Revista Espaola de Herpetologa

Journal of the Spanish Herpetological Society (AHE)

Volume 25 (2011)

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 BASIC & APPLIED HERPETOLOGY
REVISTA ESPAOLA DE HERPETOLOGA

Spanish Herpetological Society (AHE)

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Basic & Applied Herpetology (Editors)

Manuel E. Ortiz Santaliestra (Amphibians) Ana Perera Leg (Reptiles)
Instituto de Investigacin en Recursos Cinegticos CIBIO
(IREC). CSIC-UCLM-JCCM Campus Agrrio de Vairo.
Ronda de Toledo, s/n. Rua Padre Armando Quintas-Castro
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manuele.ortiz@uclm.es perera@mail.icav.up.pt

Asociacin Herpetolgica Espaola

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 BASIC & APPLIED HERPETOLOGY
REVISTA ESPAOLA DE HERPETOLOGA

CONTENTS
Volume 25 (2011)

Reviews Pag.
GUEST CONTRIBUTION: Geometric morphometrics in herpetology: modern tools for enhan- 5
cing the study of morphological variation in amphibians and reptiles
A. Kaliontzopoulou

Research papers
Embryonic development of kidneys in viviparous Typhlonectes compressicauda (Amphibia, 33
Gymnophiona)
M. Bastit, J.-M. Exbrayat
Population estimators and adult sex ratio for a population of Bolitoglossa altamazonica 43
(Caudata: Plethodontidae)
D.L. Gutirrez-Lamus, J.D.Lynch, G.C. Martnez-Villate
Behavioural responses of Iberian midwife toad tadpoles (Alytes cisternasii) to chemical stimulus 55
of native (Natrix maura and Squalius pyrenaicus) and exotic (Procambarus clarkii) predators
V. Gonalves, S. Amaral, R. Rebelo
Reproductive cycles in Bufo mauritanicus (Schlegel, 1841) in a wet area of Beni-Belad (Jijel, 65
Algeria)
O. Kisserli, S. Doumandji, J.-M. Exbrayat
Age structure of Levant water frog, Pelophylax bedriagae, in Lake Slkl (Western Anatolia, 73
Turkey)
K. iek, M. Kuma, D. Ayaz, A. Mermer, .D. Engin
Population size, habitat use and movement patterns during the breeding season in a popula- 81
tion of Perezs frog (Pelophylax perezi) in central Spain
G. Snchez-Montes, I. Martnez-Solano
A re-analysis of the molecular phylogeny of Lacertidae with currently available data 97
P. Kapli, N. Poulakakis, P. Lymberakis, M. Mylonas
Biometry and pholidosis of Thamnophis scaliger: an atypical example of sexual dimorphism 105
in a natricine snake
M. Feriche, S. Reguera, X. Santos, E. Mocio-Deloya, K. Setser, J.M. Pleguezuelos

Cover illustration: Mauritanian toad (Bufo mauritanicus), Sidi Ifni, Morocco (see article by
Kisserly et al. in this volume). Author: Luis Garca Cardenete.
 BASIC & APPLIED HERPETOLOGY
REVISTA ESPAOLA DE HERPETOLOGA

CONTENIDOS
Volumen 25 (2011)

Revisiones Pag.
ARTCULO INVITADO: Morfometra geomtrica en herpetologa: nuevas herramientas para 5
promover el estudio de la variacin morfolgica en anfibios y reptiles
A. Kaliontzopoulou

Artculos de investigacin
Desarrollo embrionario de los riones en Typhlonectes compressicauda vivparas (Amphibia, 33
Gymnophiona)
M. Bastit, J.-M. Exbrayat
Estimadores de poblacin y razn de sexos en una poblacin de Bolitoglossa altamazonica 43
(Caudata: Plethodontidae)
D.L. Gutirrez-Lamus, J.D.Lynch, G.C. Martnez-Villate
Respuestas conductuales de las larvas de sapo partero ibrico (Alytes cisternasii) a los estmulos qu- 55
micos de depredadores nativos (Natrix maura y Squalius pyrenaicus) y exticos (Procambarus clarkii)
V. Gonalves, S. Amaral, R. Rebelo
El ciclo reproductor de Bufo mauritanicus (Schlegel, 1841) en el humedal de Beni-Belad 65
(Jijel, Algeria)
O. Kisserli, S. Doumandji, J.-M. Exbrayat
Estructura de edad de la rana verde levantina, Pelophylax bedriagae, en el lago Slkl 73
(Anatolia occidental, Turqua)
K. iek, M. Kuma, D. Ayaz, A. Mermer, .D. Engin
Tamao poblacional, uso del espacio y patrones de movimiento durante el periodo repro- 81
ductor en una poblacin de rana verde comn (Pelophylax perezi) en Espaa central
G. Snchez-Montes, I. Martnez-Solano
Reanlisis de la filogenia molecular de los Lacertidae usando los datos disponibles en la actualidad 97
P. Kapli, N. Poulakakis, P. Lymberakis, M. Mylonas
Biometra y folidosis de Thamnophis scaliger: un ejemplo atpico de dimorfismo sexual en un 105
colbrido natricino
M. Feriche, S. Reguera, X. Santos, E. Mocio-Deloya, K. Setser, J.M. Pleguezuelos

Ilustracin de portada: macho de sapo moruno (Bufo mauritanicus), Sidi Ifni, Marruecos
(vase artculo de Kisserly et al. en este volumen). Autor: Luis Garca Cardenete.
 Guest contribution Basic and Applied Herpetology 25 (2011): 5-32

Geometric morphometrics in herpetology: modern tools

for enhancing the study of morphological variation
in amphibians and reptiles
Antigoni Kaliontzopoulou1,2
1
CIBIO/UP, Centro de Investigao em Biodiversidade e Recursos Genticos, Campus Agrario de Vairo, 4485-661 Vairo, Portugal.
2
Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, Iowa 50011, USA.

* Correspondence: CIBIO/UP, Centro de Investigao em Biodiversidade e Recursos Genticos, Campus Agrario de Vairo, 4485-661
Vairo, Portugal. Phone: +351 252660411, Fax: +351 252661780, E-mail: antigoni@mail.icav.up.pt
Received: 30 August 2011; received in revised form: 8 October 2011; accepted: 10 October 2011.

The use of geometric morphometrics for studying phenotypic variation in amphibians and reptiles has visibly increased in the last
decade. These modern tools provide a robust statistical framework to study organismal shape while preserving the geometric pro-
perties of the studied structures and thus improve our capacity for investigating patterns of morphological variation, and unders-
tanding their ecological and historical causes. Their application in herpetology has shed new light to the remarkable diversity obser-
ved among extant and extinct amphibians and reptiles. Here I first briefly consider the historical emergence of geometric mor-
phometric methods, trying to provide a practical guide for herpetologists interested in implementing these tools to their investi-
gation. I then review the wide array of published studies using geometric morphometrics to investigate morphological patterns in
amphibians and reptiles. Across different investigation fields, an emergent pattern is the existence of general similarities, but also
profound differences, among members of higher taxonomic groups. Size-shape allometry is a common pattern in many groups,
but remarkable variation of allometric trajectories exists among closely related taxa. Sexual dimorphism has been extensively stu-
died in reptiles, but less so in amphibians, while the contrary is true for phenotypic plasticity. The use of geometric morphome-
trics has allowed the detection of potentially adaptive shape patterns and the investigation of their causes. Finally, these methods
have been invaluable in the study of fossils and have provided a better understanding of the paleobiology of extinct taxa.

Key words: adaptation; allometry; geometric morphometrics; paleontology; phenotypic plasticity; sexual dimorphism.

Morfometra geomtrica en herpetologa: nuevas herramientas para promover el estudio de la variacin morfolgica en
anfibios y reptiles. El uso de la morfometra geomtrica para estudiar la variacin fenotpica en anfibios y reptiles ha aumenta-
do notablemente durante la ltima dcada. Esta nueva herramienta proporciona un marco estadstico slido para estudiar la
forma de los organismos preservando las propiedades geomtricas de las estructuras analizadas, mejorando as la comprensin
de los factores ecolgicos e histricos que explican los patrones de variacin morfolgica. Su aplicacin en herpetologa propor-
ciona una nueva forma de explorar la diversidad morfolgica de anfibios y reptiles tanto actuales como extintos. En esta revisin
comienzo examinando la secuencia histrica que llev a la aparicin de la morfometra geomtrica, tratando de ofrecer tambin
una gua prctica para aquellos herpetlogos interesados en incorporar esta herramienta en su investigacin. Despus reviso un
amplio muestrario de trabajos en los que la morfometra geomtrica se usa para estudiar patrones morfolgicos en anfibios y
reptiles. Una pauta que emerge repetidamente es la existencia de similitudes generales, pero tambin de profundas diferencias,
entre los miembros de los grupos taxonmicos de mayor rango. La existencia de una relacin alomtrica entre tamao y forma
es comn en muchos grupos, pero tambin se observa una variabilidad considerable en las trayectorias alomtricas entre taxo-
nes hermanados. El dimorfismo sexual se ha estudiado extensivamente en reptiles, pero no tanto en anfibios, mientras que con
la plasticidad fenotpica ocurre lo contrario. El uso de la morfometra geomtrica permite la deteccin de variaciones adaptati-
vas en los patrones morfolgicos y la investigacin de sus causas. Finalmente, estos mtodos tienen un valor incalculable para el
estudio de organismos fsiles y proporcionan una mejor compresin de su paleobiologa.

Key words: adaptacin; alometra; dimorfismo sexual; morfometra geomtrica; paleontologa; plasticidad fenotpica.
6 KALIONTZOPOULOU
Morphology is undoubtedly one of the
main components of an organisms phenoty-
pe. As such, morphological traits have always
been at the centre of attention of biological
research, comprising important pieces of evi-
dence for a wide variety of investigation
fields. From traditional taxonomy and the
modern school of systematics, through
physiology, development, ecology, biogeo-
graphy, and all the way to modern evolutio-
nary biology, practically all biological fields
include questions and hypotheses related to
how morphological variation emerges and
how it is distributed across temporal and
spatial scales. Geometric morphometrics
(GM) revolutionised the way we measure,
study and perceive morphological diversity
(ROHLF & MARCUS, 1993; ADAMS et al.,
2004). By establishing a solid mathematical
basis for the study of organismal shape, while
preserving the geometric properties of the
structures of interest, GM methods provide a
powerful tool for depicting and studying
morphological variation in a more realistic
and integrated manner than previous mor-
phometric tools (BOOKSTEIN, 1996; ROHLF,
2000a). Amphibians and reptiles, being
excellent model organisms, have been the
subject of extensive morphological research,
often serving as paradigmatic cases in the
study of phenotypic variation. Accordingly,
over the past two decades, herpetologists
have taken advantage of the modern toolkit
of GM to further enhance our understan-
ding of the astonishing morphological
variety that exists across amphibian and rep-
tile taxa. Since the early development of GM
tools, most extant and several extinct
herpetofaunal groups have been investigated
using these methods.
This review aims at putting together an
up-to-date account of how GM methods
have been implemented in herpetology until
today, also providing a comprehensive basis
for herpetologists interested in exploring
these methods in their research. Although I
have tried to include all studies to which I
had access and provide examples from all her-
petofaunal groups, this is not meant to be an
exhaustive account from a taxonomical pers-
pective, but rather to give insight on how
GM methods have advanced our understan-
ding of the patterns and processes underlying
the phenotypic diversification of our study
organisms.
GEOMETRIC MORPHOMETRICS: WHY AND HOW?
The birth of GM or how GM is different
Morphometrics, the quantitative study of
biological shape variation and its covariation
with other variables (BOOKSTEIN, 1991;
DRYDEN & MARDIA, 1998), may be seen as
the successful outcome of the long-standing
interest in organismal form. Ever since
Aristotle biologists have been intrigued by the
huge diversity of forms we encounter in natu-
re and the causes and processes that create it.
However, such interest was only put on a
quantitative basis with the statistical advances
made during the 19th and 20th centuries,
which led to the emergence of biometry as a
formal discipline (SOKAL & ROHLF, 1995;
SLICE, 2005; MITTEROECKER & GUNZ,
2009). The development of statistical tools for
analysing multivariate data opened a new
door to the description and study of complex
phenotypes. This was achieved through the
quantification of a number of linear distances,
 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY
counts, ratios or angles, describing the proper-
ties of a morphological trait of interest
(ADAMS et al., 2004; SLICE, 2005). The appli-
cation of multivariate statistics on the above
biometric variables then allowed for the tes-
ting of specific biological hypotheses about an
organisms multivariate phenotype (i.e. tradi-
tional morphometrics; MARCUS, 1990).
However, morphometrics still suffered some
shortcomings which troubled morphometri-
cians and urged for solutions. These problems
deserve our attention, since their resolution
was the basic motivation for the development
of the field of GM and they provide direct
insight into how this new methodology differs
from traditional morphometrics.
The birth of GM is tightly linked to four
considerations related to biological form: size,
homology, shape description and visualisa-
tion. With size and its effect on other mor-
phological traits being of central importance
to the evolution of all living organisms
(GOULD, 1966), it was soon evident that in
order to study biological form, a full mathe-
matical definition of size was in order, due
both to practical and to theoretical reasons
(BOOKSTEIN, 1989a; SLICE, 2005). Numerous
solutions were proposed (see ROHLF &
BOOKSTEIN, 1987 for review and compari-
son), but no solid argument could definitely
support the use of a single method. A similar
problem existed as to the homology of the
studied traits, in the sense of the operational,
reproducible definition of the quantities to be
measured and compared (BOOKSTEIN, 1982;
SLICE, 2005). Yet an additional concern regar-
ded the selection of variables used for shape
description; since the geometric relationships
between linear measurements were not inclu-
ded in the dataset, one could neither predict
7
nor guarantee the statistical differentiation of
shapes known to be different (BOOKSTEIN,
1982, 1996; SLICE, 2005). Finally, since the
geometry of the studied objects was not cap-
tured, morphometric data could not be effec-
tively used to directly visualise shape variation
related to other biological variables of interest
(ADAMS et al., 2004). All the above adversities
led to the development of GM methods.
What is GM and how to use it
The revolution in morphometrics
(ROHLF & MARCUS, 1993) started by advan-
ces in the application of outline and landmark
tools for the geometrical study of organismal
shape (BOOKSTEIN, 1986; ROHLF, 1986) and
the simultaneous development of statistical
theory for shape analysis (KENDALL, 1984,
1985). The combination of landmark techni-
ques for capturing organismal form with a
newly introduced, statistically robust shape
theory led to the growth of a set of morpho-
metric methods that preserved the geometric
properties of the studied objects, namely GM
(SLICE, 2005; MITTEROECKER & GUNZ, 2009).
The raw data used for GM consist of outline
data describing the bounding edge of the
structure of interest or, more frequently, of
Cartesian coordinates of landmark locations
(SLICE, 2005; MITTEROECKER & GUNZ, 2009).
Outline data were the first to be used, but
they were later largely abandoned, especially
after the introduction of semi-landmark
methods that incorporate boundary curve
information directly into landmark-based
analyses (BOOKSTEIN, 1997; MITTEROECKER
& GUNZ, 2009). Since two- and three-
dimensional landmark-based GM methods
are more frequently used today, I will concen-
8 KALIONTZOPOULOU
trate on these methods for a practical review.
However, the interested reader can explore the
published bibliography on outline methods
(see among others ROHLF & ARCHIE, 1984;
FERSON et al., 1985; ROHLF, 1986, 1990;
ADAMS et al., 2004).
Landmark-based GM analysis begins with
the definition of landmarks (Table 1, Fig. 1a).
Simplistic as this may seem, it is a central part
of biological inference: Landmarks are the
points at which ones explanations of biologi-
cal processes are grounded (BOOKSTEIN,
1991). It is through the definition of land-
marks, based on biological intuition and pre-
vious observation of the organisms of interest,
that the biologist will manage to fully capture
the shape of interest. Once landmarks have
been defined, these are digitised in a number
Figure 1: Removing non-shape variation from
landmark coordinates through Generalized
Procrustes Analysis (GPA: ROHLF & SLICE,
1990). Once landmarks have been defined (a) and
digitised in a number of specimens, their coordi-
nates are first translated (b) by placing their cen-
troid to the origin of a Cartesian system. Then
they are scaled (c) to unit centroid size. Finally,
they are rotated (d) using a least-squares criterion.
This way landmark coordinates are superimposed
to a common coordinate system and non-shape
variation is removed.
of specimens, resulting in a collection of
Cartesian coordinates. These coordinates still
include non-shape information (size, orienta-
tion and position) and need to be mathemati-
cally processed in order to obtain shape varia-
bles. The dominant procedure today uses
shape variables lying in Kendalls shape space
(or most frequently an approximation in a
space tangent to the mean shape of the stu-
died sample), Procrustes distance being the
associated metric (Table 1). While several
methods were proposed in the past for obtai-
ning shape variables from landmark coordina-
tes, methods using Kendall's space have been
proven to be the most powerful and statisti-
cally robust (ROHLF, 1999, 2000a,b, 2003).
In order to obtain shape variables, landmark
configurations are first superimposed using a
least-squares procedure, namely Generalized
Procrustes Analysis (GPA, Table 1, ROHLF &
SLICE, 1990). GPA removes variation due to
digitizing location, scale and orientation
through the optimal translation (Fig. 1b), sca-
ling (Fig. 1c) and rotation (Fig. 1d) of land-
mark coordinates. The specimen points alig-
ned through this procedure can then be pro-
jected into a linear shape space tangent to
Kendall's shape space (Table 1; ROHLF, 1999;
SLICE, 2001, 2005; MITTEROECKER & GUNZ,
2009), where the Euclidean distances between
observations closely approximate Procrustes
distances in Kendalls space. One should be
aware that, in addition to GPA, several other
superimposition techniques exist, with poten-
tial benefits in particular datasets (SIEGEL &
BENSON, 1982; BOOKSTEIN, 1991; ZELDITCH
et al., 2004; SLICE, 2005).
Once non-shape variation has been remo-
ved, the superimposed landmark coordinates
of specimens not differing in shape will per-
 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY 9

Table 1: Glossary of terms frequently used in geometric morphometrics*.

Term Description

Allometry Shape change associated to size change.

Centroid size The size measure used in GM. It is calculated as the square root of the sum of squared dis-
tances of each landmark from the centroid of the landmark configuration.
Consensus configuration A landmark configuration intended to represent the central tendency (for example mean
shape) of an observed sample. Often a consensus configuration is computed to optimize
some measure of fit to the full sample: in particular, the Procrustes mean shape is computed
to minimize the sum of squared Procrustes distances from the consensus landmarks to those
of the sample.
Deformation grid The visual representation of a shape transformation as modelled using the thin-plate spline,
based on D'Arcy Thompson's idea of using grids to represent a shape difference.
Generalized Procrustes A generalized superimposition method that works by minimising the partial Procrustes dis-
Analysis (GPA) tance over all sampled shapes by a least-squares fitting procedure.
Kendall's shape space The geometric setting for analyses of Procrustes distances among arbitrary sets of landmarks.
Each point in this shape space represents the shape of a configuration of points in some
Euclidean space, irrespective of size, position, and orientation.
Landmark A specific point on a biological form or image of a form located according to some
rule. BOOKSTEIN (1991) recognised three categories of landmarks, depending on the
criteria used for their definition: Type I landmarks correspond to points clearly defined
by some local property, such as the juxtaposition of different tissues; Type II landmarks
are defined purely based on geometric, not biological, properties, such as the maxi-
mum curvature of a structure; finally, Type III landmarks are the less robustly defined
ones, since they are located in relation to other points in the structure. Type III land-
marks can be incorporated in GM analyses, but are not in fact considered true land-
marks and caution should be taken when using them.
Partial warps An auxiliary structure for the interpretation of shape changes and shape variation in sets of
landmarks. They are eigenvectors of the bending energy matrix that describes the net local
information in a deformation along each coordinate axis.
Principal warps Eigenfunctions of the bending-energy matrix interpreted as actual warped surfaces (thin-
plate splines) over the picture of the original landmark configuration.
Procrustes distance Approximately, the square root of the sum of squared differences between the positions of the
landmarks in two optimally (by least-squares) superimposed configurations at centroid size.
This is the metric for Kendall's shape space, and thus the distance measure used in GM.
Procrustes residuals The set of vectors connecting the landmarks of a specimen to corresponding landmarks in
the consensus configuration after a Procrustes fit. The sum of squared lengths of these vec-
tors is approximately the squared Procrustes distance between the specimen and the consen-
sus in Kendall's shape space. The partial warp scores are an orthogonal rotation of the full
set of these residuals.
Relative warps Principal components of partial warp scores. In a relative warps analysis, the parameter can
be used to weight shape variation by the geometric scale of shape differences.
Shape The geometric properties of a configuration of points that are invariant to changes in trans-
lation, rotation, and scale.
Thin-plate spline An interpolation function used to model the difference in shape between two objects by
minimising the bending energy of the deformation. It provides a unique solution to the
construction of D'Arcy Thompson-type deformation grids for data in the form of two land-
mark configurations.
* Compiled and augmented from SLICE et al. (1996) and ZELDITCH et al. (2004).
10 KALIONTZOPOULOU
fectly coincide. In turn, specimens of different
shape will present at least some differences in
landmark positions. The largest the shape dif-
ference between specimens, the largest the dif-
ference in the positions of homologous land-
marks after superimposition. Such shape dif-
ference is quantified through the Procrustes
distance metric, which allows for statistical
comparisons and hypothesis testing (SLICE,
2005; MITTEROECKER & GUNZ, 2009). In
this sense, then, Procrustes residuals (i.e. land-
mark coordinates after superimposition) can
be used as shape variables to investigate shape
variation. However, Procrustes residuals suffer
the statistical adversities of not being a full-
rank set of variables (due to superimposition)
and of being non-Euclidean in nature, which
frequently complicates their statistical treat-
ment since they cannot be subjected to analy-
sis using linear models. While this adversity
can be overcome through projection into a
tangent, Euclidean space, the usual approach
is to perform a series of mathematical opera-
tions to model shape variation. This is done
using the thin-plate spline. The thin-plate
spline is an interpolation technique borrowed
for use in morphometrics from the fields of
computational surface theory and computer
graphics (BOOKSTEIN, 1989b, 1991). Imagine
that the shape of interest, represented by a
configuration of landmarks, lies on an infini-
tely thin, flat, metal plate of infinite size. The
change into another shape can be obtained
through a set of vertical displacements of the
metal plate in a direction perpendicular to its
surface, one Cartesian coordinate at a time.
By minimising the energy necessary to bend
the metal plate between two shapes (bending
energy) we obtain a criterion for parsimo-
niously describing and modelling shape chan-
ge. The descriptors resulting from the applica-
tion of the thin-plate spline are partial warps
(Table 1), which are in fact the eigenvectors of
the bending-energy matrix and are orthogonal
components describing shape variation accor-
ding to spatial scale. The partial warp scores
(together with the uniform components of
shape variation) of each individual can then
be used as shape variables for multivariate sta-
tistical analyses (SLICE, 2005).
In addition to providing Euclidean shape
variables for statistical hypothesis testing, the
thin-plate spline is an essential tool for visuali-
sing shape variation in an integrated and intui-
tive manner (SLICE, 2005). Since the thin-
plate spline is in fact an interpolation function,
it can be used to map the deformation in
shape between two objects (BOOKSTEIN,
1991). This is done through a mathematically
formal realization of D'Arcy Thompson's idea
of transformation grids (THOMPSON, 1917), a
solution long sought by morphometricians
(BOOKSTEIN, 1996). These maps of shape
change, referred to as deformation grids (Table
1), use a visual representation of a wire mesh
to depict the bending necessary to transform
one shape into another, a procedure known as
warping (Fig. 2). This is one of the most
important advances provided by GM
methods: since the geometry of the studied
objects is preserved throughout the analysis,
shape differences between objects can be
directly described in terms of differences in the
deformation grids representing these objects
(ADAMS et al., 2004; SLICE, 2005;
MITTEROECKER & GUNZ, 2009). Several soft-
ware packages are available for conducting all
the above GM analyses, performing statistical
comparisons and visualising the results (see
Appendix 1).
 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY
Figure 2: How the thin-plate spline can be used to
visualize shape differences found from landmark-
based GM methods. In this example, shapes A
and B are being compared. Once landmark confi-
gurations have been aligned through superimposi-
tion (C), shape differences between them can be
visualised as the deformation caused on a wire
mesh which is bended from one shape to the
other (D). Linear vectors on the landmarks repre-
sent the direction of change for each of them from
shape A into shape B (exaggerated five-fold to
enhance visualisation).
GM IN HERPETOLOGY:
A TAXONOMICAL ACCOUNT
Herpetologists have been increasingly
using GM techniques to study morphological
variation in amphibians and reptiles over the
last decade. Focusing on the studies conside-
red here, a visible increase in the use of GM
methods for studying extant or extinct
amphibians and reptiles is observed after the
11
year 2000, from less than five publications per
year between 2000 and 2003 to an average of
about 13 publications per year after 2007
(Fig. 3). Among amphibians, both anurans
and urodeles have been investigated, but GM
methods have not yet been applied to the
best of my knowledge for studying caeci-
lians. Among reptiles, saurians are the most
studied group, with a total of 25 publications,
followed by chelonians (19 studies). Other
groups are visibly less explored, with only
three studies in snakes and three in crocodiles,
while, as far as I am aware, amphisbaenians
are still to be studied. GM tools are also exten-
sively used for studying extinct amphibian
and reptile taxa, with an important contribu-
tion to the total number of studies (Fig. 3)
and remarkable results (see below).
STUDYING PHENOTYPIC VARIATION:
FIELDS OF APPLICATION
The integrated study of shape through
GM motivated an explosion of morphologi-
cal investigation in amphibians and reptiles
and expanded our knowledge of patterns of
variation and their causes. In the following
section, I provide a question-based review of
published studies, aiming at describing gene-
ral shape patterns observed across herpeto-
faunal taxa and discussing their potential cau-
ses as seen by herpetologists.
Allometric patterns: shape change due to size
GM tools have been a cornerstone contribu-
tion to the study of development and ontogene-
tic shape change. By enhancing shape quantifi-
cation and shape change visualisation, GM
methods have provided the possibility not only
12 KALIONTZOPOULOU
Figure 3: Yearly evolution of the number of publi-
cations using GM methods to analyse morphologi-
cal variation in extant and extinct amphibians and
reptiles. Data retrieved from Google Scholar and Isi
Web of Science. *Records concerning 2011 include
studies published or in press until July 31.
of accurately describing complex shapes, but
also of understanding exactly how shape chan-
ges throughout an organisms development
(LAWING & POLLY, 2010). Most studies investi-
gating allometric relationships, both in an onto-
genetic and static context of allometry (i.e.
GOULD, 1966), indicate that large part of the
shape variation observed is attributable to size-
shape allometry (see MONTEIRO & ABE, 1997;
BIRCH, 1999; BONNAN, 2007; SMITH &
COLLYER, 2008; PIRAS et al., 2010; CHIARI &
CLAUDE, 2011; IVANOVI et al., 2011 for illus-
trative examples from different groups).
Caution is advised to avoid conceptual misun-
derstandings: while GM methods remove size
variation through GPA or similar superimposi-
tion procedures, the allometric dependence of
shape on size still remains in the data and can be
investigated (MONTEIRO, 1999). Statistically,
since shape variables produced through GM
techniques are size-free, any significant associa-
tion with size (either represented by centroid
size or any other size variable) indicates devia-
tion from isometry (ZELDITCH et al., 2004).
Extensive research about ontogenetic shape
change at different developmental stages has
been carried out mainly in amphibians, revea-
ling both similarities and profound differences
among groups. Pre-metamorphic shape onto-
geny of the chondrocranium in anurans seems
to generally follow a common pattern, at least
in species of the genera Bufo, Pelodytes, Rana
and Telmatobius (LARSON 2002, 2004, 2005;
CANDIOTI, 2008; GARRIGA & LLORENTE, in
press). In these anuran species, general patterns
of skull development include the reduction of
the sensory capsules and a hypermetric or iso-
metric growth of trophic structures (LARSON,
2002, 2004), in line with predictions made for
all tetrapods (EMERSON & BRAMBLE, 1993).
Interestingly, these studies also attest that the
development of the chondrocranium does not
seem to be tightly linked to that of the hind
limb, thus rendering Gosner stages a deve-
lopmental staging system frequently used in
anurans (GOSNER, 1960) a relatively poor
indicator of chondrocranial differentiation
(LARSON, 2002). Similarly, species of newts
studied show generally congruent patterns, the
skull base and rostral portion being the areas
more profoundly modified throughout
ontogeny (IVANOVI et al., 2007, 2008).
However, while general trends appear relatively
uniform, allometric trajectories frequently vary
among closely related species (LARSON, 2005;
IVANOVI et al., 2007, 2008), providing a
potentially important mechanism of morpho-
logical differentiation, both in extant and
extinct taxa (WITZMANN et al., 2009). The
same is true for body shape variation before
and through metamorphosis in newts (VAN
BUSKIRK, 2009; IVANOVI et al., 2011),
although further studies should investigate the
generality of the results obtained for Triturus,
since remarkable variation of allometric pat-
terns has been observed in some cases (VAN
 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY
BUSKIRK, 2009). Finally, caution should be
taken when extrapolating between groups or
even populations of the same species, since
allometric trajectories of amphibian shape have
been found to present radical modifications in
both plastic and adaptive responses to environ-
mental variation (see below).
Allometric shape variation has been also
extensively investigated in reptiles, again reve-
aling concordance of general trends, but also
significant variation between closely related
species. In lizards, the general pattern of skull
allometry, both in an ontogenetic (MONTEIRO
& ABE, 1997; KALIONTZOPOULOU et al.,
2008; RAIA et al., 2010) and static (BRUNER &
COSTANTINI, 2007; COSTANTINI et al., 2010;
LJUBISAVLJEVI et al., 2011; ZUFFI et al., 2011)
context, includes a relative shortening of the
anterior area and an enlargement of the poste-
rior region. Interestingly, some studies have
reported a lack of an allometric relationship
between head shape and size in some lizard
species (VIDAL et al., 2005, 2006); however,
these results should be considered with cau-
tion, since in the above studies, allometry was
investigated by bivariate regression of the first
principal component of shape variation on
centroid size, thus potentially providing an
incomplete view of allometric patterns. In fact,
multivariate regression of all shape variables
(i.e. partial warps) on centroid size or other
size measures are better suited for investigating
size-shape relationships in a GM context
(KLINGENBERG, 1996; MONTEIRO, 1999),
since size may significantly contribute to the
observed shape patterns even without being
the main source of variation (captured by prin-
cipal components analysis). As for amphi-
bians, allometric trajectories of the skull have
been found to vary extensively among species
13
(LJUBISAVLJEVI et al., 2010), but intraspecific
variation seems to be less common.
Regarding other reptile groups, size varia-
tion also seems to be a main determinant of
shape variation in turtles, including ontogene-
tic, static and evolutionary allometric effects
(CLAUDE et al., 2003, 2004; DEPECKER et al.,
2006; ANGIELCZYK, 2007; MYERS et al., 2007;
NISHIZAWA et al., 2010; ANGIELCZYK et al.,
2011; CHIARI & CLAUDE, 2011). Different
characters show varying degrees of variation in
allometric trajectories. For example, evolutio-
nary allometry of skull shape seems to be cons-
trained, and similar to intraspecific allometry
(CLAUDE et al., 2004), while extensive varia-
tion is observed between ecologically distinct
groups in the shoulder girdle (DEPECKER et al.,
2006). The trait most frequently studied in
turtles, the shell, also shows varying degrees of
allometric modifications. For instance, CHIARI
& CLAUDE (2011) described substantial modi-
fication of growth trajectories between two
closely related lineages of Galpagos tortoises,
while the same seems to be the case for the
miniaturised species of emydine turtles
(ANGIELCZYK, 2007). Other reptile groups
have been less investigated; however, extensive
variation seems to exist in skull ontogenetic
patterns of crocodiles (MONTEIRO et al., 1997;
PIRAS et al., 2010), while size variation seems
to be a moderate source of skull shape varia-
tion as compared to other factors in the
rhynchocephalians (JONES, 2008).
Sexual dimorphism
Sexual dimorphism (SD) is a ubiquitous
feature of many animal taxa and the applica-
tion of GM has importantly enhanced our
understanding of the proximate and evolutio-
14 KALIONTZOPOULOU

