Published in the United States of America

-2017 • VOLUME 11 • NUMBER 2-

AMPHIBIAN & REPTILE

CONSERWION

amphibian-reptile-conservation.org

ISSN: 1083-446X

*

• Vk

elSSN: 1525-9153

♦

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).

Copyright: © 2017 Proano and Perez. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommer-
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.

1

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.

2

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

a

-

DMSO 5% - RT

03.26 ±4.00

b

-

DMSO 5% - 4C

19.48 ±21.73

b

10.99 ±2.98

DMSO 10% - RT

10.73 ± 13.00

b

-

DMSO 10% - 4C

23.17 ±27.13

b

10.43 ±4.64

DMSO 15% - RT

02.44 ±3.13

b

-

DMSO 15%-4C

07.90 ± 8.96

b

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).

PF

(M ± SD x 10 7 sperm/ml)

PT

(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

a

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

b

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.

4

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.

Literature Cited

Adams GP, Ratto MH, Collins CW, Bergfelt DR. 2009.
Artificial insemination in South American camelids
and wild equids. Theriogenology> 71(1): 166-175.
Al-Anzi B, Chandler D. 1998. A sperm chemoattractant
is released from Xenopus egg jelly during spawning.
Developmental Biology 198(2): 366-375.

Barnett S, Cover J, Wright K. 2001. Amphibian Hus¬
bandry and Housing. Pp. 35-61 In: Amphibian Med¬
icine and Captive Husbandry. Editors, Wright K,
Whitaker B. Krieger Publishing Company, Malabar,

Florida, USA. 570 p.

Beesley SG, Costanzo JP, Lee RE. 1998. Cryopreserva¬
tion of spermatozoa from freeze-tolerant and -intol¬
erant anurans. Cryobiology 37(2): 155-162.

Bishop PJ, Angulo A, Lewis JP, Moore RD, Rabb GB,
Garcia Moreno J. 2012. The Amphibian Extinction
Crisis - what will it take to put the action into the
Amphibian Conservation Action Plan? S.A.P.I.EN.S
5(2): 96-111. Available: https://sapiens.revues.

org/1406 [Accessed: 22 July 2017],

Bolanos F, Castro F, Cortez C, De la Riva I, Grant T,
Hedges B, Heyer R, Ibanez R, La Marca E, Young
B, et al. 2008. Amphibians of the Neotropical realm.
Pp. 92-105 In: Threatened Amphibians of the World.
Editors, Stuart S, Hoffmann M, Chanson J, Cox N,
Berridge R, Ramani P, Young B. IUCN. Lynx Edi-
cions, Barcelona, Spain; IUCN, Gland, Switzerland;
and Conservation International, Arlington, Virginia,
USA. 776 p. Available: http://www.amphibians.org/
publications/threatened-amphibians-of-the-world/
[Accessed: 22 July 2017],

Browne R, Clulow J, Mahony M, Clark A. 1998. Suc¬
cessful recovery of motility and fertility of cryopre¬
served cane toad (Bufo marinus) sperm. Cryobiol¬
ogy 37(4): 339—45.

Browne RK, Clulow J, Mahony M. 2002. The short-term
storage and cryopreservation of spermatozoa from
hylid and myobatrachid frogs. CryoLetters 23(2):
129-136.

Carolsfeld J, Godinho HP, Zaniboni Filho E, Harvey
BJ. 2003. Cryopreservation of sperm in Brazilian
migratory fish conservation. Journal of Fish Biology
63(2): 472^189.

Clulow J, Vance L, Trudeau, Kouba A. 2014. Amphibian
declines in the twenty-first century: Why we need
assisted reproductive technologies. Pp. 275-316
In: Reproductive Science in Animal Conservation:
Progress and Prospects. Editors, Holt WV, Brown
JL, Comizzoli P. Springer Science and Business
Media, New York, New York, USA. 549 p.

Della Togna G. 2015. Structural and functional charac¬
terization of the Panamanian golden frog ( Atelopus
zeteki ) spermatzoa - Impact of medium osmolality
and cryopreservation on motility and cell viability.
Ph.D. Dissertation, University of Maryland, College
Park, Maryland, USA. 192 p.

Dumont J. 1972. Oogenesis in Xenopus laevis (Daudin).
I. Stages of oocyte development in laboratory main¬
tained animals. Morphology 136(2): 153-179.

Fuller B. 2004. Cryoprotectants: The essential anti¬
freezes to protect life in the frozen state. CryoLetters
25(6): 375-388.

Hammerstedt R, Graham J, Nolan J. 1990. Cryopreserva¬
tion of mammalian sperm: What we ask them to sur¬
vive. Journal of Andrology 11(1): 73-88.

Kouba AJ, Lloyd RE, Houck ML, Silla AJ, Calatayud
N, Trudeau VL, Clulow J, Molinia F, Langhorne

Amphib. Reptile Conserv.

5

August 2017 | Volume 11 | Number 2 | e143

Proano and Perez

C, Della Togna G, et al. 2013. Emerging trends for
biobanking amphibian genetic resources: The hope,
reality and challenges for the next decade. Biologi¬
cal Conservation 164: 10-21.

Mansour N, Lahnsteiner F, Patzner RA. 2010. Motility
and cryopreservation of spermatozoa of European
common frog, Rana temporaria. Theriogenology
74(5): 724-732.

Michael SF, Jones C. 2004. Cryopreservation of sperma¬
tozoa of the terrestrial Puerto Rican frog, Eleuthero-
dactylus coqui. Cryobiology 48(1): 90-94.

Mugnano J, Costanzo P, Beesley S, Lee R. 1998. Evalua¬
tion of glycerol and dimethyl sulfoxide for the cryo¬
preservation of spermatozoa from the wood frog
(Rana sylvatica). CryoLetters 19: 249-254.

Paskova V, Hilscherova K, Blaha L. 2011. Teratogenic¬
ity and embryotoxicity in aquatic organisms after
pesticide exposure and the role of oxidative stress.
Reviews of Environmental Contamination and Toxi¬
cology 211: 25-61.

Rastogi R, Pinelli C, Polese G, D’Aniello B, Chieffi-
Baccari G. 2010. Hormones and reproductive
cycles in Anuran amphibians. Pp. 171-186 In: Hor¬
mones and Reproduction of Vertebrates. Volume
2-Amphibians. Editors, Norris DO, Lopez KH. Aca¬
demic Press, Amsterdam, Netherlands. 240 p.

Rubinsky B. 2003. Principles of low temperature cell
preservation. Heart Failure Reviews 8: 277-284.

Sargent MG, Mohun TJ. 2005. Cryopreservation of
sperm of Xenopus laevis and Xenopus tropicalis.
Genesis 41(1): 41—46.

Viveiros ATM, Godinho HP. 2009. Spenn quality and
cryopreservation of Brazilian freshwater fish spe¬
cies: A review. Fish Physiology and Biochemistry
35(1): 137-150.

Wright K. 2001. Restraint techniques and euthanasia.
Pp. 111-122 In: Amphibian Medicine and Captive
Husbandry. Editors, Wright K, Whitaker B. Krieger
Publishing Company, Malabar, Florida, USA. 570 p.

Will MA, Clark NA, Swain JE. 2011. Biological pH buf¬
fers in IVF: Help or hindrance to success. Journal
of Assisted Reproduction and Genetics 28: 711-724.

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.

6

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).

Copyright: ©2017 Poe and Ryan. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-
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.

Amphib. Reptile Conserv.

2

July 2017 | Volume 11 | Number 2 | e141

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.

3

July 2017 | Volume 11 | Number 2 | e141

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.

5

July 2017 | Volume 11 | Number 2 | e141

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

0

0

0

0

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

July 2017 | Volume 11 | Number 2 | e141

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.

7

July 2017 | Volume 11 | Number 2 | e141

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.

8

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.

9

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

Amphib. Reptile Conserv.

10

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.

11

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-

12

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.

Literature Cited

Alfoldi J, Palma FD, Grabherr M, Williams M, Kong L,
Mauceli E, Russell P, Lowe CB, Glor RE, Jaffe JD,

Amphib. Reptile Conserv.

