventeen potential breed¬

ing pools were located around a reservoir, including one

natural pool along a stream (potential breeding pool num¬

ber 5, abbreviated as PBP#5) and sixteen artificial irriga¬

tion pools for agriculture (PBP#l-4, PBP#6-17) (Fig. 1).

The irrigation pools were scattered along the forest edge

in mix-crop plantations, and most pools were shallow

(water depth from 5-30 cm, the deepest one, PBP#14,

90 cm) with aquatic vegetation. Shores of the pools con¬

sisted of either rocky walls with crevices or dense ter¬

restrial vegetation (Fig. 2). No newts were found in the

reservoir, moving streams, or pools that were connected

to streams (PBP#5). In addition, no newts were found

in the mining sediment pools or pools close to the tin

mining site (PBP#2). These same habitats were occu¬

pied by other amphibian species, including Aquixalns

sp., Dianrana pleuraden, Duttaphrynus melanostictus,

and Kaloula verrucosa. In addition, loaches {Misgnrnus

anguillicaudatus ) were found in some pools (PBP#14,

16, and 17).

Field behavioral observations

Six males and one female of I yangi were observed after

dusk from 20.00h May 17 th to Ol.OOh the next day, in

which all males were found at the bottom of irrigation

pools of plantations (one in PBP#11, one in PBP#12,

and four in PBP#13), while a female was found crossing

the newly plowed plantation not far from pool #13. No

behavior patterns that might be interpreted as territorial

or aggressive (such as biting or chasing) were observed

among males in pool #13; and interactions were limited

to nudging (and perhaps sniffing) one another’s snouts

and bodies. After placing the female into pool #13, the

closest male soon approached her and made several brief

contacts with his snout to her head. He then moved for¬

ward to a position in front of the female, coiling his body

into a “C”-shape and holding it next to his body. The

female showed no interest and moved away (Fig. 3).

Amphib. Reptile Conserv.

November 2017 | Volume 11 | Number 2 | e145

Wang et al.

In...

it #

%W.

SjBfP

■ i V f

Fig. 3. Heterosexual encounters of Tylototriton yangi in the breeding pools near Yangjiatian Reservior, Gejiu, Yunnan Province,

China. Clockwise from top left: a) male approaching a much larger female; b) male following the female; c) male coiling up and

blocking female’s path; d) male folding its tail toward the female; e) female swimming away; f) male following. Photographs by

Kai WANG.

Another seven males were observed at night from (6)

May 27 th to May 28 th (two in pool# 11, one in #13, and

four in #14), all of which were on the substrate in water

and not on land, and five larvae were found in pool #17. (7)

Captive behavioral observations

As with all newts, sperm transfer in Tylototriton is accom- (8)

plished by means of a spermatophore, placed on the sub¬

strate by the male and then is taken up into the cloaca of (9)

the female (Houck and Arnold 2003). Pre-spermatophore

deposition courtship behavior patterns were identical to

those observed in the field, and were the same for the two

captive trials despite differences in water depth. Males

were not observed to clasp females in amplexus. Here we

provide an ethogram of the behavior patterns observed

before spermatophore deposition in our two-males/one

female trios (actual deposition was not observed) (Fig.

4).

(1) Swim away: the female turns or moves away from

an approaching male.

(2) Nudging among males: males get distracted by other

males’ movements and nudge (sniff?) the head and

lateral body of other males; but they quickly lose

interest and move away from each other.

(3) Follow: the male rapidly moves after the female as

she moves away from him.

(4) Approach: the males move toward the female when

she is stationary.

(5) Male touch: the male makes repeated contacts with

his head to the female’s head, lateral body, espe¬

cially her orange warts, and the lateral aspect of the

proximal portion of her tail.

Female nudge: with the pair in close proximity, the

female turns her head toward the male and nudges

(sniffs?) him with her snout.

Male rub: the male repeatedly rubs his snout and

cheek horizontally and laterally on the head and

lateral aspect of the female’s body, especially her

orange warts.

Tail tremble: the female trembles her tail when the

male rubs her body with his cheek.

Tail fan: the male moves forward and turns to place

his body in front of the female. The male then curls

the posterior part of his body and folds his tail

inward in a “S”-shaped posture, with the tip of his

tail is close to its base. He then rapidly undulates or

fans the distal portion of his tail laterally in a fluid

movement toward the female for 3—4s.

Eggs and larval morphology

Eggs were laid individually, not adhered to plants, on

the floor of the container, or to one another, even though

alternative oviposition materials were available in the

containers. The animal pole was dark and the vegetal

pole was white (Fig. 5a), and cleavage was observed in

most embryos about 24 hours after their initial discovery.

Since different sexes were kept separately except during

the heterosexual encounter trials, and no actual mating

occurred during the heterosexual encounters, females

must have mated and so acquired sperm in the field prior

to capture. At room temperature (20-25 °C), the hatch¬

ling period was 15 days.

Newly hatched larvae were between 10-12 mm in

total length with large eyes; one pair of balancers was

present on the lower aspect of the sides of the head; small

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November 2017 | Volume 11 | Number 2 | e145

Reproductive biology of Tylototriton yangi

Fig. 4. Pre-spermatophore courtship behavior pattern of Tylototriton yangi in captivity. Clockwise from top-left: a) male nudging

the side of the female’s head with his snout; b) male nudging the side of the female’s body; c) male blocking female’s path and

beginning to fold his tail; and d) male fanning the tip of his tail toward the female’s head. Photographs by Kai WANG.

forelimb buds were present with very indistinctive toes;

individuals had large abdominal yolk sacs; three pairs of

gills were present, all of which were well-developed and

were the same length as the head; tail fins were relatively

deep (dorsal fin began from anterior part of the body,

which runs for about three-fourths of the total length;

ventral fin began from the posterior edge of the yolk sac,

which runs about one-third of the total length). The dor¬

sal surface of the body was yellowish brown and speck¬

led with small dark dots, which formed two lateral bands

running along the dorsal midline as well as the mid-lat¬

eral line. Speckled patterns also occurred on the tail fins.

The gills were light pink and somewhat translucent, and

the yolk sac was bright yellow with very few speckled

patterns on the upper edges (Fig. 5b).

About five days after hatching, three toes showed on

the distal end of the forelimbs and the tail fins were more

developed (Fig. 5c). Through the development, the col¬

oration of the larvae got darker, and the gills and the tail

fin continued to grow. Later-stage larvae were brownish

yellow with dark speckled patterns, possessed high tail

fins and long gills, which were also speckled (Fig. 5d).

Older pre-metamorphic larvae began to show some adult

morphology, in which the head was less pointed, dorsal

coloration became dark brown with developing light-col¬

ored patches along dorsolateral line, and the tail fins and

gills were less translucent (Fig. 5e). Right before meta¬

morphosis, larvae resembled adults in morphology: col¬

oration became black, the head broadened and showed

some trace of ridges, mid-dorsal orange ridge started to

show, and a series of small orange warts became distinct

dorsolaterally (Fig. 5f). Gills eventually disappeared, and

the metamorphosis was completed in approximately 115

days (Fig. 5g).

Discussion

Review of courtship behaviors of

Tylototriton verrucosus group

Significant differences in pre-spermatophore-deposi-

tion courtship behavior have been reported among dif¬

ferent populations of Tylototriton verrucosus sensu lato

from India (Roy and Mushahidunnabi 2001; Deuti and

Hedge 2007), upper Myanmar (Boulenger 1920), south¬

west China (unpubl. data), and from the pet-trade with

unknown locality (Sparreboom 2014). For the Indian

populations, Roy and Mushahidunnabi (2001) reported

that individual newts display extensive nose rubbing,

tail fanning, and ventral amplexus (the male clasps the

female’s forelimbs with his forelimbs, with his dorsal

side facing her ventral side). Similar amplexus behav¬

ior was also observed for the upper Myanmar population

(Boulenger 1920). However, Sparreboom (1999, 2014)

reported only tail fanning behavior in T. cf. verruco¬

sus for pet-trade individuals from an unknown locality,

and he did not observe extensive nose rubbing or ven¬

tral amplexus. For the topotypic individuals of T. verru-

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November 2017 | Volume 11 | Number 2 | e145

Wang et al.

Fig. 5. Developmental series from fertilized embryos to newly metamorphosed juvenile of Tylototriton yangi. Clockwise from the

upper left: a) fertilized embryos of T. yangi, embryos sank to the bottom of water, and were not adhesive to plants, the bottom of

the container, or to one another; b) newly hatched larvae with one pair of balancers 6-day post-hatch; c) larva 17-days post-hatch,

in which the forelimbs became visible; d) larva 50-days post-hatch; e) larva 75-days post-hatch; f) pre-metamorphic larva 95-days

post hatch; g) newly metamorphosed individual 115-day post hatch. Photographs by Kai WANG and Guangyu LI.

cosus from southwestern Yunnan Province, China, Yuan

observed nose-rubbing and tail-fanning behavior, but not

ventral amplexus (unpubl. data).

Recently, several new species have been described

from the T. verrucosus complex, including T. himalaya-

nus from Nepal (Khatiwada et al. 2015) and T. shanorum

from northern Myanmar (Nishikawa et al. 2014). Given

the close geographic distance between the type localities

of the two newly described species and the localities of

previously identified T. cf. verrucosus populations with

different courtship behaviors from India and Myanmar

(Boulenger 1920; Roy and Mushahidunnabi 2001), dif¬

ferences in courtship behavior among these two popula¬

tions may represent differential behaviors of T. himalaya-

nus and T. shanorum respectively, and ventral amplexus

may be a characteristic behavioral pattern that differenti¬

ates T. himalayanus and T. shanorum from T. verrucosus

sensu stricto.

In contrast, Hernandez (2016) reported ventral

amplexus during courtship in T. verrucosus sensu stricto.

However, the reference Hernandez cited describes court¬

ship behavior of T. verrucosus populations from Thailand

(Humphrey and Bain 1990), which, based on Hernan¬

dez’s book, are now considered as T. uyenoi Nishikawa,

Khonsue, Pomchote, Matsui 2013, instead of T. verru¬

cosus sensu stricto. Furthermore, the photographic evi¬

dence of ventral amplexus of T. verrucosus sensu stricto

that Hernandez (2016) reported is of pet-trade individu¬

als in France with no known locality information; and

based on the external morphology of the individuals in

the photo, these individuals should be identified as T.

shanorum, as Hernandez suggested in his own book.

Therefore, we recommend that further behavioral stud¬

ies are needed to confirm the courtship behavior of T.

verrucosus sensu stricto using topotypic individuals of

the species.

Comparative reproductive biology of

Tylototriton yangi

Based on our results, the reproductive biology of Tylo¬

totriton yangi differs substantially from what is known

for other species of the T. verrucosus group, especially in

terms of courtship behavior and egg morphology (Table

1). The courtship behavior of T. yangi is most similar to

those of Indian populations of T. cf. verrucosus, in which

they all court in water, exhibit tail-fanning movements,

and display extensive nudging and rubbing behaviors

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Reproductive biology of Tylototriton yangi

Table 1. Differential reproductive biology of members of the Tylototriton verrucosus group. absent; +: present.

Courtship behavior displayed by males

Characteristics of eggs/

clutches

Species

Source

Nose-

Sniffing

rubbing

Tail

fanning

Ventral

amplexus

Courtship

site

Eggs singular or

forming clusters

Adhesive

layer of

eggs

Tylototriton yangi

Present study

Aquatic

Singular

Tylototriton shanjing

Ziegler et al. 2008; Li et

al. 2012

Mainly

Terrestrial

Singular,

sometimes small

clusters

Tylototriton cf.

verrucosus

Boulenger 1920; Roy

and Mushahidunnabi

2001; Deuti and Hedge

2007; Sparreboom 2014

Aquatic

Singular,

sometimes small

clusters

Tylototriton

kweichowensis

Hu 1994; Tian et al.

1998

Aquatic

Singular

Tylototriton

taliangensis

Fleck 1997; Fei et al.

2006; pers. comm.

Aquatic

Singular

(Roy and Mushahidunnabi 2001). However, the Indian

population of T. cf. verrucosus displays ventral amplexus

during its courtship (Roy and Mushahidunnabi 2001),

which was not observed in the courtship of T. yangi in our

study. Compared to populations of T. cf. verrucosus from

the pet-trade with unknown localities, Tylototriton yangi

displays extensive nose rubbing and nudging (sniffing?)

behavior prior to tail fanning, which were not observed

in pet-trade T. cf. verrucosus (Sparreboom 1999, 2014).