nary causes of shape SD. Surprisingly, studies
using GM methods to address SD in amphi-
bians are very limited. Some studies investiga-
ted the effect of sex on shape and reported
significant SD (IVANOVI et al., 2007, 2008,
2009), but in these cases sex was treated as a
side variable, rather than being the main focus
of interest. This markedly contrasts with the
extensive investigation of shape SD using GM
in reptiles, and particularly in lizards. As is true
for allometric patterns (see above), the analysis
of lizard SD using GM has focused mainly on
head and skull shape and has revealed a gene-
ral resemblance of global patterns, but also sig-
nificant variation across groups, although the
number of studies is visibly skewed towards
the lacertids. Indeed, head/skull shape is
sexually dimorphic in all lacertid lizards that
have been examined (i.e. Algyroides:
LJUBISAVLJEVI et al., 2011; Dalmatolacerta
and Dinarolacerta: LJUBISAVLJEVI et al., 2010;
Lacerta: BRUNER et al., 2005; COSTANTINI et
al. 2007; Podarcis: KALIONTZOPOULOU et al.,
2007, 2008; LJUBISAVLJEVI et al., 2010; RAIA
et al., 2010). In all of the aforementioned
genera, sexual shape variation is mainly located
at the posterior region of the head, males
always presenting a more enlarged parietal
(dorsally) and tympanic (laterally) areas as
compared to females (Fig. 4). The same trend
is also observed in other phylogenetically dis-
parate lizard groups, such as iguanids of the
genus Liolaemus (VIDAL et al., 2005) and
Tarentola geckos (ZUFFI et al., 2011).
Additionally, in all the above examples, inves-
tigation of the proximate causes of shape SD
indicated that males and females follow com-
mon allometric slopes when considering the
relationship between head shape and size (as
represented by centroid size). Interestingly,
however, when size variation is taken into
account, some studies indicate size-indepen-
dent differentiation of head shape between the
sexes (i.e. difference of allometric regression
intercepts, KALIONTZOPOULOU et al., 2008),
while others indicate size SD as the only sour-
ce of shape SD (BRUNER et al., 2005;
LJUBISAVLJEVI et al., 2010). This, together
with the variation of allometric slopes obser-
ved among closely related species
(Ljubisavljevi et al., 2010) and the complete
lack of head shape dimorphism observed in
some instances (MONTEIRO & Abe, 1997),
indicates that important variation may exist
across species, urging for further investigation.
Figure 4: Typical pattern of head shape sexual
dimorphism in lacertid lizards, characterised by a
relative enlargement of the posterior region and
reduction of the anterior area in males. In this case,
the transformation of female (filled symbols, conti-
nuous line) to male (open symbols, dashed line)
shape in Podarcis bocagei is represented through
deformation grids for the dorsal (top) and lateral
(bottom) view of the head (modified from
KALIONTZOPOULOU et al., 2008). Shape differences
are exaggerated five-fold to enhance visualisation.
 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY
A promising line of research may be the elucida-
tion of the functional significance of head shape
SD as captured by GM methods, in order to
provide further evidence to the long-standing
hypothesis of sexual selection acting on bite
force as the main determinant of the observed
morphological patterns (HERREL et al., 2007;
HUYGHE et al., 2009; KALIONTZOPOULOU et
al., in press).
The investigation of SD using GM is rela-
tively limited in turtles and snakes, but some
general conclusions may be drawn. In turtles,
some studies have reported significant SD in
shell shape, which is the only trait that has been
examined. However, there is a marked discor-
dance among authors regarding the compari-
son between traditional and geometric mor-
phometrics for quantifying shape in turtles;
while some authors use GM as a powerful
method for capturing patterns invisible to tra-
ditional approaches, such as SD in hatchlings
(VALENZUELA et al., 2004; LUBIANA &
FERREIRA JNIOR, 2009), others report a wea-
ker SD in geometric shape as compared to that
observed using linear measurements (CHIARI
& CLAUDE, 2011). Such a disagreement may
be due to differences in the degree and direc-
tion of shape SD in different taxa, a possibility
supported by the limited data available
(VALENZUELA et al., 2004; CEBALLOS &
VALENZUELA, 2011). A different pattern is
observed in snakes, although the reduced num-
ber of studies once again hinders the extraction
of definite conclusions. The available studies
indicate that head/skull shape SD in snakes is
non-existent or of relatively low importance, at
least as compared to other sources of variation,
such as geographic locality (MANIER, 2004;
SMITH & COLLYER, 2008) or phylogenetic sig-
nal (GENTILLI et al., 2009). Clearly, patterns of
15
shape SD and its variation in turtles and snakes
needs to be further investigated in the future.
Shape evolution: adaptation and phenotypic
plasticity
The search for causal factors that may
explain the extensive morphological variation
we observe in many animal groups has always
been a fascinating field of investigation. GM
methods have enhanced our capacity of descri-
bing shape variation and associating it with
both ecological factors and performance mea-
sures, thus providing evidence of the adaptive
potential of certain shape traits. Extensive her-
petological research has focused on the search
for such variation, shedding new light on the
way amphibians and reptiles respond morpho-
logically to environmental disparity.
GM-based studies of the evolution of turtle
shell shape provide an exemplar system to the
study of adaptation. The shell represents a fun-
damental component of the turtle phenotype
and it is recognised as one of the most remarka-
ble novelties among tetrapods (BURKE, 1989).
Moreover, the shape of the shell has been shown
to present an important heritable component,
thus holding strong evolutionary potential
(MYERS et al., 2006). At the same time, turtles
have diversified to occupy a wide range of eco-
logical niches, while preserving a basic body
plan, thus constituting exceptional model orga-
nisms for studying the consequences of ecologi-
cal diversification on morphological traits.
Several components of habitat use have been
shown to directly influence turtle body shape.
The differentiation between aquatic and terres-
trial life is undoubtedly one of the main causes
of turtle phenotypic diversification, being
reflected in the shape of the carapace (CLAUDE
16 KALIONTZOPOULOU
et al., 2003; RIVERA & CLAUDE, 2008;
STAYTON, 2011), the plastron (CLAUDE et al.,
2003; ANGIELCZYK et al., 2011), the skull
(CLAUDE et al., 2004) and the shoulder articu-
lation (DEPECKER et al., 2006). Diet is an addi-
tional niche dimension involved in skull diffe-
rentiation in turtles (CLAUDE et al., 2004),
while anti-predatory strategies, specifically as
represented by plastral kinesis (PRITCHARD,
2008), seem to be a main factor of shell diffe-
rentiation, at least among emydines
(ANGIELCZYK et al., 2011). By contrast, phylo-
genetic inertia seems to play a subsidiary role in
turtle shape differentiation, at least in compari-
son with ecological factors (CLAUDE et al.,
2003, 2004; ANGIELCZYK et al., 2011).
Although less frequent, intraspecific studies of
shape variation also support the importance of
habitat effects for turtle morphology. Not only
does the shell of aquatic turtles differ from that
of terrestrial ones, it is also highly susceptible to
the characteristics of water flow. Indeed, RIVERA
(2008) showed that freshwater turtles of the
genus Pseudemys inhabiting fast-flowing water
regimes present a significantly more streamlined
shell, while those inhabiting slow-flowing regi-
mes are more domed (Fig. 5). Further suppor-
ting an adaptive explanation for the observed
patterns, the shape typical of fast-flowing regi-
mes importantly reduced drag during swim-
ming. Interestingly, a trade-off was also shown
to exist between this hydrodynamic efficiency
and mechanical strength. Flattened, hydrody-
namic shells were more fragile (RIVERA &
STAYTON, 2011), a pattern observed also betwe-
en aquatic and terrestrial emydids (STAYTON,
2011). The functional relevance of shell shape
has also been confirmed in terms of swimming
speed, where slider turtles with a relatively wider
and shorter plastron attained higher speeds than
elongated ones (MYERS et al., 2007).
A wide range of studies have also used
GM to decipher the effects of environmental
variation on the morphology of amphibians.
As is common for this group (WELLS, 2007),
multiple studies support the existence of
extensive phenotypic plasticity in both head
and body shape. Temperature (JORGENSEN &
SHEIL, 2008), predation risk (JOHNSON et al.,
2008; VAN BUSKIRK, 2009; HOSSIE et al.,
2010), competition (GARRIGA & LLORENTE,
in press), as well as numerous other habitat
components (VAN BUSKIRK, 2009) have been
shown to produce plastic responses on larval
body shape and ontogenetic shape allometry.
Furthermore, body shape has been shown to
Figure 5: Three-dimensional shell shape variation
observed between male Pseudemys turtles inhabi-
ting fast-flowing and slow-flowing water environ-
ments, as visualised through a PCA analysis of the
three-dimensional coordinates of the carapace.
Individuals of both sexes from lotic habitats are
more stream-lined, a shape modification that has
also been shown to reduce drag, providing empi-
rical evidence for an adaptive causation of the
observed differences (modified with permission
from RIVERA, 2008). Deformation grids represent
the extreme values observed across the first (A, B)
and second (C, D) principal component axes.
 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY
be tightly related to swimming performance
in anuran larvae (DAYTON et al., 2005;
ARENDT, 2010), although the plastic shape
modifications observed due to predation do
not seem to enhance speed, but may rather be
related to tail lure anti-predator display
(JOHNSON et al., 2008).
While plasticity studies are dominated by
anurans, the investigation of evolutionary
shape change as a response to environmental
factors is clearly skewed towards salamanders,
and particularly those of the genus Plethodon,
which have served as an important model
system for the evolutionary application of
GM tools. Character displacement due to
competition has been shown to be a major
force of head shape diversification in this
genus, which has been associated to both bio-
mechanical (ADAMS & ROHLF, 2000) and
behavioural (ADAMS, 2004) modifications.
Undoubtedly, head shape is a trait with
strong evolutionary potential in this salaman-
der group, an observation corroborated by
the existence of an important heritable com-
ponent (ADAMS, 2011). However, the detai-
led studies conducted also reveal that compe-
titive interactions, while repeatable across
space when the same pair of species is invol-
ved (ADAMS, 2010), are also characterised by
important variability (ADAMS et al., 2007;
ARIF et al., 2007; MYERS & ADAMS, 2008),
indicating that extrapolation from one pair of
species to another can be problematic. While
species interactions seem to be of central
importance in shaping morphological varia-
tion in Plethodon, other factors are also invol-
ved. For example, MAERZ et al. (2006) repor-
ted significant intraspecific variation associa-
ted to trophic polymorphism. Diet also
seems to be significantly associated to shape
17
variation of the hyobranchial skeleton of anu-
ran larvae (CANDIOTI, 2006). Other patterns
of shape variation in salamanders concern
potential adaptation to structural environ-
ment, where shape variation among closely
related taxa also involves the modification of
shape allometries. In a very interesting con-
trast of evolutionary patterns, JAEKEL &
WAKE (2007) and ADAMS & NISTRI (2010)
respectively described how divergence or con-
vergence of ontogenetic allometries can be
modified to produce foot shapes that match
the structural environments of Bolitoglossa
and Hydromantes salamanders. These two
examples nicely illustrate how GM can be
used to describe potentially adaptive mor-
phological variation (foot webbing) and asso-
ciate it to the functional advantages gained by
certain shape modifications (capacity for
climbing), while also remarking on the
importance of ontogenetic trajectories for
understanding the evolution of shape and
other traits (KLINGENBERG, 2010).
Several examples also illustrate the ecomor-
phological relevance of head/skull shape in
lizards. While visibly less integrated than the
examples on Plethodon above, the available
studies provide evidence to the importance of
different ecological factors for shape evolution
in various lizard taxa. In an insightful theoreti-
cal consideration of convergence in multiple
niche dimensions, HARMON et al. (2005) used
GM on head shape in Anolis lizards, together
with other character sets, to test the hypothe-
sis that if multidimensional convergence really
occurs in response to different aspects of the
environment, different character systems will
show different patterns of convergence among
species. In fact, character systems differed and
variation in head shape was suggested to be
18 KALIONTZOPOULOU
due to differences in diet, aggressive or anti-
predatory behaviour among habitat types
(HARMON et al., 2005). The hypothesis that
diet may profoundly influence head shape in
lizards is also supported by the observation
that parallel and convergent evolution occurs
between groups specialised in a certain type of
diet (STAYTON, 2005, 2006), while the same
observation stands for crocodiles (PIERCE et al.,
2008). Nevertheless, the strength of such an
influence seems to vary across taxonomic
levels, since similar studies among gekkotans
(DAZA et al., 2009) and the rhynchocephalians
(JONES, 2008) indicate that, although head
shape is associated to diet, phylogenetic affi-
nity visibly dominates over feeding behaviour
as a factor of skull shape differentiation.
Considering other factors, both habitat type
(KALIONTZOPOULOU et al., 2010) and
insularity (BNCIL et al., 2010; RAIA et al.,
2010) have been shown to influence head
shape in lacertid lizards, but these observations
should be further tested in other lizard groups.
Systematics, taxonomy and phylogenetic signal
Due to their increased effectiveness for cap-
turing shape variation, GM methods have been
used for species discrimination and for descri-
bing morphological variation between closely
related, and in many cases cryptic, taxa. For
instance, LEACH et al. (2009) used GM on
cranial horn shape of the coast horned lizard
species complex, combined with a large num-
ber of other biologically meaningful traits, to
characterise the process of lineage formation in
this group. In a similar approach, CHIARI &
CLAUDE (2011) used GM to study carapace
size and shape variation in Galpagos tortoises
and confirmed the morphological differentia-
tion between two genetically distinct lineages,
which had been described to differ morpholo-
gically using linear methods. GM tools have
also been used to analyse intraspecific geogra-
phic variation (MANIER, 2004; VIDAL et al.,
2005; CLEMENTE-CARVALHO et al., 2008;
SMITH & COLLYER, 2008), investigate the
strength of phylogenetic signal in shape data
(GENTILLI et al., 2009) and examine the degree
of concordance between phylogenetic related-
ness and morphological similarity (IVANOVI et
al., 2008, 2009). From a purely taxonomical
perspective, JAMNICZKY & RUSSELL (2004)
used GM to investigate the batagurine pro-
cess, a potential diagnostic character of the
turtle family Bataguridae, while VIEIRA et al.
(2008) investigated the morphological diffe-
rentiation between colour morphs of a toad
population to examine the taxonomical impli-
cations of the observed polymorphism.
While the above studies pose biologically
meaningful questions and most use GM in a
statistically robust framework to test specific
hypotheses, the use of shape characters in
phylogeny and systematics has been questioned
extensively (ADAMS & ROSENBERG, 1998;
ROHLF, 1998; KLINGENBERG & GIDASZEWSKI,
2010) and caution is advised when moving in
this area of investigation. This predicament lies
on both practical and theoretical grounds and
is mainly associated to the use of shape varia-
bles (either partial warps or their principal
components) as cladistic characters (ADAMS et
al., 2011). The main difficulty presented is that
of transforming continuously varied, multivaria-
te data as shape into discrete character states for
parsimony inference (ROHLF, 1998; MONTEIRO,
2000). Additional problems regard the effect of
a reference form, which is of central importan-
ce to the operations necessary to obtain shape
 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY
variables, and which has been repeatedly
shown to deeply influence the results obtained,
thus rendering GM shape variables of doubtful
usefulness for phylogenetic inference
(BOOKSTEIN, 1994; ADAMS & ROSENBERG,
1998). This of course does not mean that orga-
nismal shape as described by GM methods can-
not be analysed in a phylogenetic context, or
used to investigate the morphological affinity of
closely related species and compare it to their
known phylogenetic relationships. Moreover,
GM methods often provide a useful tool for
obtaining further evidence to support or reject
phylogenetic hypotheses (as implemented for
example in PIRAS et al., 2010) or to complement
the evidence provided by molecular studies
(CLEMENTE-CARVALHO et al., 2011) but caution
should be taken for correct implementation.
Paleontology
GM-based methods have been of great uti-
lity in paleontology, providing an innovative
view of morphological patterns in extinct
amphibian and reptile taxa (ELEWA, 2004).
Practically all the aforementioned fields have
partners in paleontological research, which
implement GM as a powerful tool to obtain
shape data from samples that frequently suffer
in terms of structural quality (BASZIO &
WEBER, 2002; ANGIELCZYK & SHEETS, 2007).
New methods have been developed for the
identification of fossils and used to specify the
structural position of paleontological findings
in snakes (POLLY & HEAD, 2004), as well as
being implemented as an indirect tool of esti-
mating the size of extinct taxa (HEAD et al.,
2009). Furthermore, GM methods have aided
the investigation of sexual dimorphism in
archosaurs, providing evidence for potentially
19
dimorphic structures (BARDEN & MAIDMENT,
2011) and allowing corroboration of the results
through comparison with their extant relatives
(i.e. Alligator; PRIETO-MARQUEZ et al., 2007;
BONNAN et al., 2008). Numerous studies have
taken advantage of the tool-kit of GM to cha-
racterise morphological disparity patterns and
investigate their temporal variation and to exa-
mine macroevolutionary trends (CANUDO &
CUENCA-BESCS, 2004; STAYTON & RUTA,
2006; PIERCE et al., 2009a; YOUNG & LARVAN,
2010; YOUNG et al. 2010). As for extant groups,
shape variation has also been investigated in the
light of its functional implications, thus provi-
ding a deeper understanding of the paleobio-
logy and paleoecology of archosaur taxa
(BONNAN, 2004, 2007; PIERCE et al., 2009b;
BONNAN et al., 2010). Finally, a very interes-
ting contribution in terms of the originality of
the studied shapes is that of RODRIGUES &
FARIA DOS SANTOS (2004), who used GM to
investigate the variation of sauropod tracks.
Amphibians and reptiles as models for the deve-
lopment of new methods
The multivariate nature of GM data fre-
quently challenges statistical methods and has
stimulated morphometricians to extend exis-
ting methods for studying complex shapes.
Apart from addressing biological questions,
several authors have used amphibians and rep-
tiles as model organisms to develop new
methodological approaches that utilise the
toolbox of GM and further enhance our capa-
city of investigating morphological variation.
For example, MONTEIRO (1999) used GM
data on sexual and ontogenetic variation of the
skull of tegu lizards to provide a comprehensi-
ve review of how multivariate regression tech-
20 KALIONTZOPOULOU
niques can be implemented to understand the
processes and causes underlying shape changes.
Along the same lines, MAGWENE (2001) used
turtle shells as an example to illustrate how
growth fields as characterised by a set of growth
vectors could be studied and compared in
order to understand the developmental proces-
ses involved in ontogenetic shape change. In a
conceptually similar approach, COLLYER &
ADAMS (2007) examined character displace-
ment in Plethodon salamanders (ADAMS, 2004)
to exemplify the use of shape change vectors for
the study of two-state multivariate phenotypic
change, an approach later generalized to multi-
state phenotypic trajectories (ADAMS &
COLLYER, 2009). Also trying to characterise the
direction of phenotypic change, STAYTON
(2006) proposed a new method for quantifying
data disparity patterns and used it to test for
convergence in skull shape among herbivorous
lizards. In the field of quantitative genetics of
shape, MYERS et al. (2006) generalized the uni-
variate approximation of shape heritability
based on Procrustes distance (MONTEIRO et al.,
2002) for application with unequal sample
sizes among families, thus complementing
other existing multivariate methods (i.e.
KLINGENBERG, 2003; KLINGENBERG &
MONTEIRO, 2005), and used it to study pat-
terns of plastron shape heritability in slider tur-
tles. A very promising methodology for the
field of biomechanics was put forward by
PIERCE et al. (2008) and STAYTON (2009), who
joined shape theory and finite element models
to study the mechanical properties of the croco-
dilian skull and three-dimensional turtle shell
shape correspondingly. Finally, although not
directly linked to the study of shape variation, it
is worth mentioning the use of GM tools as a
means of standardising specimen position for
studying colour patterns in salamanders
(ASHLOCK et al., 2003; COSTA et al., 2009).
CONCLUDING REMARKS
The aforementioned studies illustrate the
wide range of questions for which GM may
be implemented, facilitating the integrated
study of shape variation and its causes using
amphibians and reptiles as model organisms.
Many of these studies have compared tradi-
tional morphometric methods to GM and
have reached the conclusion that GM tech-
niques are frequently more powerful than
linear measurement data for detecting and
describing organismal shape variation
(VALENZUELA et al., 2004; BONNAN et al.,
2008; KALIONTZOPOULOU et al., 2008;
ARENDT, 2010). This is both an advantage
and a potential pitfall. On one hand, GM
methods are expected to be statistically more
powerful in detecting subtle shape variation,
since the number of variables analysed is
usually much higher than that examined in
traditional morphometrics. On the other,
caution should be taken in the implementa-
tion of such a sensitive tool, to keep with
strong biological inference and avoid mis-
sing focus of biological hypotheses. As
modern technological resources become
increasingly available for use in biological
sciences, a vast amount of new techniques
for obtaining shape data can be explored,
including for example computerised tomo-
graphy (CT) scans and three-dimensional
surface scanning. Herpetologists have until
now taken full advantage of the technical
and statistical tools available for the analysis
of shape variation, providing new insights to
the evolution of shape and frequently put-
 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY
ting forward new methods for data analysis.
Nevertheless, the exploration of GM
methods for understanding shape variation
in amphibians and reptiles is still an open
field, with promising perspectives for futu-
re contributions. I hope the above review
has provided an informed view of the ques-
tions that have been explored and the ans-
wers obtained, and point to directions for
further inquiry.
Acknowledgement
I am grateful to the editors of Basic and
Applied Herpetology for giving me the
opportunity to write this review. D. Adams,
M.A. Carretero, G. Rivera and two anony-
mous reviewers provided useful comments on
previous versions of the manuscript. Special
thanks to G. Rivera for allowing permission
to reproduce figure 5 of his article (as Fig. 5
here). This work was supported by a post-
doctoral grant (SFRH/BPD/68493/2010)
from Fundao para a Cincia e Tecnologia
(FCT, Portugal).
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 GEOMETRIC MORPHOMETRICS IN HERPETOLOGY
APPENDIX 1:
SOFTWARE RESOURCES
Several software packages have been develo-
ped for the application of GM methods, inclu-
ding data acquisition (digitising landmarks),
obtaining shape variables and performing
exploratory analyses and hypothesis-testing. A
detailed account of all available software, as
well as numerous other useful resources, can be
found in the SUNY Morphometrics webpage
developed and maintained by F.J. Rohlf
(http://life.bio.sunysb.edu/morph). All pro-
grams are freely available for download. Here I
briefly provide an account of the main softwa-
re packages more frequently used in recent
publications. Apart from the software mentio-
ned below, all GM procedures can also be
carried out in R language (R DEVELOPMENT
CORE TEAM, 2010), with an extremely high
flexibility for data visualisation and analysis.
The book Morphometrics with R (CLAUDE,
2008) is the essential guide for this purpose.
The tps series (ROHLF, 2011): Developed
since the early days of GM, this is a series of
software packages that aid the user in treating
different aspects related to the acquisition
and analysis of GM data. Rather than an
integrated package, this is a series of pro-
grams, each thought for carrying out specific
operations or answering relevant biological
questions. In this sense, the tps programs are
organised conceptually, depending on the
operations or statistical analyses of interest.
Different programs provide utility operations
(tpsUtil) and landmark acquisition (tpsDig),
superimposition methods (tpsSuper), mode-
lling shape variation through the thin-plate
spline and related methods (tpsRelW;
tpsSplin) and a wide array of more specific
31
exploratory and statistical procedures such as
the visualization of thin-plate splines on trees
(tpsTree), regression of shape onto indepen-
dent variables and regression-related hypo-
thesis testing (tpsRegr) and two-block partial
least squares analysis (tpsPLS). Although
some users might find the separation in diffe-
rent programs troublesome, this is a very
question-driven software series, specifically
designed for answering shape-related ques-
tions and accompanied by extremely tho-
rough help pages that provide the user with
both a conceptual and mathematical unders-
tanding of the operations involved. Both 2D
and 3D analyses are supported. Designed for
use in Windows, but will run without pro-
blems in Linux using Wine.
MorphoJ (KLINGENBERG, 2011): A user-
friendly integrated software package that pro-
vides a platform for the most important types
of analyses usually carried out with GM data.
These include 2D and 3D Procrustes supe-
rimposition with and without object
symmetry, utility operations, generation of
covariance matrices, matrix correlation, prin-
cipal components analysis, two-block partial
least squares, canonical variate and linear dis-
criminant analyses, regression analysis, map-
ping shapes onto a phylogeny and calculation
of phylogenetic independent contrasts and
analyses related to the quantitative genetics of
shape. Written in Java, it is a practically plat-
form-independent program that will run on
all Windows, Macintosh OS X and Linux.
IMP (SHEETS, 2000): This is a set of six
basic and several other auxiliary programs for
analysing GM data. It will perform all the
usual GM operations, including the genera-
tion of shape coordinates, principal compo-
nents analysis, canonical variate analysis,
32 KALIONTZOPOULOU
shape regression and pair-wise multivariate
shape comparisons. Both 2D and 3D analy-
ses are supported. It is based on MATLAB,
but will work without this software being ins-
talled on the computer. It is a Windows-
based program, but will apparently run well
on MAC and Linux through emulators.
Morphologika (O' HIGGINS & JONES,
2006): A set of integrated tools for examining
size and shape variation among objects descri-
bed by configurations of both 2D and 3D land-
mark coordinates. It enables generalised
Procrustes fitting of configurations, tangent
space projection, PCA of shape or size and
shape, multivariate regression of shape on an
independent variable, visualization of size and
shape variations by warping of the mean or
computation of transformation grids. As for the
tps-series, shape variables can be exported and
more mainstream multivariate analyses are to be
carried out in external statistical software.
Although still frequently used, morphologika is
no longer maintained and has rather been repla-
ced by the EVAN toolbox (http://www.evan-
society.org), which is more oriented to 3D
shape analysis.
 Basic and Applied Herpetology 25 (2011): 33-42

Embryonic development of kidneys in viviparous

Typhlonectes compressicauda (Amphibia, Gymnophiona)
Michel Bastit, Jean-Marie Exbrayat*
Universit de Lyon, Biologie Gnrale, Universit Catholique de Lyon, EPHE Reproduction et Dveloppement Compar, Lyon, France.

* Correspondence: Universit de Lyon. UMRS 449. Biologie Gnrale, Universit Catholique de Lyon, EPHE Reproduction et Dveloppement
Compar, 25 rue du Plat, F-69288 Lyon Cedex 02, France. Phone: (33) 4 72 32 50 36, Fax: (33) 4 72 32 50 66. E-mail: jmexbrayat@univ-catholyon.fr
Received: 1 February 2011; received in revised form: 24 June 2011; accepted: 13 September 2011.

The embryonic development of organs within Gymnophiona is still poorly known. In Typhlonectes compressicauda,
a caecilian amphibian expressing a derived viviparous reproductive mode, development can be divided into 34 sta-
ges and is characterized by a metamorphosis occurring between stages 30 and 33. At stages 18-19, Wolffian ducts
appear in the anterior part of the embryo. At stage 23, a pair of pronephric kidneys is clearly visible with several
nephrostomes that empty into the coelomic cavity. At stage 30, pronephroi that were observed at the level of the
second and fifth vertebrae are now observed between the 24th and 28th vertebrae. At this stage, several mesoneph-
ric tubules appear between the 29th and 32nd vertebrae, and mesonephric tissue is observed in the posterior part
of the body. At stage 31, pronephroi begin to disappear. The mesonephroi have now proximal and distal tubules.
At stages 32-33, two mesonephroi are visible as a pair of lengthened layers, representing the definitive kidneys.

Key words: Amphibia; development; Gymnophiona; mesonephros; pronephros.

Desarrollo embrionario de los riones en Typhlonectes compressicauda vivparas (Amphibia, Gymnophiona).

La ontognesis de los rganos en Gymnophiona es an poco conocida. En la cecilia vivpara Typhlonectes compressicau-
da el desarrollo se divide en 34 estadios, y se caracteriza por una metamorfosis que aparece entre los estadios 30 y 33.
Durante los estadios 18-19 los conductos de Wolff aparecen en la parte anterior del embrin. En el estadio 23, se
pueden apreciar claramente un par de riones pronfricos con varios nefrostomas que vierten a la cavidad celmica.
En el estadio 30, los pronefros que antes se podan apreciar al nivel de la segunda y quinta vrtebras aparecen ahora
entre las vrtebras 24 y 28. En este estadio, varios tbulos mesonfricos aparecen entre las vrtebras 29 y 32, mien-
tras que en la parte posterior del cuerpo se puede observar el tejido mesonfrico. En el estadio 31, el pronefros comien-
za a desaparecer, mientras que el mesonefros presenta tbulos tanto proximales como distales. En los estadios 32-33,
pueden apreciarse dos mesonefros con forma de capas alargadas, en lo que representa los riones definitivos.