13

July 2017 | Volume 11 | Number 2 | e141

Poe and Ryan

Ray DA, Boissinot S, Shedlock AM, Botka C, Cas-
toe TA, Colboume JK, Fujita MK, Moreno RG, ten
Hallers BF, Haussler D, Heger A, Heiman D, Janes

DE, Johnson J, de Jong PJ, Koriabine, MY, Lara
M, Novick PA, Organ CL, Peach SE, Poe S, Pol¬
lock DD, de Queiroz K, Sanger T, Searle S, Smith
JD, Smith Z, Swofford R, Turner-Maier J, Wade J,
Young S, Za-'dissa A, Edwards SV, Glenn TC, Sche-
nider CJ, Losos JB, Lander ES, Breen M, Ponting

CP, Lindblad-Toh K. 2011. The genome of the green
anole lizard and a comparative analysis with birds
and mammals. Nature 477(7366): 587-591.

Castaneda R, de Queiroz K. 2011. Phylogenetic relation¬
ships of the Dactyloa clade of Anolis lizards based
on nuclear and mitochondrial DNA sequence data.
Molecular Phylogenetics and Evolution 61(3): 784-
800.

Crawford AJ, Lips KR, Bermingham E. 2010. Epidemic
disease decimates amphibian abundance, species
diversity, and evolutionary history in the highlands
of central Panama. Proceedings of the National
Academy of Sciences 107(31): 13,777-13,782.

Donnelly MA, Crother BI, Guyer C, Wake MH, White
ME. 2005. Ecology and Evolution in the Tropics, A
Herpetological Perspective. University of Chicago
Press, Chicago, Illinois, United States. 675 p.

Glor RE, Laport R. 2012. Are subspecies of Anolis lizards
that differ in dewlap color and pattern also geneti¬
cally distinct? A mitochondrial analysis. Molecular
Phylogenetics and Evolution 64: 255-60.

Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayes¬
ian inference of phylogenetic trees. Bioinformatics
17(8): 754-755.

Kohler G. 2011. A new species of anole related to Ano¬
lis altae from Volcan Tenorio, Costa Rica (Reptilia,
Squamata, Polychrotidae). Zootaxa 3120: 29-42.

Lanfear R, Calcott B, Ho SYW, Guindon S. 2012. Par-
titionfinder: Combined selection of partitioning
schemes and substitution models for phylogenetic
analyses. Molecular Biology and Evolution 29:
1,695-1,701.

Lotzkat S, Hertz A, Bienentreu JF, Kohler G. 2013. Dis¬
tribution and variation of the giant alpha anoles
(Squamata: Dactyloidae) of the genus Dactyloa in
the highlands of western Panama, with the descrip¬
tion of a new species formerly referred to as D.
microtus. Zootaxa 3626(1): 1-54.

McCune B, Grace JB. 2002. Analysis of Ecological Com¬

munities. MjM Software, Gleneden Beach, Oregon,
USA. 304 p.

Mielke PW. 1984. Meteorological applications of per¬
mutation techniques based on distance functions.
Pp. 813-830 In: Editors, Krishnaiah PR, Sen PK.
Handbook of Statistics , Volume 4. North-Holland,
Amsterdam. 990 p.

Poe S, Latella IM, Ay ala-Varela F, Yanez-Miranda C,
Torres-Carvajal O. 2015. A new species of phena-
cosaur Anolis (Squamata: Iguanidae) from Peru and
a comprehensive phylogeny of Dactyl oa-c\adQ Ano¬
lis based on new DNA sequences and morphology.
Copeia 103(3): 639-650.

Poe S, Scarpetta S, Schaad EW. 2015. A new species of
Anolis from Panama. Amphibian & Reptile Conser¬
vation 9( 1): 1-13.

Poe S, A Nieto-Montes de Oca A, Torres-Carvajal O, de
Queiroz K, Velasco JA, Truett B, Gray LN, Ryan MJ,
Kohler G, Ayala-Varela F, Latella IM. 2017. A phy¬
logenetic, biogeographic, and taxonomic study of all
extant species of Anolis (Squamata; Iguanidae). Sys¬
tematic Biology doi: 10.1093/sysbio/syx029. [Epub
ahead of print],

Rambaut A, Suchard MA, Xie D, Drummond AJ. 2014.
Tracer vl.6. Available: http://beast.bio.ed.ac.ulc/
Tracer [Accessed: 14 June 2017],

Savage JM. 1974. Type localities for species of amphib¬
ians and reptiles described from Costa Rica. Revista
de Biologia Tropical 22(1): 71-122.

Savage JM, Talbot JJ. 1978. The giant anoline lizards of
Costa Rica and western Panama. Copeia 1978(3):
480-492.

Savage JM. 2002. The Amphibians and Reptiles of Costa
Rica: A Herpetofanna between Two Continents
between Two Seas. University of Chicago Press,
Chicago, Illinois, United States. 934 p.

Simpson GG. 1961. Principles of Animal Taxonomy.
Columba University Press, New York, New York,
United States. 247 p.

StataCorp. 2013. Stata Statistical Software: Release 13.
StataCorp LP, College Station, Texas, USA. Avail¬
able: http://www.stata.com/ [Accessed: 14 June
2017],

Taylor EH. 1956. A review of the lizards of Costa Rica.
University of Kansas Science Bulletin 38: 1-320.

Wiley EO. 1978. The evolutionary species concept
reconsidered. Systematic Zoology 27(1): 17-26.

William EE, Rand AS, O’Hara RJ. 1995. A computer
approach to the comparison and identification of
species in difficult taxonomic groups. Breviora 502:
1 ^ 7 .

Amphib. Reptile Conserv.

14

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

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
0 5
5 3
5 2
5 4
5 4
5 2
5 2

5 0
5 0
5 5
5 5
0 5
0 5
0 5
0 5

5

1

0

0

2

3
1

4

Amphib. Reptile Conserv.

15

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.

16

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).

Copyright: © 2017 Burger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-
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.

17

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.

18

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.

19

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

20

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.

21

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

Amphib. Reptile Conserv.

22

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.

Amphib. Reptile Conserv.

23

July 2017 | Volume 11 | Number 2 | e142

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

Amphib. Reptile Conserv.

24

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).

Amphib. Reptile Conserv.

25

July 2017 | Volume 11 | Number 2 | e142

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

Amphib. Reptile Conserv.

26

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.

Literature Cited

Ashton RE, Ashton PS. 2008. The Natural History and
Management of the Gopher Tortoise (Gopherus
polyphemus). Krieger Publishing Company, Mala¬
bar, Florida, USA. 275 p.

Bidwell RD, Ryan CM. 2006. Collaborative partnership
designs: The implications of organizational affilia¬
tion for watershed partnerships. Society and Natural
Resources 19: 827-843.

Bonney R, Cooper CB, Dickinson J, Kelling S, Phillips
T, Rosenberg KV, Shirk J. 2009. Citizen science: A
developing tool for expanding science knowledge
and scientific literacy. BioScience 59: 977-984.

Boyd HP. 1991. A Field Guide to the Pine Barrens of
New Jersey: Its Flora, Fauna, Ecology, and Historic
Sites. Plexus Pub., Medford, New Jersey, USA. 420
P-

Amphib. Reptile Conserv.

27

July 2017 | Volume 11 | Number 2 | e142

Burger et al.

Figure 9. Sometimes many volunteers are
necessary for a project, either digging up a
hibernation site (Fig 9a), clearing open areas
for sun penetration, or digging up an old septic
line to prevent collapses and injuries to snakes.

Burger J. 1989a. Incubation temperature has
long-term effects on behavior of young
Pine Snakes ( Pitnophis melanoleucus).

Behavioral Ecology and Sociobiology
24: 201-208.

Burger J. 1989b. Following of conspecifics
and avoidance of predator chemical cues
by Pine Snakes ( Pitnophis melanoleu¬
cus). Journal of Chemical Ecology 15:

799-806.

Burger J. 1990. Response of hatchling Pine Snakes ( Pitu-
ophis melanoleucus) to chemical cues of sympatric
snakes. Copeia 1990: 1,160-1,163.

Burger J. 1991a. Effects of incubation temperature on
behavior of hatchling Pine Snakes: Implications
for reptilian distribution. Behavioral Ecology and
Sociobiology 28: 297-303.

Burger J. 1991b. Response to prey chemical cues by
hatchling Pine Snakes ( Pituophis melanoleucus):
Effects of incubation temperatures and experience.
Journal of Chemical Ecology 17: 1,069-1,078.