In addition to differences in courtship behavior, Tyloto¬

triton yangi also differs from all populations of T. ver¬

rucosus sensu lato in egg morphology, in which eggs of

T. yangi do not possess an adhesive outer layer, whereas

those of the latter are adhesive and attached to aquatic

vegetation (Roy and Mushahidunnabi 2001; Deuti and

Hedge 2007; Wang, pers. observ.).

For other species, Tylototriton yangi differs from T.

shanjing by courtship site (aquatic vs. mainly terrestrial),

showing extensive nudging (sniffing?) and nose-rubbing

behavior, and non-adhesive, singular eggs (vs. adhesive

eggs sometimes in small clutches) (Ziegier et al. 2008;

Li et al. 2012), and from T. kweichowensis, T. taliangen-

sis, and T. pseudoverrucosns by showing extensive nose

rubbing behavior and absence of ventral amplexus (Hu

1994; Fleck 1997; Tian et al. 1998; Fei et al. 2006; Her¬

nandez 2016).

In contrast, recently Hernandez (2016) reported ven¬

tral amplexus during courtship in T. yangi , without refer¬

ences or photographic evidence, and he noted males of

the species would develop rugose nuptial pads on their

forelimbs during the breeding season, as in the amplec-

tant salamandrid Pleurodeles. However, such amplexus

behavior and the development of nuptial pads during

breeding season were not observed during our field or

captive observations. Further study is needed to confirm

the presence of amplexus behavior in T. yangi.

Importance of chemical communication in

courtship of Tylototriton

In newts and salamanders, olfactory signals are involved

in intersexual recognition both within and among species

(Dawley 1984, 1986). The extensive snout nudging and

rubbing behavior patterns that we observed in male T.

yangi suggests that they may obtain olfactory information

from females during courtship: nudging may be sniffing.

It may be that glands on the heads and in the warts of

these newts show sexual dimorphism in glandular prod¬

ucts, enabling discrimination between the sexes. On the

other hand, Li et al. (2012) suggested that T. shanjing did

not show any sniffing or nudging behavior and seemed

to rely on visual cues at the beginning stage of courtship.

Given these apparent differences in cues used in recogni¬

tion processes among Tylototriton species and examples

of behavioral isolation through chemical recognition in

desmognathine salamanders (Tilley et al. 1990; Verrell

and Mabry 2000; Mabry and Verrell 2004), it is possi¬

ble that behavioral isolation also is present among spe¬

cies in the genus Tylototriton. Further work is needed to

determine whether these behavioral differences, occur¬

ring before spermatophore deposition and at a time when

species recognition might be expected to occur, result in

decreased successes of heterospecific encounters (Verrell

and Mabry 2003). Continued work on systematics and

reproductive biology will surely reveal more about pat¬

tern and process in the evolutionary history of the genus

Tylototriton generally, and the T. verrucosus group spe¬

cifically.

Conservation of Tylototriton yangi

Our field observations indicate that scattered permanent

ponds and other permanent bodies of stationary water are

used for reproduction by T. yangi. Not all available water

Amphib. Reptile Conserv.

November 2017 | Volume 11 | Number 2 | e145

Wang et al.

Figure 6. Habitat destruction of Tylototriton yangi in southern Yunnan Province, China, a) Coal mining site at Yangjie, Mengzi,

Yunnan Province, China; b) illegal tin mining at the type locality of T. yangi in Gejiu, Yunnan, China; c) Deforestation and

infrastructure constructions at the type locality of T. yangi in Gejiu, Yunnan, China. Photographs by Kai WANG.

sources were occupied by newts during the duration of

this study (e.g., the reservoir, and PBP#10, PBP#15, and

PBP#16), and some pools (e.g., PBP#13 and PBP#14)

were used by more newts than the others. These differ¬

ences in pool use may be due to ecological factors such

as nearby canopy coverage, amount of aquatic vegeta¬

tion, water depth, food availability, and predation risk.

We found the most newts in deep pools (30-50 cm in

depth) with no large aquatic predators (e.g., large fish),

some but not dense aquatic vegetation and dense sur¬

rounding terrestrial vegetation. These may be key factors

for breeding site selection by T. yangi. Further studies

are needed to determine the details of factors that affect

breeding-site selection.

Having a restricted range in southern Yunnan Prov¬

ince of China, Tylototriton yangi faces a number of seri¬

ous anthropogenic challenges. Habitat loss, especially

of breeding habitat, is the greatest threat to the species

(Hernandez 2016). Heavy tin/coal mining and accom¬

panying deforestation were observed at our field sites

during this study. This contaminated remaining poten¬

tial breeding ponds and split terrestrial habitats into frag¬

mented patches (Fig. 6). In addition to the habitat loss,

illegal collections are the second most serious threats to

the persistence of local populations of T. yangi. Local

people harvest breeding adults from May to July every

year, which are then dried and sold for traditional medi¬

cines. In addition, individuals are collected and sold alive

as exotic pets in the illegal pet-trade. In fact, T. yangi ,

which was confused with T. kweichowensis, was the most

common species of Tylototriton sold in the U.S. market

before the official importation ban of Asian newts (Row-

ley et al. 2016), and illegally collected animals have also

reached European countries such as France, Germany,

and Russia (Hernandez 2016).

Because of these anthropogenic challenges, we rec¬

ommend increasing attention to the conservation of the

endemic species, Tylototriton yangi. Specifically, we rec¬

ommend: 1) adding T. yangi to the List of Endangered

Species of China as a Class II nationally protected spe¬

cies; 2) increasing law enforcement of the Wildlife Pro¬

tection Act of China during the breeding season of the

species from May to August, especially increasing patrol

frequency in the pet markets and traditional medicine

markets in Mengzi and Gejiu of Honghe Prefecture, Yun¬

nan, China, 3) conserving existing adult habitats, partic¬

ularly at the type locality in Gejiu, through restoration of

natural plant communities and construction of artificial

breeding ponds; and 4) initiating captive-breeding pro¬

grams in research institutions in China, giving hope for

subsequent release of newts to augment natural popula¬

tions. Lastly, following the recommendation by Fei et al.

(2012) and IUCN assessment criteria (extent of occur¬

rence estimated to be < 20,000 km 2 , severely fragmented,

and inferred continued decline in extent of occurrence

and area of occupancy), we recommend the listing of T.

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November 2017 | Volume 11 | Number 2 | e145

Reproductive biology of Tylototriton yangi

yangi as Vulnerable under IUCN assessment criteria.

Acknowledgements. —We would like to thank Mr.

Jiajun Zhou for providing the locality information, Mr.

Qiang Li for his great assistance in the held, Dr. Kevin

Messenger, Dr. Max Sparreboom, and Dr. Gernot Vogel

for providing and translating literature for us, Ms.

Jingting Liu for editing photographs, and Dr. Jesse Brun¬

ner for providing insightful comments on the manuscript.

This research was generously supported by the Under¬

graduate Herpetological Research Grant from Chicago

Herpetological Society and the MHS Grant in Herpeto¬

logical Conservation and Research from Minnesota Her¬

petological Society.

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Reproductive biology of Tylototriton yangi

Kai Wang is a Ph.D. graduate student at University of Oklahoma, with a bachelor’s

degree in general zoology from Washington State University. His research focuses on

the natural history, taxonomy, phylogeography, and evolution of reptiles and amphibians

from China and neighboring countries in Southeast Asia.

Zhiyong Yuan is Full Lecturer in the College of Forestry, Southwest Forestry University,

Kunming, China. He recieved his Ph.D. in zoology from the Kunming Institute of

Zoology, Chinese Academy of Sciences. His research focuses on the taxonomic revision,

speciation, biogeography, and conservation of the herpetofauna from southern China.

Guanghui Zhong is a herpetologist at the Sichuan Academy of Forestry. He obtained

his Master’s degree from Chengdu University of Technology in 2016. Mr. Zhong is

interested in taxonomy, biogeography, morphology, behavior and field research of

reptiles and amphibians.

Guangyu Li is an amphibian enthusiast and conservation advocator. He has successfully

bred many native amphibians of China and continues to contribute his knowledge of

captive breeding to conservation research. Mr. Li obtained his bachelor and master’s

from Tsinghua University in electrical engineering.

Paul A. Verrell is an ethologist and herpetologist. He earned his Ph.D. in animal behavior

in England (1983) and then moved to the U.S. (1986), where he is an Associate Professor

in the School of Biological Sciences at Washington State University. Verrell’s research

focuses largely on the function and evolution of sociosexual behavior in animals, and he

has studied this in isopods, spiders, salamanders, snakes, lizards, fishes, and frogs. He

makes occasional forays into studying the behavior of undergraduate students.

Amphib. Reptile Conserv.

November 2017 | Volume 11 | Number 2 | e145

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

11(2) [General Section]: 44-50 (e146).

Temperature sex determination, incubation duration, and

hatchling sexual dimorphism in the Espailola Giant Tortoise

(Chelonoidis hoodensis) of the Galapagos Islands

1j *Ana Sancho, 2i William H. N. Gutzke, -Howard L. Snell, 4 Solanda Rea, 5 Marcia Wilson,

and 6 ’ 7 Russell L. Burke

1 Pontficia Universidad Catolica del Ecuador, Escuela de Ciencias Biologicas, Apartado 17-01-2184, 170517 Quito, ECUADOR 2 Memphis State

University, Department of Biology/, Memphis, Tennessee 38152, USA 3 University of New Mexico, Biology> Department, Museum of Southwestern

Biology’, Albuquerque, New Mexico 87131, USA 4 Fundacion Charles Darwin, Isla Santa Cruz, Galapagos, ECUADOR 5 U.S. National Park Service,

Chihuahuan Desert I&MNetwork, Las Cruces, New Mexico 88003, USA 6 Department of Biology’, IIof sir a University, Hempstead, New York 11549,

USA 1 American Littoral Society, Northeast Chapter, 28 West 9th Road, Broad Channel, New York 11693, USA

Abstract. —Sex determination (SD) mode is documented in only 26% of turtle species; temperature dependent

sex determination (TSD) is common but not ubiquitous. SD mode is documented for only five tortoise species;

all of these have TSD with the la pattern. Temperature dependent sex determination was reported in Galapagos

tortoises (Chelonoidis nigra complex) in 1991 based solely on a personal communication. Here we report TSD

pattern, incubation duration, and hatchling sexual dimorphism in the Espahola Giant Tortoise (Chelonoidis

hoodensis) of the Galapagos Islands based on experiments conducted in 1986-87. We found strong evidence

for Type la TSD, a pivotal incubation temperature of 28.3 °C, and a range for transition temperatures of 25.2-31.4

°C. We also found longer incubation durations for male than for female hatchlings, and describe a new method

for sex identification for hatchling tortoises. These results have important implications for incubation of eggs

for head-starting captive breeding, especially for conservation purposes, and for interpretation of data from

natural nests. We caution against the assumption that all C. nigra complex species have similar pivotal or

transitional temperature ranges, and encourage evaluation of more species in this group.

Resumen. —El modo de determinacion sexual (DS) solamente se ha documentado para el 26% de las especies de

tortugas; la determinacion del sexo por la temperatura (DST) en las tortugas es comun pero no es generalizada.

Se conoce el modo SD solamente para cinco especies de tortugas; todas ellas tienen el modo de DST. Se

reporto en 1991 la determinacion TSD para las tortugas de Galapagos (complejo Chelonoidis nigra), sobre la

base de una comunicacion personal. En este trabajo reportamos el patron de DST, la duracion de la incubacion

y el dimorfismo sexual a la eclosion en Chelonoidis hoodensis (la Tortuga Gigante de Espahola de las Islas

Galapagos), sobre la base de experimentos realizados entre 1986-87. Nosotros encontramos firme evidencia

para el DST tipo la, una temperatura pivotal de incubacion de 28.3 °C y un rango de temperaturas transicionales

de 25.2-31.4 °C. Tambien detectamos que los periodos de incubacion hasta la eclosion de tortugas machos

fueron mas prolongados en comparacion con las hembras. Estos resultados tienen implicaciones ventajosas

e importantes para la incubacion de los huevos y para la interpretacion de datos tornados de nidos naturales.

Sugerimos evitar el inferir que todas las especies del complejo C. nigra tengan rangos de temperaturas

transicionales similares y sugerimos la evaluacion de mas especies dentro de este grupo.