Key words: Amphibia; desarrollo; Gymnophiona; mesonephros; pronephros.

In vertebrates, the development of excre-
tory organs is characterized by the formation
of a first pair of primitive kidneys, the pro-
nephroi, situated in the anterior part of the
body cavity (BALINSKY, 1975). Behind each
pronephros a second type of kidney, the
mesonephros, develops generally separated
from the pronephros by a space without renal
structures. The mesonephros is the definitive
kidney in agnates, cartilaginous and bony fis-
hes, and amphibians. The pronephros is cha-
racterized by the presence of nephrostomes
that connect the nephrons to the coelomic
cavity. Each pronephros degenerates during
the embryonic development except in some
cyclostomes and teleosteans (BRACHET, 1935;
BALINSKY, 1975). Wolffian ducts, a pair of
primary ureters that collect renal tubules
34 BASTIT & EXBRAYAT
from excretory organs, appear in amphibians
after the development of pronephroi.
Particularly in amphibians, both open
and closed nephrons with or without glome-
ruli appear in the mesonephros (PORTER,
1972; BALINSKY, 1975). In caecilians (order
Gymnophiona), very few works have been
devoted to the embryonic development of
kidneys. Previous work on this subject has
been published by SEMON (1892), who stu-
died the structure and development of uroge-
nital system in Ichthyophis glutinosus, BRAUER
(1900, 1902), who investigated kidney deve-
lopment in Hypogeophis sp., and WAKE
(1970), who studied the ontogeny of kidneys
in embryos of Hypogeophis sp. and Gymnopis
multiplicata, as well as the adult kidney of
several species. SAKAI et al. (1986) studied the
structure and ultrastructure of Typhlonectes
compressicauda kidneys, CARVALHO &
JUNQUEIRA (1999) the adult kidneys in
Siphonops annulatus, WROBEL & S (2000)
the structure and ultrastructure of Ichthyophis
kohtaoensis young larvae kidneys, and
MBJERG et al. (2004) the kidneys of adult
Geotrypetes seraphini. Whereas in most
amphibians pronephroi degenerate just after
the mesonephroi start to function (BALINSKY,
1975), in the studied caecilians it might be
possible that pronephroi do not degenerate at
this time and that both pro- and mesoneph-
ros could form a unique kidney, called holo-
nephros, which would be the supposed pri-
mitive situation in vertebrates.
We describe for the first time the develop-
ment of both pronephroi and mesonephroi
in the phylogenetically derived caecilian T.
compressicauda in order to add data to the
knowledge of kidney development in caeci-
lians, to understand the relationships between
pronephroi and mesonephroi in basal and
derived caecilians, and to understand the evo-
lutionary relationships, based on kidney mor-
phology and ontogeny, between caecilians
and other amphibians.
Typhlonetctes compressicauda (Gymnophiona:
Typhlonectidae) is a viviparous caecilian
amphibian living in South America. The
studied specimens live in French Guiana, in
swamped areas. Breeding occurs during the
rainy season, from December to June, and
pregnancy lies until September or October,
months when newborn are observed in the
field (EXBRAYAT, 1986). The embryonic
development has been previously divided in
34 stages (SAMMOURI et al., 1990). At the
beginning of the development, embryo
develops from the yolk mass followed by an
intrauterine hatching at stages 25-26.
Embryo escapes from the mucous envelope
and moves freely in the uterine lumen.
Foetal teeth develop on the lower jaw ena-
bling embryo to grasp the uterine secretions
and epithelial cells from the uterine wall
(HRAOUI-BLOQUET et al., 1994; HRAOUI-
BLOQUET & EXBRAYAT, 1996; EXBRAYAT &
HRAOUI-BLOQUET, 2006). Metamorphosis
occurs at stages 30-33, and at that time
juveniles resemble adults. Gills develop as a
pair of blades surrounding the embryo.
Gills are narrowly crushed against the uteri-
ne wall, participating to the constitution of
a placental structure (HRAOUI-BLOQUET &
EXBRAYAT, 1994; EXBRAYAT & HRAOUI-
BLOQUET, 2006).
MATERIALS AND METHODS
Embryos were obtained from gravid
females belonging to a collection of ani-
 KIDNEY DEVELOPMENT IN TYPHLONECTES COMPRESSICAUDA 35

mals retrieved from Kaw, a village situated RESULTS

80 km southwest of Cayenne, French
Guiana, in 1979, 1980 and 1982 (Table 1). At early embryonic stages (18-19), at the
Animals were euthanized by immersion in level of the fifth somite, in a well anterior
MS 222; embryos at several stages of deve- position, several undifferentiated cells dispo-
lopment were fixed in Bouins fluid, measu- sed as a crown are the first indications of the
red by placing them on graph paper, embed- Wolffian ducts (Fig. 1a).
ded in paraffin, then sectioned in 5 m-thick At stages 22-23 (Fig. 1b) certain proneph-
pieces and stained with haemalum-eosin, ric tubules are observed laterodorsally to each
Masson-Goldners trichrome or Romeiss Wolffian duct. The lumen of the Wolffian
azan in order to obtain several views of duct is lined with a single layer of cubic cells.
morphological structures, according to Several nephrons are observed near the
EXBRAYAT (2001). To prepare the stains, Wolffian ducts, with nephrostomes opening
haematoxylin and heamalum were purcha- into the coelomic cavity. The lumen of the
sed from RAL (Clichy, France), eosin renal tubules is covered with a single layer of
from Acros Organics (Morris Plains, New cubic cells without any apical specialization
Jersey, USA), iron alum, acid fuschin and and nephrostomes are lined with a single
azocarmine G from Sigma-Aldrich (Saint layer of ciliated cubic cells. In the posterior
Louis, Missouri, USA), fast green from part, the lumen of the Wolffian duct becomes
Edward Gurr (London) and aniline blue circular, lined with a single layer of cubic
from Merck (Whitehouse Station, New cells. Several flexuous renal tubules develop
Jersey, USA). from Wolffian ducts. Blood cells can agglo-
merate into a single glomerulus, and several
Table 1: Length and developmental stage ranges of blood vessels are observed near the tubules.
Typhlonectes compressicauda embryos used for the This posterior part corresponds to the begin-
study of kidney development, according to ning of mesonephros development (Fig. 1b).
Sammouri et al. (1990). At stage 24 (Figs. 1c, 1d), a pair of pro-
nephroi is observed at the level of the second to
Developmental stage Length (mm) N the fifth vertebrae. They are composed of well
18 Not measured 1
differentiated renal tubules lined with cubic
19 Not measured 1 cells containing vacuoles. Two to four nephros-
21-22 7 1 tomes open into the coelomic cavity are also
23 8-10 3
observed. Some tubules are wider than others.
24 9-10 5
25 10-11 4 The widest tubules, corresponding to proximal
26-27 13-14 3 ones, are lined with cubic cells with abundant
28 18-24 2 cytoplasm, some of which possess an apical
29 27-30 3
30 33-34 2 brush border directed to the lumen. The narro-
31 37-40 2 west tubules, corresponding to the interme-
32 63-70 4 diate and distal ones, are lined with cubic cells,
33 95-100 2
with or without microvilli, whose cytoplasm is
36 BASTIT & EXBRAYAT
less developed than that of cells lining the
widest tubules. Several capillaries extending
between tubules can be observed in the pro-
nephros. Capillaries are still not completely
built and each one appears as a single cavity
filled with blood cells. In the caudal part, the
nephric tissue is still not well developed.
At stages 25-26, pronephroi are very well
developed with a single glomerulus (Fig. 1e).
In the posterior part, nephrons continue to
develop, and glomeruli begin to appear
(Fig. 1f ). Wolffian ducts are lined with a sin-
gle layer of columnar stereociliated cells.
At stages 27-28, nephrostomes are obser-
ved within the pronephroi. Each nephrosto-
me empties into the coelomic cavity (Fig. 2a).
Nephrostomes are lined with ciliated cells.
Mesonephros is well developed frontally (Fig.
2b), and continue to develop in the posterior
part. The tubules reach the corresponding
Figure 1: Sections of anterior kidney of early
embryonic stages of Typhlonectes compressicauda.
(a) Stage 19. Cross section stained with haema-
lum-eosin. (b) Stage 22. Longitudinal section
stained with Masson-Goldners trichrome. The
insert shows a cross section stained with haema-
lum-eosin. (c) (d) Stage 24. Cross sections stai-
ned with haemalum-eosin. (e) Stage 26.
Longitudinal section stained with haemalum-
eosin. (f ) Stage 26. Longitudinal section stained
with Romeiss azan. Gl: glomerulus, mesoneph:
mesonephros, neph: nephrostome, NT: neural
tube, proneph: pronephros, Tub: renal tubule,
WD: Wolffian duct.
Wolffian duct. Renal tubules are lined with
brush border cells.
At stage 29, each pronephros is well deve-
loped, bearing large nephrons with several
capillaries forming a single common ventral
Malpighian body. The mesonephros begins
to be observed, containing renal tubules with
complete nephrons whose glomeruli are
more or less developed.
At stage 30, at the beginning of the meta-
morphosis, all the organs seem to move
towards the posterior part of the embryos
body. The heart that was previously observed
at the level of the third and fourth vertebrae
is now observed at the level of the 23rd and
24th vertebrae. Both pronephroi and meso-
nephroi are also displaced. Pronephroi are
now observed between the 24th and 28th
vertebrae and mesonephroi, situated behind
the pronephroi, are observed behind the 29th
 KIDNEY DEVELOPMENT IN TYPHLONECTES COMPRESSICAUDA 37

Figure 2: Sections of the anterior kidney of

late embryonic stages of Typhlonectes compres-
sicauda stained with Romeiss azan. (a) Stage
27. Longitudinal section showing proneph-
ros. (b) Stage 27. Longitudinal section sho-
wing mesonephros. (c) Stage 30.
Longitudinal section showing pronephros
and mesonephros. (d) Stage 32. Longitudinal
section showing mesonephros with a glome-
rulus. (e) Stage 32. Longitudinal section sho-
wing in detail a glomerulus. (f ) Stage 33.
Longitudinal section. CC: coelomic cavity,
Gl: glomerulus, L: liver, Lu: lung, mesoneph:
mesonephros, neph: nephrostome, proneph:
pronephros, Tub: renal tubule.

vertebra. The mesonephros is not well separa-
ted from the pronephros, and this temporary
situation resembles a holonephros (Fig. 2c).
The mesonephric nephrons are smaller than
the pronephric ones. The posterior part of
the mesonephros is still not differentiated.
At stage 31, the nephrostomes decrease in
size but their general shape is still recognizable.
The pronephros is always observed. The meso-
nephros continues to develop and appears to
be segmented into four masses of tissue. Under
the first renal tubules, already developed, new
glomeruli are in construction. It is remarkable
to note that the anterior part of the mesoneph-
ros still contains nephrostomes.
At stage 32 (Fig. 2d), mesonephroi contai-
ning voluminous glomeruli (Fig. 2e) appear par-
tially segmented, but at this stage the pro-
nephros is no longer observed. The anterior tip
of each kidney is situated at the level of the heart,
but it cannot be identified as a pronephros using
anatomical and histological criteria because
neither open nephrostomes nor wide tubules are
observed. The posterior part of the kidneys is
almost completely differentiated.
At stage 33 (Fig. 2f ), kidneys (meso-
nephroi) are at the end of their development.
They appear as two bands of tissue situated
between the heart and the cloaca. In their
anterior parts, three or four voluminous
nephrons connect to the Wolffian ducts, but it
is difficult to recognize them as a pronephros.
Nephrons possess now the definitive structure
that will be found in adult individuals.
Glomeruli are about 90 m in diameter.
In adults, the kidneys (mesonephroi)
appear as two parallel bands the tip of them
being observed at the level of stomach, clearly
well behind the heart, suggesting the degene-
ration of pronephros.
38 BASTIT & EXBRAYAT
DISCUSSION
For the first time, the development of kid-
neys has been described in T. compressicauda.
Kidneys have been described in several caeci-
lian species (SPENGEL, 1876; SEMON, 1892;
WIEDERSHEIM, 1879; BRAUER, 1902;
BRACHET, 1935; CHATTERJEE, 1936; GARG
& PRASAD, 1962; WAKE, 1970; SAKAI et al.,
1986, 1988a,b; CARVALHO & JUNQUEIRA,
1999; WROBEL & S, 2000; MBJERG et al., several capillaries that occur between tubules,
2004). In T. compressicauda, the examination
of kidney development shows the formation
of a pair of pronephroi characterized by
nephrostomes, large tubules, and small capi-
llaries irrigating tubules. In Hypogeophis sp.,
BRAUER (1902) described the presence of
very well differentiated nephrostomes
throughout both pronephroi and meso-
nephroi. In this species, eight or nine tubules
develop, although 12 tubules can be observed
at the initial stages of development. These
tubules develop between the 13th and 24th
vertebrae, but immediately degenerate. In the
posterior part, tubules develop constituting
the mesonephros, which is separated from
the pronephros. In small I. kohtaoensis larvae
studied by WROBEL & S (2000), a large
pronephros is observed caudally to the bran-
chial region, overlapping in its posterior part
with the mesonephros.
In embryos of Gymnopis sp. and
Hypogeophis sp., WAKE (1970) observed that
tubules opened into the coelomic cavity through
nephrostomes. Inversely, in adult S. annulatus
studied by CARVALHO & JUNQUEIRA (1999),
the kidneys were segmented in the rostral
part, with presence of nephrostomes. This
situation could indicate the presence of a per-
sistent pronephros in this species, but no
ontogenetic study is available in this case. In
Hypogeophis sp., pronephroi appear at the level
of the fourth somite (BRAUER, 1902; BRACHET,
1935; WAKE, 1970), while in I. kohtaoensis it is
located just under the gills (WROBEL & S,
2000). In I. kohtaoensis (WROBEL & S,
2000) and Hypogeophis sp. (BRAUER, 1902;
BRACHET, 1935), Malpighian corpuscle of
each pronephros was unique, consisting of a
vessel derived from the aorta and divided into
like in T. compressicauda.
After a phase at which pronephroi and
mesonephroi are observed together, in a very
close position but without overlapping like in
I. kohtaoensis (WROBEL & S, 2000), pro-
nephroi of T. compressicauda disappear at
metamorphosis. Several nephrostomes can be
observed in the anterior part of mesonephroi,
like in other caecilians (SPENGEL, 1876;
BRAUER, 1902; CARVALHO & JUNQUEIRA,
1999; MBJERG et al., 2004). With the excep-
tion of T. compressicauda, no information is
available on kidney structure during caecilian
metamorphosis. In Hypogeophis sp., pro-
nephroi degenerate when mesonephroi beco-
me active (BRAUER, 1902; BRACHET, 1935), but
authors reporting these findings do not specify
if this is coincident with metamorphosis.
In T. compressicauda, mesonephroi are
segmented in several masses. This situation is
different from that of adult G. seraphini,
whose mesonephroi are segmented in a fron-
tal position only (MBJERG et al., 2004).
SAKAI et al. (1986, 1988a,b) gave structural
and ultrastructural descriptions of adult kid-
neys in T. compressicauda and observed the
presence of several more or less degenerated
nephrostomes. Their results are coincident
with those of SPENGEL (1876) and BRAUER
 KIDNEY DEVELOPMENT IN TYPHLONECTES COMPRESSICAUDA
(1902) for Hypogeophis sp., or those of
MBJERG et al. (2004) for adult G. seraphini,
whose kidneys are long mesonephroi with
both ventral tubules open into the coelomic
cavity and closed tubules. On the contrary,
WAKE (1970) observed numerous adult cae-
cilian species and did not see any nephrosto-
mes open into the coelom.
Several comparative data about embryo-
nic development of kidneys in amphibians
have been published (BRACHET, 1935;
GIPOULOUX, 1986; DITRICH
LAMETSCHWANDTNER, 1992; GIPOULOUX &
CAMBAR, 1995; RICHTER, 1995; MBJERG et al.,
2000; DRAWBRIDGE et al., 2003). Kidneys of
caecilians, and particularly of T. compressicauda,
possess a lot of common features with the
kidneys of both anurans and urodeles
(LAMETSCHWANDTNER et al., 1978; SAKAI et
al., 1986). Several differences also exist bet-
ween caecilians and other amphibians. In
caecilians, kidneys are elongated like other
organs, which is considered to be an adap-
tation to the burrowing habits of these
animals (TAYLOR, 1968). Kidneys of caeci-
lians also present a segmental disposition
(WAKE, 1970) that does not exist in anu-
rans or urodeles.
The development of kidneys is similar
across all amphibian groups. Pronephroi
develop between the third and fourth somites
in urodeles, and between the third and fifth
somites in anurans. They present nephrosto-
mes that open into the coelomic cavity.
Pronephroi are then replaced with functional
mesonephroi at metamorphosis (GIPOULOUX,
1986; GIPOULOUX & CAMBAR, 1995).
Epithelial cells of the mesonephros first con-
tain osmiophilic granules that disappear at
metamorphosis, indicating a change in kid-
39
ney activity at that time (MBJERG et al.,
2000). The ablation of pronephroi in larvae
of several anuran species provokes an impor-
tant oedema followed by death (CAMBAR,
1947), which suggests that these first kidneys are
functional at least during a part of larval deve-
lopment. In larval Ambystoma mexicanum,
HAUGAN et al. (2010) proposed that pro-
nephroi were important for modification of
urine. In Bufo viridis, each pronephros is a
single convoluted tubule open into the coelo-
& mic cavity through three nephrostomes
(MBJERG et al., 2000), and urine is formed
by filtration from an external glomerulus.
In basal caecilians, such as the genus
Ichthyophis, pronephroi and mesonephroi
overlap, looking like a continuous kidney
(SEMON, 1892; WROBEL & S, 2000) and
resembling a holonephros, the theoretical pri-
mitive kidney of vertebrates. Yet, even in
Ichthyophis spp., pronephroi degenerate
during development, like in other caecilians
such as Hypogeophis sp., Gymnopis sp. (WAKE,
1970), G. seraphini (MBJERG et al., 2004)
and T. compressicauda (this work), as well as in
anurans and urodeles. In the caecilians whose
kidney development has been studied, pro-
nephroi and mesonephroi are separated from
each other, either closely or with a large space
without any renal formation. These data could
be indicating a trend towards the separation of
pronephroi and mesonephroi within the order
Gymnophiona. Siphonops annulatus can be an
exception, as it shows indications of persistent
pronephroi in adults (CARVALHO &
JUNQUEIRA, 1999), but no developmental data
are available for this species.
In conclusion, the development and structu-
re of T. compressicauda kidneys resemble that of
other amphibians (BRACHET, 1935; GIPOULOUX
40 BASTIT & EXBRAYAT
& CAMBAR, 1995), with morphological diffe-
rences related to burrowing habits. Within cae-
cilians, kidney development and structure of
adult kidneys present variations from one species
to another. Like other organs, variations could
be related to the taxonomic position, with basal
Asiatic Ichthyophiidae and American
Rhinatrematidae showing pronephros and
mesonephros in a closer position than modern
taxa such as Typhlonectidae (WILKINSON &
NUSSBAUM, 2006; EXBRAYAT & RAQUET, 2009),
and more specifically such as T. compressicauda.
Acknowledgement
Authors thank Fondation Singer-Polignac
who supported collection of animals in French
Guiana. Authors also thank Mrs. Marie-
Thrse Laurent for her technical assistance.
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 Basic and Applied Herpetology 25 (2011): 43-54

Population estimators and adult sex ratio for a population of

Bolitoglossa altamazonica (Caudata: Plethodontidae)
Doris Laurinette Gutirrez-Lamus1,*, John Douglas Lynch1, Germn C. Martnez-Villate2
1
Instituto de Ciencias Naturales, Amphibian's Laboratory, Universidad Nacional de Colombia, Bogot, Colombia.
2
Faculty of Veterinary Medicine and Husbandry, Fundacin Universitaria San Martn, Sede Bogot, Colombia.

* Correspondence: Cra. 4 # 24-59 apt. 1504, Bogot, Colombia. Phone: 57-1-4794101, E-mail: dlgutierrezl@unal.edu.co
Received: 17 March 2011; received in revised form: 10 October 2011; accepted: 3 November 2011.

Bolitoglossa altamazonica is the species of plethodontid salamander with the widest distribution in the tropics.
However, while aspects related to population size, survival rates, recruitment, sex ratios along with other life
history traits are well documented for temperate salamanders, such information is relatively scarce for tropi-
cal species. We conducted an intensive capture-recapture study on a population of B. altamazonica. We used
the Jolly-Seber method to estimate three parameters (size, recruitment and survivorship) for the population
as a whole, as well as for males, females and juveniles separately. All these parameters varied monthly for each
class and the entire population. Juvenile recruitment occurred between november and july. Survivorship of
juveniles increased when there was no recruitment at all. The adult sex ratio during the breeding season was
significantly biased towards females (up to 1:3 depending on the month).

Key words: population size; recruitment; salamanders; sex ratio; survivorship.

Estimadores de poblacin y razn de sexos en una poblacin de Bolitoglossa altamazonica (Caudata:

Plethodontidae). Bolitoglossa altamazonica es la especie de pletodntido con distribucin ms amplia en los
trpicos. Sin embargo, mientras que los aspectos relacionados con el tamao de la poblacin, tasa de super-
vivencia, reclutamiento, razn de sexos y otras caractersticas de la historia vital estn bien documentados en
salamandras de zonas templadas, la informacin para especies tropicales es relativamente escasa. Llevamos a
cabo un estudio intensivo de marcaje-recaptura en una poblacin de B. altamazonica. Utilizamos el mtodo
de Jolly-Seber para estimar tres parmetros (tamao, reclutamiento y supervivencia) tanto para el conjunto
de la poblacin como para machos, hembras y juveniles por separado. Todos los parmetros estimados expe-
rimentaron variaciones mensuales en cada una de las clases as como en el conjunto de la poblacin. El reclu-
tamiento de los juveniles sucedi entre noviembre y julio. La supervivencia de los juveniles se increment
cuando no exista reclutamiento. La razn de sexos de la poblacin adulta durante el periodo reproductor
estuvo significativamente sesgada en favor de las hembras (hasta 1:3 dependiendo del mes).

Key words: razn de sexos; reclutamiento; salamandras; supervivencia; tamao de poblacin.

Reports on declining and disappearing and differentiating problematic declines from

amphibian populations have received a great
deal of attention in recent years (PECHMANN &
WILBUR, 1994). These population fluctuations
are believed to be the result of natural events
(PECHMANN & WILBUR, 1994) or environ-
mental perturbations (POUNDS et al., 1997),
natural fluctuations in populations is an issue
of particular difficulty in applied ecology
(PECHMANN et al., 1991). One-third of all
amphibian species worldwide is endangered or
threatened with extinction (STUART et al.,
2004). Efforts to understand the causes of this
44 GUTIRREZ-LAMUS ET AL.
alarming decline, known as the global amphi-
bian crisis, have focused primarily on frogs;
comparatively little attention has been paid to
salamanders (LIPS, 1998; PARRA-OLEA et al.,
1999; WHITFIELD et al., 2007). A reason for
this bias includes the fact that most salaman-
ders are secretive in nature, so populations
trends may not be as apparent as in frogs, but
the global amphibian crisis, usually discussed
in terms of frogs, clearly involves neotropical
salamanders as well (ROVITO et al., 2009).
Declining trends are impossible to detect
without long-term abundance-based data on
population densities collected by using a con-
sistent methodology. Although those data
sets are exceptionally rare, they are critical to
understand the full extent of the global
amphibian crisis (WHITFIELD et al., 2007).
An assessment of the status and conservation
of amphibians requires an expanded, regional
perspective (HECNAR & MCLOSKEY, 1996);
however, few estimates of amphibian natural
history parameters exist against which to
judge the extent of additional mortality
(BLACKWELL et al., 2004).
Population size indicates how the repro-
ductive health for a given population is, and
survivorship often explains a large portion of
an individuals lifetime reproductive success
(OLGUN et al., 2001). Therefore, analyses of
the variation of life history parameters such
as survival rates and recruitment over time
are of major importance not only for unders-
tanding the life history of a species (FLATT et al.,
1997), but also for providing an expanded
regional perspective for conservation and
management (BLACKWELL et al., 2004).
In ecological studies, the adult sex ratio,
defined as the proportion of reproductive
females and males within the breeding popu-
lation, is considered a key parameter in
understanding sexual selection, mating beha-
viour, and population dynamics (KVARNEMO
& AHNESJ, 2002). According to classical sex
allocation theories, in natural populations a
balanced sex ratio should be maintained in
the long term by a selective advantage to the
parents producing the rarest sex (RANTA et
al., 2000). However, in amphibians, the rela-
tive numbers of sexually active males and
females show large interspecific variations
depending on the mode of reproduction,
mating system and resource distribution
(ZUG et al., 2001).
All neotropical salamanders belong to the
tribe Bolitoglossini (family Plethodontidae);
these salamanders have direct development of
young inside terrestrially laid eggs (WAKE,
1966). The supergenus Bolitoglossa contains
about two-thirds of plethodontid species and
about 40% of all species of salamanders
(AMPHIBIAWEB, 2011). Bolitoglossa altamazo-
nica occurs on the eastern slopes of the Andes
from Venezuela and Colombia, through
Ecuador, Peru and Bolivia, and as far east as eas-
tern Brazil. Because of its wide distribution and
presumed large populations, B. altamazonica is
listed as Least Concern in the IUCN Red List
(AZEVEDO-RAMOS et al., 2009).
The main goals of this study were to exa-
mine three population parameters (size, sur-
vivorship and recruitment) for a population
of B. altamazonica and to evaluate whether
the adult sex ratio is balanced and constant
through time. The present study contributes
to the knowledge of amphibian life history by
documenting temporal population parame-
ters of a population of B. altamazonica. This
dataset provides the best opportunity to exa-
mine changes in salamander populations over
 POPULATION ECOLOGY OF A NEOTROPICAL SALAMANDER
time, thus serving as a reference for compari-
son to populations in other localities from
Colombia and along its distribution range.
MATERIALS AND METHODS
The study site, Jardn Botnico de
Villavicencio, is a local natural reserve since
1983 located in Villavicencio City, Meta,
Colombia (04 09' 09'' N, 73 39' 15'' W) at
640 m above sea level. It is a humid tropical
forest (HOLDRIDGE et al., 1971) with mean
annual rainfall, temperature and relative
humidity of 4531 mm, 25.9C and 76%, res-
pectively. The rainfall regime is unimodal
with a maximum peak of rains from May to
June. The dry season usually extends from
December to March (data recorded from the
nearest weather station: Vanguardia
Instituto de Hidrologa, Meteorologa y
Estudios Ambientales de Colombia).
At the study site, we established a 7300 m2-
plot. We captured salamanders within this plot
by visual encounter (CRUMP & SCOTT, 1994;
ANGULO et al., 2006). Two experienced resear-
chers looked for salamanders during eight
nights (19:00-02:00), monthly from April
2008 to November 2008 with an additional
sampling performed in January 2009. All sur-
veys were conducted at night because the focal
species is nocturnal and surface activity occurs
at night. We marked animals using freeze bran-
ding with liquid nitrogen (DAUGHERTY, 1976).
Individual identification was achieved by com-
binations of marks in fifteen body locations
(modified from NISHIKAWA & SERVICE, 1988),
five on each side of the body and five mid-dor-
sally according to the following distribution:
anterior and posterior to the fore limb, mid-
body, anterior and posterior to the hind limb.
45
We measured snout-vent length (SVL: tip of
the snout to posterior margin of the vent) using
a vernier calliper after restraining the salaman-
ders in a plastic bag. Adults were sexed in
accordance with their size (SVL) and secondary
characters; during the breeding period, mature
males had an enlarged, disc-like mental gland
in the anterior region of the lower jaw (SVL:
34.58-44.86 mm) whereas mature females
were gravid (SVL: 36.34-57.84 mm). We used
these SVLs as a reference for sexing individuals
out of the breeding season; however, because of
the big overlap in size between sexes, only large
females (SVL > 44.86 mm) and small males
(SVL < 36.34 mm) could be sexed in the field.
All specimens with an SVL below 34.00 mm
without sexual secondary characters were clas-
sified as juveniles. We returned all animals to
the spot where we captured them after data
collection and marking.
The primary goal of the mark-recapture
analysis was to estimate population size, survi-
val and recruitment. In an open population
that is affected by mortality and migration,
variable survival rates are more biologically
realistic than fixed ones (DONNELLY & GUYER,
1994; KREBS, 1999). Survival can be estimated
using standard mark-recapture methodology
(LEBRETON et al., 1992). Local survivorship
represents the probability of surviving from
month (i) to month (i + 1), and is affected by
both mortality and permanent emigration.
Local recruitment is the number of new ani-
mals in the population at time i per animal in
the population at time i - 1 (PRADEL, 1996),
and it includes in situ reproduction and immi-
gration. We included sex and age as grouping
factors in the model selection procedure to
allow for testing sex- and age-specific effects
on the parameters of interest. We analyzed
46 GUTIRREZ-LAMUS ET AL.

mark-recapture data using Cormack Jolly- that did not have standard errors extremely
Seber (CJS) and Pradel survival and recruit- high or low fixed. We used the Jolly-Seber ori-
ment extensions of Program MARK version ginal model (JOLLY, 1965; SEBER, 1965) to
5.0 (WHITE & BURNHAM, 1999). We used an estimate population size, since program Mark
overall model selection procedure before para- never reached numerical convergence. Adult
meter estimation. Ten a priori candidate sex ratio was expressed as the relative propor-
models offered different biological representa- tions of estimated mature males and females,
tions of the role of capture probability among and deviations from a 1:1 ratio were tested
sexes and ages (adult or juvenile). Rather than through a Chi-square test.
including all possible permutations in the pri-
mary model selection procedure, we initially RESULTS
tested four models to evaluate sex differences
in detectability and survival; then, we tested We captured 880 individuals of B. alta-
four additional models accounting for possible mazonica, 244 of which were recaptured for a
age differences in these variables. Parameters in total of 1124 captures in nine months.
the candidate models were either allowed to Monthly captures regardless of sex or age ran-
vary over time or to remain constant. Model ged from 64 to 184 individuals, and fluctua-
selection was based on the small-sample ted following the same pattern as rainfall,
Akaikes information criterion (AICc; with a peak of captured salamanders in June
BURNHAM & ANDERSON, 2002). Some para- (Fig. 1). Because of the intense sampling, fre-
meters were estimated after 15 000 simula- quency of captures of new individuals decli-
tions using Markov chain Monte Carlo ned rapidly over time, in spite of which we
(MCMC), keeping all the other parameters captured new individuals in all months.
Table 1: Model rankings to evaluate sex and age effects on survival (Phi) and detectability (p), using the
Cormack-Jolly-Seber (CJS) extension, and age and time effects on survival and recruitment (f), using the
Pradel extension, in a population of Bolitoglossa altamazonica. Models are listed in decreasing order of support
using Akaikes Information Criterion. g = gender/age-dependent, t = time-dependent.