Burger J. 1998a. Effects of incubation temperature on
behavior of hatchling Pine Snakes: Implications for
Survival. Behavioral Ecology and Sociobiology 43:
11-18.

Burger J. 1998b. Anti-predator behavior of hatchling
pine snakes: Effects of incubation temperature and
simulated predators. Animal Behavior 56: 547-553.

Burger J. 2006. Whispers in the Pines: A Naturalist in the
Northeast. Rutgers University Press, New Bruns¬
wick, New Jersey, USA. 352 p.

Burger J. 2007. The behavioral responses of emerging
Pine Snakes ( Pituophis melanoleucus) in the New
Jersey Pine Barrens. Journal of Herpetology 22:
425-433.

Burger J, Gochfeld M. 1985. Behavioral development:
Nest emergence of young Pine Snakes (Pituophis
melanoleucus). Journal of Comparative Psychology
99: 150-159.

Burger J, Zappalorti RT. 1986. Nest Site Selection by
Pine Snakes, Pituophis melanoleucus , in the New
Jersey Pine Barrens. Copeia 1986(1): 116-121.

Burger J, Zappalorti RT. 1988a. Habitat use in free-rang¬
ing Pine Snakes Pituophis melanoleucus in the New
Jersey Pine Barrens. Herpetologica 44: 48-55.

Burger J, Zappalorti RT. 1988b. Effects of incubation
temperature on Pine Snake development: Differen¬
tial vulnerability of males and females. American
Naturalist 132: 492-505.

Burger J, Zappalorti RT. 1989. Habitat use by Pine
Snakes ( Pituophis melanoleucus) in the New Jersey
Pine Barrens: Individual and sexual variation. Jour-

Amphib. Reptile Conserv.

28

July 2017 | Volume 11 | Number 2 | e142

Stakeholder contributions to conservation of threatened Northern Pine Snakes

nal of Herpetology 23: 68-73.

Burger J, Zappalorti RT. 1991. Nesting behavior of Pine
Snakes ( Pitnophis m. melanoleucus ) in the New
Jersey Pine Barrens. Journal of Herpetology 25:
152-160.

Burger J, Zappalorti RT. 1992. Philopatry and nesting
phenology of Pine Snakes Pituophis melanoleucus
in the New Jersey Pine Barrens. Behavioral Ecology
and Sociobiology 30: 331-336.

Burger J, Zappalorti RT. 2011a. The Northern Pine
Snake (Pituophis melanoleucus ) in New Jersey: Its
Life History, Behavior and Conservation. Pp. 1-56
In: Reptiles: Biology, Behavior, and Conservation.
Nova Science Publishers, Inc. New York, New York,
USA. 226 p.

Burger J, Zappalorti RT. 2011b. Effects of handling,
marking, and recapturing Pine Snakes (Pituophis m.
melanoleucus) from the New Jersey Pine Barrens.
Journal of Environmental Indicators 6: 17-32.

Burger J, Zappalorti RT. 2015. Hibernation site philop¬
atry in Northern Pine Snakes (Pituophis melano¬
leucus) in New Jersey. Journal of Herpetology 49:
245-251.

Burger J, Zappalorti RT. 2016. Conservation and protec¬
tion of threatened Pine Snakes (Pituophis melano¬
leucus) in the New Jersey Pine Barrens, USA. Her-
petological Conservation and Biology) 11: 304-314.

Burger J, Zappalorti RT, Gochfeld M. 1987. Develop¬
mental effects of incubation temperature on hatch¬
ling Pine Snakes Pituophis melanoleucus. Compar¬
ative Biochemistry and Physiology 87(A): 727-732.

Burger J, Zappalorti RT, Gochfeld M, Boarman W, Caf-
frey M, Doig V, Garber S, Mikovsky M, Safina C,
Saliva J. 1988. Hibernacula and summer dens of
Pine Snakes (Pituophis melanoleucus) in the New
Jersey Pine Barrens. Journal of Herpetology 22:
425^133.

Burger J, Boarman W, Kurzava L, Gochfeld M. 1991.
Effect of experience with Pine (Pituophis mela¬
noleucus) and King (Lampropeltis getulus) snake
odors on Y-maze behavior of Pine Snake hatchlings.
Journal of Chemical Ecology 17: 79-87.

Burger J, Zappalorti RT, Dowdell J, Hill J, Georgiadis T,
Gochfeld M. 1992. Subterranean predation on Pine
snakes (Pituophis melanoleucus). Journal of Herpe¬
tology 26: 259-263.

Burger J, Zappalorti RT, Gochfeld M, DeVito E. 2007.
Effects of off-road vehicles on reproductive success
of pine snakes (Pituophis melanoleucus) in the New
Jersey pinelands. Urban Ecosystems 10: 275-284.

Chess C, Purcell K. 1999 Public participation and the
environment: Do we know what works? Environ¬
mental Science & Technology 33: 2,685-2,692.

Conant R, Collins JT. 1998. A Field Guide to Reptiles and
Amphibians of Eastern and Central North America.
Third Edition, Expanded. Houghton Mifflin Com¬
pany, New York, New York, USA. 640 p.

Conrad CC, Hilchey KG. 2011. A review of citizen sci¬
ence and community-based environmental monitor¬
ing: Issues and opportunities. Environmental Moni¬
toring and Assessment 176: 273-291.

Cooper CB, Dickinson J, Phillips T, Bonney R. 2007.
Citizen science as a tool for conservation in residen¬
tial ecosystems. Ecology and Society 12: 1-11.

Dickinson JL, Zuckerberg B, Bonter, DN. 2010. Citizen
science as an ecological research tool: Challenges
and benefits. Annual Review of Ecology, Evolution,
and Systematics 41:149-172.

Florida Fish and Wildlife Commission. 2012. Gopher
Tortoise management Plan (Gopherus polyphemus).
Florida Fish and Wildlife Commission, Tallahassee,
Florida, USA. 224 p.

Gerald GW, Bailey MA, Holmes JN. 2006a. Movements
and activity range sizes of Northern Pine Snakes
(Pituophis melanoleucus melanoleucus) in middle
Tennessee. Journal of Herpetology 40: 503-510.

Gerald GW, Bailey MA, Holmes JN. 2006b. Habitat uti¬
lization of Pituophis melanoleucus melanoleucus
(Northern Pine Snakes) on Arnold Air Force Base
in middle Tennessee. Southeastern Naturalist 5:
253-264.

Gibbons JW, Scott DE, Ryan TJ, Buhlmann KA, Tuber-
ville TD, Metts BS, Greene JL, Mills T, Leiden Y,
Poppy S, Winne CT. 2000. The global decline of rep¬
tiles, deja vu. BioScience 50: 653-666.

Glowinski SL, Moore FR. 2014. The role of recreational
motivation in the birding participation-environmen¬
tal concern relationship. Human Dimensions Wild¬
life 19: 219-233.

Golden DM, Jenkins D. 2003. Northern Pine Snake,
Pituophis melanoleucus melanoleucus. Pp. 193-200
In: Endangered and Threatened Wildlife of New Jer¬
sey. Editors, Beans BE, Niles L. Rutgers University
Press, New Brunswick, New Jersey, USA. 328 p.

Golden DM, Winkler P, Woerner P, Fowles G, Pitts W,
Jenkins D. 2009. Status assessment of the Northern
Pine Snake (Pituophis m. melanoleucus) in New Jer¬
sey: An evaluation of trends and threats. New Jersey
Department of Environmental Protection, Division
of Fish and Wildlife, Endangered and Nongame Spe¬
cies Program. Trenton, New Jersey, USA. 53 p.

Himes IG, Hardy LM, Craig D. 2002. Growth Rates and
Mortality of the Louisiana Pine Snake. Herpetology
36: 683-687.

Lawrence A. 2006. ‘No personal motive?’ Volunteers,
biodiversity, and the false dichotomies of participa¬
tion. Ethics, Place, and Environment 9: 279-298.

National Research Council (NRC). 1983. Risk Assess¬
ment in the Federal Government: Managing the
Process. National Academy Press, Washington, DC,
USA. 191 p.

National Research Council (NRC). 1996. Understand¬
ing Risk: Informing Decisions in a Democratic Soci¬
ety. The National Academies Press, Washington DC,

Amphib. Reptile Conserv.

29

July 2017 | Volume 11 | Number 2 | e142

Burger et al.