Keywords. Turtle, reproduction, egg, conservation, life history, husbandry

Citation: Sancho A, Gutzke WHN, Snell HL, Rea S, Wilson M, Burke RL. 2017. Temperature sex determination, incubation duration, and hatchling

sexual dimorphism in the Espahola Giant Tortoise ( Chelonoidis hoodensis) of the Galapagos Islands. Amphibian & Reptile Conservation 11(2)

[General Section]: 44-50 (el46).

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-

reptiie-conservation.org>.

Received: 10 February 2017; Accepted: 21 August 2017; Published: 18 December 2017

*Deceased

Correspondence. 3 snell@unm.edu A solanda.rea@fcdarwin.org.ec 5 marcia wilsontynps.gov 6 biorlb@hofstra.edu (corresponding author)

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e146

Sancho et al.

Introduction

Sex determination (SD) mode is documented in only

86 (26%) of the approximately 335 known turtle spe¬

cies; temperature dependent sex determination (TSD)

is common but is not ubiquitous (The Tree of Sex Con¬

sortium 2014a, b). In the family Testudinidae (tortoises,

ca. 57 extant species, TTWG 2017), SD mode is docu¬

mented for only five species: Testudo hermanni (Eende-

bak 1995), T. graeca (Pieau 1971), Gopherus agassizii

(Spotila et al. 1994), G. polyphemus (Burke et al. 1996;

Demuth 2001), and Malacochersus tornieri (Ewert et

al. 2004); all have TSD Type la. Two other Testudinidae

(“Geochelone elephantopus ” - Chelonoidis nigra com¬

plex and “G. gigantea ” = Aldabrachelys gigantea) were

reported as TSD in Janzen and Paukstis (1991), however

both reports were based on unpublished data. The source

data for C. nigra complex was unclear but presumably

based on unpublished work by Sancho (1988) (Janzen,

pers. comm.).

Chelonoidis is the largest tortoise genus (ca. 15 extant

species, TTWG 2017); all Chelonoidis species are South

American and most (10-12) Chelonoidis species are in

the C. nigra complex (Galapagos giant tortoises) (van

Dijk et al. 2014; Poulakakis et al. 2015; TTWG 2017).

Populations of Galapagos giant tortoises have been

greatly reduced, in some cases to extinction, due to pre¬

dation by humans and by interactions with introduced

species (MacFarland et. al. 1974a, b). Captive rearing

of several Chelonoidis species for repatriation to their

islands of origin has been an important part of Galapagos

conservation programs (Cayot et al. 1994; Cayot 2008).

These programs have become increasingly sophisticated,

now including genetic analyses (Russello et al. 2010;

Milinkovitch et al. 2013) and studies of the impact that

repatriations have on vegetation (Gibbs et al. 2008).

The discovery that sex is determined by incubation

temperature in most turtles has been of interest to the

coordinators of Galapagos giant tortoise conservation

programs for decades. This is because detailed knowl¬

edge of the effects of incubation temperature on hatch¬

ling sex could help managers avoid obvious pitfalls, such

as producing all males, and to deliberately manipulate

sex ratios (Vogt 1994). However, SD studies of Chelo¬

noidis have not progressed because sexually dimorphic

characteristics typically take many years to develop and

it is unacceptable to conduct risky procedures on individ¬

uals so valuable to conservation. Therefore, the develop¬

ment of quick, easy, and harmless ways to identify the

sex of hatchlings (e.g., Burke et al. 1994; Mrosovsky et

al. 1999; Valenzuela et al. 2004; Martinez-Silvestre et al.

2015) are potentially very valuable.

Typically, investigations of TSD target four param¬

eters: 1) the TSD pattern (Ewert and Nelson 1991), 2)

the pivotal (threshold; Bull et al. 1982) temperature, (=

the constant incubation temperature that results in 1:1

offspring sex ratios, Mrosovsky and Pieau 1991), 3) the

transitional range of incubation temperatures (TRT) (=

the range of constant incubation temperatures that pro¬

duce both sexes), and 4) the temperature-sensitive period

(TSP) (= portion of the incubation period during which

incubation temperature can affect hatchling sex, Bull and

Vogt 1981). We sought to identify the SD mode, pivotal

temperature, and TRT of the Espanola Giant Tortoise

(i Chelonoidis hoodensis) of the Galapagos Islands and

develop ways to identify hatchling sex using external

morphology and incubation duration. This species has

been the subject of long term conservation efforts (Gibbs

et al. 2014). Espanola Giant Tortoises were reduced to

just 15 individuals by 1960; these were brought into cap¬

tivity 1963-1974 and became the parents of thousands of

offspring (Cayot et al. 1994; Cayot 2008; Marquez et al.

1991). Nearly 1,500 offspring have been released onto

Espanola, and successful reproduction was first observed

starting in 1990 (Marquez et al. 1991; Cayot et al. 1994;

Cayot 2008). Although C. hoodensis remains Critically

Endangered (CITES I, IUCN Red List), this is clearly an

example of a highly successful chelonian head-starting

program, despite low levels of genetic variation (Mil¬

inkovitch et al. 2013).

Materials and Methods

Incubation of eggs at different temperatures

A total of 189 Chelonoidis hoodensis eggs laid in 1986

were incubated at three temperatures: 25.5,29.5, and 33.5

°C (67 eggs at each temperature) at the Galapagos Rear¬

ing Center, Puerto Ayora, Santa Cruz, Galapagos, Ecua¬

dor. Eggs were placed in plastic boxes with damp ver-

miculite; boxes were covered and placed in incubation

chambers at constant temperatures. Boxes were rotated

inside the incubators once per week to avoid effects of

any thermal gradients in the chamber (Gutzke and Pauk¬

stis 1983). Incubation data were also collected from six

additional tortoise hatchlings incubated and hatched ear¬

lier in the same facility.

Sex identification

Hatchling sex was identified in three ways: by direct

gross observation of gonads, histological examination

of gonads, and by laparoscopy. The gonads from 35

young tortoises that died of natural causes were exam¬

ined via both direct gross observations of gonads and

histological examinations of gonads. In some cases, the

gonads were removed and fixed soon after the tortoise’s

death. However, most samples came from tortoises that

were preserved intact either in formalin or alcohol. The

gonads were embedded in Paraplast, cut at 5 pm thick¬

ness and stained with Harris’ Hematoxylin and Eosin yel¬

low stains. The histological procedures are described in

Sancho (1988). Samples from tortoises fixed in alcohol

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e146

Temperature sex determination in the Espanola Giant Tortoise

produced very poor histological sections and the gonads

could not be identified. Fixations in formalin was also

poor, but the gonads could be identified (Sancho 1988).

Laparoscopies were performed on 15 additional young

tortoises; using standard surgical techniques. A small

incision was made in the inguinal pocket just anterior to

tortoises’ hind legs to permit examination of the gonads.

After observation, the skin was sutured and bathed with

an antiseptic solution. We also counted the number of

large dorsal scales in the tails of these individuals.

We assessed SD mode and estimated both pivotal tem¬

perature and TRT using the program TSD 4.0.3 (Giron-

dot 1999, 2012; Godfrey et al. 2003) as in Burke and

Calichio (2014). This program uses a maximum likeli¬

hood approach with a rather simple mathematical equa¬

tion to compare the fit of observed data to four different

sex determination models (genotypic sex determination,

TSD IA, IB, and II) and uses Akaike Information Cri¬

terion (AIC) to rank the different models by penalizing

for more parameters. The minimum data requirement for

the TSD 4.0.3 program is sex ratio data from at least two

constant temperature incubation experiments in which

both sexes are produced.

Results

The juvenile gonads of Chelonoidis

hoodensis

We examined the tortoise gonads both macroscopi-

cally and histologically; there was complete agreement

between sex identification according to the gross mor¬

phology and the histology of gonads (Sancho 1988). The

characteristics of juvenile gonads in C. hoodensis were

similar to those of other turtles (Gutzke and Bull 1986),

they consisted of two parts, the cortex and the medulla.

The testicles of the juvenile tortoises (of up to two years

of age) were white cylindrical structures of 7 to 8 mm

in length, located on the ventral surface of the kidney.

Testicles had a uniform reticular pattern of vasculariza¬

tion and the cortex was thin. Males lacked Muellerian

ducts (or oviducts). Ovaries in juvenile tortoises, in con¬

trast, were longer, thicker and flatter than testicles (mean

length 11 mm). Vascularization was restricted to the

medulla and the cortex was thick. In females, sex identi¬

fication was aided by the presence of Muellerian ducts.

Germ cells were found in the medulla of males and

in the cortex of females (Sancho 1988). Germ cells were

rounder and larger than the somatic cells of the gonads.

In one individual, germ cells were found both in the cor¬

tex and the medulla; in this embryo sex was not yet deter¬

mined.

Effect of the temperature of incubation on

sex determination

For unknown reasons, many embryos died during early

incubation and others died during the last stages of incu¬

bation or at the time of hatching. Ten of the 11 hatchlings

(91% male, hatch rate = 16.4%) from eggs incubated at

25.5 °C were identified as males, one was a female. At

29.5 °C, 27 (hatch rate = 40.3%) tortoises hatched and

survived. We were able to identify sex in only 15 of

these. Five of the 15 sexable hatchlings from eggs incu¬

bated at 29.5 °C were identified as males, 10 were female

(33% male). All of the eggs incubated at 33.5 °C died

during early development.

Results of the Hill and logistic models (program TSD

4.0.3) were indistinguishable using AIC (both AIC val¬

ues = 8.99, Akaike weights = 0.50, goodness of fit <

0.001). This is strong evidence for Type la TSD. The

logistic model predicted a pivotal incubation temperature

of 28.3 °C (S.E. = 0.24), and a range for transition tem¬

peratures (TRT) of 25.2 °C (S.E. = 0.56)-31.4 °C (S.E. =

0.55). The Hill model predicted a pivotal incubation tem¬

perature of 28.3 °C (S.E. = 0.25), and a range for transi¬

tion temperatures (TRT) of 25.2 °C (S.E. = 0.24)-31.5

°C (S.E. =0.29).

Incubation duration for male hatchlings ranged from

125-167 days (x = 141.7) and incubation duration for

female hatchlings ranged from 111-122 (x = 118.9).

Incubation duration for males was significantly longer

than for females (t = 4.24, d.f. = 18, two tailed P < 0.001).

The number of large dorsal scales in the tails of hatch¬

lings identified as males ranged from 4-7 (n - 10, x =

4.9), females ranged from 2-5 (n — 10, x = 3.7). Male

hatchlings had significantly more large dorsal scales on

their tails than did females (t = 2.48, d.f. = 18, two tailed

P = 0.023).

Discussion

Our finding that the Espanola Giant Tortoise ( Chelonoi¬

dis hoodensis ) has TSD is not surprising because this

was reported by Sancho (1988) and is well known by

the managers in charge of the Galapagos Tortoise Cap¬

tive Breeding Program (Marquez et al. 1999; Burke,

pers. obs.). However, we have added considerable detail

to previously vague reports, including the pivotal tem¬

perature and the range for transition temperatures. These

findings can inform captive breeding programs and field

studies. For example, this type of information has been

used in other species to predict hatchling sex ratios in

natural nests (Georges et al. 1994; Delmas et al. 2008;

Grosse et al. 2014).

Our finding that eggs incubated at female-produc¬

ing temperatures and eggs incubated at male-produc¬

ing temperatures differed in incubation duration is also

not surprising, because the negative correlation between

incubation temperature and incubation duration is well

known for many turtles (e.g., Yntema 1978; Mrosovsky

andYntema 1980; Booth 1998). However, although this

knowledge is commonly used in studies of sea turtles

(e.g., Mrosovsky et al. 1999) to predict sex ratios of natu-

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e146

Sancho et al.

ral nests, we could find no similar studies in other turtles.

We suggest that incubation duration could be used more

commonly to predict sex in both artificially incubated

eggs and eggs incubated in situ.

We consider our results indicating that female C.

hoodensis had fewer large scales on the dorsal aspects

of their tails interesting but needing additional investiga¬

tion, especially a standardization of the method of count¬

ing tail scales. If the number of tail scales is sexually

dimorphic, this technique could provide an extremely

convenient way to sex hatchlings, and could be poten¬

tially valuable to many studies. We point out that incu¬

bation temperature is known to affect many hatchling

characteristics, such as survivorship, body size, locomo¬

tor performance, and growth (e.g., Janzen 1993; Roosen-

burg and Kelley 1996; Demuth 2001). In addition, Burke

et al. (1994), Valenzuela et al. (2004), and Lubiana and

Junior (2009) found significant sexual dimorphisms in

body size and shape in hatchling turtles, while tail length

is commonly sexually dimorphic in turtles as well (e.g.,

Casale et al. 2005).