Extension Model AICc Delta AICc Model likeli- Deviance

AICc weight hood

CJS (Sex) Phi(t) p(t) 203.87 0.00 0.669 1.00 20.85

Phi(g*t) p(g*t) 206.41 2.53 0.188 0.28 12.81
Phi(g*t) p(t) 207.84 3.97 0.092 0.137 18.52
Phi(t) p(g*t) 209.09 5.21 0.049 0.074 19.76

CJS (Age) Phi(t) p(g*t) 1763.93 0.00 0.882 1.00 239.94

Phi(g*t) p(g*t) 1768.23 4.299 0.103 0.116 229.49
Phi(t) p(t) 1772.19 8.263 0.014 0.016 264.82
Phi(g*t) p(t) 1777.36 13.426 0.001 0.001 253.37

Pradel Phi (t) p(g*t) f(g*t) 5444.219 0.00 0.984 1.00 242.66
Phi (t) p(g*t) f(t) 5452.48 8.26 0.016 0.016 257.28
 POPULATION ECOLOGY OF A NEOTROPICAL SALAMANDER
Figure 1: Monthly variation in the number of captu-
red salamanders (black line) and its relationship with
rainfall values (grey bars).
From the set of models used to evaluate
sex effects on survival and detectability, the
model that fitted better into our capture-
recapture history was that considering both
parameters to vary over time without effects
of sex (Table 1). Thus, we did not include in
subsequent analyses the effects of sex on these
two variables. The second set of models, used
to evaluate age effects on survival and detec-
tability, supported the time-varying detecta-
bility, revealing differences between ages in
detectability but not in survival (Table 1).
Finally, the comparison of the two models to
evaluate age and time effects on recruitment
showed that this parameter varied as a func-
tion of both factors (Table 1).
Population size estimates fluctuated bet-
ween months, ranging from 582 in May to a
maximum of 2165 individuals in October.
Survivorship also fluctuated between
months, with the highest trustable estimate
in July. Although the estimated survivorship
for September was higher than that of July,
we disregarded it because of its large standard
error; such a high value for survivorship is a
mathematical artefact produced by the large
47
number of individuals marked in October in
contrast with the low number of individual
marked during September (Table 2).
Using the maximum number of salaman-
ders estimated by the Jolly-Seber method, the
population density within the studied plot
would be 0.23 salamanders / m2. For density
estimation, we used the population size obtai-
ned in October (highest value) because a good
number of specimens were already marked by
that month (752), and salamanders were
highly active because of favourable weather
conditions, allowing us to calculate population
density without underestimations.
For estimating juvenile population para-
meters, we used 412 marked specimens and
69 recaptures, for a total of 480 captures. We
identified an individual as juvenile in May
(SVL = 34.6 mm), but when we recaptured it
30 days later it showed a visible and completely
developed mental gland (SVL = 38.24 mm).
Between-months recruitment was highly
variable (Table 3). The highest juvenile
recruitment occurred in June and July, while
Table 2: Population size (N) and survival (Phi) esti-
mates ( SE) for a population of Bolitoglossa altama-
zonica. No data from January were calculated becau-
se, due to the absence of sampling in December, they
did not meet the Jolly-Seber method requirement of
consecutive samplings.
Month N Phi
April - 0.342 ( 0.107)
May 582 ( 253) 0.847 ( 0.140)
June 1348 ( 350) 0.779 ( 0.125)
July 1344 ( 280) 0.880 ( 0.162)
August 1267 ( 275) 0.830 ( 0.171)
September 776 ( 150) 1.950 ( 0.196)
October 2165 ( 896) 0.825 ( 0.362)
November 1298 ( 560) 0.281 ( 17.995)
January - -
48 GUTIRREZ-LAMUS ET AL.

Table 3: Juvenile and adult recruitment (f) and detectability (p) ( SE) in a population of Bolitoglossa altamazonica.

Month Adults Juveniles

f p f p

May 1.145 ( 0.276) 0.174 ( 0.102) 7.558 ( 1.709) 0.242 ( 0.000)

June 0.825 ( 0.219) 0.152 ( 0.050) 0.061 ( 0.124) 0.238 ( 0.174)
July 0.122 ( 0.274) 0.136 ( 0.037) 0.320 ( 0.127) 0.105 ( 0.037)
August -0.008 ( 0.004) 0.127 ( 0.033) 0.011 ( 0.223) 0.111 ( 0.0321)
September 0.268 ( 0.123) 0.200 ( 0.045) 0.006 ( 0.062) 0.077 ( 0.017)
October 0.407 ( 0.182) 0.120 ( 0.027) 0.006 ( 0.089) 0.071 ( 0.016)
November 0.327 ( 0.227) 0.082 ( 0.039) 0.024 ( 0.093) 0.064 ( 0.015)
January 0.027 ( 1.085) 0.104 ( 0.021) 0.011 ( 0.130) 0.124 ( 0.055)

no new juveniles entered the population bet- meters (PLEDGER et al., 2003). Numerous
ween September and October. We did not authors have suggested that this assumption is
take into account juvenile recruitment estima- met as capture probability is likely to vary
tes in May because of the high standard error. among demographic groups over time
We performed estimates for adults from 468 (WILLSON et al., 2011). The mistake of assu-
individuals, 175 of which were recaptured for ming constant capture probability when varia-
a total of 643 captures. Adult recruitment was tion actually exists can bias estimates of abun-
highest in June and dropped along with the dance or recruitment (POLLOCK et al., 1990;
rainfall; in August-September no new adults BAILEY et al., 2004). Imperfect detectability
entered the population (Table 3). also extends to the estimation of survival
Using the 100 males and 245 females cap- (MAZEROLLE et al., 2007). Adjusted population
tured during the breeding season, which runs estimates, which estimate the true population
from January to July (GUTIRREZ-LAMUS, based on capture-recapture techniques, are
2009), we found a significantly female-biased labour-intensive but may yield a more accurate
sex ratio that ranged between months from picture of the number of salamanders present
1:2 to 1:3 (Table 4). in a population (JUNG et al., 2000).
Table 4: Chi-square test to analyse the sex ratio obtai-
DISCUSSION
ned in a population of Bolitoglossa altamazonica. The
null hypothesis is a one-to-one sex ratio. P-values in
The results from this study are the first of bold face are significant.
its kind for B. altamazonica and contribute to
the knowledge about this population in Month N (males) N (females) Sex ratio 2 (d.f. = 1) P
regards to its size, survivorship, recruitment
April 17 31 1:1.8 4.0833 0.0433
and adult sex ratio. May 17 46 1:2.7 13.3492 0.0000
The key estimates for many ecologists using June 29 67 1:2.3 15.0417 0.0003
the CJS model on capture-recapture data are July 23 64 1:2.8 19.3218 0.0000
the survival rates, with the capture probabilities January 14 37 1:2.6 10.3726 0.0013
Total 100 245 1:2.45 60.9420 0.0000
often viewed as little important nuisance para-
 POPULATION ECOLOGY OF A NEOTROPICAL SALAMANDER
According to recruitment estimates, most
juveniles enter the population in July, and
recruitment gradually decreases during subse-
quent months along with rainfall. Juvenile
recruitment was almost null between August
and October, but in November new young
individuals entered the population again.
Nevertheless, such results can be sample size
artefacts because we marked young indivi-
duals during those months when recruitment
estimated by Pradel method was close to zero.
The method does not provide estimates for
the first month of sampling. For this reason,
we believe that juvenile recruitment occurs
continually from November through July.
The intensity of recruitment depends on the
effective size of the breeding population and
the survivorship of the eggs (DUELLMAN &
TRUEB, 1994), as well as the volume of cap-
tures. According to the estimates for both
adults and juveniles, most recruitment occurs
between June and July, when rainfall reaches
maximum levels. On the other hand, new
adults do not enter the population when
rainfall decreases (August-September).
Several reasons could have caused the fluc-
tuations in our estimates; first, our inability to
find and mark brooding females; second, sam-
pling during rainy nights when activity
notably decreased; third, the behavioural ther-
moregulation demonstrated in plethodontids
(SPOTILA, 1972; FEDER, 1982), consisting in
that animals select optimal temperatures by
moving to preferred parts of the gradient. If
optimal temperatures were achieved in the lit-
ter, we could hardly find animals there.
Fourth, the cutaneous gas exchange may
account for more than 90% of the exchange in
plethodontids, which might force animals to
use microhabitats with adequate humidity and
49
temperature in order to minimize the risk of
water loss (SPOTILA 1972). Salamander captu-
re probabilities are influenced by a number of
factors, including site-specific characteristics,
weather conditions and hour of day (JUNG et
al., 2000). Nevertheless, long-time monito-
ring studies in species such as Ambystoma
maculatum (BLACKWELL et al., 2004) and
Ambystoma tigrinum (WHITEMAN &
WISSINGER, 2005) have revealed fluctuations
in population size between years.
After comparing rainfall data and animal
abundance, we detect an obvious relationship
between population density and weather condi-
tions. We obtained the greatest values for captu-
res, population size and recruitment when rain-
fall levels were high. It has been generally accep-
ted that moisture, as expressed in the amount
and distribution of rainfall, exerts the greatest
influence on the distribution of organisms in
tropical environments (AUBERT DE LA RE et al.,
1957; RICHARDS, 1957 in VIAL, 1968). Similar
responses to annual distribution of rainfall have
been reported for Bolitoglossa subpalmata (VIAL,
1968), Batrachoseps spp. (HENDRICKSON, 1954;
ANDERSON, 1960) Aneides lugubris
(ROSENTHAL, 1957) and Ensatina spp.
(STEBBINS, 1954).
Available information on population den-
sities of salamander comes from a limited
number of studies that employ a variety of
techniques (VIAL, 1968). Among neotropical
salamanders, densities have been reported
only for B. subpalmata, ranging from 0.0756
to 0.9097 individuals / m2 (VIAL, 1968),
being the present study the second report so
far. In general, there is a great variation in
local population densities for plehodontids;
TEST & BINGHAM (1948) reported 0.0496
individuals / m2 of Plethodon cinereus, and
50 GUTIRREZ-LAMUS ET AL.
then BURTON & LIKENS (1975) estimated for
the same species densities ranging from
2.3670 to 2.5830 individuals / m2. Estimated
densities for other salamanders include
0.0070 individuals / m2 in Plethodon yonahlossee,
0.0220 individuals / m2 in Plethodon jordani
(GORDON et al., 1962), 0.4180-0.8440 indi-
viduals / m2 in Plethodon glutinosus
(SEMLITSCH, 1980), 0.4051-0.4989 indivi-
duals / m2 in A. lugubris (ANDERSON, 1960),
0.10-0.25 individuals / m2 in Aneides aeneus
(GORDON, 1952) and 0.1482-0.1729 indivi-
duals / m2 in Ensatina eschscholtzii (STEBBINS,
1954). Unfortunately, most reports on popu-
lation densities of plethodontids were calcula-
ted before herpetologists could deal with
imperfect detection through more sophistica-
ted approaches, which ultimately served to
avoid repercussions of poor detection on the
assessment of population size, population
density and any other vital rate.
Survivorship is dependent upon finding an
adequate refuge to obtain protection from pre-
dators and desiccation (SMYERS et al., 2002).
HUSTING (1965) reported survival rates of 0.72
for males and 0.60 for females in a Michigan
population of A. maculatum, and BLACKWELL et
al. (2004) also reported high survival rates in a
population of this species from Alabama, which
led them to the conclusion that adult survival
was the largest contributor to population growth
in A. maculatum. In the most comprehensive
study of survivorship of plethodontids, ORGAN
(1961) calculated life tables for five species of
Desmognathus and showed that there was a pro-
gressive increase in early survival rate from the
most aquatic species, D. quadramaculatus, to the
most terrestrial one, D. wrighti.
Survivorship estimates for B. altamazonica
including all captured animals are quite high
and did not vary too much. However, we
must take into account that we calculated
apparent survivorship and not real survival
rates, because without conducting a more
exhaustive study it was not possible to attri-
bute losses in the population to deaths or
emigration accurately. Generally, adult survi-
val is likely to be more important than
recruitment for population persistence,
because it determines how long a population
can persist without recruiting new indivi-
duals (SCHMIDT et al., 2005).
The sex ratios obtained during the present
study showed a predominant proportion of
females in the breeding population, which is
in agreement with data reported for several
species of plethodontid salamanders like
Plethodon vehiculum, Plethodon dunni
(DUMAS, 1956), P. yonahlossee (POPE, 1950),
D. quadramaculatus (O RGAN , 1961) and
A. maculatum (BLACKWELL et al., 2004). This
unbalanced sex ratio cannot be attributed to
differential mortality, as we found homoge-
neity across sexes and ages in survival rates. A
possible reason to explain our result would be
that B. altamazonica males do not exhibit
philopatry. On the contrary, other pletho-
dontids such as P. cinereus (TEST &
BINGHAM, 1948), A. aeneus (GORDON,
1952), Desmognathus fuscus, Desmognathus
carolinensis, D. wrighti (ORGAN, 1961) and
Eurycea wilderae (BRUCE, 1988) show sex
ratios unbalanced in favour of males. Finally,
for B. subpalmata (VIAL, 1968) and
Bolitoglossa nicefori (ORTEGA et al., 2009)
there is no bias between sexes, even though
they belong to the same genus as our study
species. Differences in sex ratios for species
belonging to the same genus have been also
reported in Plethodon and Desmognathus.
 POPULATION ECOLOGY OF A NEOTROPICAL SALAMANDER
Although sex ratio varied slightly among
months, there was on average three females per
male. In some species of Desmognathus, varia-
tions in sex ratio over time are due to the fact
that females move to the aquatic habitat during
brooding. That is not the case for B. altamazonica,
whose nesting microhabitats offer optimal con-
ditions and are commonly used also by non-
reproducing members of the population. Such
a pattern of habitat use has been described also
for B. subpalmata (VIAL, 1968).
Data reported here are a baseline essential
for evaluating population changes over time,
and for assessing relationships between sala-
mander populations and environmental fac-
tors. The present study can be used as a star-
ting point for comparison to other B. altama-
zonica populations along the wide range of
distribution of the species; such comparisons
will be useful when planning mitigation or
restoration projects.
Acknowledgement
The environmental manager of Jardn
Botnico de Villavicencio and E. Gomez pro-
vided access to the study place. R. Moreno-
Arias and two anonymous reviewers provided
helpful comments that greatly improved this
manuscript. This project complies with all
laws of Colombia and was conducted under
CORMACARENAs scientific research per-
mit (authorization No 000884), with funds
provided by Divisin de Investigaciones sede
Bogot - National University of Colombia
(No 8003167) and equipments donated by
IDEA WILD. This paper is from a portion of
a M. Sc. Dissertation completed under the
direction of J.D. Lynch and N. Ruiz-Rodgers
at Universidad Nacional de Colombia.
51
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 Basic and Applied Herpetology 25 (2011): 55-64

Behavioural responses of Iberian midwife toad tadpoles (Alytes

cisternasii) to chemical stimulus of native (Natrix maura and
Squalius pyrenaicus) and exotic (Procambarus clarkii) predators
Vera Gonalves1,*, Sandra Amaral2, Rui Rebelo2
1
Department of Evolutionary Biology Leo Pardi, University of Florence, Florence, Italy.
2
Department of Animal Biology and Centre for Environmental Biology, F.C.U.L., Lisbon, Portugal.

* Correspondence: Department of Evolutionary Biology Leo Pardi, University of Florence, Via Romana 17, 50125 Florence, Italy.
Phone: +055 2288200, Fax: +055 2288309-222565, Email: vera.goncalves@unifi.it
Received: 17 March 2011; received in revised form: 1 July 2011; accepted: 25 July 2011.

Predation can be an important force of selection, resulting in the evolution or learning of antipredator defen-
ces in amphibian larvae. In the laboratory, we compared the behavioural responses of the tadpoles of Alytes
cisternasii subjected to the chemical stimulus of an exotic predator, Procambarus clarkii, with the responses to
the chemical stimuli of two of its native predators, the snake Natrix maura and the fish Squalius pyrenaicus,
which employ different predation strategies. Tadpoles reacted more intensely to N. maura and then to P. clarkii,
with no significant responses to S. pyrenaicus. The alteration in the use of the vertical axis of the aquaria was
the antipredator behaviour more frequently used towards both native and exotic predators, and the adopted
behaviour was adequate to the activity period and predation strategy of each predator. Alytes cisternasii tad-
poles reacted to P. clarkii, a predator introduced about 20 years ago in the study area. These reactions may
result from a micro-evolutionary process, but may also be a fortuitous response to a non-familiar cue. We can
also not discard the possibility of learning by tadpoles since, due to the reproductive characteristics of this
species, it was not possible to collect egg masses before their release in the aquatic environment.

Key words: alien predators; Alytes cisternasii; antipredator behaviour; chemoreception; invasions; Procambarus clarkii.

Respuestas conductuales de las larvas de sapo partero ibrico (Alytes cisternasii) a los estmulos qumicos de depredado-
res nativos (Natrix maura y Squalius pyrenaicus) y exticos (Procambarus clarkii). La depredacin es una importante
presin selectiva que resulta en la evolucin o el aprendizaje de conductas antipredatorias en larvas de anfibios.
Comparamos en el laboratorio las respuestas conductuales de larvas de Alytes cisternasii expuestas a los estmulos qu-
micos procedentes de un depredador extico, Procambarus clarkii, con las respuestas a los estmulos qumicos de dos
de sus depredadores naturales, la culebra viperina (Natrix maura) y el cacho (Squalius pyrenaicus), los cuales utilizan
estrategias de depredacin diferentes. Las larvas reaccionaron de manera ms intensa a N. maura, seguida de P. clarkii,
sin que existieran respuestas significativas a S. pyrenaicus. La alteracin en el uso de la columna de agua fue la conduc-
ta antipredatoria ms utilizada ante los depredadores, ya fueran nativos o exticos, adecuando las larvas su comporta-
miento al periodo de actividad y a la estrategia de cada depredador. Las larvas de A. cisternasii reaccionaron ante los
estmulos procedentes de P. clarkii, un depredador introducido tan solo hace 20 aos en el rea de estudio. Estas reac-
ciones podran ser resultado de un rpido proceso micro-evolutivo, pero tambin ser una respuesta fortuita mostrada
ante un estmulo novel para las larvas. Tampoco podemos descartar la posibilidad de aprendizaje por parte de las lar-
vas ya que, dadas las caractersticas reproductoras de la especie, no fue posible colectar masas de huevos antes de que
las larvas eclosionaran y entraran en contacto directo con el medio acutico.

Key words: Alytes cisternasii; conducta antipredatoria; depredadores alctonos; invasiones; Procambarus clarkii;
quimiorrecepcin.
56 GONALVES ET AL.
Native and alien predators can be an
important force of selection in natural systems,
resulting in the evolution or learning of anti-
predator defences by prey populations. Several
studies have demonstrated that larval amphi-
bians are able to innately recognize and res-
pond to coexisting native predators (KATS et al.,
1988; SIH & KATS, 1994; KIESECKER &
BLAUSTEIN, 1997). In larval amphibians, the
development of these defences may include
changes in life history, morphology or beha-
viour (SKELLY & WERNER, 1990; LARDNER,
2000). Antipredator behaviour may include
the reduction of activity levels, alterations in
the use of different microhabitats or an incre-
ased use of refuges (KATS et al., 1988; SKELLY
& WERNER, 1990; KIESECKER et al., 1996;
KIESECKER & BLAUSTEIN, 1997; RELYEA,
2004; RICHTER-BOIX et al., 2007). Since pre-
dators differ in their predation strategies, prey
frequently exhibit specific antipredator beha-
viour (RELYEA, 2001, 2004). Thus, when
facing an introduced predator for the first
time, nave native prey may exhibit no antipre-
dator behaviour (KIESECKER & BLAUSTEIN,
1997; NYSTRM et al., 2001; POLO-CAVIA et al.,
2010), or may show behavioural modifications
of the antipredator tactics that have evolved as
a response to native predators, and that may be
inefficient against introduced predators
(GAMRADT & KATS, 1996).
In Portugal, a series of studies have shown
that an invading predator, the American red
crayfish, Procambarus clarkii (Girard, 1852),
predates egg masses and larvae of Southwest
Iberian amphibians (CRUZ & REBELO, 2005)
and that this exotic species may exclude several
species of these amphibians from their repro-
duction habitats (CRUZ et al., 2006). It is also
known that Iberian midwife toad tadpoles,
Alytes cisternasii Bosc, 1879, show some beha-
vioural modifications when faced with P. clarkii,
namely by modifying their use of stream bed
refuges and by fleeing to the margins during the
night (GONALVES et al., 2007). Alytes cisternasii
is an Iberian endemism commonly found in
semi-arid regions. After fertilization, the male
carries the string of eggs on its hind legs in the
terrestrial environment for about three weeks,
after which it deposits the eggs in the water,
mainly in small temporary streams (MRQUEZ,
1992). In the SW of Portugal these tadpoles
take from 3 to 5 months to metamorphose and
are subject to predation by a diverse array of
aquatic predators (R. Rebelo, personal observa-
tion). The types of predator-related stimuli to
which these tadpoles are sensitive are not yet
clearly identified. However the closely related
species Alytes muletensis is known to react to
chemical cues of the viperine snake Natrix
maura (SCHLEY & GRIFFITHS, 1998).
In aquatic ecosystems, chemical cues from
predators are particularly important for prey
in assessing predation risk (KATS & DILL,
1998). The chemicals to which prey respond
may originate from predator-specific odours
and/or from cues that are released by distur-
bed, injured or consumed conspecifics
(HETTYEY et al., 2010).
The objective of the present work is to com-
pare the antipredator behaviour of these tadpoles
in the presence of chemical cues from P. clarkii,
and from two of its native predators, the Iberian
chub Squalius pyrenaicus (Gnther, 1868) and
the viperine snake Natrix maura (Linnaeus,
1758). The native predators employ different
predation strategies: while the fish is a generalist
omnivore that actively searches for prey in the
bottom of streams as well as in the water
column (BLANCO-GARRIDO et al., 2003), the
 ANTIPREDATOR BEHAVIOUR OF A. CISTERNASII TADPOLES
viperine snake is a sit-and-wait predator that
mostly preys at the bottom or margins of ponds
and streams (GONZALO et al., 2008). The pre-
dation strategy of P. clarkii is intermediate it
actively searches for prey, but effectively only at
the bottom of the water bodies (CRUZ &
REBELO, 2005). We expected that tadpoles of
A. cisternasii would show appropriate antipreda-
tor responses to the chemical cues of their two
natural predators, being their similarity with
those elicited by the cues of the recently arrived
P. clarkii incompletely known.
MATERIALS AND METHODS
We used data from AMARAL (2004),
obtained in February 2004 (experiment 1),
and performed a similar experiment in April-
May 2005 (experiment 2). Part of our 2005
results were the subject of a previous paper
(GONALVES et al., 2007), concerning diffe-
rences between seasons. The present experi-
ments differed in the native predator used to
test tadpoles experiment 1 tested the effects
of S. pyrenaicus, while experiment 2 tested the
effects of N. maura.
All the animals involved in the experi-
ments were captured in the small streams of
the Field Station of the CBA the Herdade
da Ribeira Abaixo (Serra de Grndola, Baixo
Alentejo, SW Portugal; 380628.57N;
83414.56W). Tadpoles and fishes were
captured with dip-nets; crayfishes were cap-
tured with baited funnel traps and snakes
were captured by hand.
The Portuguese territory south of the
Tagus River is a region where no native cray-
fish ever existed (ALMAA, 1991). Tadpoles
for both experiments belonged to a popula-
tion that is in contact with P. clarkii since the
57
middle of the 1990s. For each tadpole tested
we measured head length (HL, mm) and
identified the developmental stage (GOSNER,
1960) (Table 1). These were compared
among treatments with a Kruskal-Wallis test.
Before the experiments, tadpoles were kept
separately in the biotherium of the field sta-
tion in PVC aquaria filled with spring water
for one week, under a 12:12 light-dark pho-
toperiod, and fed ad libitum with cooked let-
tuce and commercial fish food. The water
temperature was kept at 10-14C (experi-
ment 1) or 16-18C (experiment 2).
Experiments took place in opaque PVC
aquaria (40 x 60 x 37.5 cm), with the floor
covered with rocks placed in order to mimic a
stream bed. Each aquarium was filled with 35
litres of spring water, and we suspended an
opaque cage slightly sunk at the surface in the
centre of each aquarium. This cage was made
with a plastic bottle of 1.5 litres. Its ends were
cut and then covered with green net of fine
mesh (2 mm) and the lateral walls were pier-
ced, allowing for the circulation of water.
Number of replicates was as follows: experiment
1, empty cage (control treatment), 9 replicates;
cage with an individual of P. clarkii (alien pre-
dator treatment), 10 replicates; cage with an
individual of S. pyrenaicus (native predator tre-
atment) 10 replicates; experiment 2, empty
cage (control), 15 replicates; cage with an indi-
vidual of P. clarkii (alien predator treatment)
15 replicates; cage with an individual of N. maura
(native predator treatment) 15 replicates. For
the trials, the empty cages and the cages with
each of the three different predatory species
were randomly distributed by the experimental
aquaria, and five tadpoles were released in each
aquarium right after cage placement. The order
of the replicates was random.
58 GONALVES ET AL.
Both experiments ran in two series, first
diurnal and then nocturnal. During the
night, the tadpoles were observed with a low
intensity lantern, having been verified in pre-
liminary tests that this did not affect tadpole
behaviour (AMARAL, 2004). Each tadpole,
fish and crayfish was used only once; howe-
ver, the individuals of the diurnal series were
used in the corresponding nocturnal series.
Due to the difficulty to maintain N. maura in
captivity, the same two individuals were used
in the several replicates. Neither tadpoles nor
predators were fed during the experiments;
each replicate lasted a maximum of 12 hours.
The choice of the behaviours to record was
based on those described for other species
(KATS et al., 1988; KIESECKER et al., 1996;
KIESECKER & BLAUSTEIN, 1997; NYSTRM et al.,
2001; ALTWEGG, 2002). After being released
in the experimental aquarium, tadpoles were
given a 30 minute period for acclimatization.
Then, we recorded at minutes 30, 45 and 60
the following parameters, all consisting in tad-
Table 1: Head length (HL) and developmental stage, according to GOSNER (1960), of tadpoles from
experiments 1 and 2. HL values correspond to mean standard deviation. For the developmental stage
the modal class is presented.
Variable Treatment / predator
HL Control
P. clarkii
S. pyrenaicus
N. maura
Developmental Control
stage P. clarkii
S. pyrenaicus
N. maura
pole counts: use of refuges (under the crevices
formed by the stones of the aquarium floor,
tadpole totally visible vs. not visible or partially
visible), activity (active vs. inactive, activity
being defined as any manifestation of move-
ment when the observer saw the tadpole for
the first time), margin use (touching the wall
of the aquarium or the wall of the cage vs. not
touching marginal surfaces) and three varia-
bles representing the vertical microhabitat use,
use of the substratum, use of the water
column and use of the surface.
For each replicate, we calculated the average
number of tadpoles observed engaged in each
behaviour, using the records obtained at 30, 45
and 60 minutes. After checking for normality
(using Kolmogorov-Smirnov and Lilliefors
tests), the absolute frequencies of each behaviour
were compared with one-way analyses of the
variance (ANOVAs), followed by Fishers least
significant difference (LSD) post-hoc tests.
Instead of using the time of the day as an addi-
tional factor in the analysis, the results of diurnal
Experiment 1 (Winter) Experiment 2 (Spring)
Amaral (2004) Present study
17.71 1.81 15.92 1.89
(N = 45) (N = 75)
17.26 1.77 15.78 1.85
(N = 50) (N = 75)
17.90 1.51
(N = 50)
15.26 2.13
(N = 75)
25 41
25 41
27
37
 ANTIPREDATOR BEHAVIOUR OF A. CISTERNASII TADPOLES
and nocturnal trials were analysed separately
because, as stated above, we used the same indi-
viduals for both trials. Therefore, the results of
both trials were not independent from each
other. All analyses were performed with the pro-
gram Statistica version 5.5 (StatSoft).
RESULTS
Experiment 1
There were no differences among treatments in
tadpole length (H2, 145 = 2.88, P > 0.05) but there
was a difference in developmental stage (H2, 145 =
11.09, P < 0.01); tadpoles from the S. pyrenaicus
treatment were in a more advanced stage (Table 1),
presenting slightly longer hind limbs than those
shown by tadpoles from the other treatments.
During the day there were no significant dif-
ferences between the three treatments (control vs.
alien predator vs. native predator) in any of the
behaviours recorded. During the night there
were significant differences for the behaviour use
of refuge (F2, 26 = 5.18, P < 0.05). Tadpoles sub-
jected to P. clarkii cues decreased the use of refu-
ge compared with tadpoles subjected to the con-
trol treatment (LSD test, P < 0.01) (Fig.1).
Figure 1: Use of refuges by tadpoles during the
diurnal and nocturnal periods in experiment 1,
when they were subjected to chemical cues from
an alien predator (P. clarkii) and a native predator
(S. pyrenaicus). Values in the ordinate axis refer to
the average ( standard error) number of tadpoles
manifesting the specified behaviour. Lower case
letters (a, b) refer to groups significantly different
(P < 0.05) as defined by post-hoc tests.
59
Experiment 2
There were not differences among treatments
either in tadpole length (H2, 225 = 0.64, P > 0.05) or
in developmental stage (H2, 225 = 3.80, P > 0.05).
During the day, there were significant
differences between treatments in the
behaviours activity, use of the water
column and margin use (Table 2, Fig. 2).
All the differences were found between
tadpoles subjected to N. maura cues and
those subjected to the other treatments.
Tadpoles subjected to N. maura cues were
less active, decreased the use of the water
column and increased the use of margins
(LSD test, P < 0.05 for all mentioned
variables and pair wise comparisons invol-
ving N. maura).
During the night, there were significant
differences for the use of the substratum and
for the use of the surface (Table 2, Fig. 3).
Tadpoles subjected to N. maura cues decrea-
sed the use of the substratum and increased
their permanence at the surface of the experi-
mental aquarium (LSD test, P < 0.05 for all
mentioned variables and pair wise compari-
sons involving N. maura).
60 GONALVES ET AL.
Figure 2: Recorded behaviours of tadpole (a) acti-
vity, (b) use of the water column and (c) margin
use during the diurnal period in experiment 2,
when they were subjected to chemical cues from
an alien predator (P. clarkii) and a native predator
(N. maura). Values in the ordinate axis refer to the
average ( standard error) number of tadpoles
manifesting the specified behaviour. Lower case
letters (a, b) refer to groups significantly different
(P < 0.05) as defined by post-hoc tests.
b c
DISCUSSION
The alteration in the use of the vertical
axis of the aquaria by tadpoles seems to be the
most common antipredator behaviour shown
as a response to the chemical cues from nati-
ve and alien predators in our experiments,
except for S. pyrenaicus, towards which we
did not find significant responses. In the
experiment 2, as expected, tadpoles presented
strong antipredator responses to N. maura
during both nocturnal and diurnal periods.
During the day tadpoles decreased their acti-
vity and fled from the water column towards
the margins, whereas at night tadpoles remai-
ned more frequently at the surface. In the
laboratory, N. maura presents both nocturnal
a
and diurnal activity, using mainly the areas of
the aquaria close to the substratum during
the day and those close to the margins at
night (S. Scali, unpublished data). Therefore,
tadpoles diminished the probability of being
found in the same microhabitat as their natu-
ral predator. The diurnal reduction of activity
might also be an adaptive behaviour towards
a predator that visually locates its prey
(HAILEY & DAVIES, 1986).
The behavioural responses to P. clarkii
were best observed in the experiment 1, with
the decrease of the use of refuge during the
night. However, this pattern was not found
in the experiment 2, which may be related to
the more advanced developmental stages of
the tadpoles that were used in this experi-
 ANTIPREDATOR BEHAVIOUR OF A. CISTERNASII TADPOLES 61

Table 2: Results of one-way ANOVAs to compare among treatments tadpole behaviours recorded
during the day and during the night in experiment 2. Values in bold indicate significant differences
among treatments.