USA. 264 p.

New Jersey Department of Environmental Protection
(NJDEP). 2002. DEP Commissioner Campbell
Announces Olf-Road Vehicle Policy Reinforces Ban
on Public Lands; Seeks Maximum Fines, Additional
Sanctions for Illegal Use (News Release 10/02/02).
Available: http: //www. state. nj. us/dep/ne wsrel/

releases/02_0095.htm [Accessed: 03 July 2017],

New Jersey Pinelands Commission (NJPC). 2009. The
Pinelands National Reserve. New Jersey Pinelands
48 Commission, New Lisbon, New Jersey. Avail¬
able : http: //www. state .nj. us/pinelands/index, shtml
[Accessed: 03 July 2017],

Miller GJ, Smith LL, Johnson SA, Franz R. 2012. Home
range size and habitat selection in the Florida Pine
Snake {Pitiiophis melanoleucus mugitus). Copeia
2012(4): 706-713.

Paavola J, Hubacek KI. 2013. Ecosystem services, gov¬
ernance, and stakeholder participation: An introduc¬
tion. Ecology and Society 18: 42^-7.

Pittinger CA, Bachman R, Barton AL, Clark JR, deFur
PL, Ells SJ, Slimac MW, Wentzel RS. 1998. Amulti-
stakeholder framework for ecological risk manage¬
ment: Summary from a SETAC technical workshop.
Environmental Toxicology and Chemistry Supple¬
ment 18, 1. Available: https://c.ymcdn.com/sites/
www.setac.org/resource/resmgr/publications_and_
resources/ermsummbklet.pdf [Accessed 03 July
2017],

Powell R, Conant R, Collins JT. 2016. Peterson Field
Guide to Reptiles and Amphibians of Eastern and
Central North America. Fourth Edition, Houghton
Mifflin Company, New York, New York, USA. 512

P-

Presidential/Congressional Commission on Risk Assess¬
ment and Risk Management (PCCRARM). 1997.
Framework for Environmental Health Risk Manage¬
ment. Final report, Volume 1. Decision Focus, Inc.,
Washington DC, USA. 213 p.

Rudolph DC, Schaefer RR, Burgdorf SJ, Duran M, Con¬
ner RN. 2007. Pine Snake ( Pitiiophis ruthveni and
Pitiiophis melanoleucus lodingi) hibernacula. Jour¬
nal of Herpetology 41: 560-565.

Schwartz V, Golden DM. 2002. Field Guide to Reptiles
and Amphibians of New Jersey. New Jersey Divi¬
sion of Fish and Wildlife, Trenton, New Jersey,
USA. 87 p.

United States Environmental Protection Agency
(USEPA). 1992. Framework for ecological risk
assessment. Risk Assessment Forum, Washington
DC, USA. EPA/630/R-92-001.

United States Environmental Protection Agency
(USEPA). 1998. Guidelines for ecological risk
assessment. Risk Assessment Forum, Washington
DC, USA.EPA/630/R-92-001.

Webb JK, Shine R, Pringle RM. 2005. Canopy removal
restores habitat quality for an endangered snake in a
fire suppressed landscape. Copeia 2005(4): 894-900.

Young JC, Jordan A, Searle KR, Butler A, Chapman
DS, Simmons P, Watts AD. 2013. Does stakeholder
involvement really benefit biodiversity conserva¬
tion? Biological Conservation 158: 359-370.

Zappalorti RT, Burger J. 1986. On the importance of dis¬
turbed sites to habitat selection in Pine Snakes in the
Pine Barrens of New Jersey. Environmental Conser¬
vation 12: 358-361.

Zappalorti RT, Burger J, Burkett DW, Schneider DW,
McCort MP, Golden DM. 2014. Fidelity of north¬
ern pine snakes {Pitiiophis m. melanoleucus) to nat¬
ural and artificial hibernation sites in the New Jersey
Pine Barrens. Journal of Toxicology and Environ¬
mental Health 77: 1,285-1,291.

Zappalorti R, Burger J, Peterson, F. 2015. Home range
size and distance traveled from hibernacula in
Northern Pine Snakes in the New Jersey Pine Bar¬
rens. Herpetologica 71: 26-36.

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.

30

July 2017 | Volume 11 | Number 2 | e142

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.

31

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.

32

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).

Copyright: © 2017 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-
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.

33

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.

34

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.

35

November 2017 | Volume 11 | Number 2 | e145

Wang et al.

In...

a

it #

b

%W.

c

1W

SjBfP

mm

'

■ 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

Amphib. Reptile Conserv.

36

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-

Amphib. Reptile Conserv.

37

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

Amphib. Reptile Conserv.

38

November 2017 | Volume 11 | Number 2 | e145

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.

39

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.

Amphib. Reptile Conserv.

40

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.

Literature Cited

Anderson J. 1871. Description of a new genus of newts
from western Yunnan. Proceedings of the Zoological
Society of London 1871: 423-425.

Barley AJ, White J, Diesmos AC, Brown RM. 2013. The
challenge of species delimitation at extremes: Diver¬
sification without morphological change in Philip¬
pine Sun Skinks. Evolution 67(12): 3,556-3,572.
Blair WF. 1962. Evolution at populational and interpop-
ulational levels: Isolating mechanisms and interac¬
tions in anuran amphibians. Quarterly Review of
Biology 39: 333-334.

Boulenger GA. 1920. Observations sur un batracien
urodele d’Asie, Tylototriton verrucosus Anderson.
Bulletin de la Societe Zoologique de France 45:
98-99.

Dawley EM. 1984. Recognition of individual sex, and
species odours by salamanders of the Plethoden glu-
tinosus-P. jordani complex. Animal Behavior 32:
353-361.

Dawley EM. 1986. Behavioral isolating mechanisms in
sympatric terrestrial salamanders. Herpetologica
42(2): 156-164.

Deuti K, Hedge VD. 2007. Handbook of Himalayan Sal¬
amander. Nature Books India, Delhe, India. 50 p.

Fei L, Hu S, Ye C, Huang Y. 2006. Fauna Sinica,
Amphibia Volume 1. General accounts of Amphibia
Gymnophiona and Urodela. Science Press, Beijing,
China. 471 p. [In Chinese],

Fei L, Ye C, Jiang J. 2012. Colored Atlas of Chinese
Amphibians and their Distributions. Science Press,
Beijing, China. 619 p. [In Chinese],

Fleck J. 1997. Nachzucht von Tylototriton taliangensis.
Elaphe 5: 86. [In German],

Gong D, Mou M, Li X, Teng J, Zhang K. 2008. Repro¬
ductive biology of Tylototriton wenxianensis. Chi¬
nese Journal of Zoology 43(4): 48-55. [In Chinese],
Hernandez A. 2016. Crocodile Newts: The genera E chi -
notriton and Tylototriton. Edition Chimaria, Frank¬
furt, Germany. 415 p.

Houck LD, Arnold SJ. 2003. Courtship and mating

behavior. Pp. 383-424 In: Reproductive Biology and
Phylogeny of Urodela. Editor, Sever BGM. Taylor &
Francis, England. 627 p.

Hou M, Li P, Lv S. 2012. Morphological research devel¬
opment of genus Tylototriton and primary confirma¬
tion of the status of four cryptic populations. Journal
ofHuangshan University 14: 61-65. [In Chinese],

Hu S. 1994. Observation of reproductive behavior of
Guizhou knobby newts, Tylototriton kweichowensis.
Journal of Bijie University A. 8-10. [In Chinese],

Humphrey SR, Bain JR. 1990. Endangered Animals of
Thailand. Sandhill Crane Press, Inc., Gainesville,
Florida, USA. 468p.

Khatiwada JR, Wang B, Ghimire S, Vasudevan K, Paudel
S, Jiang J. 2015. A new species of the genus Tylo¬
totriton (Amphibia: Urodela: Salamandridae) from
Eastern Himalaya. Asian Herpetological Research
6: 245-256.

Le D, Nguyen T, Nishikawa K, Nguyen S, Pham A, Mat-
sui M, Bernardes M, Nguyen TQ. 2015. A new spe¬
cies of Tylototriton Anderson, 1871 (Amphibia: Sal¬
amandridae) from Northern Indochina. Current Her¬
petology? 34(1): 67-74.