Our results on pivotal temperature, transitional tem¬

peratures, and incubation duration should not be assumed

to be identical in other Chelonoidis, even other C. nigra

complex species. Variation in TSD patterns can occur

between closely related turtle species (Bull et al. 1982;

Ewert et al. 1994; Ewert et al. 2004) and even within a

species (Ewert et al. 2005).Because of the diverse nesting

microhabitats used by C. nigra complex species (Burke,

pers. obs.), there may be considerable diversity in pivotal

temperatures, TRT, and TSR

Acknowledgements. —We thank Eugenia M. del

Pino, Pontificia Universidad Catolica del Ecuador,

Escuela de Ciencias Biologicas, for her pivotal role in

this project. We also thank the Charles Darwin Research

Station (CDRS), Dr. Gunther Reck, Director of the

CDRS in 1986, and the Servicio del Parque Nacional

Galapagos (SPNG) and Ing. Humberto Ochoa, SPNG

Superintendent in 1986, for allowing us to collaborate in

the giant tortoise conservation programs. We thank Lie.

Cruz Marquez and other members of the Department of

Herpetology of the Charles Darwin Research Station for

their help, and Thomas Fritts for many interesting dis¬

cussions. The Servicio del Parque Nacional Galapagos

issued the permits for the collection of tissue samples

and for their transport to Quito. M. Girondot was again

extraordinarily helpful with analytical assistance and the

use of his software.

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Peichel CL, Pennell MW, Perrin N, Ross L, Valen¬

zuela N, Vamosi JC. 2014a. Tree of sex: A database

of sexual systems. Scientific Data 1: 140015. doi:

http://dx.doi.org/10.1038/sdata.2014.15

The Tree of Sex Consortium, Ashman T-L, Bachtrog D,

Blackmon H, Goldberg EE, Hahn MW, Kirkpatrick

M, Kitano J, Mank JE, Mayrose I, Ming R, Otto SP,

Peichel CL, Pennell MW, Perrin N, Ross L, Valen¬

zuela N, Vamosi JC. 2014b. Data from: Tree of sex:

A database of sexual systems. Diyad Digital Reposi¬

tory. doi: http://dx.doi.org/10.5061/dryad.vl908

Turtle Taxonomy Working Group [Rhodin AGJ, Iver¬

son JB, Bour R, Fritz U, Georges A, Shaffer HB,

van Dijk PP], 2017. Dirties of the World: Anno¬

tated Checklist and Atlas of Taxonomy, Synonymy,

Distribution, and Conservation Status. (8 th Edition).

In: Editors, Rhodin AGJ, Iverson JB, van Dijk PP,

Saumure RA, Buhlmann KA, Pritchard PCH, Mit-

termeier RA. Conservation Biology of Freshwa¬

ter Turtles and Tortoises: A Compilation Project of

the IUCN/SSC Tortoise and Freshwater Turtle Spe¬

cialist Group. Chelonian Research Monographs 7:

1-292. doi: 10.3854/crm.7.checklist.atlas.v8.2017

Valenzuela N, Adams DC, Bowden RM, Gauger AC.

2004. Geometric morphometric sex estimation for

hatchling turtles: A powerful alternative for detect¬

ing subtle sexual shape dimorphism. Copeia 2004:

735-742.

Van Dijk PP, Iverson JB, Rhodin AGJ, Shaffer HB, Bour

R. 2014. Turtles of the World. 7 th Edition. Annotated

checklist of taxonomy, synonomy, distribution with

maps, and conservation status. Chelonian Research

Monographs 5. doi: 10.3854/crm.5.000.checklist.

V7.2014

Vogt RC. 1994. Temperature controlled sex determina¬

tion as a tool for turtle conservation. Chelonian Con¬

servation and Biology 1: 159-162.

Yntema CL. 1978. Incubation times for eggs of the tur¬

tle Chelydra serpentina (Testudines: Chelydridae) at

various temperatures. Herpetologica 34: 274-277.

Ana Sancho (1965-2009) was an Ecuadorian biologist with an MBA specialized in project

management, dedicated her work to the conservation of biodiversity, particularly in the Galapagos

Islands. One of her research projects showed the link between Galapagos giant tortoises’ sex and

their eggs’ incubation temperature. Later on, as Fishing Officer for South America at the NGO

Traffic, she researched and coordinated the publication of the Report of Fishery activities and

Trade of Patagonian Toothfish, which was presented at the Commission for the Conservation of

Antarctic Marine Living Resources; as well as the Report on Sea Cucumber Trade in the Galapagos

Islands. Between 2004 and 2008, she worked as coordinator of the UNDP/GEF project for the

Control of Invasive Species in the Galapagos Archipelago. Among her main achievements was

the establishment of a trust fund to control invasive species of the archipelago, which raised over

$15 million. Her last professional activity was as coordinator of the proj ect for the Implementation

of Early Warning Systems and Natural Risk Management in 2009. She published several books

and was part of Ecuador’s official delegations in conservation events around the world. Apart

from her extraordinary professional legacy, her friends and family remember her for her love and

determination.

William H. N. Gutzke was a well-known herpetologist who studied embryonic development

and phenotypic plasticity of reptiles and amphibians at both Memphis State University and the

University of Memphis. Bill completed his Ph.D. (1984) on the influence of environmental factors

on eggs and hatchlings of painted turtles (Chrysemys picta) and did post-doctoral work with James

Bull at the University of Texas. He subsequently published 30+ articles in scientific journals,

mentored four Ph.D. students, two Master’s students, and at least 60 undergraduates. Bill Gutzke

passed away in 2004.

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e146

Temperature sex determination in the Espanola Giant Tortoise

Howard L. Snell is a professor in the Biology Department of the University of New Mexico

and Curator of the Herpetology Division of the Museum of Southwestern Biology, also at

UNM. Howard and his wife Heidi started work in the Galapagos Islands as volunteers from

the US Peace Corps at the Charles Darwin Research Station in 1977. They continued visiting

the archipelago through 2004. Within that interval they were variously based at Colorado

State University, Texas Christian University, and Memphis State University before settling at

the University of New Mexico in 1986. Howard worked with the Charles Darwin Foundation /

Research Station as Program Leader for Reptiles, Vice President for North America, Program

Leader for Vertebrate Ecology & Monitoring, and Director of Science Programs.

Solanda Rea became part of the Charles Darwin Research Station in 1983 when she

started working as Herpetology Assistant with the Giant Tortoise Breeding Program. She

currently works with the Visiting Scientists Program and has a key role managing the sample

exportation process. In addition, Solanda has been in charge of the meteorological station

since 1994, ensuring the collection and registration of data which is an important tool in the

analysis of environmental events that influence the Galapagos Islands.

Marcia Wilson is the program manager for the National Park Service (NPS) Chihuahuan

Desert Inventory and Monitoring (l&M) Network. She has been working with the NPS I&M

program since 2003. Prior to her time with NPS, she was Deputy Chief for the Branch of

Migratory Birds Research at Patuxent Wildlife Research Center (PWRC) where she conducted

research on wintering migratory birds in southern Mexico. Her first position with PWRC was

as Leader of the Puerto Rico Research Group. She was responsible for the captive-breeding

program and the wild flock management of the endangered Puerto Rican Parrot. She began

her career as Head of the Charles Darwin Terrestrial Ecology Department located on the

Galapagos Islands of Ecuador.

Russell L. Burke is the Donald E. Axinn Distinguished Professor of Ecology at Hofstra

University in New York. He has been conducting research on reptiles for over 30 years,

mostly focusing on the ecology and conservation of turtles. He has published 50+ scientific

articles, numerous publications for the general public, and mentored 28 Master’s students.

Each year he runs a large citizen science project exploring the ecology of Diamondback

Terrapins in Jamaica Bay, New York, and he regularly takes groups of college students to the

Galapagos islands for field ecology classes.

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e146

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

11(2) [General Section]: 51-58 (e147).

Diversity, threat, and conservation of reptiles from

continental Ecuador

1A3 ’ 5 Carolina Reyes-Puig, 46 Ana Almendariz C., and ^Omar Torres-Carvajal

1 Museo de Zoologlct, Escuela de Ciencias Biologicas, Pontificia Universidad Catolica del Ecuador, Avenida 12 de Octubre 1076 y Roca, Casilla

Postal 17-01-2184, Quito, ECUADOR 2 Seccion de Herpetologici, Instituto Nacional de Biodiversidad, Calle Rumipamba 341 y Av. De Los Shyris,

Casilla Postal 17-07-8976, Quito, ECUADOR 3 Instituto de Zoologla Terrestre, Universidad San Francisco de Quito USFO, Colegio de Ciencias

Biologicas y Ambientales COC1BA, Diego de Robles y Via Interoceanica, 170901, Ointo, ECUADOR 4 Instituto de Ciencias Biologicas, Escuela

Politecnica Nacional, Casilla Postal 17-01-2759, Quito, ECUADOR

Abstract .—Ecuador is one of the most reptile-diverse countries in the world, with 464 currently recognized

species. Similar to other taxa, reptiles in Ecuador face important conservation challenges because of

anthropogenic activities. Using distribution data of nearly 90% of the species of reptiles from continental

Ecuador, as well as information on ecosystem protection status and anthropogenic activities, we present the

first comprehensive quantitative study of reptile conservation in Ecuador. While species richness is higher

in northwestern Ecuador and the central-northern Amazon, the conservation priority areas identified in this

study also include the central Pacific coast, southwestern Ecuador, and the central-southern Amazon. Similar

areas have been identified by previous studies as conservation gaps. Thus, our study reinforces the idea of

protecting those areas to improve the conservation of biodiversity in continental Ecuador.

Keywords. Conservation priority areas, endemism, importance, opportunity, species distribution models

Citation: Reyes-Puig C, Almendariz C A, Torres-Carvajal 0. 2017, Diversity, threat, and conservation of reptiles from continental Ecuador. Amphibian

& Reptile Conservation 11(2) [General Section]: 51-58 (e147).

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-

reptiie-conservation.org>.

Received: 14 June 2017; Accepted: 08 December 2017; Published: 21 December 2017

Introduction

Compared to other groups of terrestrial vertebrates, rep¬

tiles have been subject to relatively few conservation

studies leading to the identification of either global or

local threats. Similar to amphibians, some authors (e.g.,

Gibbons et al. 2000; Todd et al. 2010) conclude that rep¬

tiles face six significant threats at a global scale: habitat

loss and degradation, introduced invasive species, pol¬

lution, disease, unsustainable use, and climate change;

however, those studies are mostly descriptive and their

sampling of taxa is poor. Only recently was the conser¬

vation of reptiles analyzed at a global scale. Based on

a worldwide sample of 1,500 species (-14.6% of total),

Bohm et al. (2013) concluded that nearly 20% of spe¬

cies of reptiles are threatened with extinction, whereas

another 20% could not be evaluated because of lack of

data (Data Deficient). Moreover, a recent global analysis

of the distribution of terrestrial tetrapods including 99%

of all species of reptiles revealed that reptiles are not as

well represented as mammals and birds under current

conservation schemes (Roll et al. 2017).

Tropical areas have been identified as facing the most

dramatic rates of habitat loss, as well as having high

percentages of threatened reptile species (Bohm et al.

2013). With an area of only 284,000 km 2 , Ecuador is a

tropical megadiverse country crossed by two biodiver¬

sity hotspots, Tumbes-Choco-Magdalena and the Tropi¬

cal Andes (Mittermeier et al. 2004; Myers et al. 2000). To

date 464 species of reptiles have been recorded in Ecua¬

dor (Torres-Carvajal et al. 2017), which represents the

highest reptile diversity in the world when considering

species number per unit area. Nonetheless, a comprehen¬

sive, quantitative study of diversity and conservation of

reptiles in Ecuador is lacking.

In this study, we generate species distribution mod¬

els for nearly 90% of species of reptiles from continen¬

tal Ecuador based on distribution data from collections

and the literature to assess (i) general patterns of diver¬

sity and endemism, (ii) threats, and (iii) priority areas for

their conservation.

Correspondence. ^Carolinareyes.88@hotmail.com, 6 ana.almendariz@epn.edu.ec, 1 omartorcar@gmail.com (Corresponding author)

Amphib. Reptile Conserv.

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Reyes-Puig et al.