Behaviour Source of variation Diurnal trials Noctural trials

Sum of d.f. F P Sum of d.f. F P
squares squares

Refuge use Treatment 34.53 2 1.76 0.18 6.53 2 0.52 0.60

Error 412.67 42 264.27 42
Activity Treatment 4.04 2 4.03 <0.05 0.31 2 0.44 0.65
Error 21.07 42 14.93 42
Water column Treatment 4.58 2 3.81 <0.05 1 2 0.69 0.51
Error 25.2 42 3 42
Substratum Treatment 76.84 2 2.27 0.11 88.31 2 4.89 <0.05
Error 710.26 42 378.93 42
Surface Treatment 86.58 2 2.21 0.12 98.8 2 4.62 <0.05
Error 824 42 449.2 42
Margins Treatment 12 2 4.31 <0.05 21.38 2 1.23 0.30
Error 10 42 363.6 42

ment. As the tadpoles of A. cisternasii appro-
ach metamorphosis, they tend to spend more
time near the surface and margins of the
aquaria (GONALVES et al., 2007), therefore
reducing their presence at the substratum,
where refuges were located.
According to our results, A. cisternasii tad-
poles showed unique behavioural alterations
in the presence of P. clarkii, different from
those shown in the presence of two of their
natural predators (based in our results, it is
questionable whether S. pyrenaicus really
constitutes a danger to these tadpoles). This
could be an adequate behaviour to compen-
sate for the predation strategy of P. clarkii,
which is a nocturnal tactile predator, active at
the substratum level (HARPER et al., 2002).
However, we have no way to positively ascri-
be this response as an antipredator behaviour
adopted towards this alien species or simply
as a reaction to a new, unknown cue. To cla-
rify the meaning of this finding, future expe-
riments will have to assess the effects of che-
mical cues of non-predatory novel stimuli
that may modify tadpole behaviour by, for
instance, providing cues of food availability.
Still, in the case these responses are new
and specific to P. clarkii, they could be the
result of selection. There are several reported
cases of prey rapid evolution in response to
selection from predator invaders (e.g.
SCHLAEPFER et al., 2005; STRAUSS et al.,
2006) but, to our knowledge, the shortest
time period reported for this to happen in
amphibian species is 50-60 years
(KIESECKER & BLAUSTEIN, 1997). Another
species of the same genus, A. muletensis, was
shown to be responsive to the chemical cues
of N. maura, an introduced predator in
Majorca island (MOORE et al., 2004), but
the introduction of this snake in the island
is supposed to have taken place more than
2000 years ago. Our period of coexistence of
less than 30 years is quite short in evolutio-
62 GONALVES ET AL.

Figure 3: Recorded behaviours of tadpole (a) use of the substratum and (b) use of the surface during the noc-
turnal period in experiment 2, when they were subjected to chemical cues from an alien predator (P. clarkii)
and a native predator (N. maura). Values in the ordinate axis refer to the average ( standard error) num-
ber of tadpoles manifesting the specified behaviour. Lower case letters (a, b) refer to groups significantly
different (P < 0.05) as defined by post-hoc tests

a b

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before hatching and the individuals used in
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that have never been in contact with P. clarkii
when faced with this species.
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 Basic and Applied Herpetology 25 (2011): 65-71

Reproductive cycles in Bufo mauritanicus (Schlegel, 1841)

in a wet area of Beni-Belad (Jijel, Algeria)
Omar Kisserli1, Salaheddine Doumandji2, Jean-Marie Exbrayat3,*
1
Laboratoire de Pharmacologie et Phytochimie, Facult des Sciences, Universit de Jijel, Ouled Aissa, Jijel, Algeria.
2
Laboratoire de Zoologie Agricole et Forestire, Institut National Agronomique. El - Harrach, Alger, Algeria.
3
Universit de Lyon, Laboratoire de Biologie Gnrale, Universit Catholique de Lyon et Reproduction et Dveloppement Compar, Ecole
Pratique des Hautes Etudes, Lyon, France.

* Correspondence: Universit de Lyon. UMRS 449. Biologie Gnrale, Universit Catholique de Lyon, Reproduction et
Dveloppement Compar, EPHE 25 rue du Plat, F-69288 Lyon Cedex 02, France. Phone: +33 472325036, Fax: +33 472325066
E-mail: jmexbrayat@univ-catholyon.fr
Received: 1 February 2011; received in revised form: 1 July 2011; accepted: 17 July 2011.

Bufo mauritanicus is an anuran amphibian living in North Africa. Reproductive cycles of this species are not
well known, especially in populations living in Algeria. This study is devoted to the knowledge of the repro-
ductive cycle in a population living in the wet area of Beni Belad, under a Mediterranean climate characte-
rized by two rainy seasons, January until May and September until December. The examination of the gonads
of both sexes allowed us to describe continuous cycles in males and females.

Key words: Bufo mauritanicus; reproduction; sexual cycle.

El ciclo reproductor de Bufo mauritanicus (Schlegel, 1841) en el humedal de Beni-Belad (Jijel, Algeria).
Bufo mauritanicus es un anuro que habita en el Norte de frica. Los ciclos reproductivos de esta especie han
sido poco investigados, especialmente en lo que se refiere a las poblaciones de Argelia. Este estudio profundi-
za en el conocimiento del ciclo reproductor de una poblacin de B. mauritanicus en la zona hmeda de Beni
Belad, en Argelia, caracterizada por un clima mediterrneo con dos estaciones lluviosas, de enero a mayo y
de septiembre a diciembre. El estudio de las gnadas de los dos sexos nos ha permitido describir la existencia
de ciclos reproductivos continuos tanto en machos como en hembras.

Key words: Bufo mauritanicus; ciclo sexual; reproduccin.

Reproductive cycles of amphibians have GUEYDAN-BACONNIER et al., 1984a,b; PUJOL,

been studied in many species (NEYRAND DE
LEFFEMBERG & EXBRAYAT, 1995; EXBRAYAT et
al., 1998) although in general sexual cycles
have been more investigated in males than in
females. The reproductive cycles of African spe-
cies are rather little known. For example, some
studies detected continuous annual cycles in
several species (Bufo regularis, Ptychadena mac-
carthyensis, P. oxyrhynchus, Phrynobatrachus cal-
caratus, Xenopus laevis; DELSOL et al., 1980,
1981, 1995; GUEYDAN-BACONNIER, 1980;
1985; PUJOL & EXBRAYAT, 1996, 2000, 2001;
EXBRAYAT et al., 1998; DU PREEZ et al., 2005;
VAN WYK et al., 2005). On the other side, dis-
continuous male and female cycles of repro-
duction have also been described in the vivipa-
rous Nectophrynoides occidentalis and other rela-
ted species (LAMOTTE & TUCHMAN-
DUPLESSIS, 1948; LAMOTTE et al., 1964,
GAVAUD, 1976, 1977; XAVIER, 1986).
Bufo mauritanicus is a nocturnal anuran
endemic to North Africa, and it is found all
66 KISSERLI ET AL.
across the Maghreb, including Algeria,
Morocco, and Tunisia (BONS & GENIEZ,
1996; SCHLEICH et al., 1996). Despite its wide
distribution and relative abundance, few
works have been devoted to the biology of B.
mauritanicus, including a review in SCHLEICH
et al. (1996) and a study by GUILLON et al.
(2004) quoting data related to various ecological
aspects, and where the rarity of B. mauritanicus
in semi-arid areas of Morocco was remarked.
The reproductive onset of this species depends
greatly on the local conditions (SCHLEICH et
al., 1996). KISSERLI & EXBRAYAT (2006)
reported preliminary data about the male
reproductive cycle in this species. In males,
sexual cycle is continuous with an increase in
the number of spermatozoa in August, and a
minimum in April, just after the breeding
period, revealed by the presence of fertilized
eggs in the field (KISSERLI & EXBRAYAT, 2006).
However, information regarding female repro-
ductive cycles is still lacking. The aim of the
present work was to analyse in detail the repro-
ductive cycle and variations in the gonadal tis-
sues of both male and female B. mauritanicus
along the year and its relationships to environ-
mental factors, namely precipitation. The
analysis of hormones present during folliculo-
genesis was also investigated in order to
understand hormonal regulation of female
reproductive features.
MATERIALS AND METHODS
The studied animals were randomly cap-
tured in the wet area of Beni-Belaid (Jijel,
Algeria) throughout the year. This area is
limited by the Mediterranean Sea in its nor-
thern part, by an agricultural area in its sou-
thern part, by Oued El-Kebir to the West and
by Zhour Oued and pond areas to the East.
Altitude in this locality varies from 0 to 10 m
above sea level. This area is submitted to two
rainy seasons, one from January until May
and other from September until December,
and one dry season, from June until August.
In total, 12 males and 27 females were collec-
ted across the year as follows: three males and
11 females during the wet season between
January and May; four males and eight fema-
les during the dry season (between June and
August) and five males and eight females
during the wet season between September
and December. This limited sampling did
not threaten the population studied.
Animals were euthanized with tricaine
mesylate (MS 222). Sexual organs were
immediately fixated with 10% formalin after
dissection. Left gonads were included with
paraffin, cut in 5 to 7 m thick sections using
a micrometer and stained with the Romeiss
azan according to Exbrayat (2001).
Histochemical stainings were used to cha-
racterize the chemical composition of tissues.
For that, frozen sections of gonads (14 m
thick) were stained with Sudan black in order
to detect lipids (MARTOJA & MARTOJA,
1967). Additional paraffin sections were also
stained with Periodic Acid Schiff (PAS) and
alcian blue-PAS in order to detect the presen-
ce of neutral and acidic carbohydrates accor-
ding to MARTOJA & MARTOJA (1967).
The detection of 17- estradiol in ovaries
was performed using immunohistochemical
techniques. For that, sections were first incu-
bated with H2O2 (Fluka, Buchs, Switzerland)
in order to eliminate endogenous peroxida-
ses, and then with bovine serum albumin
(Sigma, St. Louis, Missouri, USA) to elimi-
nate non-specific reactions. After this, a spe-
 BUFO MAURITANICUS REPRODUCTIVE CYCLE 67

cific antibody directed against 17- estradiol DISCUSSION

(Euromedex, Souffelweyersheim, France) was
applied on the sections. After rinsing, immu-
noreactions were visualized with a streptavi-
din-biotin amplification test (Kit LSAB 2,
Dako, Glostrup, Denmark) using amino
ethyl carbazole (AEC) as a chromogen.
Presence of hormone was labelled by a red
precipitate. Controls were performed by dele-
ting the first antibody. The observation of
adjacent tissues was used as internal controls.
RESULTS
The histological study of both male and
female reproductive organs of B. mauritani-
cus showed that spermatogenesis, oogenesis
and folliculogenesis were continuous, and
comparable to the ones observed in most
African anurans. Like other amphibians, ste-
roid hormones were found in the follicle wall
(FERNANDEZ & RAMOS, 2003).
In several African anurans (B. regularis,
P. maccarthyensis, P. oxyrhynchus, P. calcaratus
or X. laevis), sexual cycles are continuous in

The histological examination of the sections

of testis revealed the presence of all the cellular
categories of spermatogenesis throughout the
year (Fig. 1a). The spermatozoa were anchored
in Sertoli cells, ready to be released in the light of
the seminiferous tube. Presence of lipids was
detected in Leydig-like cells (Fig. 1b).
The folliculogenic dynamic was described
in ovaries. Seven stages have been found (sta-
ges Ia, Ib, II, III, IVa, IVb, V). Stages IVa an
IVb were the vitellogenic stages at which the
oocyte was increasingly filled with yolk plate-
lets (Figs. 2a,b). Stage V was the final stage of
maturation at which ovulation can occur. In
addition, several atretic follicles were obser-
ved. In each stage, the oocyte was surrounded
with a more or less thick vitelline membrane
that was PAS positive showing the presence
of mucopolysaccharides (Fig. 2c). Presence of
lipids was detected in the cortical part of previ-
tellogenic and vitellogenic oocytes (Fig. 2d). Figure 1: Cross sections of Bufo mauritanicus testis.
The immunohistochemical detection of 17- (a) All the spermatogenetic categories are present.
FS: Spermatozoa fascia, LTS: Lumen of the semini-
estradiol revealed the presence of this hor-
ferous tube, SP: Spermatid. Bar = 50 m.
mone in the follicular cells and connective (b) Detection of lipids on frozen sections stained
theca in both previtellogenic and vitellogenic with Sudan black. CL: Leydig-like cells, FS:
stages (Figs. 2e,f ). Spermatozoa fascia. Bar = 50 m.
68 KISSERLI ET AL.

Figure 2: Cross sections of Bufo mauritanicus ovary. (a) Cross section of B. mauritanicus ovary showing previ-
tellogeic follicles. C: cytoplasm, N: nucleus, n: nucleoli, St Ia: stage Ia follicle, St II: stage II follicle,
TC: connective theca. (b) B. mauritanicus ovary showing vitellogenic follicles. St IVa: oocyte in early vitelloge-
nesis, St V: oocyte at the end of vitellogenesis. (c) Presence of PAS positive material in the cortical part of the
vitellogenic oocytes and mucopolysaccharidic nature of vitellin membrane (mv). (d) Detection of lipids on fro-
zen sections stained with black Sudan. L: Lipids, VO: Vitellogenic oocyte. (e) Immunohistochemical detection
of 17-estradiol in follicles, during previtellogenesis. St. II: stage II follicle. The arrow indicates the presence of
labelled follicle cells. (f) Immunohistochemical detection of 17-estradiol in follicles during vitellogenesis. The
arrow indicates the presence of labelled follicle cells. Bar = 50 m.
 BUFO MAURITANICUS REPRODUCTIVE CYCLE
both males and females (DUMONT, 1972;
DELSOL et al., 1980, 1981, 1995; GUEYDAN-
BACONNIER, 1980; GUEYDAN-BACONNIER et
al., 1984a,b; PUJOL, 1985; HAUSSEN &
RIEBESELL, 1991; PUJOL & EXBRAYAT, 1996,
2000, 2001; EXBRAYAT et al., 1998;
SANCHEZ & VILLECCO, 2003; DU PREEZ et
al., 2005; VAN WYK et al., 2005). A conti-
nuous reproductive cycle has also been observed
in the African caecilian Boulengerula taitanus
living in Kenya (MEASEY et al., 2008). In the
viviparous toad N. occidentalis living in
Nimba Mount, Guinea, sexual cycles are dis-
continuous in males (GAVAUD, 1976, 1977)
as well as in females (LAMOTTE & TUCHMAN-
DUPLESSIS, 1948; LAMOTTE et al., 1964,
XAVIER, 1986).
Our results extent the results found in
males of B. mauritanicus by KISSERLI &
EXBRAYAT (2006) to females regarding to the
continuous nature of the reproductive cycle
of this species. In several African toads inclu-
ding B. mauritanicus, gonads are ready to
produce germ cells when external conditions
are favourable. In African toads living in
semi-arid areas, reproduction is narrowly lin-
ked to rainfalls coupled with a brutal decrea-
se of temperature (GUILLON et al., 2004).
Only in N. occidentalis and other member of
genus Nectophrynoides (XAVIER, 1986), sexual
cycles are discontinuous. This fact is certainly
related to viviparity or ovoviviparity, modes
of reproduction protecting embryos from
external conditions during their develop-
ment. Reproduction of B. mauritanicus
depends on local climate (SCHLEICH et al.,
1996) and breeding is observed when rain
falls down. When precipitations are insuffi-
cient, reproduction does not occur during
several successive years (SCHLEICH et al.,
69
1996). This situation can be compared to
that of B. regularis. When this species is living
in a semi-arid area, it is ready to breed
throughout the year, rainfall being the relea-
sing factor of breeding (PUJOL, 1985; PUJOL
& EXBRAYAT, 1996, 2001). So, reproduction
of B. mauritanicus is an additional example of
adaptation to seasonal variations in an
African amphibian.
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72
 Basic and Applied Herpetology 25 (2011): 73-80

Age structure of Levant water frog, Pelophylax bedriagae, in

Lake Slkl (Western Anatolia, Turkey)
Kerim iek*, Meltem Kuma, Diner Ayaz, Ahmet Mermer, . Deniz Engin
Ege University, Faculty of Science, Biology Department, Zoology Section, Bornova-Izmir, Turkey

* Correspondence: Ege University, Faculty of Science, Biology Department, Zoology Section, 35100, Bornova-Izmir, Turkey. Phone:
+90 (232) 3112409, Fax: +90 (232) 3881036. E-mail: kerim.cicek@ege.edu.tr, kerim.cicek@hotmail.com
Received: 8 June 2011; received in revised form: 23 September 2011; accepted: 23 September 2011.

During the present study, we obtained data via skeletochronology on the age structure of Levant water frog,
Pelophylax bedriagae, in Lake Slkl (Manisa, Turkey). The mean snout-vent length was 56.1 mm (SD = 7.7)
for males and 64.5 mm (SD = 14.8) for females. While both sexes reached sexual maturity following their second
hibernation, the modal age was two years for males and three years for females. The average age of the adult popu-
lation was 2.50 years (SD = 0.65, range= 2-4) in males and 2.95 years (SD = 0.99, range = 2-5) in females.
Furthermore, the threatening factors of Lake Slkl population were outlined.

Key words: ; Pelophylax bedriagae; skeletochronology; Turkey; Western Anatolia.

Estructura de edad de la rana verde levantina, Pelophylax bedriagae, en el lago Slkl (Anatolia occidental, Turqua).
Durante el presente estudio obtuvimos datos, mediante un anlisis esqueletocronolgico, sobre la estructura de edades
de la rana verde levantina, Pelophylax bedriagae, en el lago Slkl (Manisa, Turqua). La longitud media hocico-cloaca
fue de 56,1 mm (SD = 7,7) en machos y 64,5 mm (SD = 14,8) en hembras. Mientras que en ambos sexos la madurez
sexual se alcanz tras la segunda hibernacin, la edad modal fue dos aos para los machos y tres aos para las hembras.
La edad media en la poblacin adulta fue 2,50 aos (SD = 0,65, rango = 2-4) en los machos y 2,95 aos (SD = 0,99,
rango = 2-5) en las hembras. Por ltimo, se indicaron algunas de las amenazas que afectan a la poblacin estudiada.

Key words: occidental; edad; esqueletocronologa; Pelophylax bedriagae; Turqua.

Palearctic water frogs of the genus
Pelophylax Fitzinger, 1843 contain 22 taxa
and are widely distributed in Eurasia (Beerli,
1995; Frost, 2011). The Levant water frog,
Pelophylax bedriagae, is distributed across the
eastern Mediterranean (PAPENFUSS et al.,
2008; FROST, 2011). In Turkey, the species is
widespread along the western and southern
parts of Anatolia (PAPENFUSS et al., 2008).
Pelophylax bedriagae is threatened by habi-
tat loss as a consequence of wetland drainage,
pollution, drought and urbanization of coastal
areas (PAPENFUSS et al., 2008; AMPHIBIAWEB,
2011). It is also threatened by collection for
human consumption, being the individuals
collected from Turkey exported to western
Europe (BARAN et al., 1992; PAPENFUSS et al.,
2008). Its populations tend to decrease, in
spite of which the species is included in the
category of Least Concern in the Red List of
the International Union for Conservation of
Nature (PAPENFUSS et al., 2008).
Determining the age structure of popula-
tions is essential in understanding the popu-
lation dynamics and breeding of species.
Regarding amphibians and reptiles, skele-
tochronology is a widely used method for age
estimation (CASTANET & SMIRINA, 1990;
74 IEK ET AL.
CASTANET et al., 1993; SMIRINA, 1994).
Skeletochronology is based on the observa-
tion of annual growth rings found in bones;
these rings are formed during the hibernation
phases, and are commonly known as lines of
arrested growth (LAGs) (CASTANET et al.,
1977). In many amphibians and reptiles, ske-
letochronology is a non-destructive techni-
que as it can be performed on the phalanges,
without sacrificing the individuals (SMIRINA,
1972; HEMELAAR, 1985; CASTANET &
SMIRINA, 1990). The aim of the present study
was to assess, through the skeletochronologi-
cal study of phalanges, the age structure of
P. bedriagae population inhabiting Lake
Slkl in order to retrieve information
about growth, size at maturity and longevity.
MATERIALS AND METHODS
The study site, Lake Slkl, is located on
the northeastern slope of Mount Spil
(38.565035 N, 27.532617 E, 612 m above
seal level), in Manisa (Western Anatolia,
Turkey). The surface area of the lake is nearly
1.58 ha and its depth 2-4 m. Three amphi-
bian species other than P. bedriagae inhabit
the lake: Pseudopidalea viridis, Lissotriton vul-
garis and Triturus karelinii.
A total of 40 individuals (four juveniles,
14 males, and 22 females) were captured on
May 4th 2010, at the end of the breeding sea-
son, from Lake Slkl. The sex of all indivi-
duals was determined through the observa-
tion of vocal sacs and nuptial pads present in
males, and their snout-vent lengths (SVL)
were measured to the nearest 0.1 mm using
digital calipers. The second phalange of the
fourth toe of the right hind limb was sectio-
ned and stored in 70% alcohol. Then, the
individuals were released in the same place
they were captured.
The bones were fixed in 10% buffered
formalin for 24 hours and then left in tap
water for 24 hours. Decalcification was per-
formed with 5% nitric acid for 2-4 hours
according to the size of the bone, after which
bones were kept in tap water for another 24
hours in order to wash the acid. Tissue sam-
ples were embedded in paraffin and 15-micron
thin transversal sections were obtained from
the middle of the diaphysis. The sections
were stained by immersion in Ehrlichs hema-
toxylin for 20 minutes. LAGs were counted
by two observers who were blind to the iden-
tification of the individuals. All mount pre-
parations were photographed with an
Olympus LC20 Soft Imaging System
(Olympus, Mnster, Germany) and observed
with a light microscope. For each phalange,
we selected at least three cross sections from
the mid-diaphyseal level, the area of the bone
with the smallest marrow cavity. The diame-
ters of medullary cavity (MC), metamorpho-
sis line (ML), resorption line (RL), each visi-
ble LAG, and periosteal outer margin were
measured in each individual in order to esti-
mate endosteal resorption. When diameter of
innermost visible LAG was > 2 standard
deviations greater than its group mean, the
first LAG was considered as eroded, being the
innermost visible LAG actually the second
one (e.g. TSIORA & KYRIAKOUPOLOU-
SKLAVOUNOU, 2002; GUARINO et al., 2008).
The age of maturity was identified using
the first decreasing interval between LAGs,
which is supposed to indicate the attainment
of sexual maturity as proposed by
KLEINENBERG & SMIRINA (1969) and widely
used by many authors (e.g. SMIRINA, 1994;
 AGE OF PELOPHYLAX BEDRIAGAE FROM WESTERN ANATOLIA 75

TSIORA & KYRIAKOPOULOU-SKLAVOUNOU,
2002; YILMAZ et al., 2005; GUARINO et al.,
2008; GL et al., 2011). Besides, the sexual
maturity was confirmed when possible
through the observation of the sexual secon-
dary characters mentioned above. Longevity
was determined as the age of the oldest indi-
vidual of each sex in the population.
SVL and age structure were compared
between sexes with a t-test and a Mann
Whitneys U-test, respectively. Growth was
estimated by the equation of von
BERTALANFFY (1983), which has been pre-
viously used in several studies on amphibians
(ARNTZEN, 2000; MIAUD et al., 2001;
COALNICEANU & MIAUD, 2003, SARASOLA-
PUENTE et al., 2011). The growth formula
was applied as used by HEMELAAR (1988):
SVLt = SVLmax - (SVLmax - SVLmet) e-K(t - t met )
where SVLt is the average length at age t,
SVLmax the average maximum length, SVLmet
the average length at metamorphosis, t the
number of growing season experienced (age),
tmet the intercept with time axis (time of meta-
morphosis), and K the growth rate coeffi-
cient. Units are given in growth increase per
Table 1: Snout-vent length mean values and ranges related to each sex and age, as estimated by skele-
tochronology, in Pelophylax bedriagae from Lake Slkl.
year. SVLmax, K, tmet and their standard errors
were estimated through the nonlinear least-
square regression with R version 2.12.2 (R
DEVELOPMENT CORE TEAM, 2011). The
alpha level was set to 0.05.
RESULTS
The mean SVL was 42.4 mm (SD = 7.76;
N = 4) in juveniles, 56.1 mm (SD = 7.68;
N = 14) in males and 64.5 mm (SD = 4.61;
N = 22) in females (Table 1). The females were
larger than the males (t34 = 2.25; P = 0.031).
LAGs were seen in all phalangeal sections
and their sharpness was different. They were
very clear in 29 out of the 40 studied indivi-
duals (Fig. 1a), barely perceptible in another
nine, and different in thickness in the remai-
ning two specimens (Fig. 1b). Furthermore,
double resting lines (DL) were observed in
three individuals (Fig. 1c). The first LAG
was entirely resorbed by the endosteal bone
in 16 individuals (one juvenile, seven males
and eight females) and partially resorbed by
the endosteal bone in the rest of the exami-
ned individuals.
The average age was 2.50 years (SD = 0.65;
range 2-4) in males and 2.95 years (SD = 0.99;

Age (years) Juveniles Males Females

N Mean (SD) Range N Mean (SD) Range N Mean (SD) Range

1 3 38.6 (1.3) 37.0-39.3

2 1 53.9 8 52.6 (3.2) 50.4-60.0 8 59.8 (11.4) 45.8-83.1
3 5 58.2 (8.4) 50.7-72.2 10 64.0 (14.3) 52.2-86.2
4 1 73.2 1 64.2
5 3 78.6 (22.0) 53.2-92.4

Total 4 42.4 (7.8) 37.0-53.9 14 56.1 (7.7) 50.4-73.2 22 64.5 (14.6) 45.8-92.4
76 IEK ET AL.

Figure 1: Phalangeal cross-sections of Pelophylax bedriagae from Lake Slkl (Western Anatolia). (a) five
year-old male (73.5 mm). (b) two year-old female (53.9 mm). (c) four year-old female (56.0 mm). (d) 1 year-
old juvenile (SVL = 37.0 mm). dl: double resting lines, EB: endosteal bone, fc: food canal, MC: marrow
cavity, rl: resorption line. Arrows point to lines of arrested growth and resorption lines. Bar = 100 m.

range 2-5) in females (Table 1). No statistical DISCUSSION

difference was observed between males and
females in terms of age structure (U = 115.50;
P = 0.128). Although both sexes commonly
reached sexual maturity after their second
hibernation, some females reached sexual
maturity after their third hibernation.
Snout-vent length at metamorphosis
(SVLmet) was estimated as 26 mm (SD = 12.36;
N = 14), by measuring newly metamorpho-
sed individuals at the end of June from Lake
Slkl (Fig. 1d). According to the von
Bertalanffy equation, the SVLmax was calcula-
ted as 90.22 (SE = 31.89), K as 0.30 (SE = 0.33)
and tmet as 0.03 (SE = 0.87) (Fig. 2).
This study revealed that the estimated
average age and longevity of P. bedriagae
from Lake Slkl were, respectively, 2.50
and 4 years in males and 2.95 and 5 years in
females. The individuals reached sexual
maturity after their second hibernation, and
the modal age was two years for males and
three years for females.
The previous studies reported that the
first LAG generally disappeared (fully or
partially) after reaching sexual maturity
(e.g. ROZENBLUT & OGIELSKA, 2005;
KYRIAKOPOULOU-SKLAVOUNOU et al., 2008;
 AGE OF PELOPHYLAX BEDRIAGAE FROM WESTERN ANATOLIA 77

SOCHA & OGIELSKA, 2010, GL et al.,

2011). The first LAG of Lake Slkl popu-
lation was entirely or partially resorbed by
the endosteal bone. Besides, double resting
lines in some individuals were also detected.
The double resting lines in the bone are
generally formed as a result of either a short
activity period during hibernation
(HEMELAAR & VAN GELDER, 1980), a starva-
tion period, an exposure to cold temperatu-
res during the activity period (SMIRINA et
al., 1986) or other environmental factors
(SOCHA & OGIELSKA, 2010). Figure 2: Growth curve of Pelopghylax bedriagae from
Table 2 shows some age and growth para- Lake Slkl. SVLt= 90.2 - (90.2 - 26.0) e-0.30(t - 0.03).
meters of other Pelophylax species. In this
genus the maximum longevity reported was Slkl, the maximum longevity was higher
12 years in Caucasian populations (namely in females than in males, as previously repor-
for P. esculentus and P. lessonae) (SHALDYBIN, ted by many other researchers working with
1976; ALEXANDROVSKAYA & KOTOVA, 1986), green frogs (TSIORA & KYRIAKOPOULOU-
while in European populations it was 11 SKLAVOUNOU, 2002; ERIMI, 2005;
years. Generally, longevity tends to increase KYRIAKOPOULOU-SKLAVOUNOU et al., 2008;
in northern or mountain populations and it SOCHA & OGIELSKA, 2010; GL et al.,
tends to decrease in southern or lowland 2011). Nevertheless, we must consider that
populations (SMIRINA, 1994). In Lake sampling at Lake Slkl was performed on

Table 2: Overall range of ages of adult individuals and values by sexes of mean population age, age at sexual
maturity (MAT) and growth rate coefficient (K) of some Pelophylax species studied by skeletochronology.
M: males, F: females.