Li J, Liu A, Li X, Liu X, Jing K. 2012. The breeding
ecology of red knobby newts, Tylototriton shanjing.
Chinese Journal'of Zoology 47: 8-15. [In Chinese],

Mabry M, Verrell PA. 2004. Stifled sex in sympatry:
patterns of sexual incompatibility among desmog-
nathine salamanders. Biological Journal of the Lin-
nean Society 82: 367-375.

Marshall JC, Arevalo E, Benavides E, Sites JL, Sites JL
Jr. 2006. Delimiting species: Comparing methods
for Mendelian characters using lizards of the Sce-
loporus grammicus (Squamata: Phrynosomatidae)
complex. Evolution 60: 1,050-1,065.

Nishikawa K, Khonsue W, Pomchote P, Matsui M.
2013. Two new species of Tylototriton from Thi-
land (Amphibia: Urodela: Salamandridae). Zootaxa
3737(3): 261-279.

Nishikawa K, Matsui M, Rao D. 2014. A new species
(Amphibia: Urodela: Salamanderidae) from central
Myanmar. Natural History Bulletin of the Siam Soci¬
ety 60(1): 9-22.

Nishikawa K, Rao D, Matsui M, Eto K. 2015. Taxo¬
nomic relationship between Tylototriton daweisha-
nensis Zhao, Rao, Liu, Li, and Yuan, 2012 and T.
yangi Hou, Li, and Lu, 2012 (Amphibia: Urodela:
Salamandridae). Current Herpetology’ 3'4(1): 61-1 A.

Phimmachak S, Aowphol A, Stuart BL. 2015. Morpho¬
logical and molecular variation in Tylototriton (Cau-
data: Salamanderidae) in Laos, with description of a
new species. Zootaxa 4006: 285-310.

Rissler LJ, Apodaca JJ. 2007. Adding more ecology into
species delimitation: Ecological niche models and
phylogeography help define cryptic species in the
black salamander ( Aneides flavipunctatus). System¬
atic Biology 56(6): 924-942.

Amphib. Reptile Conserv.

41

November 2017 | Volume 11 | Number 2 | e145

Wang et al.

Nussbaum RA, Brodie ED Jr, Yang D. 1995. A taxo¬
nomic review of Tylototriton verrucosus Anderson
(Amphibia: Caudate, Salamandridae). Herpetolog-
ica 51: 257-268.

Rowley JJL, Shepherd CR, Stuart BL, Nguyen TQ,
Hoang HD, Cutajar TP, Wogan GOU, Phimmachak
S. 2016. Estimating the global trade in Southeast
Asian newts. Biological Conservation 199: 96-100.

Roy D, Mushahidunnabi M. 2001. Courtship, mating and
egg-laying in Tylototriton verrecosus from the Dar¬
jeeling district of the Eastern Himalayas. Current
Sciences 81: 693-695.

Rundle HD, Nosil P. 2005. Ecological speciation. Ecol¬
ogy Letters 8 (3): 336-352.

Sites JW, Marshall JC. 2004. Operational criteria for
delimiting species. Annual Review of Ecology, Evo¬
lution, and Systematics 35:199-227.

Sparreboom M. 1999. Haltung von Nachzucht Tylototri¬
ton verrucosus. Elaphe 7(2): 20-24. [In German],

Sparreboom M. 2014. Salamanders of the Old World:
The Salamanders of Europe, Asia and Northern
Africa. KNNV Publishing, Zeist, Netherlands. 431

P-

Tian Y, Sun A, Li S. 1998. Studies on reproductive ecol¬
ogy of Tylototriton kweichowensis Fang and Chang.
Sichuan Journal of Zoology 17: 60-64. [In Chinese],

Tilley S, Verrell PA, Arnold S. 1990. Correspondence
between sexual isolation and allozyme differen¬
tiation: A test in the salamander Desmognathus
ochrophaeus. Proceeding of National Academy of
Sciences of the United States of America 87: 2,715-
2,719.

Topfer-Hofmann G, Cordes GD, Helversen OV. 2000.
Cryptic species and behavioral isolation in the Par-
dosa lugubris group (Araneae, Lycosidae), with
description of two new species. Bulletin of the Brit¬
ish Arachnological Society 11(7): 251-21 A.

Verrell PA, Mabry M. 2000. The courtship of plethod-
ontid salamanders: Form, function, and phylogeny.
Pp. 371-380 In: The Biology of Plethodontid Sala¬
manders. Editors, Bruce RC, Jaeger RG, Houck LD.
Plenum Press, New York, New York, USA. 485 p.

Verrell PA, Mabry M. 2003. Sexual behaviour of the
Black Mountain dusky salamander ( Desmognathus
welteri ), and the evolutionary history of courtship
in the Desmognathinae. Journal of Zoology London
260: 367-376.

Ziegler T, Hartmann T, Straeten KV, Karbe D, Bohme
W. 2008. Captive breeding and larval morphology
of Tylototriton shanjing Nussbaum, Brodie & Yang,
1995, with an updated key of the genus Tylototriton
(Amphibia: Salamandridae). Der Zoologische Gar¬
ten 77(4): 246-260.

Amphib. Reptile Conserv.

42

November 2017 | Volume 11 | Number 2 | e145

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.

43

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).

Copyright: © 2017 Sancho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-
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.

44

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.

45

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.

46

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.

Literature Cited

Booth DT. 1998. Effects of incubation temperature on
the energetics of embryonic development and hatch¬
ling morphology in the Brisbane River Turtle Emy-
dura signata. Journal of Comparative Physiology B
168: 399^104.

Bull JJ, Vogt RC. 1981. Temperature-sensitive periods
of sex determination in emydid turtles. Journal of
Experimental Zoology 218: 435-440.

Bull JJ, Vogt RC, McCoy CJ. 1982. Sex determining
temperatures in turtles: A geographic comparison.
Evolution 36: 326-332.

Burke RL, Calichio AM. 2014. Temperature sex determi¬
nation in the Diamond-backed Terrapin, Malaclemys
terrapin. Journal of Herpetology 48: 466-470.

Burke RL, Jacobson EJ, Degayner M, Guillette JL. 1994.
Non-invasive sex identification of juvenile gopher
and desert tortoises (Genus: Gopherus). Amphibia-
Reptilia 15: 183-189.

Burke RL, Ewert ME, McLemore JB, Jackson DR. 1996.
Temperature-dependent sex determination and
hatching success in the gopher tortoise ( Gopherus
Polyphemus'). Chelonian Conservation and Biology
2 : 86 - 88 .

Casale P, Freggi D, Basso R, Argano R. 2005. Size at
male maturity, sexing methods and adult sex ratio
in loggerhead turtles ( Caretta caretta) from Italian
waters investigated through tail measurements. The
Herpetological Journal 15: 145-148.

CAYOT LJ. 2008. The restoration of giant tortoise and
land iguana populations in Galapagos. Galapagos
Research 65: 39-43.

Cayot LJ, Snell HL, Llerena W, Snell HM. 1994. Con¬
servation biology of Galapagos reptiles: Twenty-five
years of successful research and management. Pp.
297-305 In: Captive Management and Conserva¬
tion of Amphibians and Reptiles. Editors, Murphy
JB, Adler K, Collins JT. Society for the Study of
Amphibians and Reptiles, Ithaca, New York, USA.
408 p.

Delmas V, Prevot-Julliard A-C, PIEAU C, GIRON¬
DOT M. 2008. A mechanistic model of tempera¬
ture-dependent sex determination in a chelonian,
the European pond turtle. Functional Ecology 22:
84-93.

Demuth JP. 2001. The effects of constant and fluctuat¬
ing incubation temperatures on sex determination,
growth, and performance in the tortoise Gopherus
polyphemus. Canadian Journal of Zoology 79:
1,609-1,620.

Eendebak BT. 1995. Incubation period and sex ratio of
Hermann’s tortoise, Testudo hermanni boettgeri.
Chelonian Conservation and Biology 1: 227-231.

Ewert M, Nelson C. 1991. Sex determination in turtles:
Diverse patterns and some possible adaptive values.
Copeia 1991: 50-69.

Ewert MA, Etchberger CR, Nelson CE. 2004. Turtle sex
determining modes and TSD patterns, and some
TSD pattern correlates. Pp. 21-32 In: Temperature
Dependent Sex Determination in Vertebrates. Edi¬
tors, Valenzuela N, Lance VA. Smithsonian Books,
Washington, DC, USA. 200 p.