Materials and Methods

Data collection

We obtained locality data points for 406 species of rep¬

tiles from three local museum databases—Museo de

Zoologia at Pontificia Universidad Catolica del Ecua¬

dor (QCAZ), Museo Ecuatoriano de Ciencias Natura-

les (MECN), Museo de Historia Natural Gustavo Orces

at Escuela Politecnica Nacional (MEPN)—, HerpNET,

Global Biodiversity Information Facility (GBIF), as well

as from the literature. We validated each data point in

ArcMap v. 10.2 (ESRI2013) and removed taxonomically

incongruent records (e.g., localities along the Pacific

coast for species known to occur exclusively east of the

Andes). Duplicate points (for the same species), as well

as points <2 km close to each other were also removed to

avoid oversampling bias in the analyses.

Species distribution maps

We used Maxent, a technique based on the principle of

maximum entropy to construct species distribution mod¬

els (SDMs) for those species (n = 287) with > 10 locality

data points (Elith et al. 2011; Phillips et al. 2006; Renner

and Warton 2013). As predictor variables, we used spe¬

cies presence data (i.e., geographical coordinates) and

bioclimatic variables from Worldclim 1.4 (http://www.

worldclim.org), which are based on temperature and

precipitation data at ~1 km 2 spatial resolution (Hijmans

et al. 2005). After removing highly correlated (r > 0.8)

variables, selected explanatory variables were Tempera¬

ture Seasonality, Annual Precipitation, Precipitation Sea¬

sonality, and Minimum Temperature of Coldest Month.

Additionally, we included the ombrothermic index,

ombrothermic index of the driest bimonth, and the ter¬

rain ruggedness index, which have been used in previous

studies of distributional patterns in the Andes (Killeen

et al. 2007; Tovar et al. 2013). To construct the models,

we set the convergence threshold to 0.00001, maximum

iterations to 1,000, and the regularization parameter to

1. SDMs with AUC (Area Under Curve) values below

0.7 were discarded (Elith and Leathwick 2007). SDMs

for those species with 5-9 locality data points were

constructed in Bioclim (Busby 1991; type output: true/

false). After removing highly correlated (r > 0.8) vari¬

ables, selected explanatory variables were Annual Mean

Temperature, Mean Diurnal Range, Temperature Season¬

ality, Maximum Temperature of Warmest Month, Mini¬

mum Temperature of Coldest Month, Annual Precipita¬

tion, Precipitation of Warmest Quarter, and Precipitation

of Coldest Quarter.

The distribution of species with four localities (;n =

43) and species with rejected SDMs (i.e., AUC < 0.7)

was delimited with minimum convex polygons. For spe¬

cies with fewer than four localities {n = 76), a 1 km 2 buf¬

fer was constructed around their presence data points.

Conservation priority areas

To identify priority areas for the conservation of reptiles

we employed the Toolbox developed by Rios-Franco et

al. (2013) for ArcMap. This method integrates three cri¬

teria—threat, importance, and opportunity. We used it

to identify regions outside the National Protected Areas

System (PANE for its initials in Spanish) with maximum

threat and importance values that show opportunity to be

considered as priority areas for the conservation of rep¬

tiles in continental Ecuador.

According to the threat criterion, those areas with

human activities are the most vulnerable. We generated a

raster file with values from 0 (non-threatened zones) to 1

(highly threatened zones) based on the results of a short

survey to reptile experts that included questions on risks,

distances and intensity of threats, such as roads, oil fields,

mines, and human settlements (Appendix). Areas that are

easy to access pose a major threat to species because they

represent great opportunities for humans to exploit natu¬

ral resources (Sanderson et al. 2002). For this reason, we

also created a file with geographic information on human

settlements, roads, navigable rivers and terrain slope.

The toolbox calculates the access probability from each

of these elements assuming that a single person walks at

a maximum speed of three km/h on a flat terrain without

road access (Rios-Franco et al. 2013).

The importance criterion prioritizes areas based on

richness, endemism, and threatened species and ecosys¬

tems. We generated richness, endemism, and threat maps

by overlapping the distributions of (i) all species of rep¬

tiles included in this study (see Species distribution maps

above), (ii) endemic species, and (iii) threatened species.

Details on the threat status of the reptiles from Ecuador

will be published elsewhere. To identify threatened eco¬

systems, we generated a raster file with values between 0

and 1, where values close to 1 correspond to natural eco¬

systems that are well represented within the PANE, and

values close to 0 correspond to the opposite (i.e., threat¬

ened ecosystems). The importance criterion was sum¬

marized in a raster file with values of 0-1, where val¬

ues close to 1 represent areas with high levels of species

richness, endemism, threatened species, and threatened

ecosystems.

The opportunity criterion identifies areas with poten¬

tial to be established as areas of conservation priority.

Since 2008 the Ecuadorian government established the

“Socio Bosque” program (SBP) to pay farmers and indig¬

enous communities that voluntarily protect their native

forests. We overlapped the threat and importance raster

files with an “opportunity” file containing SBP areas, as

well as private reserves and remnant vegetation.

Results

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e147

Diversity, threat, and conservation of reptiles from Ecuador

0 40 80 160

Figure 1. Maps of richness (left), endemism (center), and threat (right) for species of reptiles from continental Ecuador. Gradient

values correspond to number of species.

Species richness, endemism and threat

Two regions in continental Ecuador have the highest

numbers of species of reptiles. The most diverse region

includes the central and northern Amazonian territo¬

ries; however, northwestern Ecuador—Choco and adja¬

cent Andean slopes—is highly diverse as well (Fig. 1).

Endemism is mostly concentrated in northwestern Ecua¬

dor, with large numbers of endemic species also pres¬

ent both on western and eastern Andean slopes. Simi¬

larly, the highest numbers of threatened species occur in

northwestern Ecuador, followed by the Andes in south¬

ern Ecuador (Fig. 1).

Areas of conservation priority

The Pacific lowlands are more accessible to humans than

any other regions in continental Ecuador. In contrast,

according to the threat criterion, human activities that

threaten reptiles are widespread mostly along the Andes

and adjacent lowlands, with a slightly higher concentra¬

tion in southern Ecuador (Fig. 2). The areas selected by

the importance criterion based on species richness, ende¬

mism, and threat are described above; regarding threat¬

ened ecosystems, a large part of the Pacific lowlands,

as well as Andean slopes in southern Ecuador are the

least represented by the PANE. The central and southern

Amazon include the areas with the greatest potential to

be established as areas of conservation priority, most of

them represented by SBP forests (Fig. 2).

Conservation priority areas were selected based on

three of 12 possible solutions (Table 1). Accordingly,

four areas were identified as the most important for the

conservation of reptiles in continental Ecuador (Fig.

3): (1) the northwestern slopes of the Andes in Pichin-

cha and Santo Domingo de los Tsachilas provinces that

include the Mindo-Nambillo Protected Forest, remnant

Toachi-Pilaton vegetation, and SBP forest; (2) a central-

south Amazonian area mostly in Morona Santiago prov¬

ince that includes remnant vegetation within the Kutuku

and Shaimi cordilleras and SBP forest; (3) the southern

Figure 2. Maps of anthropogenic threat (left), importance (center), and opportunity (right), the three criteria used in this study to

identify priority areas for the conservation of reptiles in continental Ecuador. SBP = Socio-Bosque protected forest, OPA = Other

protected areas, PANE = National Protected Areas System.

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e147

Reyes-Puig et al.

Conservation initiatives

Priority areas

0 30 60 90 120

Figure 3. Map of priority areas for the conservation of reptiles in continental Ecuador.

Andean slopes and adjacent lowlands in Azuay and El

Oro provinces that include the Molleturo and Molle-

pungo forests; and (4) the central Pacific coast in Manabl,

Santa Elena and Guayas provinces that includes remnant

vegetation in the Chongon-Colonche cordillera, as well

as SBP areas.

Discussion

With three species per 2,000 km 2 , Ecuador is the most

reptile-diverse country in the world if country area is

accounted for. The highest diversity of reptiles is located

in the central and northern Amazon, as well as the Ecua¬

dorian Choco and adjacent Andean slopes. This pat¬

tern of species richness is concordant with other ani¬

mal and plant taxa, both at local (Lessmann et al. 2014)

and continental scales (Bass et al. 2010; Jenkins et al.

2013; Myers et al. 2000), which highlights the biologi¬

cal importance of these areas. Nonetheless, this pattern

should not be taken as definitive because a considerable

percentage of Ecuador’s biodiversity has been discov¬

ered in recent years, and not necessarily from the most

diverse regions. Nearly 10% of species of reptiles from

Ecuador have been described or reported in this century.

Of these, nearly 35% were discovered in southern Ecua¬

dor, which remains a largely undersampled area that has

also been repeatedly identified as an area of conservation

priority (this study; Cuesta et al. 2017; Lessmann et al.

2014; Tapia-Armijos et al. 2015).

Unlike other terrestrial vertebrates and plants

(Gonzalez-Palacios et al. 2015; Lessmann et al. 2014;

Menendez-Guerrero and Graham 2013), the conserva¬

tion status and threats to reptiles from continental Ecua¬

dor remain poorly studied. For example, the IUCN Red

List of Threatened Species (http://www.iucnredlist.org)

lists -25% of the species of reptiles from continental

Ecuador (i.e., excluding the Galapagos islands), of which

17% are Data Deficient. Moreover, recent conservation¬

planning studies based on a variety of taxa do not include

data on reptiles (Lessmann et al. 2016; Lessmann et al.

2014), with only one recent study including 112 species

of reptiles for the first time (Cuesta et al. 2017). Here we

present the first comprehensive quantitative study of rep¬

tile conservation in continental Ecuador including distri¬

bution data of nearly 90% of the species of reptiles from

continental Ecuador, as well as information on ecosys¬

tem protection status and anthropogenic activities that

might affect reptile populations negatively.

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e147

Diversity, threat, and conservation of reptiles from Ecuador

Table 1. Solutions to identify areas of conservation priority for reptiles from continental Ecuador. Selected solutions are marked

with an asterisk.

Solution

Importance

Threat

Opportunity

State protected

High

yes

High

yes

High

yes

High

Medium

yes

High

Medium

yes

High

Medium

yes

Medium

High

yes

Medium

High

yes

Medium

High

yes

Medium

yes

Medium

yes

Medium

yes

We identified parts of the northwestern slopes of the

Andes, central-south Amazonian area, southwestern

Andean slopes and adjacent lowlands, and the central

Pacific coast as priority areas for the conservation of rep¬

tiles in continental Ecuador. These areas partially over¬

lap with some of the Marxan-defined areas reported by

Lessman et al. (2014) based on 809 species of amphib¬

ians, birds, mammals, and plants; and Cuesta et al.

(2017) based on 744 species of amphibians, birds, rep¬

tiles (112 species), and plants. Thus, in addition to iden¬

tifying those areas that are priorities for the conservation

of reptiles, our study also supports the conservation of

general areas that would benefit a larger number of ani¬

mals and plants in continental Ecuador. Unfortunately,

some of these areas are severely threatened. For example,

Tapia-Armijos et al. (2015) reported that -46% of south¬

ern Ecuador’s original forests had been converted into

pastures and other anthropogenic land cover types by

2008. Similarly, deforestation and extinction in western

Ecuador has long been documented (Dodson and Gentry

1991). In conclusion, our study provides further evidence

demanding the establishment of protected areas in cer¬

tain regions of continental Ecuador that remain unpro¬

tected and under anthropogenic threat.

Acknowledgements. —We thank A. Merino-Viteri for

help with SDMs, and both S. Espinosa and S. Ron for

reviewing an earlier version of this manuscript. Special

thanks to M. Martins, U. Roll, F. Kraus, S. Meiri, and

R Uetz for filling out the surveys; as well as M. Yanez-

Munoz for access to the MECN specimen database. This

work was supported by Pontificia Universidad Catolica

del Ecuador and Secretarla de Educacion Superior, Cien-

cia, Tecnologla e Innovacion (SENESCYT) under the

“Area de Noe” Initiative (Pis: S.R. Ron and O. Torres-

Carvajal).

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nebo AV, Woolmer G. 2002. The Human Foot¬

print and the Last of the Wild. BioScience 52(10):

891-904.

Tapia-Armijos MF, Homeier J, Espinosa Cl, Leuschner

C, de la Cruz M. 2015. Deforestation and forest

fragmentation in south Ecuador since the 1970s -

Losing a hotspot of biodiversity. PLoS ONE 10(9):

e0133701.