Species Origin Rangea Meana (M-F) MATa (M-F) K (M-F) Reference

P. caralitanus Anatolia 2-10 2-3 ERIMI (2005)

P. ridibundus Russia 4-11 SHALDYBIN, 1976; ALEXANDROVSKAYA &
KOTOVA, 1986; SMIRINA, 1994
Georgia 2-7 2.78-4.03 2 GOKHELASHVILI & TARKHNISHIVILI (1994)
Anatolia 1-7 3.90-3.72 2-4 YILMAZ et al. (2005)
Anatolia 2-8 2 GL et al. (2011)
Anatolia 4-11 3-4 GL et al. (2011)
Greece 1-5 2.96-3.73 1 KYRIAKOPOULOU-SKLAVOUNOU et al. (2008)
P. esculentus complex Romania 4-10 5.0-6.7 4 0.22-0.28 COGLNICEANU & MIAUD (2003)
Poland 2-7 4.1 2-3 0.76-0.59 SOCHA & OGIELSKA (2010)
P. epeirotica Greece 1-5 2.82-3.22 1 0.88-0.49 TSIORA & KYRIAKOPOULOU-SKLAVOUNOU (2002)
P. bedriagae Anatolia 2-5 2.50-2.95 2 0.30 Present study
78 IEK ET AL.
a single day and at the end of the breeding
season, and older individuals might have alre-
ady come back to their refuges, thus causing
a bias in estimation of age structure and an
underestimation of maximum longevity.
The growth rate estimated in P. bedriagae
population from Lake Slkl (0.30) is lower
than that of most European Pelophylax spe-
cies (0.22-0.88; Table 2), which could be
related to habitat quality, environmental con-
ditions, and predator pressure. Lake Slkl
is surrounded by cherry orchards. The wastes
of agricultural pesticides used in orchards are
mixed with rainwater or spilled directly to
the lake. In addition, the owners of the
surrounding orchards use the water of the
lake for irrigation. Another element of pres-
sure is predation from the common carp
(Cyprinus carpio), which has been repeatedly
introduced to the lake. These fishes may
reach body sizes up to 40-50 cm.
In conclusion, when the Lake Slkl
population of P. bedriagae is compared with
other Caucasian and European Pelophylax
populations, the age composition resembles
that of the European ones. Furthermore,
there are anthropogenic factors that threaten
this population. Younger average age of the
studied population as well as lower longe-
vity, survival and growth rate in comparison
with other Pelophylax populations could be
caused by impoverished habitat quality and
environmental conditions, as well as by pre-
dator pressure.
Acknowledgement
We thank Derek H. Ogle for your help in
using R version 2.12.2 and Ivelin Mollow for
reviewing English style.
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 Basic and Applied Herpetology 25 (2011): 81-96

Population size, habitat use and movement patterns

during the breeding season in a population of Perezs frog
(Pelophylax perezi) in central Spain
Gregorio Snchez-Montes1, Iigo Martnez-Solano2,*
1
Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain.
2
Instituto de Investigacin en Recursos Cinegticos, CSIC-UCLM-JCCM, Ciudad Real, Spain.

* Correspondence: Instituto de Investigacin en Recursos Cinegticos (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real,
Spain. Phone: +34 926 295 450 ext. 6255, Fax: +34 926 295 451, E-mail: inigomsolano@irec.csic.es
Received: 5 July 2011; received in revised form: 10 November 2011; accepted: 10 November 2011.

Information about demography and habitat use is key for the effective management of amphibian populations, because
it is the basis for the long-term monitoring of endangered species and provides insights about the processes by which com-
mon species thrive in heavily transformed habitats. A capture-mark-recapture study was performed on a population of
Perezs frog (Pelophylax perezi) in central Spain. The study area includes three breeding sites at a maximum distance of 700
metres in a straight line. The aim of the study was to obtain information about demographic parameters, habitat use and
movement patterns in this locality during the 2010 breeding season. In one of the breeding sites (Laguna de Valdemanco)
we estimated a population of 173 frogs: 91 males (95% confidence interval: 51-130) and 82 females (95% CI: 21-144).
In the second major breeding site, an adult population of 62 males (95% CI: 42-83) and 17 females (95% CI: 10-24)
was estimated. Areas of activity of 21 frogs captured more than twice, based on calculation of the minimum area poly-
gons defined by their different capture locations, ranged from 1.28 to 2763.75 m2 (median = 97.94 m2). All recorded
movements took place in the vicinities of the site where the frogs were first captured (mean distance standard deviation
= 49 41 m, maximum distance = 168 m), with the exception of a male that moved between two breeding sites 273
metres apart. These preliminary results can be applied to management strategies for this and other co-distributed species.

Key words: amphibians; central Spain; demography; dispersal; habitat use; population size.

Tamao poblacional, uso del espacio y patrones de movimiento durante el periodo reproductor en una poblacin
de rana verde comn (Pelophylax perezi) en Espaa central. Los estudios sobre demografa y uso del espacio resultan
esenciales para la gestin de poblaciones de anfibios, ya que fundamentan el seguimiento a largo plazo de especies
amenazadas y permiten comprender los mecanismos mediante los cuales especies comunes proliferan en medios fuer-
temente alterados. En este trabajo presentamos resultados de un estudio de captura-marcaje-recaptura en una pobla-
cin de rana verde comn (Pelophylax perezi) en Espaa central. El rea de estudio alberga tres puntos de reproduc-
cin, separados por distancias mximas de 700 metros en lnea recta. El objetivo del estudio fue estimar parmetros
demogrficos y analizar patrones de uso del espacio y movilidad durante la temporada de reproduccin en 2010. En
uno de los puntos (Laguna de Valdemanco) se estim una poblacin de 173 ranas: 91 machos (intervalo de confian-
za al 95%: 51-130) y 82 hembras (IC 95%: 21-144). En otro punto de reproduccin estimamos una poblacin adul-
ta de 62 machos (IC 95%: 42-83) y 17 hembras (IC 95%: 10-24). Se estimaron las reas de actividad de 21 ejem-
plares en base al clculo de los polgonos de rea mnima definidos por los puntos de captura, con valores entre 1.28
y 2763.75 m2 (mediana = 97.94 m2). Todos los movimientos detectados se produjeron en las inmediaciones del lugar
inicial de captura (media: 49 m, desviacin estndar: 41 m, distancia mxima: 168 m), excepto un macho que se
desplaz entre dos puntos de reproduccin distantes entre s 273 m. Estos resultados preliminares son aplicables al
diseo de estrategias de gestin para sta y otras especies codistribuidas.

Key words: anfibios; demografa; dispersin; Espaa central; tamao poblacional; uso del espacio.
82 SNCHEZ-MONTES & MARTNEZ-SOLANO
Several studies have described the spatial
organization of different amphibian species as
metapopulations (SJGREN-GULVE & RAY,
1996; MARSH & TRENHAM, 2001; GREEN,
2003; but see also SMITH & GREEN, 2005).
Structured this way, amphibian populations can
subsist and even thrive in areas under heavy
human pressure, like rural areas characterized
by a mosaic-like landscape, provided that a well-
connected network, including both aquatic bre-
eding sites and terrestrial habitats, exists
(MAZEROLLE, 2001; BAUER et al., 2010). A key
factor is connectivity, which requires knowledge
on the dispersal abilities of the species involved,
such that if distances between breeding sites
and/or between aquatic and terrestrial habitats
exceed certain thresholds, re-colonization dyna-
mics cannot counteract local extinctions, com-
promising the long-term survival of popula-
tions (SJGREN GULVE, 1994; CARLSON &
EDENHAMN, 2000). Thus, information about
habitat use and demography is key for the effec-
tive management of amphibian populations
(WELLS, 2007). For instance, based on these
studies, areas that are important for the connec-
tivity of different population nuclei in a region
can be identified and the critical distances above
which populations can be considered isolated
from a demographic perspective can be assessed
(FORTUNA et al., 2006; COMPTON et al., 2007).
Detection of demographically isolated popula-
tions is crucial, since they are more prone to
extinction caused by purely stochastic processes
(SJGREN GULVE, 1994; CUSHMAN, 2006).
Several studies have provided data on
demographic parameters (ADAMA &
BEAUCHER, 2006; KAYA et al., 2010), spatial
movements (SJGREN-GULVE, 1998a,b) and
habitat use (BLOMQUIST & HUNTER, 2009) in
amphibian populations worldwide (see review
in WELLS, 2007), but data on Iberian amphi-
bians are comparatively scarce (MALKMUS,
1982; DAZ PANIAGUA & RIVAS, 1987; LIZANA
et al., 1989; GARCA-PARS et al., 2004).
Capture-mark-recapture studies require conti-
nued, intensive sampling effort through many
years, but they can provide very detailed infor-
mation about the demography of populations,
which is a fundamental aspect to take into
account for their management (MARSH &
TRENHAM, 2001; BLACKWELL et al., 2004;
SCHMIDT et al., 2005; ERISMIS, 2011). Most of
these studies attempt to understand the basic
requirements of endangered species with the
aim of designing conservation strategies to
avoid their extinction (RICHTER & SEIGEL,
2002; CONROY & BROOK, 2003). But a com-
plementary approach to the problem of amphi-
bian declines may involve understanding the
processes by which other species maintain posi-
tive demographic trends and thrive in the same
environments where others disappear.
Perezs frog Pelophylax perezi (Seoane,
1885) is a good model in this respect. This
species is endemic from the Iberian Peninsula
and southern France, where it is almost uni-
formly distributed and locally very abundant
and, therefore, it is catalogued as Least
Concern under the IUCN extinction risk
criteria (LLORENTE et al., 2002; BOSCH et al.,
2008). Although there are some studies about
biometry (GOS & ARIAS, 2009) and popula-
tion age-structure as assessed by skeletochro-
nology (PATN et al., 1991; ESTEBAN et al.,
1996), there is no information about their
typical home ranges or habitat use and its
general biology is poorly known (EGEA-
SERRANO, 2009). In this paper, we present
preliminary results about the demography,
areas of activity and movement patterns
 DEMOGRAPHY AND HABITAT USE IN PELOPHYLAX PEREZI 83

during the breeding season of a population of
P. perezi in an area in the north of the provin-
ce of Madrid (central Spain) in 2010. The
specific objectives of the study were:
1) To estimate the population size, apparent
survival and the probability of recapture of
individuals of P. perezi in the study area
using a capture-mark-recapture method.
2) To record the distances travelled by indivi-
duals during the breeding season and to
calculate their areas of activity.
3) To investigate possible differences in demogra-
phic parameters (population size, apparent sur-
vival, probability of recapture) and movement
patterns between sexes and breeding sites.
4) To provide biometric (body mass and snout-
vent length) data and analyze their relations-
hip with demographic parameters (popula-
tion size, apparent survival, probability of
recapture), and movement patterns.
MATERIALS AND METHODS
Study area
The study area has an extension of 0.5 km2
and is located near the town of
Valdemanco, Madrid, 1140 m above sea level
(UTM: x = 30T 445213 E, y = 4522623 N,
included in the 10 x 10 km UTM grid VL42)
(Fig. 1). For practical purposes, it was delimi-
ted using some of the existing trails, which
surround an area with lower cattle pressure
(which is, with mining, the main land use in
the area) than adjacent lands. There are no
artificial barriers potentially affecting amphi-

Figure 1: Map of the study area near Valdemanco, in Madrid (central Spain). The inset in the upper right cor-
ner shows the location of the study area in the Iberian Peninsula and in the region of Madrid. The trails that
delimit the study area are highlighted with a dotted white line. Indicated are the locations of the three main
breeding sites: Laguna de Valdemanco, the mining pond and the water trough.
84 SNCHEZ-MONTES & MARTNEZ-SOLANO
bian dispersal. The study area includes two
main water bodies where a rich amphibian
community breeds, including Pleurodeles
waltl, Triturus marmoratus, Alytes cisternasii,
Pelobates cultripes, Bufo calamita, Hyla arborea
and Pelophylax perezi (MARTNEZ-SOLANO,
2006). In general, it is a well-preserved area, with
occasional minor impacts derived from cattle
(grazing, eutrophication) and human activities
(land filling, trash dumping, introduction of
exotic species).
The largest pond, Laguna de Valdemanco,
is an epigenic and semi-permanent aquatic
system of 12 800 m2 of extension, with a
maximum depth of 1 m. It is included in the
Catlogo de Embalses y Humedales de la
Comunidad de Madrid (COMUNIDAD DE
MADRID, 2004). Adjacent meadows are
usually flooded during the winter and early
spring. Abundant and tall aquatic vegetation
dominates in the spring (Carum verticillatum,
Juncus acutiflorus, Ranunculus fluitans,
Eleocharis palustris). In the north bank there
are some willow trees (Salix sp.) and in the
vicinities of the pond, the gum rockrose
(Cistus ladanifer) predominates.
The other pond is an abandoned mining site
between 35 and 55 years old (see satellite images
at COMUNIDAD DE MADRID, 2011). It is smaller
(2100 m2), but deeper (maximum depth: 1.7 m)
and with a longer hydroperiod than Laguna de
Valdemanco, from which it is separated by 700
metres in a straight line (Fig. 1). The margins of
this pond are more abrupt, and aquatic vegeta-
tion is less abundant. In the east bank there are
some willow trees (Salix sp.) and gum rockroses
(Cistus ladanifer). During the course of the pre-
sent study, we detected the presence of the red
swamp crayfish (Procambarus clarkii) in this
pond, although densities were low.
Apart from these two ponds, we detected
breeding activity of P. perezi in a small (2 m
long, 50 cm deep) water trough in the study
area (Fig. 1). This site presents running water
throughout the year, with no vegetation other
than green algae, and is 233 m apart from
Laguna de Valdemanco and 706 m apart
from the mining pond (linear distances).
Sampling methods
In order to monitor the frog population
in the study area, we performed night sur-
veys, focused on breeding sites, with all the
water surface and shores sampled homogene-
ously. Frogs were captured by hand or with
the help of dip nets. We also surveyed terres-
trial habitats, mostly along the trails delimi-
ting the study area, but also covering the rest
of the study area in search of adult frogs acti-
vely dispersing (in nights with appropriate
climatic conditions: warm temperatures, high
humidity, little or no wind) or hiding under
rocks or other refuges during the day. In all
cases, we recorded temperature, number of
researchers involved in the surveys and total
time spent. Data were obtained from a total
of 44 field visits during the period of activity
of the frogs (March-October 2010).
The differences in hydroperiod of the
ponds conditioned the maximum number of
visits in each case. A total of 11 surveys were
performed in Laguna de Valdemanco between
April 6th (when the first P. perezi individuals
were detected in the pond after hibernation)
and June 11th 2010 (when only a few adults
remained in the pond, just before it dried up).
The mining pond was visited 18 times, star-
ting on May 18th and until October 30th, when
only a few isolated puddles remained. The
 DEMOGRAPHY AND HABITAT USE IN PELOPHYLAX PEREZI
water trough was visited five times between
July 21st and October 25th. The sampling was
completed with 10 surveys on terrestrial habi-
tats. The frequency of the visits was irregular,
since the goal of the study was to capture and
mark as many frogs as possible within a single
breeding season, but on average the study area
was visited once every five days.
The location of each captured frog was
recorded with accuracy equal to or lower
than five metres using a Garmin Etrex GPS
device (Garmin International Inc., Olathe,
Kansas, USA). For all specimens, we recor-
ded sex, snout-vent length (SVL) and body
mass (measured with a Pesola MS 1000 scale
(Pesola AG, Baar, Switzerland) with a preci-
sion of 0.2 g). When individuals were cap-
tured for the first time, we marked them
with an 8 mm AVID M.U.S.I.C. chip
(EzID, Greeley, Colorado, USA) including a
unique identity code that was dorsally inser-
ted under the skin using a hypodermic nee-
dle (AVID Single Use Disposable Syringe
monoject). Chips were read using an AVID
Minitracker II RS232 reader device. All spe-
cimens were released back in their place of
capture after marking.
Biometry
We tested for differences in body mass and
SVL between sexes and between breeding sites
(excluding the water trough due to low sample
size). In the case of frogs captured more than
once, the values recorded in their first capture
were used for the analyses. The variable body
mass did not adjust to a normal distribution,
so we used non-parametric tests (Mann-
Whitneys U) to analyze differences in body
mass between sites and sexes, whereas parame-
85
tric tests (Students t) were used to test for dif-
ferences in SVL, since this variable did adjust
to a normal distribution. We also explored the
effects of the interaction sex*locality for SVL
data. For all analyses, P-values < 0.05 were con-
sidered significant.
In recaptured frogs, we also calculated mass
variation between the first and the last captu-
re, taking into account the time elapsed betwe-
en captures (= body mass variation per day).
Differences were expressed as a proportion of
the body mass at the time of the first capture.
We tested for differences between sexes using
non-parametric (Mann-Whitneys U) tests,
and between breeding sites using parametric
(Students t) tests using SPSS 15.0.
Estimates of demographic parameters
No dispersal was detected between the
two main breeding sites during the activity
period in 2010, so in our analyses they were
considered as independent, open popula-
tions, and population sizes of both sites
(Laguna de Valdemanco and the mining
pond) were thus estimated separately. The
number of captured frogs at the third site
(water trough) was too small (see Results), so
this site was not included in the analyses.
We used the free software MARK 6.0
(WHITE & BURNHAM, 1999) to estimate
demographic parameters. MARK allows to
test the fit of different models to observed data
(in this case, the encounter history of each
individual), and assess which model (or
models) is the best according to the Akaike
Information Criterion corrected for finite
sample sizes (AICc, AKAIKE, 1974; BURNHAM
& ANDERSON, 2002). The AICc assigns a
score to each model on the basis of the amount
86 SNCHEZ-MONTES & MARTNEZ-SOLANO
of variance explained, penalized by the num-
ber of parameters in the model. To estimate
population sizes, MARK uses the Jolly-Seber
method (JOLLY, 1965; SEBER, 1965) in the
POPAN subroutine (SCHWARZ & ARNASON,
1996). This method shares the typical assump-
tions of capture-mark-recapture (CMR) stu-
dies in open populations and is based on the
estimation of four main parameters: 1) appa-
rent survival of individuals between capture
events (phi), 2) probability of capture in each
survey (p), 3) rate of entrance of new indivi-
duals to the study area between two sampling
occasions (pent) and 4) the population size
(N). In all cases, N and the other parameters
are treated as dependent variables. We tested
different models, analyzing the behaviour of
the parameters phi, p and pent in four diffe-
rent, albeit complementary ways, considering
them as: a) constant, b) time-dependent, c) sex-
dependent and d) time-and-sex-dependent.
Estimates for N, phi and p were calculated for
adult frogs along with their 95% confidence
intervals (95% CI) for each pond, by avera-
ging the best models. The average of each
parameter is calculated as the weighted avera-
ge of the values estimated by the different
models, where each model has a weight inver-
sely proportional to its AICc. We also used
MARK 6.0 to test for correlations between
population parameters (phi and p) and biome-
tric variables (body mass and SVL, separately).
Movement patterns and areas of activity
The distances covered by each individual
captured at least once after the initial capture
were calculated using the software ArcGIS 9.2.
Additionally, for frogs captured twice or more
after their initial capture, areas of activity
during the breeding season were estimated as
the minimum convex polygon (MCP) defined
by their recorded locations (KIE et al., 1996).
ArcGIS was also used to calculate the 50, 90
and 95 Kernel areas (i.e. the areas with 50%,
90% and 95% probability of including an
individual). Absolute measures of distances
covered and areas of activity were standardized
with respect to the time interval in which they
were recorded (and thus represent daily distan-
ces or areas, respectively), in order to 1) allow
direct comparison of movements between sexes
and to 2) test for possible correlations between
movement patterns and biometry (body mass
and SVL). These analyses were performed with
the software SPSS 15.0 for Windows.
We tested for differences in the variables
mean daily distance (= mean of the distan-
ce values per each elapsed day) and mean
daily area (= mean of the area of activity
values per day) between the two main ponds
and between both sexes using non-parametric
Mann-Whitneys U tests, since none of them
was normally distributed when using all the
locations together, nor when the two ponds
were analyzed separately. We also used
Spearmans correlation coefficient to analyze
linear relationships between movement pat-
terns and biometry (body mass and SVL) in
all observations pooled together.
RESULTS
Sampling effort
Total sampling effort, measured as per-
sons*hours, was 296 (150 at the mining pond,
83 at Laguna de Valdemanco and 63 in diur-
nal and nocturnal transects along trails). All
captured frogs were found at or in the imme-
 DEMOGRAPHY AND HABITAT USE IN PELOPHYLAX PEREZI
diate vicinity of the three breeding sites (the
two ponds and the water trough). Day and
night surveys in terrestrial habitats were
unsuccessful, although we found juveniles
and adults of other species, like B. calamita,
H. arborea and P. cultripes.
During the 34 surveys performed at bre-
eding sites, we recorded a number of 207
captures (including recaptures), 94 of them
at Laguna de Valdemanco, 111 at the
mining pond and two at the water trough.
We marked 129 different individuals (73 at
Laguna de Valdemanco, 55 at the mining
pond and one at the water trough). Mean
number of captures per unit of sampling
effort was 1.16 per person*hour in Laguna
de Valdemanco and 0.74 per person*hour
in the mining pond. We marked more
males than females or juveniles (78 males:
43 in L. Valdemanco, 34 in the mining
pond and one in the water trough; 42 fema-
les: 30 in L. Valdemanco and 12 in the
mining pond; and nine juveniles: all of
them at the mining pond).
Recapture rates per individual were in
general low: 62.79% of sampled individuals
were captured only once, 20.93% twice,
10.08% three times, 5.43% four times and
a single individual was captured five times.
Recapture rates were significantly lower in
Laguna de Valdemanco (mean = 0.30; SD = 0.57)
than in the mining pond (mean = 1.02; SD =
1.15) (Mann-Whitneys U: U = 1322.0; N
(L. Valdemanco) = 73, N (mining pond) = 55;
P < 0.001). Recapture rates in juveniles
(mean = 0.33; SD = 0.71) were non-signifi-
cantly lower than in adults (mean adult males =
0.63, SD = 0.87; mean adult females = 0.62,
SD = 1.08) (Kruskal-Walliss K: H2 = 6.213;
P = 0.469).
87
Biometry
Body mass and SVL were strongly correla-
ted (Spearmans Rho: Rho = 0.970; N = 124;
P < 0.001). Very significant biometric diffe-
rences were found between sexes, with fema-
les being significantly larger than males in
body mass (median / inter-quartile amplitude:
males = 19.0 / 10.5 g, females = 50.2 / 25.4 g;
Mann-Whitneys U: U = 170.5; N: males = 73,
females = 42; P < 0.001) and SVL (mean SD:
males = 63.3 8.4 mm, females = 82.02 10.02
mm; Students t: t118 = -10.769; P < 0.001). No
differences between individuals of the same
sex from Laguna the Valdemanco and the
mining pond were found in body mass
(males: median / inter-quartile amplitude:
L. Valdemanco = 19.0 / 10.4 g, mining pond
= 19.0 / 13.1 g; Mann-Whitneys U: U = 613.5;
N: L. Valdemanco = 39, mining pond = 33;
P = 0.735. Females: median / inter-quartile
amplitude: L. Valdemanco = 51.2 / 27 g,
mining pond = 42.1 / 22.4 g; Mann-
Whitneys U: U = 143.0; N: L. Valdemanco = 30,
mining pond = 12; P = 0.314) or SVL (males:
mean SD: L. Valdemanco = 62.0 6.7 mm,
mining pond = 64.9 10.1 mm; Students t:
t75 = -1.025; P = 0.309. Females: mean SD:
L. Valdemanco = 81.8 10.7 mm, mining
pond = 82.5 8.6 mm; Students t: t40 = -0.193;
P = 0.848). We found no significant effect of the
interaction sex*locality in SVL (data not shown).
Calculation of body mass differences rela-
tive to initial body mass in individuals captu-
red more than once revealed that females suf-
fered in general stronger losses. However,
gains of more than 20% with respect to body
mass at the time of first capture were also recor-
ded in individuals from both sexes (Fig. 2).
When differences relative to initial body mass
88 SNCHEZ-MONTES & MARTNEZ-SOLANO
Figure 2: Frequency histogram of body mass differences relative to body mass at initial capture for males
(dark bars) and females (grey bars). Values are expressed as proportions.
were time-standardized, no differences betwe-
en sexes were found (median / inter-quartile
amplitude: males = 0.0017 / 0.0117 day-1,
females = 0.0031 / 0.0131 day-1; Mann-
Whitneys U: U = 163.00; N (males) = 31, N
(females) = 12; P = 0.547). However, whereas
adult frogs in Laguna de Valdemanco expe-
rienced losses in body mass throughout the
study, frogs from the mining pond showed the
opposite trend (mean SD: adults (L.
Valdemanco): -0.0038 0.0085 day-1, adults
(mining pond): 0.0066 0.0067 day-1;
Students t: t41 = -4.492; P < 0.001). There was
no significant effect of the interaction between
sex and locality for the variable body mass
variation per day (data not shown).
Estimates of demographic parameters
According to MARK results, the best
models were those that assume that the pro-
bability of entrance (pent) is time-dependent,
whereas apparent survival (phi) and probabi-
lity of capture (p) are either constant or sex-
dependent (Table 1). More complex models
were penalized due to over-parameterization.
In order to obtain accurate estimates for the
parameters of interest, a weighted averaging
of the best models was performed according
to their AICc. Apparent survival (phi) was
high in both water bodies and there were
only slight differences between males and
females (estimated phi (95% CI): males (L.
Valdemanco) = 0.912 (0.860-0.946); females
(L. Valdemanco) = 0.932 (0.850-0.971);
males (mining pond) = 0.978 (0.968-0.985);
females (mining pond) = 0.977 (0.964-0.985)).
On the other hand, although males were
similarly detectable in both ponds, females
were easier to capture in the mining pond
than in Laguna de Valdemanco (estimated p
(95% CI): males (L. Valdemanco) = 0.253
(0.135-0.423); females (L. Valdemanco) = 0.169
Table 1: Model selection. Scores of the Corrected
Akaike Information Criterion (AICc), model weights
and number of parameters for preferred models at
each breeding site. g = gender-dependent, t = time-
dependent, and . = constant.
Pond Model name AICc AICc Num. of
Weight params.
Laguna de phi(.)p(.)pent(t) 243.2289 0.35544 13
Valdemanco phi(g)p(g)pent(t) 243.2908 0.34461 15
phi(.)p(g)pent(t) 244.4822 0.18994 14
phi(g)p(.)pent(t) 245.6365 0.10665 14
Mining pond phi(.)p(g)pent(t) 417.2786 0.68731 21
phi(g)p(g)pent(t) 420.4249 0.14254 22
phi(.)p(.)pent(t) 420.4340 0.14189 20
 DEMOGRAPHY AND HABITAT USE IN PELOPHYLAX PEREZI
(0.060-0.392); males (mining pond) = 0.225
(0.153-0.318); females (mining pond) = 0.383
(0.233 -0.559)).
We found slight differences in the rela-
tionship between demographic and biome-
tric variables as a function of site or sex. In
Laguna de Valdemanco, the best model for
estimating male population size selected by
MARK included a positive linear relations-
hip between apparent survival and SVL,
whereas the probability of capture was inde-
pendent of size (body mass and SVL). For
females, however, there was no significant
relationship between demographic and bio-
metric variables. Similarly, in the mining
pond, the best models assumed that there
was no relationship between demographic
and biometric variables (phi and p were
constant for both sexes).
Population size (N) was higher in
Laguna de Valdemanco (mean of 173 adult
frogs) than in the mining pond (79 adult
frogs), but the 95% CI was narrower in
the latter, as a result of the higher recaptu-
re rates in this site (Table 2). There were
differences in sex-ratio between ponds,
with proportions near 1:1 in Laguna de
Valdemanco, but biased toward males in
the mining pond (4:1).
Table 2: Estimates of population sizes (N) and
their 95% confidence interval (95% CI) for
adults of both sexes at Laguna de Valdemanco and
at the mining pond.
Breeding site Sex N 95% CI
Laguna de Males 91 51-130
Valdemanco Females 82 21-144
Mining pond Males 62 42-83
Females 17 10-24
89
Movement patterns and areas of activity
We recorded a total of 48 distances betwe-
en captures, 19 in Laguna de Valdemanco
(14 males and five females, including the lon-
gest distance recorded, an adult male that dis-
persed from the pond to the water trough)
and 29 in the mining pond (19 males, eight
females and two juveniles). Juveniles were
excluded from subsequent analyses due to the
low number of observations. Since a mini-
mum number of three locations are required
to estimate the area of activity for a given
individual, our dataset was reduced to sam-
ples of 21 individuals, two in Laguna de
Valdemanco and 19 in the mining pond.
Mean distances covered by adult frogs
during the breeding season were around 50
m (mean distance SD = 49 41 m, Fig. 3).
The longest distance overall was covered by a
male that was initially captured at Laguna de
Valdemanco and recaptured four months
later at the water trough, which is 273 m
apart. The largest estimated area of activity
(2763.75 m2) corresponded to a male that
was recaptured three times after the initial
capture at Laguna de Valdemanco in a time
lapse of 17 days, but median values were
much smaller (median / inter-quartile ampli-
tude: 97.94 / 199 m2, Fig. 4).
We found no significant differences bet-
ween sexes in time-standardized distances,
either in the combined dataset (median /
inter-quartile amplitude: males = 1.563 /
7.116 m day-1, females = 2.048 / 3.259 m
day-1; Mann-Whitneys U: U = 213.00; N:
males = 33, females = 13; P = 0.971) or when
both ponds were analyzed separately (Laguna
de Valdemanco: median / inter-quartile
amplitude: males = 8.257 / 18.078 m day-1,
90 SNCHEZ-MONTES & MARTNEZ-SOLANO
Figure 3: Frequency histogram of absolute distances between captures of males (dark bars) and females
(grey bars) at the study area.
females = 4.718 / 5.867 m day-1; Mann-
Whitneys U: U = 19.00; N: males = 14,
females = 5; P = 0.156. Mining pond:
median / inter-quartile amplitude: males =
1.074 / 0.845 m day-1, females = 1.701 /
1.659 m day-1; Mann-Whitneys U: U = 52.00;
N: males = 19, females = 8; P = 0.217). Time-
standardized distances recorded in Laguna de
Valdemanco were significantly larger than
those recorded in the mining pond for males
(Mann-Whitneys U: U = 46.00; N: L.
Valdemanco = 14, mining pond = 19; P = 0.001)
but not for females (Mann-Whitneys U: U =
13.00; N: L. Valdemanco = 5, mining pond = 8;
P = 0.354).
Due to the low number of observations
available for Laguna de Valdemanco, we did
not test for differences in areas of activity
across sites or between sexes within this site.
On the other hand, there were no significant
differences between time-standardized areas
of activity in males and females in the mining
pond (median / inter-quartile amplitude:
males = 4.618 / 4.95 m2 day-1, females = 3.039
/ 9.636 m2 day-1; Mann-Whitneys U: U = 34.00;
N: males = 11, females = 7; P = 0.724).
Finally, no significant correlation was found
between time-standardized distances or areas
and biometric variables (Distance-body mass:
Spearmans Rho: Rho = -0.05; N = 44; P = 0.745.
Distance-SVL: Rho = -0.078; N = 46; P = 0.607.
Area-body mass: Rho = 0.041; N = 20; P = 0.862.
Area-SVL: Rho = -0.051; N = 20; P = 0.830).
DISCUSSION
The population of P. perezi at Laguna de
Valdemanco is almost twice as big as that in the
mining pond, although uncertainty in popula-
tion size estimates is also larger in the former. In
spite of this, the difference in estimated popula-
tion sizes is not as large as could be expected
given their relative areas (Laguna de Valdemanco
has six times more water surface than the mining
pond). Differences in hydroperiod may help
explain this pattern, since Laguna de
Valdemanco dries up in early or mid summer,
whereas the mining pond maintains water
throughout most or all the summer, and diffe-
rences in hydroperiods obviously represent a
limiting factor for water dependent amphibians,
especially for species with longer tadpole stages,
like P. perezi (RICHTER-BOIX et al., 2006).
Sex ratio is close to 1:1 in Laguna de
Valdemanco, in contrast with the results in
the mining pond (4:1 in favour of males).
 DEMOGRAPHY AND HABITAT USE IN PELOPHYLAX PEREZI
Figure 4: Frequency histogram of estimated areas of activity of males (dark bars) and females (grey bars)
at the study area.
This result may be an artefact related to low
recapture rates in the former site, so these dif-
ferences must be taken with caution until
new data from subsequent breeding seasons
are available. With respect to biometry, our
results are in agreement with those summari-
zed by EGEA-SERRANO (2009) and in contrast
with REAL & ANTNEZ (1991). Size differen-
ces between sexes, with females being signifi-
cantly larger than males, were consistent
across breeding sites. Body mass variations in
both sexes are probably related to breeding
activity, with sharp losses following egg laying
in females and mostly increases in males,
which present an intense feeding activity
during the breeding season (REAL &
ANTNEZ, 1991). However, these results
have to be taken with caution, since our pro-
cedure overlooked a potential source of signi-
ficant variation in mass estimates: the water
stored in the bladder of adult frogs, which
can represent an important percentage of the
total body mass. Regarding habitat use, both
sexes seem to occupy all available space along
the shores, although females seemed to use
preferentially more protected areas, like those
with denser vegetation (mainly Salix sp., Fig. 5),
although this hypothesis has not been tho-
roughly explored yet.
91
We did not detect any individual move-
ment between the main breeding sites in our
study area (with the single exception highligh-
ted above, the male moving from Laguna de
Valdemanco to the water trough), despite the
fact that one of the ponds (Laguna de
Valdemanco) dried up one month earlier than
the other. This suggests high fidelity of adults
to their breeding sites, which could be chosen
primarily during their post-metamorphic dis-
persal stage, as documented in other
Figure 5: Map showing all recorded locations of
P. perezi at Laguna de Valdemanco (males = grey
triangles, females = white circles) along with their
corresponding Kernel 50 areas (males = continuous
grey lines, females = dotted white lines). Females
were mostly captured along the north bank, where
there are some willow trees (Salix sp.) providing
refuge. Males, in contrast, are more widely distribu-
ted and tend to occupy all the available space.
92 SNCHEZ-MONTES & MARTNEZ-SOLANO
European species of green waterfrogs
(SJGREN GULVE, 1994). As a consequence,
the disappearance of a breeding site may have
a stronger impact in populations of P. perezi
than in other, more vagile species (for exam-
ple, in our study area, Hyla arborea, I.
Martnez-Solano, J. Gutirrez & G. Snchez-
Montes, unpublished data), at least in the
short term. Nonetheless, our study covered a
single breeding season, and in the long term
some degree of interconnectedness between
breeding sites is expected, especially by means
of recently metamorphosed or immature indi-
viduals. Further monitoring of this popula-
tion will help address this question, with the
aid of genetic tools that can identify cryptic
dispersal events (JEHLE et al., 2005; ZAMUDIO
& WIECZOREK, 2007). At the moment, our
observations (the single dispersal event from
Laguna de Valdemanco to the water trough as
well as additional observations during the
2011 breeding season) suggest that the three
breeding sites in the study area are connected
by low frequency migratory events.
The frequency histogram of the distances
covered by individuals of P. perezi shows the
typical leptokurtic (asymmetric and positive)
pattern, with most observations representing
short distances (82.6% < 100 m) but also a
few observations involving longer distances
(Fig. 3). The longest displacement of an indi-
vidual of P. perezi recorded in this study was by
a male from Laguna de Valdemanco that was
recaptured, four months after its first capture,
in the water trough. These sites are not con-
nected by superficial water currents or inter-
mediate puddles, and the linear distance of
273 metres is probably an underestimate of
the actual distance covered during dispersal,
which might have followed some of the exis-
ting trails, but in any case involved crossing to
some extent a dense vegetal matrix dominated
by Cistus ladanifer. On the other hand, the
relative importance of the water trough site for
the overall breeding success of P. perezi in the
study area is unclear (vs., for instance, its rele-
vance as a foraging area). EGEA-SERRANO et al.
(2005) documented that these small, artificial
breeding sites were usually negatively selected
by the species against other alternatives like
ponds or reservoirs, but our results suggest
they may be important in a metapopulation
context. In our study area, this source of per-
manent water throughout the year allows
hibernating tadpoles to metamorphose during
the next breeding season with larger body sizes
(authors personal observations), which may
be advantageous from a fitness perspective
(BERVEN & GILL, 1983). Parentage analysis
using multilocus genotypes from samples of
tadpoles and adults in all breeding sites will
help clarify the role of this site in the breeding
success of P. perezi in the study area.
With the noted exception, all recorded
movements took place within each major
breeding site. Laguna de Valdemanco, the
larger pond, offers more water surface than
the mining pond, and correspondingly, resi-
dent frogs present longer within-pond distan-
ces between captures. In general, males seem
to move more than females (Fig. 3), but some
females also have large areas of activity (Fig.
4), although differences are not statistically
significant. In any case, our calculations, with
50% of estimated areas below or equal 100 m2
(Fig. 4), are probably underestimations of the
actual areas of activity and home ranges in
this species, and while this represents a valua-
ble first approximation, more data, based on
subsequent monitoring, will be required.
 DEMOGRAPHY AND HABITAT USE IN PELOPHYLAX PEREZI
Our study presents the first data on move-
ment patterns in P. perezi. We found a typical
pattern, with high frequency of low distance
movements within breeding ponds and very
low frequency of long dispersal events (Fig. 3),
suggesting long-term interconnectedness bet-
ween breeding sites in the study area. Males
and females of P. perezi seem to have similar
areas of activity during the breeding season,
mainly restricted to the ponds where they
breed (Fig. 4). These data, while still prelimi-
nary, will be important in understanding fine-
scale population dynamics in this species,
including regional patterns of connectivity
between breeding sites.
Acknowledgement
The help of Garazi Rodrguez was key
throughout the study. Thanks as well to Juan
Zorrilla and Jos Serrano for their orientation
and to every person who collaborated in the
fieldwork: Esperanza and Jaime Iranzo,
Ignacio Urbn, Fernando A. Fernndez, Jorge
Gutirrez, Jaime Prez, Antonio Carvajal,
Ernesto Recuero, Cristina Grande, Carlos
Pedraza, Daniele Salvi and Alfredo Ortega
and Javier Castro (Agentes Forestales
Comarca V, Torrelaguna). A. Montori and
two anonymous reviewers provided useful
comments on the manuscript. Legal permits
for conducting fieldwork were provided by
Consejera de Medio Ambiente, Comunidad
de Madrid. This study was funded by project
CGL2008-04271-C02-01-BOS (Ministerio
de Ciencia e Innovacin, Spain, PI: I.
Martnez-Solano). Legal regulations and ethi-
cal considerations related to work with live
animals were strictly followed during the
course of this study.
93
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A re-analysis of the molecular phylogeny of Lacertidae