Amphib. Reptile Conserv.

47

December 2017 | Volume 11 | Number 2 | e146

Temperature sex determination in the Espanola Giant Tortoise

Ewert MA, Hatcher RE, Goode JM. 2004. Sex deter¬
mination and ontogeny in Malacochersus tornieri,
the pancake tortoise. Journal of Herpetology 38:
291-295.

Ewert MA, Jackson DR, Nelson CE. 1994. Patterns of
temperature-dependent sex determination in turtles.
Journal of Experimental Zoology 270: 3-15.

Ewert MA, Lang JW, Nelson CE. 2005. Geographic vari¬
ation in the pattern of temperature-dependent sex
determination in the American snapping turtle ( Che-
lydra serpentina ). Journal of Zoology 265: 81-95.

Georges A, Limpus C, Stoutjesduk R. 1994. Hatchling
sex in the marine turtle Caretta caretta is determined
by proportion of development at a temperature, not
daily duration of exposure. Journal of Experimental
Zoology, > 270: 432^144.

Gibbs JP, Marquez C, Sterling EJ. 2008. The role of
endangered species reintroduction in ecosystem res¬
toration: tortoise-cactus interactions on Espanola
Island, Galapagos. Restoration Ecology 16: 88-93.

Gibbs JP, Hunter EA, Shoemaker KT, Tapia WH, Cayot
LJ. 2004. Demographic outcomes and ecosystem
implications of giant tortoise reintroduction to Espa¬
nola Island, Galapagos. PLoS One 9(10): el 10742.
doi: 10.1371/journal.pone.0110742.

Girondot M. 1999. Statistical description of temperature
dependent sex determination using maximum likeli¬
hood. Evolutionary Ecology Research 1: 479-486.

Girondot M. 2012. 2012. TSD (Version 3.0.4). CNRS
et Universite' Paris-Sud, UMR-8079-Ecologie,
Syste'matique et Evolution, Department d’Ecologie
des Populations et des Communaute's, Groupe de
Conservation des Populations et des communaute's,
France.

Godfrey MH, Delmas V, Girondot M. 2003. Assessment
of patterns of temperature-dependent sex determi¬
nation using maximum likelihood model selection.
Ecoscience 10: 265-272.

Grosse AM, Crawford BA, Maerz JC, Buhlmann KA,
Norton T, Kaylor M, Tuberville KM. 2014. Effects
of vegetation structure and artificial nesting habitats
on hatchling sex determination and nest survival of
diamondback terrapins. Journal of Fish and Wildlife
Management 6: 19-28.

Gutzke WH, Bull JJ. 1986 Steroid hormones reverse sex
in turtles. General and Comparative Endocrinology
64: 368-372.

Gutzke WHN, Paukstis GL. 1983. Influence of the hydric
environment on sexual differentiation of turtles.
Journal of Experimental Zoology 226: 467-469.

Janzen FJ. 1993. The influence of incubation temperature
and family on eggs, embryos, and hatchlings of the
smooth softshell turtle ( Apalone mutica). Physiolog¬
ical Zoology 66: 349-373.

Janzen FJ, Paukstis GL. 1991. Environmental sex deter¬
mination in reptiles: Ecology, evolution and experi¬
mental design. The Quarterly Review of Biology 66:

149-179.

Lubiana A, Ferreira Junior PD. 2009. Pivotal tempera¬
ture and sexual dimorphism of Podocnemis expansa
hatchlings (Testudines: Podocnemididae) from
Bananal Island, Brazil. Zoologia (Curitiba) 26:
527-533.

MacFarland CG, Villa J, Toro B. 1974a. The Galapagos
giant tortoises ( Geochelone elephant opus). Part I:
Status of surviving populations. Biological Conser¬
vation 6: 118-133.

MacFarland CG, Villa J, Toro B. 1974b The Galapagos
giant tortoises ( Geochelone elephant opus). Part II:
Conservation methods. Biological Conservation 6:
198-212.

Marquez C, Morillo G, Cayot LJ. 1991. A 25-year man¬
agement program pays off: Repatriated tortoises
in Espanola reproduce. Noticias de Galapagos 50:
17-18.

Marquez C, Corillo G, Rea S. 1999. La Crianza de Tor-
tugas Gigantes en Cautiverio: Un Manual Opera-
tivo. Fundacion Charles Darwin, Area de Investig-
acion para Protection de Animales Nativos, Quito,
Ecuador. 120 p.

Martinez-Silvestre A, Bargallo F, Grifols J. 2015. Gen¬
der identification by cloacoscopy and cystoscopy
in juvenile chelonians. Veterinary Clinics of North
America: Exotic Animal Practice 18: 527-539.

Milinkovitch MC, Kanitz R, Tiedemann R, Tapia W,
Llerena F, Caccone A, Gibbs JP, Powell JR. 2013.
Recovery of a nearly extinct Galapagos tortoise
despite minimal genetic variation. Evolutionary
Applications 6: 377-383.

Mrosovsky N, Baptistotte C, Godfrey MH. 1999. Vali¬
dation of incubation duration as an index of the sex
ratio of hatchling sea turtles. Canadian Journal of
Zoology 77: 831-835.

Mrosovsky N, Pieau C. 1991. Transitional range of tem¬
perature, pivotal temperatures and thermosensitive
stages for sex determination in reptiles. Amphibia-
Reptilia 12: 169-179.

Mrosovsky N, Yntema CL. 1980. Temperature depen¬
dence of sexual differentiation in sea turtles: Impli¬
cations for conservation practices. Biological Con¬
servation 18: 271-280.

Pieau C. 1971. Sur la proportion sexuelle chez les embry-
ons de deux Cheloniens ( Testudo graeca L. et Emys
orbicularis L.) issues d’oeufs incubes articifielle-
ment. Comptes Rendus Hebdomadaires Des Sci¬
ences De L’ Academie Des Sciences Serie D 272:
3,071-3,074.

Poulakakis N, Edwards DL, Chiari Y, Garrick RC, Rus-
sello MA, Benavides E, Watkins-Coldwell GJ, Gla-
berman S, Tapia W, Gibbs JP, Cayot LJ, Caccone A.
2015. Description of a new Galapagos giant tortoise
species ( Chelonoidis\ Testudines: Testudinidae) from
Cerro Fatal on Santa Cruz Island. PLoS ONE 10(10):
e0138779. doi: 10.1371/joumal.pone.0138779.

Amphib. Reptile Conserv.

48

December 2017 | Volume 11 | Number 2 | e146

Sancho et al.

Roosenburg WM, Kelley KC. 1996. The effect of egg
size and incubation temperature on growth in the
turtle, Malaclemys terrapin. Journal of Herpetology
30: 198-204.

Russello MA, Poulakakis N, Gibbs JP, Tapia W, Bena¬
vides E, Powell JR, Caccone A. 2010. DNA from the
past informs ex situ conservation for the future: An
“extinct” species of Galapagos tortoise identified in
captivity. PLoS One 5(1): e8683.

Sancho A. 1988. Influencia de la Temperature de Incu-
bacion en el Sexo y Parametros para el Recono-
cimiento del Sexo en la Tortuga Gigante de Gala¬
pagos (Geochelone elephantopus) e Histologia de
la Gonada Juvenif de la Iguana Ierrestre (Cono-
lopus sub cri status). Tesis de Licenciatura en Cien-
cias Biologicas. Pontificia Universidad Catolica del
Ecuador, Quito, Ecuador. 165 p.

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. 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.

49

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.

50

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).

Copyright: © 2017 Reyes-Puig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommer-
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.

51

December 2017 | Volume 11 | Number 2 | e147

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.

52

December 2017 | Volume 11 | Number 2 | e147

Diversity, threat, and conservation of reptiles from Ecuador

0 40 80 160

km

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

A?

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.

53

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.

54

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

A

High

High

yes

yes

B

High

High

no

yes

C*

High

High

yes

no

D*

High

Medium

yes

no

E

High

Medium

no

yes

F

High

Medium

yes

yes

G

Medium

High

yes

yes

H

Medium

High

no

yes

I*

Medium

High

yes

no

J

Medium

Medium

yes

yes

K

Medium

Medium

no

yes

F

Medium

Medium

yes

no

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).