Todd BD, Willson JD, Gibbons JW. 2010. The global sta¬

tus of reptiles and causes of their decline. Pp. 47-67

In: Ecotoxicology of Amphibians and Reptiles. Sec¬

ond Edition. Editors, Sparling DW, Linder G, Bishop

CA, Krest S. CRC Press, Boca Raton, Florida, USA.

944 p.

Torres-Carvajal O, Pazmino-Otamendi G, Salazar-

Valenzuela D. 2017. Reptiles del Ecuador. Version

2018.0. Museo de Zoologia, Pontificia Universidad

Catolica del Ecuador, Quito, Ecuador. Available:

http://bioweb.bio/faunaweb/reptiliaweb [Accessed:

08 December 2017],

Tovar C, Arnillas CA, Cuesta F, Buytaert W. 2013.

Diverging responses of tropical Andean biomes

under future climate conditions. PLoS ONE 8(5):

e63634.

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e147

Diversity, threat, and conservation of reptiles from Ecuador

Carolina Reyes-Puig graduated in biological and environmental sciences from Universidad

Central del Ecuador in 2012 and received a Master’s degree in conservation biology from the

Pontificia Universidad Catolica del Ecuador in 2015. She was curator of the Herpetology Section

of the Instituto Nacional de Biodiversidad (INABIO) for almost two years, and is now an assistant

professor and researcher at the Museo de Zoologia and Instituto de Zoologia Terrestre of the

Colegio de Ciencias Biologicas y Ambientales, Universidad San Francisco de Quito (USFQ).

Her interests include taxonomic relationships of morphological characters in cryptic species of

Ecuadorian herpetofauna and the spatial analysis of distribution models for species conservation.

Ana Almendariz is a researcher and the Curator of Herpetology at the Institute of Biological

Sciences at the Escuela Politecnica Nacional in Quito, Ecuador. A native of Quito, Almendariz

holds an undergraduate degree in biology and a Master’s degree in conservation and management

of natural resources. She conducts research on amphibians and reptiles throughout Ecuador and

has published extensively in her field.

Omar Torres-Carvajal graduated in biological sciences from Pontificia Universidad Catolica del

Ecuador (PUCE) in 1998, and in 2001 received a Master’s degree in ecology and evolutionary

biology from the University of Kansas under the supervision of Dr. Linda Trueb. In 2005 he

received a Ph.D. degree from the same institution with the thesis entitled “Phylogenetic Systematics

of South American Lizards of the Genus Stenocercus (Squamata: Iguama).” 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 a professor at the Department

of Biology in the same institution. He has published more than 60 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 squamates.

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e147

Reyes-Puig et al.

Appendix 1. Reptile conservation survey: risks, distances, and intensity of threats

1) On a scale from 1 to 10, where 10 is the worst, how bad do you think a primary road is for reptiles?

2) On a scale from 1 to 10, where 10 is the worst, how bad do you think a secondary road is for reptiles?

3) On a scale from 1 to 10, where 10 is the worst, how bad do you think a tertiary road is for reptiles?

4) Imagine that you were to trace a straight line, perpendicular to a road, as far as you think that road has a negative impact on

reptiles. How far would you go for a primary road?

0-5 m

10m

50 m

100 m

500 m

1 km

5) Imagine that you were to trace a straight line, perpendicular to a road, as far as you think that road has a negative impact on

reptiles. How far would you go for a secondary road?

0-5 m

10m

50 m

100 m

500 m

1 km

6) Imagine that you were to trace a straight line, perpendicular to a road, as far as you think that road has a negative impact on

reptiles. How far would you go for a tertiary road?

0-5 m

10m

50 m

100 m

500 m

1 km

7) On a scale from 1 to 10, where 10 is the worst, how bad do you think a mining area is for reptiles?

8) On a scale from 1 to 10, where 10 is the worst, how bad do you think an oil-well area is for reptiles?

9) In your opinion, what is a mine’s ratio of negative impact for reptiles?

0-5 m

10m

50 m

100 m

500 m

1 km

10) In your opinion, what is an oil-well’s ratio of negative impact for reptiles?

0-5 m

10m

50 m

100 m

500 m

1 km

11) On a scale from 1 to 10, where 10 is the worst, how bad do you think livestock husbandry and agriculture is for reptiles?

12) If you were to define a ratio of negative impact for reptiles, where livestock/agriculture facilities represent the center, how far

would you go?

0-5 m

10m

50 m

100 m

500 m

1 km

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e147

Official journal website:

amphibian-reptile-conservation.org

Amphibian & Reptile Conservation

11(2) [General Section]: 59-68 (e149).

Development of in-country live food production for

amphibian conservation: The Mountain Chicken Frog

(Leptodactylus fallax) on Dominica, West Indies

12 > 5 Daniel J. Nicholson, ^Benjamin Tapley, ^Stephanie Jayson, 1>7 James Dale, 18 Luke Harding,

19 Jenny Spencer, 4 ’ 10 Machel Sulton, ^Stephen Durand, and 112 Andrew A. Cunningham

'Zoological Society of London, Regent’s Park, London, UNITED KINGDOM 2 Oiieen Mary University of London, Mile End Road, London, UNITED

KINGDOM 3 Paignton Zoo Environmental Park, Totnes Road, Paignton, UNITED KINGDOM 4 Department of Forestry, Wildlife, and Parks;

Ministry> of Agriculture and Forestry, Roseau, COMMONWEALTH OF DOMINICA

Abstract. —Amphibian populations are in global decline. Conservation breeding programs (CBPs) are a tool

used to prevent species extinctions. Ideally, to meet biosecurity, husbandry and other requirements, CBPs

should be conducted within the species’ geographic range. A particular issue with in-country amphibian CBPs

is that of live food supply. In many areas, such as oceanic islands, commonly cultured food species used by

zoos throughout the world cannot be used, as escapes are certain to occur and could lead to the introduction

of alien, and potentially highly destructive, invasive species. Here, we describe the establishment of live food

cultures for the Critically Endangered Mountain Chicken Frog (Leptodactylus fallax) at a conservation breeding

facility on the Caribbean island of Dominica. Not all invertebrate species were suitable for long-term culture

and several species were rejected by captive L. fallax, making them unsuitable as food items. Despite the CBP

being established within a range state, it was not possible to provide a diet of comparable variety to that of wild

L. fallax. Our experiences may provide guidance for the establishment of live food culture systems for other

conservation breeding programs elsewhere.

Keywords. Captive breeding, live food culture; invertebrate husbandry, conservation breeding program, Critically

Endangered, diet

Citation: Nicholson DJ, Tapley B, Jayson S, Dale J, Harding L, Spencer J, Sulton M, Durand S, Cunningham AA. 2017. Development of in-country

live food production for amphibian conservation: The Mountain Chicken Frog ( Leptodactylus fallax) on Dominica, West Indies. Amphibian & Reptile

Conservation 11(2) [General Section]: 59-68 (e149).

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: 03 March 2017; Accepted: 21 May 2017; Published: 31 December 2017

Introduction

Amphibian populations are in decline globally, with

extinction rates now reaching over 200 times the esti¬

mated background rate (Collins 2010; McCallum 2007;

Norris 2007). Conservation breeding programs (CBPs)

are one of the tools used to mitigate amphibian extinc¬

tions (Griffiths and Pavajeau, 2008). In order to be suc¬

cessful, these programs should aim to maintain geneti-

cally-representative populations of amphibians in captiv¬

ity for future conservation translocations (Baker 2007;

Browne et al. 2011; Shishova et al. 2011). Establishing

amphibian CBPs outside the native range of a species

is considered suboptimal due to the risk of transferring

novel pathogens to the target species or from the target

species into the local environment (Cunningham et al.

2003; Walker et al. 2008; Zippel et al. 2011). Establish¬

ing a CBP within the range of the target species reduces

this risk, facilitates the provision of natural environmen¬

tal cycles with relative ease, is often more cost effective

and can also instill pride and confidence in the public

and other stake holders in the range country (Edmonds

et al. 2015; Gagliardo et al. 2008; Tapley et al. 2015a).

Amphibian husbandry capacity, however, is often lim¬

ited in the countries with the most diverse and threatened

amphibian faunas (Zippel et al. 2011). For programs in

these countries to succeed, it is essential that amphibian

husbandry methods, successful or otherwise, are dissem¬

inated for the combined benefit of amphibian conserva¬

tion.

Suboptimal husbandry or nutrition in CBPs can pro¬

duce maladapted amphibians that are unsuitable for

Correspondence. 5 danielnicholson49@gmail.com ^Stephanie.Jayson@zsl.org 1 jmmydl@gmail.com K Luke.harding@paigntonzoo.org.uk j'en-

nyspencer22@gmail.com w machelsulton@hotmail.com n durands2@dominica.gov.dm 12 A.Cunningham@ioz.ac.uk u Ben. Tapley@zsl.org

(Corresponding author)

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e149

Nicholson et al.

release (Antwis and Browne 2009; Mendelson and Altig

2016; Ogilvy et al. 2012). As the nutritional requirements

of most amphibians are unknown, suboptimal diets,

nutrition, and nutritional disease can be barriers to the

implementation of successful amphibian CBPs (Antwis

and Browne 2009; Dugas et al. 2013; Gagliardo et al.

2008; King et al. 2010; Ogilvy et al. 2012; Tapley et al.

2015b; Verschooren et al. 2011). Even when the diet is

known, it is often not possible to replicate in captivity, as

diets for captive amphibians are limited by the commer¬

cial availability of food species and the ability to estab¬

lish breeding colonies of appropriate species, as well as

difficulties in providing the prey species themselves with

suitable diets. This could have significant repercussions

for the success of amphibian CBPs (Tapley et al. 2015a).

The Critically Endangered Mountain Chicken Frog

(Leptodactylus fallax) is the largest native amphibian

species in the Caribbean and one of the world’s larg¬

est species of frog (Adams et al. 2014; Fa et al. 2010).

Leptodactylus fallax is endemic to the Caribbean islands

of Montserrat and Dominica, although it once occurred

on at least five other islands before being lost from

those through a combination of habitat loss and degra¬

dation, introduced predators, and over-collection for

food (Adams et al. 2014; Fa et al. 2010; Malhotra et al.

2007). More recently, the only two extant island popula¬

tions have been driven towards extinction by the infec¬

tious disease, amphibian chytridiomycosis (Hudson

et al. 2016a). The population of L. fallax on Dominica

declined by more than 85% in the 18 months following

the first identification of frog mortality due to chytridio¬

mycosis on the island (Hudson et al. 2016a).

In response to these disease-mediated declines on

Dominica and Montserrat, a safety net population was

established, together with a global partnership, to ensure

the survival of L. fallax (Hudson et al. 2016b). In 2007,

the Zoological Society of Fondon (ZSF), in partnership

with the Dominican Forestry, Wildlife and Parks Divi¬

sion, established a captive breeding facility in the botani¬

cal gardens of Roseau, the capital of Dominica (Fig. 1A,

IB; Adams et al. 2014; Tapley et al. 2014). A particu¬

lar issue with regards to the keeping of mountain chick¬

ens in captivity is that of food. Mountain chickens have

voracious appetites. The commonly cultured food spe¬

cies used by zoos and hobbyists throughout the world

could not be used in Dominica as escapees could lead to

the introduction of alien (and potentially highly destruc¬

tive) invasive species onto the island. Therefore, prior to

acquiring founding stock of L. fallax for the facility, it

was imperative to establish live food cultures of suffi¬

cient quantity to provide adequate nutrition for the cap¬

tive animals. Brooks Jr (1982) investigated the diet of

L. fallax on Dominica and additional prey items were

reported by Rosa et al. (2012) for the species on Mont¬

serrat. This knowledge was used to inform the species’

captive diet.

Herein we describe the methods used to establish sus¬

tainable live food cultures for L. fallax on Dominica.

Amphib. Reptile Conserv.

This may provide guidance for the establishment of sub¬

sequent live food culture systems for other range state

amphibian conservation breeding.

Methods

Initial considerations

All species selected for culture were harvested from

Dominica. Focal species were chosen because: 1) acci¬

dental release would not lead to introductions of non¬

native species; 2) acclimatization to local environmen¬

tal conditions would not be necessary; 3) purchasing and

importation costs would be eliminated; 4) availability of

stock would not be affected by delayed importation due

to tropical storms or other unforeseen circumstances; 5)

restocking of depleted cultures would be relatively sim¬

ple and cost-effective (at the cost of culture adapted spe¬

cies). As well as being local, one of the criteria for choos¬

ing a species to trial for live food culture was a perceived

ability to rapidly reproduce. Preference was given to

those species that had been documented to form part of

the wild diet of L. fallax (Brooks Jr 1982). In addition to

the species initially selected for live food culture, further

species were harvested from the wild to include more

variation in the captive diet. All substrate was purchased

from agricultural suppliers in order to reduce the likeli¬

hood of contaminating agents/animals being brought into

the facility.