with currently available data
Paschalia Kapli1,2*, Nikos Poulakakis1,2, Petros Lymberakis1, Moysis Mylonas1,2
1
Natural History Museum of Crete, University of Crete, Irakleio, Crete, Greece.
2
Department of Biology, University of Crete, Irakleio, Crete, Greece.

* Correspondence: University of Crete, Natural History Museum of Crete, Knossos Avenue, Postal Code 71409 P.O. 2208, Iraklio,
Crete, Greece. Phone and Fax: +30 28103324366. E-mail: k.pashalia@gmail.com
Received: 11 April 2011; received in revised form: 3 August 2011; accepted: 5 September 2011.

The Lacertidae is one of the most diverse and widespread lizard families throughout Eurasia and Africa.
Several studies so far have attempted to unravel the phylogeny of Lacertidae using morphological and mole-
cular data. However, the intra-family relationships remain unclear. In an effort to explore the phylogenetic
relationships within the family Lacertidae, a concatenated dataset of 5727 bp from six genes (two nuclear and
four mitochondrial) and 40 genera was assembled based on GenBank database. Phylogenetic inference analy-
ses were conducted using Maximum Parsimony (MP), Bayesian inference (BI) and Maximum Likelihood
(ML), revealing that even a combined dataset of both mitochondrial and nuclear genes is not able to resolve
the phylogenetic relationships of the Lacertidae family under the tribe level.

Key words: GenBank; Lacertidae; phylogeny.

Reanlisis de la filogenia molecular de los Lacertidae usando los datos disponibles en la actualidad. La
familia Lacertidae es una de las ms diversas y ampliamente distribuidas en Eurasia y frica. Varios estudios
han intentado hasta ahora aclarar la filogenia de los Lacertidae usando datos morfolgicos y moleculares. Sin
embargo, las relaciones dentro del grupo permanecen poco claras. En un esfuerzo por explorar las relaciones
filogenticas dentro de la familia Lacertidae, se analiz una base de datos de 5727 pares de bases para 40 gne-
ros diferentes de lacrtidos obtenidos por la concatenacin de seis genes (dos nucleares y cuatro mitocondriales),
todos ellos disponibles en la base de datos de GenBank. Los anlisis filogenticos realizados usando mtodos de
mxima parsimonia (MP), inferencia bayesiana (BI) y mxima verosimilitud (ML), revelaron que el conjunto
combinado de genes mitocondriales y nucleares utilizados no es capaz de resolver las relaciones filogenticas de
los lacrtidos a un nivel taxonmico inferior al de tribu.

Key words: filogenia; GenBank; Lacertidae.

Lacertidae is a family of small body sized
lizards distributed throughout Eurasia and
Africa. In recent years this family has been the
subject of several taxonomical studies, conside-
ring both molecular and morphological charac-
ters. ARNOLD (1989), based on morphological
characters, constructed the phylogeny of this
family and proposed the division of the
Lacertidae into two subgroups, the Primitive
Palearctic and Oriental Lacertids and the
Ethiopian and Advanced Saharo-Eurasian
forms. A series of studies (LUTZ & MAYER,
1984, 1985; MAYER & BENYR, 1995) based on
albumin-immunology, resulted in the establis-
hment of two subfamilies, Gallotiinae, which
includes two genera, Gallotia and Psammodromus,
and Lacertinae including the rest of the
Lacertidae family. HARRIS et al. (1998) and FU
98 KAPLI ET AL.
(1998, 2000) used mitochondrial DNA sequen-
ces to explore the relationships of lacertid lizards.
However, in all cases, the datasets were insuffi-
cient to reconstruct the intra-family phylogeny of
Lacertidae. In the same study, HARRIS et al.
(1998) combined morphological characters
along with molecular data and led to the division
of the family into three subfamilies: 1)
Gallotiinae, 2) Lacertinae and 3) Eremiainae.
The first two correspond to ARNOLDs (1989)
Primitive Palearctic and Oriental Lacertids,
while Eremiainae is equivalent to the Ethiopian
and Advanced Saharo-Eurasian forms and to
ARNOLDs (1973) armatured clade. A recent
work of MAYER & PAVLICEV (2007) confirmed
the division in subfamilies proposed by HARRIS et
al. (1998) and indicated the division of the sub-
family Eremiainae into two clades, Ethiopian
and Saharo-Eurasian. ARNOLD et al. (2007)
downgraded Lacertinae and Eremiainae into tri-
bes (i.e. Lacertini and Eremiadini, respectively) of
the subfamily Lacertinae. PAVLICEV & MAYER
(2009) argued that the use of the tribe as a taxo-
nomic entity causes confusion while they conclu-
ded that the polytomy of Lacertini is more likely
to be attributed to multiple cladogenesis in a geo-
logically short time than to the poor resolution of
the markers used. Despite PAVLICEV & MAYER
(2009) opinion, in the current study we follow
the systematics proposed by ARNOLD et al.
(2007) since it is more descriptive of the phylo-
geny of the family.
The aim of this study was to reassess the
phylogeny of the family using all currently
available data from GenBank. Primarily the
data used here were produced by the molecu-
lar studies mentioned before and concluded
in a dataset of four mitochondrial and two
nuclear genes from 40 genera.
MATERIALS AND METHODS
Published sequences were retrieved from
GenBank (four mitochondrial genes: 16S
rRNA, 12S rRNA, cyt b, and COI, and two
nuclear genes: c-mos and RAG-1). We built
a concatenated dataset in which each genus
is represented by one chimerical sequence of
the six genes (all accession numbers are
given in Table 1).
All genes were identified and the correspon-
ding sequences were saved to individual
FASTA-formatted files for each gene. The
poorly aligned positions for the genes 12S
rRNA and 16S rRNA were removed using the
online version of Gblocks (V. 0.91b,
CASTRESANA, 2000) under the less stringent
options (412 bp out of 1455 bp and 168 bp out
of 1010 bp were removed for 16S rRNA and
12S rRNA, respectively). The final dataset was
comprised of 5727 bp for 40 Lacertidae genera.
Representative sequences from the genus
Eumeces (chimerical sequence out of the species
E. anthracinus, E. ergegius and E. inexpectatus)
were added to each data set as outgroup.
Phylogenetic analyses
The Bayesian Information Criterion
(BIC) as implemented by jModeltest
(v.0.1.1; POSADA, 2008), was used to choose
the best-fit model of DNA substitution. The
best fit models (among 88 available) for 12S
rRNA, 16S rRNA, COI, cyt b, rag1 and c-mos
were: TPM1uf +I +G, TPM2uf +G, TIM2 +I +G,
TPM2uf +G, Trn +I +G and K80 +G, respec-
tively. For the BI analysis in the cases where
the model selected by jModeltest could not
be implemented the closest more complica-
 PHYLOGENY OF THE LACERTIDAE 99

Table 1: List of sequences used in the analyses. Genera name and GenBank accession numbers for each
gene included in the analyses are provided (12S, 16S, cyt b, CO1, rag1 and c-mos). Sequences that were
not available in GenBank are indicated as n/a.

Genera GenBank accession numbers

12S 16s Cyt b CO1 c-mos Rag1

Gallotia AF206587 AF206587 AF101224 AF206562 EF632260 EF632215

Psammodromus AF206588 AF206588 AF206535 AF206567 EF632284 n/a
Acanthodactylus AF206607 AF206607 AF206536 AF206568 EF632252 EF632207
Adolfus AF206615 AF206615 AF206539 AF206578 EF632253 EF632208
Algyroides AF206598 AF206598 AF206529 AF206557 EF632255 EF632210
Anatololacerta AJ238188 GQ142107 DQ461765 n/a DQ461743 EF632224
Dalmatolacerta AF440601 AF440616 AY278199 n/a EF632271 EF632228
Darevskia AF206597 AF206193 U88611.3 AF206552 EF632257 EF632212
Dinarolacerta AF440600 AF440615 GQ142141 n/a EF632270 EF632227
Eremias AF206604 AF206604 AF206549 AF206576 EF632259 EF632214
Heliobolus AF206608 AF206608 AF206544 AF206583 EF632262 EF632217
Hellenolacerta AF440602 AF440617 GQ142128 n/a EF632269 EF632225
Iberolacerta AF440598 AF440612 AY267242 AF206571 EF632264 EF632219
Latastia AF206609 AF206609 AF206545 AF206563 EF632272 EF632229
Meroles AF206610 AF206611 AF206540 AF206581 EF632273 EF632230
Mesalina AY035832 AF206606 FJ416173 AF206580 EF632274 EF632232
Nucras AF206612 AF206612 AF206550 AF206565 EF632276 EF632233
Atlantolacerta AF206603 AF149945 AF206537 AF206579 GQ142144 GQ142154
Ophisops AF206605 AF206605 AF206532 AF206556 EF632278 EF632235
Parvilacerta AJ238187 GQ142106 GQ142135 n/a EF632279 EF632236
Pedioplanis AF206613 AF206613 AF206546 AF206566 EF632280 EF632237
Poromera AF080368 AF080370 AF080369 n/a EF632283 EF632240
Tropidosaura AF206616 AF206616 AF206541 AF206582 EF632291 EF632248
Ichnotropis AF080365 DQ871149 AF080366 n/a EF632266 EF632221
Phoenicolacerta NC_011606 NC_011606 DQ461762 NC_011606 DQ461740 EF632226
Takydromus AB080237 AB080237 AB080237 AF206558 EF632288 EF632245
Teira AJ004884 GQ142096 GQ142121 AF372052 EF632289 EF632246
Timon AF206595 AF206595 DQ902142 AF206569 EF632290 EF632247
Zootoca AF206594 AF206594 AY714929 AF206554 EF632292 EF632249
Apathya AF145444 AF149946 GQ142127 unpublished EF632268 EF632223
Scelarcis AF206602 AF206602 AF206538 AF206570 GQ142145 GQ142155
Omanosaura AF080347 AF080352 AF080351 n/a EF632277 EF632234
Lacerta AM176577 AM176577 AM176577 AF206551 EF632267 EF632222
Podarcis AF206601 AF206601 AY234154 AF206575 EF632282 EF632239
Australolacerta FR751396 FR751396 FR751398 n/a n/a DQ871208
Holaspis n/a n/a n/a n/a EF632263 EF632218
Iranolacerta GQ142088 GQ142111 GQ142140 n/a GQ142152 GQ142162
Philochortus n/a n/a n/a n/a EF632281 EF632238
Pseuderemias n/a n/a n/a n/a EF632286 EF632243
Archaeolacerta AF206592 AF206592 GQ142126 n/a EF632256 EF632211
Scincidae NC_000888 NC_000888 NC_000888 NC_000888 AY217888 AY662634
100 KAPLI ET AL.
ted model was used (RONQUIST &
HUELSENBECK, 2003).
Phylogenetic inference analyses were con-
ducted using Bayesian Inference (BI),
Maximum Parsimony (MP) and Maximum
Likelihood (ML). Nucleotides were used as
discrete, unordered characters. BI analysis was
performed in MrBayes (v3.1; RONQUIST &
HUELSENBECK, 2003), with partitioned data-
set by genes, using the models discussed above.
The analysis was run four times with eight
chains for 107 generations and the current tree
was saved to file every 102 generations. This
generated an output of 105 trees for every run.
The -lnL stabilized after approximately 104
generations, thus the first 104 trees (10%
burn-in in Bayesian terms) of every run were
discarded as a conservative measure to avoid
the possibility of including random, subopti-
mal trees. The percentage of samples recove-
ring any particular clade in a BI analysis repre-
sents that clades posterior probability
(HUELSENBECK & RONQUIST, 2001). A majo-
rity rule consensus tree (Bayesian tree) was
then calculated from the posterior distribution
of trees, and the posterior probabilities calcula-
ted as the percentage of samples recovering any
particular clade (HUELSENBECK & RONQUIST,
2001), where probabilities 95% indicate sig-
nificant support.
MP analysis was performed with PAUP*
v.4.0b10 (SWOFFORD, 2002). This analysis
was carried out (heuristic searches) using
stepwise addition and performing tree bisec-
tion-reconnection (TBR) branch swapping
(SWOFFORD et al., 1996). Confidence in the
nodes of MP trees was assessed by 1000
bootstrap replicates (FELSENSTEIN, 1985).
The analysis was run twice with the gap con-
sidered as missing and as a fifth character.
Finally ML analysis was performed in
the online version of RaxML (STAMATAKIS
et al., 2008) using a mixed partitioned
model and the following parameters: -shape
parameters, GTR-rates, and base frequencies
estimated and optimized for each partition
(gen). Furthermore, gamma model of rate
heterogeneity was assumed for all parti-
tions while the invariable sites were esti-
mated by the analyses. Confidence in the
nodes of ML trees was assessed by 100
bootstrap replicates.
Soft vs. hard molecular polytomies
Unresolved evolutionary relationships are
considered soft polytomies in that they are
multiple dichotomous branching events
occurring in rapid succession. To differentia-
te between poorly supported clades (soft
polytomies) vs. zero-length branches (hard
polytomies), we used the likelihood ratio test
[-2(lnLHa-lnLHo)], proposed by SLOWINSKI
(2001), where LHa is the likelihood under the
alternate hypothesis (the length of branch in
question is nonnegative) and LHo is the likeli-
hood under the null hypothesis (branch has
zero-length). Using the describe trees com-
mand following our ML run (with Perform
likelihood-ratio test for zero branch lengths
selected in the likelihood settings menu),
PAUP* calculated the probability for each
likelihood ratio under the 2 distribution
with one degree of freedom. Significance for
the likelihood ratio test for each branch in
the phylogeny was determined using the per-
centage point values under the GOLDMAN &
WHELAN (2000) mixed model. We used = 0.05
as significance level to account for possible
Type I error.
 PHYLOGENY OF THE LACERTIDAE 101

RESULTS for the clade including Meroles, Ichnotropis,

Tropidosaura and Australolacerta. Finally the
Of the 5727 sites examined, 2557 were unresolved relationships of the family, accor-
variable, 1860 of which were parsimony infor- ding to the likelihood ratio test (SLOWINSKI,
mative (2690 and 1945 respectively, when the 2001), could be considered as soft polytomies
outgroup was included). The MP analysis with the exception of the branch length
when the gap was considered as missing pro- separating Poromera ( > 0.05, Fig. 1).
duced one tree with a length of 13 941 steps,
while three equally parsimonious trees with a DISCUSSION
length of 14 361 steps were produced when
the gap was treated as a fifth character. The The genera comprised in the Eremiadini
topologies recovered were the same for most of tribe (ARNOLD et al., 2007) form a subclade
the clades (differences are discussed below). within Lacertinae in all analyses. This clade
BI resulted in the topology shown in Fig.1 showed significant internal structure into
(identical topologies were recovered for each
of the four runs). All analyses recognized the
division of the family in two main clades
(Gallotiinae and Lacertinae) and the division
of the later in two subclades (Lacertini and
Eremiadini) with strong statistical support. In
the case of Lacertini no analyses managed to
resolve the relationships among the 20 genera
under study. One monophyletic group within
Lacertini was recognized by all analyses (Teira
and Scelarcis) while Algyroides and
Dinarolacerta formed a monophyletic clade
for BI and ML, whereas for MP only when
the gap was considered as a fifth character.
ML supported a sister relationship of the later
with Iberolacerta (62% bootstrap support)
while for MP when the gap was treated as a
fifth character another monophyletic group
(Lacerta and Timon) was supported (51% Figure 1: Phylogenetic relationships among the 40
bootstrap support). In the case of Eremiadini, genera included in the analyses as inferred by BI.
MP (either when the gap was treated as mis- Bayesian posterior probability values (> 0.95) are pre-
sing or as a fifth character) could not resolve sented on the nodes followed by bootstrap values
(> 50%) for ML, MP and MP* (considering the gap
adequately the relationships of the taxa under
as a fifth character). Branch lengths statistically not sig-
study. On the contrary BI and ML resulted in nificantly greater than zero are indicated with an asterisk
the same topology with the exception that (*). Eumeces anthracinus, E. ergegius and E. inexpectatus
ML supported a basal position of Pedioplanis (Scincidae) were used as outgroup (not shown).
102 KAPLI ET AL.
three groups, two of which correspond to the
division of MAYER & PAVLICEVs (2007)
Ethiopian and Saharo-Eurasian, while
Atlantolacerta appears as basal in accordance
with ARNOLD et al. (2007). Surprisingly the
Ethiopian group, which includes most of the
least studied genera of Lacertidae (SALVI et
al., 2011), appeared as the best resolved clade
of the family. It is worth noticing that the sis-
ter relationship of Australolacerta and
Tropidosaura proposed recently by SALVI et al.
(2011) is also confirmed by this study.
The genera that belong to the Lacertini
tribe appear as a monophyletic clade in all
analyses with strong statistical support,
although relationships within the clade have
low resolution. It is interesting to note that
two monophyletic clades were recognized by
all analyses, Scelarcis with Teira and
Dinarolacerta with Algyroides (Fig. 1). For
the first case PAVLICEV & MAYER (2009) pro-
pose that these two genera should be united
in one, while the case of Dinarolacerta with
Algyroides remains puzzling until further
data for the taxa will be available. The pro-
blem of reconstructing the phylogenetic rela-
tionships within Lacertini arose in all pre-
vious studies (HARRIS et al., 1998; FU, 2000;
ARNOLD et al., 2007; MAYER & PAVLICEV,
2007, PAVLICEV & MAYER, 2009) with diffe-
rent datasets. Here we show that neither a
dataset of 5727 bp (two nuclear and four
mitochondrial genes) with a wide genera
sampling is able to shed light in the rela-
tionships between Lacertini. According to
the most recent molecular phylogeny of the
taxon (PAVLICEV & MAYER, 2009) it was
assumed that the poor resolution was more
likely to reflect a rapid radiation resulting in
a polytomy than considering the markers
used inappropriate. The results of the likeli-
hood ratio test, conducted for the current
dataset, suggest that the unresolved rela-
tionships of both Eremiadini and Lacertini
are more likely to be seen as a case of a soft
polytomy. Hence before the rejection or
acceptance of PAVLICEV & MAYERs (2009)
view, two aspects could be further resear-
ched: 1) taxa sampling and 2) quantity/qua-
lity of genetic markers. As far as the first is
concerned, even though Lacertidae has been
the subject of several studies, the intra gene-
ra variation in most of the cases remains
unexplored. Taking this under consideration
it could be assumed that the unresolved rela-
tionships of the family could be attributed to
inadequate data sampling. For the near futu-
re, analyses with more species per genus
could show improvement in case we are
facing a soft and not a hard polytomy of
Lacertini. Furthermore it should be taken
into account that if we assume hard poly-
tomy for Lacertini the same hypothesis
should be extended, to some degree, to
Eremiadini since the relationships among
some of its genera remain unresolved.
Regarding the amount and the combina-
tion of the particular genetic markers there
should not be a problem of too much or too
little variation, as stated before (PAVLICEV &
MAYER, 2009). Nevertheless the combination
of multiple genes phylogeny (i.e. complete
mtDNA genome) or the application of res-
triction site-associated DNA tags (RAD tags,
BAIRD et al., 2008, EMERSON et al., 2010)
could be interesting approaches that could
provide much more detailed and extensive
information. Also the investigation for RGCs
(Rare Genomic Changes) that have become
increasingly important in systematics and
 PHYLOGENY OF THE LACERTIDAE
complement phylogenetic analyses of pri-
mary sequence data, as noted by SPRINGER et
al. (2004), could ultimately provide the most
convincing resolution of intra-Eremiadini
and -Lacertini phylogeny.
Summarizing the above we can assume
that analyses based on the current available
data are able to resolve the phylogenetic rela-
tionships on the level of subfamily and tribe.
However, resolution of the relationships
below the tribe level in Eremiadini and
Lacertini necessitates a more sophisticated
analysis and better knowledge on the intra
genera variation.
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 Basic and Applied Herpetology 25 (2011): 105-113

Biometry and pholidosis of Thamnophis scaliger: an atypical

example of sexual dimorphism in a natricine snake
Mnica Feriche1,*, Senda Reguera1, Xavier Santos2,3, Estrella Mocio-Deloya1, Kirk Setser1, Juan M. Pleguezuelos1
1
Departamento de Zoologa, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
2
Departamento de Biologa Animal, Universidad de Barcelona, Barcelona, Spain.
3
CIBIO, Centro de Investigao em Biodiversidade e Recursos Genticos, Universidade do Porto, Vairo, Portugal.

* Correspondence: Departamento de Zoologa, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain. Phone:
+34 958 243082, Fax: +34 958 243238, E-mail: monicaf@ugr.es
Received: 6 April 2011; received in revised form: 20 July 2011; accepted: 8 August 2011.

Natural-history traits of Thamnophis scaliger (Mesa Central blotched garter snake), a Mexican endemism
recently separated from Thamnophis scalaris, are almost unknown. We provide information on biometric and
pholidotic traits according to sex for a large sample of individuals, and compare these morphological data
with those available for T. scalaris, in order to place the species within the morphological context of the highly
diverse genus Thamnophis. Moreover, we examine the adaptive value of sexually dimorphic characters from an
evolutionary approach. Thamnophis scaliger appears to have fewer subcaudal and ventral scales than T. scalaris.
Our sample also suggests that T. scaliger females have larger snout-vent lengths, masses, and body condition
indexes (traits related to fecundity) than males, but that males have larger ventral and subcaudal scale numbers,
and higher absolute and relative tail lengths (traits related to sexual selection) than females. Ventral and sub-
caudal scale number is a surrogate of vertebrae number (somites). If females are longer and relatively heavier
than males but exhibit a lower number of somites, we suggest that this sexual dimorphism might be driven
by the necessity for females of having larger and consequently more robust vertebrae to anchor muscles that
have to move a heavier body.

Key words: Mxico; sexual dimorphism; Thamnophis scalaris; Thamnophis scaliger; ventral and subcaudal scale number.