Literature Cited

Bass MS, Finer M, Jenkins CN, Kreft H, Cisneros-Here-
dia DF et al. 2010. Global Conservation significance
of Ecuador’s Yasuni National Park. PLoS ONE 5(1):
e8767.

Bohm M, Collen B, Baillie JEM, Bowles P, Chanson J. et
al. 2013. The conservation status of the world’s rep¬
tiles. Biological Conservation 157: 372-385.

Busby J. 1991. Bioclim - A bioclimate analysis and pre¬
diction system. Pp. 64-68. In: Nature Conservation:
Cost Effective Biological Surveys and Data Analy¬
sis. Editors, Margules CR, Austin MP. CSIRO, Aus¬
tralia. 207 p.

Cuesta F, Peralvo M, Merino-Viteri A, Bustamante M,
Baquero F, Freile J, Muriel P, Torres-Carvajal O.
2017. Priority areas for biodiversity conservation
in mainland Ecuador. Neotropical Biodiversity 3(1):
93-106.

Dodson CH, Gentry AH. 1991. Biological extinction in
western Ecuador. Annals of the Missouri Botanical
Garden 78(2): 273-295.

Elith J, Leathwick J. 2007. Predicting species distribu¬
tions from museum and herbarium records using
multiresponse models fitted with multivariate adap¬
tive regression splines. Diversity and Distributions
13(3): 265-275.

Elith J, Phillips SJ, Hastie T, Dudlk M, Chee YE, Yates
CJ. 2011. A statistical explanation of MaxEnt for
ecologists . Diversity and Distributions 17(1): 43-57.

Gibbons JW, Scott DE, Ryan TJ, Buhlmann KA, Tuber-
ville TD, Metts BS, Greene JL, Mills T, Leiden Y,
Poppy S, Winne CT. 2000. The Global Decline of
Reptiles, Deja Vu Amphibians. BioScience 50(8):
653-666.

Gonzalez-Palacios M, Bonaccorso E, Pape§ M. 2015.
Applications of geographic information systems

Amphib. Reptile Conserv.

55

December 2017 | Volume 11 | Number 2 | e147

Reyes-Puig et al.

and remote sensing techniques to conservation of
amphibians in northwestern Ecuador. Global Ecol¬
ogy and Conservation 3: 562-574.

Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A.
2005. Very high resolution interpolated climate sur¬
faces for global land areas. International Journal of
Climatology 25(15): 1,965-1,978.

Jenkins CN, Pimm SL, Joppa LN. 2013. Global patterns
of terrestrial vertebrate diversity and conservation.
Proceedings of the National Academy of Sciences
of the United States of America 110(28): E2602-
E2610.

Killeen TJ, Douglas M, Consiglio T, Jorgensen PM,
Mejia J. 2007. Dry spots and wet spots in the
Andean hotspot. Journal of Biogeography 34(8):
1,357-1,373.

Lessmann J, Fajardo J, Munoz J, Bonaccorso E. 2016.
Large expansion of oil industry in the Ecuadorian
Amazon: biodiversity vulnerability and conser¬
vation alternatives. Ecology and Evolution 6(14):
4,997-5,012.

Lessmann J, Munoz J, Bonaccorso E. 2014. Maximizing
species conservation in continental Ecuador: A case
of systematic conservation planning for biodiverse
regions. Ecology and Evolution 4(12): 2,410-2,422.

Menendez-Guerrero PA, Graham CH. 2013. Evaluating
multiple causes of amphibian declines of Ecuador
using geographical quantitative analyses. Ecogra-
phy 36(7): 756-769.

Mittemieier RA, Robles-Gil P, Hoffmann M, Pilgrim JD,
Brooks TB, Mittermeier CG, Lamoreux JL, Fon¬
seca GAB. 2004. Hotspots Revisited: Earth’s Bio¬
logically Richest and Most Endangered Ecoregions.
CEMEX, Mexico City, Mexico. 390 p.

Myers N, Mittermeier RA, Mittermeier CG, da Fonseca
GAB, Kent J. 2000. Biodiversity hotspots for con¬
servation priorities. Nature 403(6772): 853-858.

Phillips SJ, Anderson RP, Schapire RE. 2006. Maximum
entropy modeling of species geographic distribu¬
tions. Ecological Modelling 190: 231-259.

Renner IW, Warton DI. 2013. Equivalence of MAXENT
and Poisson point process models for species dis¬
tribution modeling in ecology. Biometrics 69(1):
274-281.

Rios-Franco C, Franco P, Forero-Medina G. 2013. Tool-

r _

box para la Identificacion de Areas Prioritarias
para la Conservacidn, Modelo SIG Dinamico VI. 0.
Wildlife Conservation Society Colombia - MacAr-
thur Foundation, Santiago de Cali, Colombia. 24 p.

Roll U, Feldman A, Novosolov M, Allison A, Bauer A. et
al. 2017. The global distribution of tetrapods reveals
a need for targeted reptile conservation. Nature
Ecology & Evolution 1: 1,677-1,682.

Sanderson EW, Jaiteh M, Levy MA, Redford KH, Wan-
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.

56

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.

57

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.

58

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).

Copyright: © 2017 Nicholson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-
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.

59

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

60

In-country live food production for the Mountain Chicken Frog

<11.60m>

Side

Doorxi

/ i

A

00

Ln

3

V

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.

61

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.

62

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

63

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

Yes

Orthoptera

Giyllus assimilis

Yes

Yes

Orthoptera

Caribacusta dominica

No

Yes

Blattodea

Blaberus discoidalis

Yes

Yes

Coleoptera

Zophobas atratus

Yes

No

Gastropoda

Veronicella sloanii

No

Yes

Gastropoda

Pleurodonte den tie ns

No

Yes

Diplopoda

Leptogoniulus sp.

Yes

No

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.

64

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.

65

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.

Literature Cited

Adams SL, Morton MN, Terry A, Young RP, Dawson J,
Hudson M, Martin L, Sulton M, Cunningham AA,
Garcia G, Lopez J, Tapley B, Burton M, Gray G,
2014. Long-Term Recovery Strategy for the Criti¬
cally Endangered mountain chicken 2014-2034.
Available: http://www.amphibians.org/wp-con-

tent/uploads/2015/08/Mountain-Chicken-SAP-
2014-working-draft-PINAL.pdf [Accessed: 24
December 2017],

Antwis RE, Browne RK. 2009. Ultraviolet radiation and
Vitamin D 3 in amphibian health, behaviour, diet and
conservation. Comparative Biochemistry and Phys¬
iology - A Molecidar and Integrative Physiology
154(2): 184-190.

Baker A. 2007. Animal ambassadors: An analysis of the
effectiveness and conservation impact of ex situ
breeding efforts. Pp. 139-154 In: A Direction for
Zoos in the 21 st Century: Catalysts for Conservation.
Editors, Zimmerman A, Hatch well M, West C, Lat¬
hs R. Cambridge University Press, London, United
Kingdom. 388 p.

Brooks Jr GR. 1982. An analysis of prey consumed by
the anuran, Leptodactylus fallax , from Dominica,
West Indies. Biotropica 14(4): 301-309.

Browne RK, Wolfram K, Garcia G, Bagaturov MF and
Pereboom ZJ. 2011. Zoo based amphibian research
and Conservation Breeding Programs. Amphibian &
Reptile Conservation 5(3): 1-14 (e28).

Cockroach Species File. 2016. Taxa hierarchy -
Blaberus discoidalis. Available: http://cock-
roach.speciesfile.org/Common/basic/Taxa.

aspx?TaxonNameID=1174169 [Accessed: 23 Octo¬
ber 2016],

Collins JP. 2010. Amphibian decline and extinction:
What we know and what we need to learn. Diseases
of Aquatic Organisms 92(2-3): 93-9.

Cunningham AA, Daszak P, Rodriguez-Perez J. 2003.
Pathogen pollution, defining a parasitological threat
to biodiversity conservation. Journal of Parasitol¬
ogy 89: 78-83.

Davis SL, Davis RB, James A, Talyn BCP 2000. Repro¬
ductive behaviour and larval development of Lepto¬
dactylus fallax in Dominica, West Indies. Herpeto-
logical Review 31: 217-220.

Dugas MB, Yeager J, Richards-Zawacki CL. 2013.
Carotenoid supplementation enhances reproductive
success in captive strawberry poison frogs (Oophaga
pumilio). Zoo Biology 32(6): 655-658.