Environmental conditions

The facility in Dominica is open-sided, using a combi¬

nation of metal wires and mesh netting. This allows the

facility to closely match the ambient temperature and

humidity of Dominica without the use of climate control

methods. The facility itself therefore matches the local

temperature range of 20-30 °C throughout the year.

Species used

Since the facility’s opening in 2007, live food culture of

eight species has been attempted: three species of cricket

(i Gryllodes sigillatus , Fig. 2A; Gryllus assimilis, Fig. 2B;

Caribacusta dominica, Fig. 2C), one cockroach (Bla¬

de ms discoidalis, Fig. 2D), one beetle ( Zophobas atra-

tus. Fig. 2E), one slug ( Veronicella sloanii , Fig. 2F), one

snail (Pleiirodonte dentiens. Fig. 2G), and an assortment

of unidentified millipede species (one species repre¬

sented in Fig. 2H).

Orthoptera

Orthopterans represent a large proportion (44%) of the

known diet of L. fallax on Dominica (Brooks Jr 1982).

Cultures of two cricket species were established at the

start of the project: G. sigillatus (Fig. 2A), and C. domi¬

nica (Fig. 2C). A colony of G. assimilis (Fig. 2B) was

December 2017 | Volume 11 | Number 2 | e149

In-country live food production for the Mountain Chicken Frog

<11.60m>

Side

Doorxi

/ i

Fig. 1. (A) The Dominican mountain chicken project captive breeding and research facility, Roseau, Dominica. (B) Layout of the

conservation breeding facility. Photo: D. Nicholson.

formed four years after the facility was set up in order to

increase the variety of live food being offered to captive

L. fallax. The founding population of C. dominica was

collected from forested areas around the island. Gryllus

assimilis colonies were established from just two found¬

ers that were collected using baited bottle traps. No other

individuals of G. assimilis have been observed on the

island since the original opportunistic encounter. Gryllus

assimilis and C. dominica are native to Dominica and the

West Indies (Orthoptera Species File 2016, Weissman et

al 2009). Gryllodes sigillatus is a southeast Asian native

but is now globally distributed (Otte 2006). Individuals

used for culture were wild-caught in-country.

Housing: Orthopteran colonies were housed in clear

plastic containers measuring 52 x 36 x 38 cm, with an

open top covered with fine fly mesh to prevent escape

(Fig. 3A). Refugia, including cardboard (hens’) egg

boxes and cardboard tubes, were provided. Housing con¬

tainers were cleaned monthly (for G. sigillatus ) or twice

monthly (for G. assimilus and C. dominica) to remove

faecal waste; uneaten food was removed three times per

week.

Feeding: Orthopteran colonies were fed fresh food three

times per week. A number of different fruits and vegeta¬

bles were provided, including pumpkin (1 cm cubes), let¬

tuce (diced), cabbage (diced), and carrots (0.5 cm thick

discs, halved). Also, a teaspoon each of Seminole Feed®

Premium Perfonnance Dog Food (Seminole Feed, Flor¬

ida, USA) and Pentair® Colour Mix Fish Flake Food

(Pentair Aquatic Eco-Systems, North Carolina, USA)

were provided to each container three times per week.

These were used due to their high protein content (dog

food: 26% protein, fish food: 45% protein) and ease of

storage.

Breeding: Oviposition sites were created using a 1:1

mix of compacted sand and sphagnum peat moss placed

into (10x5x5 cm) plastic containers (margarine tubs).

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e149

Nicholson et al.

Fig. 2. Cultured species at the CBP in Dominica. (A) Gryllodes sigillatus. (B) Gryllus assimilis. (C) Caribacusta dominica. (D)

Blaberus discoidalis. (E) Zophobas atratus. (F) Veronicella sloanii. (G) Pleurodonte dentiens. (H) Leptogoniulus sp. Photos: D.

Nicholson.

These were removed from housing units after two weeks,

or sooner if hatchlings were observed (Fig. 3B). After

removal, oviposition sites were placed into separate

housing units until all 1 st instar crickets hatched and

exited the nest box. The substrate in the oviposition sites

was kept moist at all times.

Rotation: All housing units were arranged and rotated

depending on instar. Once the oldest adult crickets had

been given sufficient time to lay eggs in the allocated

oviposition site and provided with a respite and feeding

period, they were fed to the captive L. fallax population.

The associated oviposition sites were then placed in the

first housing unit of the rotation and the remaining crick¬

ets at the most advanced stage of development were pro¬

vided with an oviposition site.

Blattodea

Cockroaches are not known to be a natural prey item for

L. fallax (Brooks Jr 1982). They were, however, selected

for culture due to their durability, high fecundity, large

size, suitability to wide scale propagation and because

they are readily consumed by captive L. fallax in Europe

(B. Tapley, pers. obs.). It is not known if B. discoidalis

(Fig. 2D) is native to Dominica, but it is native to Central

America and distributed across the West Indies (Cock¬

roach Species File 2016). The founding stock was col¬

lected from a chicken shed on the island.

Housing: Cockroaches were housed in large plastic dust¬

bins (51 x 69 cm) with an open top covered with mesh

lining to prevent escape (Fig. 3A). The bins were 1/3

filled with a sphagnum peat moss substrate to facilitate

burrowing and cardboard boxes were added as refugia

(Fig. 3C). Once per month, the containers were cleaned

and the substrate was replaced.

Feeding: Cockroach colonies were fed potatoes (1 cm

cubed, approx.), citrus fruits (quartered) and dry dog food

(Seminole Feed ® Premium Performance Dog Food) ad

lib , with fresh food provided three times per week.

Breeding: The substrate used (sphagnum peat moss) pro¬

vided a sufficient breeding medium.

Coleoptera

Coleoptera comprise 7% of the known diet of wild L.

fallax (Brooks Jr 1982). Beetles were incorporated into

the culture process at the facility after the giant meal¬

worm beetle {Zophobas atratus , Fig. 2E) was found to be

breeding in the cockroach containers and was noted to be

eaten by the captive L. fallax. Zophobas atratus is native

to Central and South America, and it is believed to be

naturally occurring in Dominica (Peck 2006). Separate

colonies of this beetle were established using the method

and housing described above for the cockroaches. Both

beetle larvae and adult beetles were offered to L. fallax.

Gastropoda

Gastropods make up 18% of the known diet of wild L.

fallax (Brooks Jr 1982), which have been observed con¬

suming them (D. Nicholson, pers. obs.). Slugs {V. sloanii ,

Fig. 2F) and snails {P. dentiens , Fig. 2G) were selected

for culture as they are highly abundant and widespread

across Dominica, readily observed on nocturnal transects

and easy to capture. Veronicella sloanii was first discov¬

ered on Dominica in 2009 and is believed to have been

introduced. Pleurodonte dentiens is endemic to Domi¬

nica, Martinique, and Guadeloupe (Robinson et al. 2009).

Housing: Both gastropod species were housed in clear

plastic containers (52 x 36 x 38 cm) with open tops cov¬

ered with mesh to prevent escape (Fig 3A). All housing

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e149

In-country live food production for the Mountain Chicken Frog

Fig. 3. (A) Two rows of cricket breeding containers and cockroach breeding bins below. (B) Inside of a cricket breeding container,

including refugia, food items, and several egg laying containers, transplanted into an empty container to allow eggs to hatch. (C)

Inside view of a cockroach breeding bin, including substrate, refugia, and several food items. Photos: D. Nicholson.

units contained refugia such as cardboard egg boxes and

sections of tree bark; sphagnum peat moss substrate was

also added. Housing containers were cleaned weekly to

remove faecal waste and un-eaten food. High humidity

was maintained by misting the substrate with water, as

required to keep it damp.

Feeding: All gastropod species were fed ad lib with the

leaves of lettuce, cabbage, and spinach, with fresh food

being provided three times per week.

Diplopoda

Millipedes (Fig. 2H) are very common on Dominica and

comprise 7% of the known diet of wild L. fallax (Brooks

Jr 1982). Millipedes were, therefore, chosen for culture

at the start of the project but this was soon abandoned

as high numbers were readily available in the immediate

area of the captive breeding facility. They were, there¬

fore, collected from the wild and presented as a prey

source shortly after capture. The different millipede spe¬

cies obtained were not identified to the species level.

Provisioning of L. fallax

Up to 11 L. fallax were housed in the facility at any one

time. The captive L. fallax were fed three times per week.

Provisioning took place at night as this species is noctur¬

nal (Adams et al. 2014). Night-provisioning increased the

likelihood of successful predation and this allowed staff

to monitor the behavior, feeding rate, and health of indi¬

vidual frogs. Prey items were placed in a plastic bag and

dusted with a multivitamin and mineral supplement high

Amphib. Reptile Conserv.

in calcium and containing vitamin D 3 Nutrobal® (Vetark

Professional, Winchester, UK) before being released

into the frog pens. The amount of prey offered at each

feeding event varied depending on the condition of the

frogs. Individuals with lower than expected body weight

for their size were given more food items to encourage

weight gain. Also, before and during the breeding sea¬

son (February-September, Davis et al. 2000) the num¬

ber of prey items offered was increased to provide for

the additional energy expenditure associated with vocal¬

izing, fighting (males), egg production, and nesting. Dur¬

ing this period, 5-6 large prey items (cockroaches) or

10-12 small prey items (crickets) per frog were provi¬

sioned. The number of invertebrates offered to the frogs

was reduced by 30% during the non-breeding season

(October-January).

Preventing metabolic bone disease

Metabolic bone disease (MBD) has been reported in cap¬

tive L. fallax reared on diets supplemented with multi¬

vitamin and mineral supplements containing vitamin D 3

and calcium but not provided with ultraviolet B radiation

(UV-B) (Tapley et al. 2015b). Animals on the same diet

did not develop MBD when provided with UV-B, indi¬

cating that the disease was caused by vitamin D 3 defi¬

ciency (Tapley et al. 2015b). In most vertebrates, vitamin

D 3 is synthesized via exposure to the UV-B present in

sunlight. Uptake of ingested vitamin D 3 might not be suf¬

ficient in all species for optimal health and this appears

to be the case for L. fallax. Vitamin D 3 plays a critical

role in regulating calcium metabolism, as well as hav-

December 2017 | Volume 11 | Number 2 | e149

Nicholson et al.

Table 1. Suitability of invertebrate species captured in the wild on Dominica for live food culture for captive Mountain Chicken

Frogs.

Class or Order of live food item

Species of live food item

Sustainable population of

food item cultured?

Food item readily consumed

by L. fallax 1

Orthoptera

Gryllodes sigillatus

Yes

Orthoptera

Giyllus assimilis

Yes

Orthoptera

Caribacusta dominica

Yes

Blattodea

Blaberus discoidalis

Yes

Coleoptera

Zophobas atratus

Yes

Gastropoda

Veronicella sloanii

Yes

Gastropoda

Pleurodonte den tie ns

Yes

Diplopoda

Leptogoniulus sp.

Yes

ing important roles in organ development, muscle con¬

traction, and the functioning of the immune and nervous

systems (Wright and Whitaker 2001). To prevent MBD

in the captive L. fallax all food items were dusted with

a multivitamin and mineral supplement which is high in

calcium and contains vitamin D, (Nutrobal®, Vetark Pro¬

fessional) before being released into L. fallax pens. Pens

were also supplied with UVB emitting lamps (12% UVB

D 3 24 W Basking Lamp, Arcadia).

Results

The ability to develop sustainable invertebrate cultures

and the palatability of these as food items for L. fallax are

summarized for each species in Table 1.

Orthoptera

Gryllodes sigillatus and G. assimilis cultures were suc¬

cessful and populations of both species have yielded

approximately 50 adults per week to date (over a period

of approximately seven and 2 years, respectively). Both

species were readily consumed by captive L. fall ax. How¬

ever, although readily consumed by L. fallax , the live

culture of C. dominica had a poor outcome. The repro¬

ductive output was consistently very low, hatchlings had

high mortality rates, and adults had short lifespans. In

2015, five years after its establishment, the population

finally collapsed when ah surviving adults died without

reproducing. The species is very common across Domi¬

nica, therefore restarting the culture was not deemed via¬

ble due to the ease of collecting animals from the wild

and the unsuitability of the species for large scale pro¬

duction.