Edmonds D, Rakotoarisoa JC, Dolch R, Pramuk J,
Gagliardo R, Andreone F, Rabibisoa N, Rabemanan-
jara F, Rabesihanaka S, Robsomanitrandrasana E.
2012. Building capacity to implement conservation
breeding programs for frogs in Madagascar: Results
from year one of Mitsinjo’s amphibian husbandry
research and captive breeding facility. Amphibian &
Reptile Conservation 5(3): 57-69 (e55).

Edmonds D, Rakotoarisoa JC, Rasoanantenaina S, Sam
SS, Soamiarimampionona J, Tsimialomanana E,
Rainer Dolch Y, Rabemananjara F, Rabibisoa N,
Robsomanitrandrasana E. 2015. Captive husbandry,
reproduction, and fecundity of the golden mantella
(Mantella aurantiaca ) at the Mitsinjo breeding facil¬
ity in Madagascar. Salamandra 51(4): 315-325.

Fa J, Hedges B, Ibene B, Breuil M, Powell R, Magin C.
2010. Leptodactylus fallax. In: The IUCN Red List
of Threatened Species 2010: e.T57125Al 1586775.
Available: http://dx.doi.org/10.2305/IUCN.

UK. 201 0-2. RLTS.T57125A1 1586775. en
[Accessed: 26 December 2017],

Gagliardo R, Crump P, Griffith E, Mendelson J, Ross H,
Zippel K. 2008. The principles of rapid response for
amphibian conservation, using the programmes in
Panama as an example. International Zoo Yearbook
42(1): 125-135.

Griffiths RA, Pavajeau L. 2008. Captive breeding, rein¬
troduction, and the conservation of amphibians.
Conservation Biology 22(4): 852-861.

Gullan P, Cranston P. 2005. The Insects: An Outline of
Entomology. 3 rd edition. Blackwell Publishing, Vic¬
toria, Australia. 528 p.

Hudson MA, Young RP, D’Urban Jackson J, Orozco-
terWengel P, Martin L, James A, Sulton M, Garcia
G, Griffiths RA, Thomas R, Magin C, Bruford MW,
Cunningham AA. 2016a. Dynamics and genetics
of a disease-driven species decline to near extinc¬
tion: Lessons for conservation. Scientific Reports
6(30772): 1-12.

Hudson MA, Young RP, Lopez J, Martin L, Fenton C,

Amphib. Reptile Conserv.

66

December 2017 | Volume 11 | Number 2 | e149

In-country live food production for the Mountain Chicken Frog

McCrea R, Griffiths RA, Adams SL, Gray G, Gar¬
cia G, Cunningham AA. 2016b. In-situ itraconazole
treatment improves survival rate during an amphib¬
ian chytridiomycosis epidemic. Biological Conser¬
vation 195: 37—45.

King JD, Muhlbauer MC, James A. 2010. Radiographic
diagnosis of metabolic bone disease in captive bred
mountain chicken frogs {Leptodactylus fallax). Zoo
Biology 30(3): 254-259.

Li H, Vaughan MJ, Browne RK. 2009. A complex enrich¬
ment diet improves growth and health in the endan¬
gered Wyoming Toad (Bufo baxteri). Zoo Biology
28(3): 197-213.

Malhotra A, Thorpe R, Hypolite E, James A. 2007. A
report on the status of the herpetofauna of the Com¬
monwealth of Dominica, West Indies. Applied Her¬
petology 4: 177-194.

McCallum ML. 2007. Amphibian decline or extinction?
Current declines dwarf background extinction rate.
Journal of Herpetology 41(3): 483—491.

Mendelson JR, Altig R. 2016. Tadpoles, froglets, and
conservation: A discussion of basic principles of
rearing and release procedures. Amphibian & Rep¬
tile Conservation 10(1): 20-27 (el 16).

Norris S. 2007. Ghosts in our midst: Coming to terms
with amphibian extinctions. BioScience 57(4): 311—
316.

Ogilvy V, Preziosi RF, Fidgett AL. 2012. A brighter
future for frogs? The influence of carotenoids on the
health, development and reproductive success of the
red-eye tree frog. Animal Conservation 15(5): 480-
488.

Orthoptera Species File. 2016. Taxa hierarchy - Cari-
bacusta dominica. Available: http://orthop-
tera. speciesfile.org/common/basic/Taxa.
aspx?TaxonNameID=1126172 [Accessed: 23 Octo¬
ber 2016],

Otte D. 2006. Gryllodes sigiflatus (Walker) is a valid
species distinct from Gryllodes supplicans (Walker).
Transactions of the American Entomological Society
132(1/2): 223-227.

Peck SB. 2006. The beetle fauna of Dominica, Lesser
Antilles (Insecta: Coleoptera): Diversity and distri¬
bution. Insecta Mundi 20(3-4): 164-210.

Robinson DG, Hovestadt A, Fields A, Breure ASH.
2009. The land Mollusca of Dominica (Lesser Antil¬
les), with notes on some enigmatic or rare species.
Zoologische Mededelingen (Leiden) 83: 615-650.

Rosa GM, Bradfield K, Fernandez-Loras A, Garcia G,
Tapley B. 2012. Two remarkable prey items for a
chicken: Leptodactylus fallax (Muller 1926) preda¬
tion upon the theraphosid spider Cyrtopholis femo-

ralis (Pocock 1903) and the colubrid snake Liophis
juliae (Cope 1879). Tropical Zoology 25(3): 135-
MO.

Shishova NR, Uteshev VK, Kaurova SA, Browne RK,
Gakhova EN. 2011. Cryopreservation of hormon¬
ally induced spenn for the conservation of threat¬
ened amphibians with Rana temporaria as a model
research species. Theriogenology 75(2): 220-232.

Tapley B, Bradfield KS, Michaels C, Bungard M.
2015a. Amphibians and conservation breeding pro¬
grammes: Do all threatened amphibians belong
on the ark? Biodiversity and Conservation 24(11):
2,625-2,646.

Tapley B, Rendle M, Baines FM, Goetz M, Bradfield KS,
Rood D, Lopez J, Garcia G, Routh A. 2015b. Meet¬
ing ultraviolet B radiation requirements of amphibi¬
ans in captivity: A case study with mountain chicken
frogs ( Leptodactylus fallax) and general recommen¬
dations for pre-release health screening. Zoo Biology
34(1): 46-52.

Tapley B, Harding L, Sulton M, Durand S, Burton M,
Spencer J, Thomas R, Douglas T, Andre J, Winston
R, George M, Gaworek-Michalczenia M, Hudson
M, Blackman A, Dale J, Cunningham AA, Tapley B.
2014. An overview of current efforts to conserve the
Critically Endangered mountain chicken ( Leptodac¬
tylus fallax) on Dominica. The Herpetological Bul¬
letin 128: 9-11.

Verschooren E, Brown RK, Vercammen F, Pereboom J.
2011. Ultraviolet B radiation (UV-B) and the growth
and skeletal development of the Amazonian milk
frog ( Trachycephalus resinifictrix) from metamor¬
phosis. Journal of Physiology and Pathophysiology
2(3): 34-42.

Walker SF, Bosch J, James TY, Litvintseva AP, Antonio

J, Vails O, Pina S, Garcia G, Rosa GA, Cunningham

AA, Hole S, Griffiths R, Fisher MC. 2008. Invasive
pathogens threaten species recovery programs. Cur¬
rent Biology 18(18): 853-854.

Wright KM, Whitaker BR. 2001. Amphibian Medicine
and Captive Husbandry. Krieger Publishing Com¬
pany, Malabar, Florida, USA. 570 p.

Weissman DB, Walker, TJ, Gray, DA. 2009. The field
cricket Gryllus assimilis and two new sister species
(Orthoptera: Gryllidae). Annals of the Entomologi¬
cal Society of America 102(3): 367-380.

Zippel K, Johnson K, Gagliardo R, Gibson R, Mcfadden
M, Browne R, Martinez C, Townsend E. 2011. The
Amphibian Ark: A global community for ex situ con¬
servation of amphibians. Herpetological Conseixa-
tion and Biology 6(3): 340-352.

Amphib. Reptile Conserv.

67

December 2017 | Volume 11 | Number 2 | e149

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.

68

December 2017 | Volume 11 | Number 2 | e149