Blattodea

Live culture of B. discoidalis was successful. To date,

seven years after its establishment, the facility has main¬

tained a yield of approximately 60 cockroaches per week.

This food item was readily consumed by L. fallax.

Coleoptera

Giant mealworm beetles were successfully cultured over

six years, but consumption rates by L. fallax were low.

While both life stages of Z atratus were observed to be

predated by the captive frogs (D. Nicholson, J. Spencer,

pers. obs.), it was noted that adult beetles were promptly

regurgitated. Larval forms were almost entirely ignored,

apart from a few occasions. The culture of Z atratus was,

therefore, discontinued.

Gastropoda

Culture attempts, while successful for both species,

yielded low numbers (<10 per week) and were labor

intensive: the enclosures required a disproportionate

amount of cleaning and maintenance for the yield. Con¬

tinuous cultures of gastropods were, therefore, stopped

after approximately three years. Cultures of both gas¬

tropod species are, however, re-established during the

breeding season to supplement the diet as they are read¬

ily consumed by the captive frogs.

Diplopoda

The harvesting of millipedes was opportunistic, there¬

fore the numbers offered to the frogs as food varied as a

result. Despite being consumed by wild L. fallax (Brooks

Jr 1982), observations of feeding behavior of captive L.

fallax showed that all millipedes species were regurgi¬

tated after ingestion. The use of millipedes as a food item

was therefore stopped at the facility. It is possible that

the species of millipede provisioned in captivity is dif¬

ferent to that observed as a wild food source by Brooks

Jr (1982).

Discussion

Provision of an appropriate diet is vitally important for

amphibians in CBPs as nutrition influences health, lon¬

gevity, and reproductive output (Li et al. 2009). The

amount of space required for rearing invertebrates for a

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e149

In-country live food production for the Mountain Chicken Frog

relatively small number of frogs was considerable and

accounted for 20% of the facility’s footprint. When CBPs

are conducted in-country, the risk of introduction of alien

pest species used as live food is high, especially in island

situations. In these cases, a culture of locally-caught spe¬

cies should be developed. A range of such species was

trialled in Dominica, of which crickets G. sigillatus and

G. assimilis and the cockroach B. discoidalis proved to

be most successful. Some other species, such as gastro¬

pods, could be cultured successfully, but the labor and

other costs of doing so outweighed the ease of harvest¬

ing from the wild. Together, the live food culture, aug¬

mented by harvesting from the wild, has provided a sus¬

tainable supply of food for the maintenance of captive L.

fallax since their introduction into the facility on Dom¬

inica in 2011. Wild harvesting of live food might also

provide trace nutrients not obtained from cultured live

food, although this was not investigated in our study. The

Mountain Chicken Frog CBP on Dominica has had no

requirement for the import of food from overseas and

no evidence of nutritional disease has been observed,

although the frogs have not yet bred in the facility.

The known diet of L. fallax in the wild is varied, com¬

prising at least 30 different prey species. In the captive

breeding facility on Dominica, however, only five prey

species could be regularly provisioned. The depauper¬

ate captive diet was primarily due to three reasons: 1)

several species were unsuitable for propagation either

because of an inability to maintain large enough cultures

or because of labor requirements; 2) certain species that

could be cultured were not consumed by L. fallax in cap¬

tivity; 3) species not known to be prey items were cul¬

tured (including a non-native cricket and cockroach, both

of which were already established on Dominica). Even

if the known wild diet of L. fallax could be matched, the

diets used to culture live food are different to those eaten

by the invertebrates in the wild. It is unlikely, therefore,

that the nutritional content of cultured live food accu¬

rately represents that of the same invertebrate species

in the wild. It is possible that the cultured diet supplied

to the captive frogs is not optimal and therefore a wider

range of food species should be harvested from the wild

if captive animals are to be maintained and bred on the

island in the future. Determining the nutritional content

of the wild diet of L. fallax , rather than replicating the

food items themselves, could inform a viable alternative

of manipulating the nutritional content of cultured live

food through supplementation or gut loading.

The orthopteran, C. dominica , is thought to be one of

the key prey items for wild L. fallax and is very com¬

monly encountered on Dominica (Brooks Jr 1982); how¬

ever, we were unable to culture it successfully in large

enough numbers to be a useful food item. Possible rea¬

sons for the unsuitability of C. dominica to the culture

process could include inappropriate diet, territoriality, or

naturally low reproductive rates. The orthopteran section

of the diet therefore relied on two species, G. assimilis

and G. sigillatus , the latter believed to be a non-native

species that has become established on Dominica.

A further limitation in our ability to provide a varied

diet was the apparent unpalatability of the readily cul¬

tured Z atratus and the various unidentified millipede

species. These beetles and (certain) millipedes were

reported as being key components of the wild diet of L.

fallax (Brooks Jr 1982), but when offered to captive frogs

they were either rejected (millipede sp. and adult Z atra¬

tus) or ignored (larval Z. atratus). This might be due to

the ability of these species to produce defensive chemi¬

cals (Gullan and Cranston 2005), which could affect prey

preference in captivity in particular because the captive

frogs are provided with a readily available food supply.

It was not possible to ascertain the identity (even to the

level of genus) of the three types of millipede offered as

prey items, and only the genus of consumed millipedes

was reported by Brooks Jr (1982). Perhaps L. fallax is

very species-specific regarding millipedes and the wrong

prey items were being offered.

The unsuitability of certain invertebrate species as live

food items left the facility on Dominica heavily reliant on

non-native species which were not listed in the wild diet

of L. fallax but were easier to culture, notably G. sigilla¬

tus and B. discoidalis (Brooks Jr 1982). Gryllodes sigil¬

latus is native to Southwestern Asia but has spread rap¬

idly across the globe and is used in other CBPs where it

is non-native (Edmonds et al. 2012). Its arrival date and

how well it is established on Dominica is not known. Bla-

berus discoidalis is native to Venezuela, a country which

has exported live poultry and other agricultural products

to Dominica since establishing a trade relationship in the

late 1970s (A. James, pers. comm.; Cockroach Species

File 2016). Blaberus discoidalis was cultured in the facil¬

ity after being found in a local chicken coop. As with G.

sigillatus , the original introduction time frame for B. dis¬

coidalis is unknown but it is reasonable to suggest the

species has been present on Dominica for many years,

at least since the trade agreement with Venezuela began.

An accurate replication of the wild diet for animals in

CBPs, including those in range states, often is unachiev¬

able. For the L. fallax CBP, and programs like it, we rec¬

ommend that the focus should be towards supplying a

diversity of locally sourced prey species while, if possi¬

ble, increasing an understanding of the nutritional make¬

up of the diet in the wild. It is important to study, wher¬

ever feasible, the wild diet of any species maintained as

part of a CBP. In this case, comprehensive studies such as

Brooks Jr (1982) and additional findings (e.g., Rosa et al.

2012) were important for ascertaining potential prey spe¬

cies for culture. Establishing the wild diet and subjecting

this to detailed nutritional analyses should provide the

data required to provide an optimal diet in captivity, pos¬

sibly through manipulating the nutritional content of live

food species via supplementation or gut loading.

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e149

Nicholson et al.

Conclusion

Sustainable colonies of invertebrates were established

using locally caught species on Dominica. These colo¬

nies were productive enough to sustain a captive popula¬

tion of L. fallax. There was no need to import exotic spe¬

cies to use as live food, but the species most suitable for

culture were locally collected, non-native species. The

wild diet could not be fully replicated in captivity but

frogs did not exhibit any evidence of nutritional disease

over the six years of this study.

Acknowledgements. —The authors would like to

thank the experts who assisted with invertebrate iden¬

tification: David Gwyn Robinson, Umit Kebapgi, Dor-

rit King, and Klaus Riede. Jeff Dawson, Kevin Johnson,

Kay Bradheld, and an anonymous reviewer provided

valuable comments on the manuscript. The mountain

chicken conservation program on Dominica was funded

by the Darwin Initiative (project 13032) and the Zoologi¬

cal Society of London. The development of live food cul¬

ture on the island was also financially supported by the

Northwest of England Zoological Society.

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Nicholson et al.

Daniel Nicholson is a zoologist, conservationist, and tropical ecologist. Graduating from the University of Derby

with a Bachelor in zoology in 2012 and a MRes degree in conservation and biodiversity from the University of

Leeds in 2013. Daniel then worked as a researcher across the globe for several different institutions including the

National University of Singapore and the Australian National University. Daniel was part of the Mountain Chicken

Project on Dominica for eight months. He is now completing a Ph.D. in Evolutionary Ecology at Queen Mary

University London and the Zoological Society of London.

Benjamin Tapley is a conservation biologist and Curator of Herpetology at the Zoological Society of London.

Ben’s primary interest is the conservation breeding and captive management of amphibians and reptiles. Ben

studied Conservation Biology at the University of Surrey Roehampton and before completing his M.Sc. in

Conservation Biology at the Durrell Institute for Conservation and Ecology. Ben is currently working on Chinese

giant salamanders in China, Mountain Chicken Frogs from the Caribbean, and Megophryid frogs in Vietnam. Ben

is a Facilitator, IUCN Amphibian Specialist Group, Captive Breeding Working Group; Chair of BIAZA Reptile &

Amphibian Working Group; and Vice-Chair of the Amphibian Taxon Advisory Group, EAZA.

Stephanie Jayson is a veterinary surgeon carrying out a three-year European College of Zoological Medicine

Residency in Zoo Health Management based at the Zoological Society of London (ZSL) and the Royal Veterinary

College (RVC). She graduated from Cambridge University in 2012 with veterinary and zoology degrees and then

completed a one-year small animal internship followed by two years as an exotic pet and zoo animal practitioner.

Steph is passionate about amphibian conservation and has conducted a number of research projects and fieldwork

with Mountain Chicken Frogs at ZSL.

James Dale worked with ZSL and the Forestry division of Dominica in 2008-2009 to establish a supply of live

food for captive amphibians. He has worked as a herpetologist at Chester Zoo, Blue Planet Aquarium, and Stapeley

Water Gardens.

Luke Harding is the Curator of Lower Vertebrates and Invertebrates at Paignton Zoo and formally a senior keeper

within the Herpetology Section of the Zoological Society of London, London Zoo. He has extensive experience

in the application of behavioral science on the captive management of species and is particularly interested in

using these techniques to manage reptiles and amphibians in zoo settings. He has a long-standing involvement in

the Mountain Chicken Frog Conservation Program, and his passion for reptile and amphibian conservation has

allowed him to travel and contribute to fieldwork projects in India, South Africa, South America, Indonesia and the

Philippines, and more recently, Tanzania.

Jenny Spencer is a highly experienced zoo professional with a focus on the management of ectotherm species. A

passion for amphibians has led to her involvement with conservation initiatives both in the United Kingdom and

the Caribbean. More recently based in New Zealand, she continues her key interests of improving welfare standards

and amphibian conservation advocacy.

Machel Sulton is the Amphibian Technician working with the Dominica Forestry, Wildlife & Parks Division.

Since Machel’s childhood days he has been passionate with wildlife which led him to pursue studies within the

conservation field. He is interested in conserving the islands natural resources. Machel started off as a Forest Trainee

to understudy senior forest officers in carrying out their duties such as forest, river and coastal patrol, identifying

forest tree species, wildlife and involved in raising community awareness of biodiversity and conservation. Machel

has been heavily involved in the Mountain Chicken Project, conducting field surveys, public awareness/outreach

and event planning and also the management of captive amphibians.

Stephen Durand has been working with Dominica’s Forestry, Wildlife & Parks Division since 1981. He is currently

head of the Research and Monitoring, and Environmental Education Unit with responsibilities for a number of

research projects including; Amphibian Captive Breeding, Dominica’s Parrot Conservation, Dominica’s Sea

turtle Conservation, and the Black-capped petrel research project. Mr. Durand’s interest, commitment, dedication

and passion for environmental conservation work are tremendous, and he is very knowledgeable with respect to

Dominica’s biodiversity.

Andrew Cunningham is Deputy Head of the Institute of Zoology, Zoological Society of London, where he is

professor of Wildlife Epidemiology. He has published over 375 scientific articles, including the first definitive report

of the global extinction of a species by an infectious disease. He has led international, multi-disciplinary wildlife

disease research projects, including those that led to the discoveries of epidemic ranaviral amphibian disease in

Europe and of Batrachochytrium dendrobatidis as a cause of global amphibian declines.

Amphib. Reptile Conserv.

December 2017 | Volume 11 | Number 2 | e149