Biometra y folidosis de Thamnophis scaliger: un ejemplo atpico de dimorfismo sexual en un colbrido natri-
cino. La historia natural de Thamnophis scaliger (serpiente de jardn manchada de Mesa Central), endemis-
mo mexicano recientemente separada de Thamnophis scalaris, es casi desconocida. Presentamos informacin
sobre los rasgos biomtricos y folidticos en funcin del sexo de una amplia muestra de individuos, y com-
paramos estos datos con los disponibles para T. scalaris, con el objetivo de situar la especie en el contexto mor-
folgico de un gnero con una elevada diversidad como Thamnophis. Adems indagamos, bajo un enfoque
evolutivo, el valor adaptativo de los caracteres sujetos a dimorfismo sexual. Thamnophis scaliger parece tener
menos escamas ventrales y subcaudales que T. scalaris. Nuestros resultados tambin apuntan a que las hem-
bras de T. scaliger superan a los machos en longitud hocico-cloaca, masa e ndice de condicin corporal
(aspectos relacionados con la fecundidad), mientras que los machos superan a las hembras en nmero de esca-
mas ventrales y subcaudales y longitudes absoluta y relativa de la cola (aspectos relacionados con la seleccin
sexual). El nmero de escamas ventrales y subcaudales es un reflejo del nmero de vrtebras (somitos).
Sugerimos que la razn por la que las hembras tienen un menor nmero de somitos que los machos aun sien-
do ms largas y pesadas que stos puede ser consecuencia de la necesidad de tener vrtebras ms grandes, y
por tanto ms robustas, para anclar unos msculos que tienen que mover un cuerpo ms pesado.

Key words: Dimorfismo sexual; Mxico; nmero de escamas ventrales y subcaudales; Thamnophis scalaris;
Thamnophis scaliger.
106 FERICHE ET AL.
The genus Thamnophis (American garter
snake), distributed throughout North and
Central America between 10N and 60N
and between 5W and 140W, is the most
widely-distributed reptile in North America
(ROSSMAN et al., 1996). It is the most diverse
and studied genus of North American snakes,
comprising 31 species (DE QUEIROZ et al.,
2002). Information, however, is biased to
some species, and while a few of them have
been widely studied (more is known about T.
sirtalis than about any other American
snake), very little is known about life histo-
ries of many other species in the genus. This
unbalanced knowledge prohibits meaningful
comparative studies in morphology and
natural history from an evolutionary ecology
approach. In this sense, understanding
phylogenetic relationships among taxa requi-
res knowledge of morphological parameters
in all species (REEDER & WIENS, 1996).
The present study characterizes morpho-
logical traits of Thamnophis scaliger, a small,
poorly-known species endemic to central
Mexico (ROSSMAN et al., 1996), previously
considered a subspecies of T. scalaris and
recently elevated to the rank of species
(ROSSMAN et al., 1996; ROSSMAN & LARA-
GNGORA, 1997). Its position within the
genus Thamnophis, however, remains unclear.
In a mitochondrial DNA analysis of 29 spe-
cies within the genus (DE QUEIROZ et al.,
2002) each of two specimens of T. scaliger
from localities separated by approximately
100 km were placed in different clades. One
specimen nested with T. scalaris, in agree-
ment with morphological evidence
(ROSSMAN & LARA-GNGORA, 1997), while
the other nested within the sister group to the
clade containing T. exsul and T. errans. This
result suggests that T. scaliger may be polyty-
pic, and indicates a need for additional data
to quantify morphological variability. In view
of this confusing situation, we compare mor-
phological data collected from T. scaliger with
those available for T. scalaris in order to place
the species within a morphological diversity
context in the genus Thamnophis. Moreover,
we provide supplementary data for biometric
and pholidotic traits according to sex for a
large sample of individuals, and examine the
adaptive value of some characters from an
evolutionary approach. Previously, basic mor-
phological information for T. scaliger was
provided only by ROSSMAN et al. (1996) and
ROSSMAN & LARA-GNGORA (1997).
MATERIALS AND METHODS
Thamnophis scaliger occurs in the Distrito
Federal and the states of Mxico,
Aguascalientes and Michoacn (19-22N, 98-
102W; elevation 2230-3000 m above sea
level; QUINTERO-DAZ et al., 1999; CANSECO-
MRQUEZ et al., 2007) in the northern por-
tion of the Mexican Transverse Volcanic axis
and the southern fringe of the Mesa Central.
The study area is situated near Atlacomulco,
north-western Mxico State (19.70N,
99.87W; 2500-2700 m above sea level), in
the highlands of the Central Volcanic Belt of
Mxico, near and within the valley of the Ro
Lerma (Fig. 1). The study area for the sample
considered here occupies 74 hectares of pastu-
re for cattle and sheep and fields for various
crops. Climate in this area is cool and humid,
but while maximum daily temperatures are
relatively independent of the season, daily
 BIOMETRY AND PHOLIDOSIS OF THAMNOPHIS SCALIGER
Figure 1: Location of the study area in Mexico State,
Mexico (arrow), and known range (circles) of the
Mesa Central blotched garter snake, Thamnophis
scaliger (modified from ROSSMAN et al., 1996).
minimum temperatures are variable (mean
minimum and maximum temperatures are
0.1-20.2C for January and 8.8-21.2C for
July). Rainfall is highly seasonal, with 69%
(570 of 830 mm) of annual precipitation
falling between June and September, and with
monthly precipitation during the driest por-
tion of the year (January to May) averaging
3% of the annual total (Comisin Nacional
del Agua station 00015139-Atlacomulco;
approximately 5 to 15 km away from study
sites; data recorded from 1961 to 1992).
Thamnophis scaliger inhabits pastures near
streams and margins of crop fields; human
alteration has reduced natural vegetation to
river gallery in the valley.
Field sampling was performed daily from
June to August 2008 and 2009 by between
two and five researchers during morning
hours (10:00-13:00), the peak activity period
for the species in this area. Snakes were
collected above ground or beneath rocks and
debris by hand or with tongs and georeferen-
ced by GPS in order to release individuals at
the exact point of capture following proces-
107
sing. Samples from two nearby areas,
Lagunita (19.90N, 99.87W, 2510 m above
seal level) and La Estancia (19.86N,
99.80W; 2720 m above sea level) were taken
during the same period (although with less
sampling effort) in order to find possible
areas of syntopy between T. scaliger and T.
scalaris. Syntopy occurred only at La
Estancia, where we found three specimens of
T. scaliger and four specimens of T. scalaris.
A total of 161 (71 males, 90 females) T.
scaliger and four T. scalaris were captured.
Snakes were anaesthetized in the laboratory
(within 20 km far from any of the study
areas) with an approximate dosage of 0.8 ml/l
of isoflurane as described by MOCIO-
DELOYA et al. (2009), to obtain reliable bio-
metric and pholidotic measurements
(SETSER, 2007). Individuals were marked
with a PIT tag (TX148511B model, 8.5 x
2.1 mm, 0.1 g, 134.2 kHz, Destron-Fearing,
St. Paul, Minnesota, USA) as described by
MOCIO-DELOYA et al. (2009) before release
in order to avoid replication of data. Males
and non-gravid females were released within
one to four days after capture, whilst gravid
females were kept in the laboratory until par-
turition (between one and two weeks).
We measured snout-vent length and tail
length (SVL and TL, respectively, 1 mm),
head length and head width (HL and HW, res-
pectively, 0.1mm), and body mass ( 0.1 g;
gravid females not included in data summaries).
We used the Body Condition Index (BCI) to
compare differences in body shape between the
sexes; this index, widely used in snakes, is calcu-
lated from residuals of regression of log body
mass on log SVL (BONNET & NAULLEAU,
1994) in mature individuals of each sex.
108 FERICHE ET AL.

Pholidotic variables measured included the hed data); we have no direct data, however, allo-
numbers of ventral scales (V; following wing estimation of size at maturity for male
DOWLING, 1951), subcaudal scale pairs (SC), T. scaliger. PARKER & PLUMMER (1987), after
dorsal scale rows at mid-body (D), and the reviewing 19 studies on 10 species of small vivi-
numbers of preocular scales (PRO), postocular parous colubrids (e.g. T. scaliger), found that, on
scales (PTO), loreal scales (L), anterior tempo- average, males matured at 87% of the size of
ral scales (AT), posterior temporal scales (PT), conspecific females. As males reach 84% of the
supralabial scales (SPL) and infralabial scales maximum size of females (see Results), this
(IFL) on each side of the head (Table 1). approach is very similar to estimating sexual
Because of high ontogenetic variation in maturity in males by utilizing the difference in
snake body size, we included only mature indi- maximum size between both sexes (SHINE,
viduals in our analysis to evaluate possible 1990). Using the generalization calculated by
sexually-based differences in morphometric PARKER & PLUMMER (1987), we estimated that
variables. Females mature at 267 mm SVL (SVL male T. scaliger matured at 235 mm SVL.
of the smallest gravid female; authors unpublis- Variables not normally distributed (SVL of
the top decile of each sex) were assessed by
Table 1: Frequencies and percentage of the number nonparametric statistical tests (Mann-Whitney
of head (both sides) and dorsal scales in a population U-test). Tail length, HW and HL varied with
of Thamnophis scaliger in the Valle del Ro Lerma, body size, and comparisons between sexes were
Atlacomulco, State of Mexico. Abbreviations are
made by analysis of covariance (ANCOVA)
defined in the Materials and Methods section.
with SVL as the covariate. Although numbers
of V, SC, and V + SC are not thought to vary
Head characters (N) Possible arrangements %
over the life of a snake, larger body size has
SPL (70) 7+7 91.4 been associated with a greater number of ven-
6+7 7.1 tral scales in Coronella girondica (SANTOS &
6+6 1.4 PLEGUEZUELOS, 2003); in these variables, com-
IFL (70) 9+9 84.3
8+9 14.3 parisons between sexes were also performed
8 + 10 1.4 using ANCOVA with SVL as covariate.
PRO (70) 1+1 100 Comparisons among dorsal scale numbers (D)
PTO (68) 3+3 47.1
2+2 29.4
were assessed using 2 x 2 contingency tables.
2+3 19.1
2+1 4.4 RESULTS
L (70) 1+1 98.6
2+1 1.4
AT (69) 1+1 97.1 Thamnophis scaliger typically exhibit 7
1+2 2.9 SPL, 9 IFL, 1 PRO, 3 PTO, 1 L, 1 AT, 2 PT,
PT (69) 2+2 81.2 and 19 D (Table 1). The sample of T. scalaris
2+3 11.6
3+3 5.8 was quite small, but in average, T. scalaris
1+2 1.4 were larger and heavier than T. scaliger and
D (113) 19 70.8 females differed in number of SC: 51-56 in
17 29.2
T. scalaris, 39-48 in T. scaliger (Table 2).
 BIOMETRY AND PHOLIDOSIS OF THAMNOPHIS SCALIGER 109

Table 2: Pholidotic and biometric variables (range, mean : standard deviations) in populations of
Thamnophis scaliger and T. scalaris in the Valle del Ro Lerma, Atlacomulco, State of Mexico. For T. scaliger,
details for males and females, as well as their statistical comparison, are provided. SVL was compared using
ANOVA, while TL, HW, HL, body mass, V, SC, and V + SC were compared using ANCOVAs, with SVL as
covariate. For mass comparison the variable was log transformed, and only sexually mature individuals and no
pregnant females were considered. See Materials and Methods section for explanations of abbreviations.

Trait % T. scaliger T. scalaris

Males Females Males Females

SVL (mm) N 68 87 1 3
Range 127-369 126-439 360-500
Mean 278.4 70.1 320.2 72.0 483 431.070.0
F1,153 = 13.11; P = 0.0004
TL (mm) N 62 70 3
Range 30-93 29-85 99-125
Mean 68.2 19.9 64.8 14.4 111.7 13.0
F1,130 = 178.51; P = 0.001
HL (mm) N 37 37 3
Range 8.8-15.5 9.5-17.4 16.8-22.9
Mean 12.78 1.769 13.53 1.92 19.2 3.2
F1,72 = 2.62; P = 0.110
HW (mm) N 37 37 3
Range 5.3-11.0 5.3-11.4 10.7-16.9
Mean 8.31 1.317 8.77 1.293 12.8 3.5
F1,72 = 0.67; P = 0.41
Body mass (g) N 50 40 1 3
Range 10.5-41.7 16.2-70.3 31.6-72.6
Mean 23.2 8.7 36.4 13.9 67 46.7 22.5
F1,88 = 7.63; P = 0.007
V N 57 71 1 3
Range 134-148 129-145 138-142
Mean 141.37 3.2 135.3 3.9 140 139.7 2.1
F1,126 = 771.7; P < 0.0001
SC N 55 63 3
Range 48 - 59 39 - 48 51 - 56
Mean 53.2 2.4 44.1 2.4 53.0 2.6
F1,116 = 389.7; P < 0.0001
V + SC N 39 42 3
Range 184 - 204 170 - 192 190 - 195
Mean 194.2 4.9 179.5 5.6 192.7 2.5
F1,79 = 271.9; P < 0.0001

Adult female T. scaliger were larger and
heavier than males (Table 2), and differences
persisted when we compared SVL from the
top decile of each sex (males: 361.9 5.1
mm, range 356-369 mm, N = 7; females:
405.2 13.9 mm, range 395-439 mm, N = 9;
Mann-Whitney U test: Z = 3.334, P < 0.001).
The longest male measured 369 mm SVL, and
35% of the 87 measured females had an SVL
greater than this value. In both absolute and
110 FERICHE ET AL.
relative scores, male tail was longer, repre-
senting 19.5% of the total length in this sex
(N = 61), whereas in females the tail repre-
sented 17.0% of total length (N = 70). In
contrast, when body length was used as
covariate, body mass was higher in females
than in males (Table 2).
BCI was higher in females (0.60, N = 40)
than in males (0.53, N = 49), as demonstra-
ted by ANCOVA with sex as factor and SVL
as covariate (F1,87 = 28.40; P < 0.0001). We
found no differences between the sexes in
relative head length or width (Table 2).
The number of V + SC scales after correc-
ting by SVL was higher in males than in
females (Table 2; Fig. 2). While ventral scale
count did not overlap between sexes, SC
counts showed limited overlap (two males
and five females [5.4% of specimens] had 48
pairs of subcaudal scales; Table 2). Males and
females differed in the relationship between
body length and number of ventral scales: at
similar body length, males had more V, SC,
and V + SC than did females (Table 2; Fig 2).
There were no differences in the number of
D between sexes (2 = 0, d.f. = 1, P = 0.96).
DISCUSSION
Differences between T. scaliger and T. scalaris
ROSSMAN et al. (1996), ROSSMAN &
LARA-GNGORA (1997) and this study
demonstrated broad overlap in morphologi-
cal traits between the two species. However,
T. scaliger apparently has a shorter head and
a lower number of subcaudal scale pairs than
T. scalaris (with no overlap between species
when sex is considered), and a shorter relati-
ve tail length (the shortest in the genus;
Figure 2: Relationship between ventral + subcaudal
(V + SC) scale count and body size (SVL) by sex in
Thamnophis scaliger in the Valle del Ro Lerma,
Mexico; only individuals with complete tails consi-
dered. Females: solid circles; males: open circles. See
Table 2 for ANCOVA results.
ROSSMAN et al., 1996). This pronounced tail
length difference is due to accommodation
for the extremely long hemipenes of T. scalaris,
which, when invaginated, reach SC pair 27
(ROSSMAN & LARA-GNGORA, 1997).
Thamnophis scaliger and T. scalaris occur
in sympatry at only one of the three sampled
sites, in La Estancia (16 km NE of the main
study area). At this locality, however, the few
specimens examined had modal values for
biometric and pholidotic traits of the species
to which they belong. With due reservations
because of the small number of localities we
sampled, we suspect these species tend to be
allopatric in distribution, but, when in
syntopy, each one retains the morphology
exclusive to its species. In this sense, the size
of the two scaliger specimens cited by
RAMREZ-BAUTISTA et al. (1995) near the
study area far exceeded the largest snake
recorded in our substantial T. scaliger sam-
ple, and we suggest these specimens were
actually T. scalaris.
 BIOMETRY AND PHOLIDOSIS OF THAMNOPHIS SCALIGER
Sexual dimorphism in T. scaliger
Body size is one of the most important bio-
logical attributes of an organism, and the genus
Thamnophis includes terrestrial and aquatic
forms with maximum total lengths between
463 mm and 1626 mm (ROSSMAN et al., 1996).
Thamnophis scaliger is one of the smallest spe-
cies within the genus, larger only than T. exsul.
Within T. scaliger, females achieve greater body
length than males, a general characteristic for
this and other natricine colubrid snakes (FITCH,
1981). Female snakes that make a significant
investment in reproduction tend to attain larger
body size than males (SHINE, 1993; BONNET et
al., 2000), and T. scaliger is not an exception, as
postpartum relative clutch mass is rather high
(55% on average, authors unpublished data)
compared to values from other ovoviviparous
species (SEIGEL & FITCH, 1984).
Male snakes generally have longer tails than
females (SHINE et al., 1999) because of the need
to accommodate the hemipenes at the base of
the tail (KING, 1989). Because of this sexual
dimorphism in tail length, males also have a hig-
her number of subcaudal scales than females
and, accordingly, 94.2% (90.6-98.5%, = 0.05)
of individuals were correctly sexed based solely
on this character. Males also had a higher num-
ber of ventral scales; while this tendency appears
in 89% of species of Thamnophis (ROSSMAN et
al., 1996; SHINE, 2000), it was found in only
20.9% of 255 various snake species examined by
SHINE (2000). Female snakes generally have
relatively longer bodies than males to permit
accommodation for a large volume of
eggs/embryos, which results in a higher number
of both trunk vertebrae and ventral scales
(fecundity selection; SHINE, 1993). In contrast,
a larger proportion of male body somites are
111
devoted to the formation of the tail, which
accommodates the hemipenes. Relatively longer
tails in males are a generality in snakes (FITCH,
1981); 94% of species examined by SHINE
(2000) showed sexual dimorphism in subcaudal
scale number, with higher values in males.
Relatively longer tails in males may increase suc-
cess in mating or male-male rivalry (sexual selec-
tion; KING, 1989).
Body length and tail length are highly corre-
lated with the number of ventral and subcaudal
scales and subject to selection (SHINE, 2000). In
most snake species, the relationship between
body length and number of ventral scales is simi-
lar in males and females (SHINE, 2000). In spe-
cies sexually dimorphic in size, this similar rela-
tionship permits natural selection to alter body
proportions in either or both sexes by varying
ventral scale number, and therefore vertebrae
number (SHINE, 2000). However, this was not
found in the genus Thamnophis (SHINE, 2000)
nor in T. scaliger included in this study, since
males had more ventral and subcaudal scales (and
therefore more vertebrae) per unit body length
than did females. Despite having a lower number
of ventrals (and subcaudals and, subsequently,
ventrals + subcaudals), female T. scaliger have lon-
ger bodies than males and higher body condition
indexes (at equivalent lengths, females tend to be
thicker than males). Assuming the number of
ventrals (and the number of subcaudal pairs) to
be reflective of the number of vertebrae and that
this relationship is universal in snakes
(ALEXANDER & GANS, 1966), we presume that
the thicker sex (the female) has fewer vertebrae
than the male. According to our presumption, it
is plausible to argue that females should have lar-
ger and consequently more robust vertebrae to
anchor muscles that have to move a heavier body.
We did not test this possibility by necropsy or
112 FERICHE ET AL.
X-rays, but the alternative hypothesis i.e. fema-
le vertebrae are not larger but simply longer than
male ones is less plausible. Sexual dimorphism
in body thickness has been found in other vivipa-
rous snakes and is interpreted as an adaptation to
female reproductive function (AUBRET et al.,
2002). Although we were unable to test our argu-
ment, we suggest the existence of a functional
relationship between sexual dimorphism in body
shape and vertebrae size in snakes.
We failed to find sexual differences in head
shape or any other structures related to prey
handling. This result is surprising given the
sexual dimorphism detected in other parts of
the body. Absence of sexual variation in head
morphology may reflect the lack of functional
differences in diet between male and female
T. scaliger, since as far as we know, both sexes
forage on the same small prey (earthworms,
REGUERA et al., 2011). This conclusion agrees
with the sexual dimorphism in head observed
in species with diet variation between sexes
(VINCENT et al., 2006).
Acknowledgement
We thank Lic. J. Martnez-Lombarry for
generously offering access to his property, and
also to P. Snchez and his family for their warm
welcome and assistance throughout the course
of our fieldwork. S. Busack kindly improved the
content and style of the manuscript. Research
was conducted in accordance with SEMAR-
NAT (Mexico) research permits issued to EMD
(SGPA/DGVS/05247/08), and was partially
funded by the projects A/010196/07 and
A/010196/07 of the AECI (Spain), granted to
JMP. XS was supported by a post-doctoral grant
(SFRH/BPD/73176/2010) from Fundao
para a Cincia e a Tecnologia (FCT, Portugal).
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SHINE, R. (1990). Proximate determinants of
sexual differences in adult body size.
American Naturalist 135: 278-283.
SHINE, R. (1993). Sexual Dimorphism in
Snakes, In R.A. Seigel & J.T. Collins (eds.).
Snakes: Ecology and Behavior. McGraw-
Hill, Inc., New York, USA pp. 49-86.
SHINE R. (2000). Vertebral numbers in male
and female snakes: the roles of natural,
sexual and fecundity selection. Journal of
Evolutionary Biology 13: 455-465.
SHINE, R.; OLSSON, M.M.; MOORE, I.T.; LE
MASTER, M.P. & MASON, R.T. (1999).
Why do male snakes have longer tails
than females? Proceedings of the Royal
Society of London B 266: 2147-2151.
VINCENT, S.E.; DANG, P.D.; HERREL, A. & KLEY,
N.J. (2006). Morphological integration and
adaptation in the snake feeding system: a
comparative phylogenetic study. Journal of
Evolutionary Biology 19: 1545-1554.
114
 115

PUBLICATION GUIDELINES
AIMS AND SCOPE
Basic and Applied Herpetology (B&AH) is the official journal of the Spanish Herpetological
Society (AHE). B&AH publishes original research papers dealing with any aspect of amphibians and
reptiles worldwide. Exceptionally, updated reviews about especially interesting issues will be accepted if
they fit with the general purpose of the journal. There is no maximum limit to the length of the papers
submitted although authors can be requested to shorten their paper if necessary. Authors can submit
short notes if these are organized around hypotheses appropriately argued and analysed quantitatively.
The editors reserve the right to publish the accepted manuscripts as original research papers or as short
notes at their convenience, regardless of the format of the original manuscript. B&AH will not accept
distribution notes or punctual or sporadic observations. This kind of papers must be submitted to the
Boletn de la Asociacin Herpetolgica Espaola
http://www.herpetologica.es/publicaciones/boletin-de-la-asociacion-herpetologica-espanola
Submission of a manuscript implies, without further acceptance by authors, that the work des-
cribed has not been published before (except in the form of an abstract), that it has not been sub-
mitted or published elsewhere, and that its content and publication in B&AH has been approved by
all co-authors. By submitting a manuscript, the authors agree that the copyright for their article is
transferred to the AHE if and when the article is accepted for publication. The copyright covers the
exclusive and unlimited rights to reproduce and distribute the article in any form of reproduction.
To minimize turnaround time, authors are encouraged to follow the instructions below.
Manuscripts not in the correct format may be returned to the authors for modification.
Instructions to authors are posted on the web page of B&AH
http://bah.herpetologica.es

MANUSCRIPT SUBMISSION
Manuscripts should be submitted preferably as e-mail attachments to the journal address:
bah@herpetologica.org
Manuscripts will be prepared using word-processing software (preferably MS Word). Please
do not submit material as PDF files.
Although less recommendable, manuscripts can also be sent through postal mail in a mass
storage device (CD-ROM, pen-drive, etc.) to one of the following addresses:
Manuel Ortiz Santaliestra. Editor, Basic and Applied Herpetology. Instituto de Investigacin en
Recursos Cinegticos UCLM-CSIC-JCCM. Ronda de Toledo s/n 13071 Ciudad Real (Spain).
Ana Perera Leg. Editora, Basic and Applied Herpetology. CIBIO. Campus Agrrio de Vairo.
Rua Padre Armando Quintas-Castro 4485-661 Vairo (Portugal).
Postal mail from Spain or Portugal should be addressed to the editor in your own country. Mail
from the rest of countries should be addressed to M. Ortiz (for papers concerning amphibians) or A.
Perera (for papers concerning reptiles). Studies with a general subject or dealing with both taxa can
be sent to either editor. It is not necessary to send a hard copy of the manuscript. The storage devi-
ce must contain only the files corresponding to the submitted paper, without duplicate files or diffe-
rent versions of the same file. Devices will not be returned to the authors regardless of the acceptan-
ce or rejection of the paper.
For the original submission, include figures and tables in the same file as the main text of the
manuscript. If high-resolution figures are necessary, send them as separate files once the paper has
116

been accepted for publication and potential corrections of such figures has been introduced. Do not
submit original high-resolution figures with the initial submission unless the originals must be seen
by the editors and the reviewers.
Authors are requested to send with the manuscript a cover letter indicating the strengths and rele-
vance of their work, and suggesting the names and contact information of at least three qualified revie-
wers. All the manuscripts will be evaluated by at least two independent reviewers. The editors reserve
the right to choose reviewers other than, or in addition to, those suggested by the authors. Duration
of the review process will vary depending on the number of manuscripts in edition, the availability of
reviewers and the time needed by reviewers and editors. Nevertheless, decisions are expected to be
communicated within a 90-day period after manuscript submission. The editors decision will be
based on the reviewers evaluations. There are three categories of response: accept after minor revision,
accept after major revision, and reject. If a paper is rejected because it requires profound changes, but
its content is considered of interest by the editors, authors will be encouraged to resubmit a corrected
version. In those cases, the resubmitted manuscript will be evaluated again by reviewers. Authors must
include with their revised manuscripts a rebuttal letter including a detailed explanation of how they
have dealt with each of the reviewers and editors comments. Revised manuscripts should be returned
to the editors as soon as possible, always within a maximum time of 60 days. After that time, revised
manuscripts can be considered a new submission and sent out for review.

GALLEY PROOFS
Shortly after a manuscript is accepted, a galley proof will be elaborated and sent to the
authors, who should return it back corrected as soon as possible. Corrections in proofs should
be limited to typographical errors. The costs of any other changes will be charged to the
authors. Corrected proofs will be published on the B&AH website. Authors will receive a PDF
copy of the article in its final version for personal use.

FORMAT AND STYLE

B&AH publishes papers in English or Spanish. However, manuscripts in English will be
given preference in the review and edition process. Manuscripts in English may include a
Spanish version of the abstract and key words. Such abstract will be added by the editors if it
is not included in the original version of the manuscript. Manuscripts in Spanish must inclu-
de an English version of the abstract and key words. Moreover, authors can include, at their
convenience, an additional translation of the abstract and key words to one of the following
languages: Portuguese, French, German or Italian.
Manuscripts must be typed double-spaced, aligned left (not justified), and using a normal font
(Times New Roman) of size 12. All paragraphs but the abstracts must be indented (1.25 cm).
Manuscripts should have line numbers (continuous for the whole document), page numbers and
wide margins (2.5 cm) throughout, including tables and figures. Use consistent punctuation; insert
only a single space between words and after punctuation. Type text without end-of-line hyphena-
tion, except for compound words. Use italics only for scientific names of genera and species.
Numbers one to nine should be written in full in the text unless they precede units of measurement
(5 mm), are designators (experiment 4), or are separated by a dash (2-3 scales). Higher numbers
should be written in Arabic numerals except at the beginning of a sentence. Close up digit numbers
except for numbers of five or more digits, in which a space should be used (4000, 45 000). Do not
use thousands separator. Even in the manuscripts in Spanish, use a decimal point, not a comma, as
 117

decimal symbol (0.2 cm). Measurement units and their abbreviations should conform to those of
the SI (Systme International dUnits). For significance tests, give the name of the test followed by
a colon, the test statistic and its value, the degrees of freedom (as a subscript to the test statistic) or
sample size (as N = x) whichever is the convention for the test, and the probability value (ANOVA:
F3,8 = 9.733; P = 0.005). The name of the test can be omitted if can be inferred from the context
(The ANOVA revealed that differences were significant (F3,8 = 9.733; P = 0.005)). Probability
values will be quoted preferably as exact values (P = 0.018), or as below the pre-established threshold
significance value (P < 0.05, P < 0.001). Use a space before and after each symbol or mathematical
operator (3.54 0.17). Do not use a space after numbers followed by an abbreviator (5%, 24C).

MANUSCRIPT SECTIONS
Manuscripts should be arranged as follows: title page, abstract page(s), text, tables, figure
captions and figures. Each section will include the following information:
Title page:
Title: short and informative. In bold characters.
Names and surnames of the authors (without initials).
Affiliations: multiple author names should be matched to affiliations by superscript numbers.
Correspondence: full postal address (including telephone and fax numbers) and e-mail address
of the corresponding author, who should be indicated with an asterisk in the list of authors.
Running title: not exceeding 50 characters.
Word count of the text (including references and excluding tables, figure captions and
figures), number of tables and number of figures in the manuscript.
Abstract page(s):
Main abstract: in the manuscript language, no more than 250 words (no more than 150
words for short notes).
Main key words: 3-6 key words arranged in alphabetical order in the manuscript langua-
ge, separated by semicolons, and preceded by the term Key words followed by a colon.
In addition, abstract page(s) should include:
a) For manuscripts in English:
Title in Spanish (optional). In bold characters.
Abstract in Spanish (optional): no more than 250 words (no more than 150 words for
short notes).
Key words in Spanish (optional): 3-6 key words arranged in alphabetical order in the
manuscript language, separated by semicolons, and preceded by the term Key words
followed by a colon.
Title in Portuguese, French, German or Italian (optional). In bold characters.
Abstract in Portuguese, French, German or Italian (optional): no more than 250
words (no more than 150 words for short notes).
Key words in Portuguese, French, German or Italian (optional): 3-6 key words arran-
ged in alphabetical order in the manuscript language, separated by semicolons, and
preceded by the term Key words followed by a colon.
b) For manuscripts in Spanish:
Title in English (mandatory). In bold characters.
Abstract in English (mandatory): no more than 250 words (no more than 150 words
for short notes).
118

Key words in English (m mandatory): 3-6 key words arranged in alphabetical order in
the manuscript language, separated by semicolons, and preceded by the term Key words
followed by a colon.
Title in Portuguese, French, German or Italian (optional). In bold characters.
Abstract in Portuguese, French, German or Italian (optional): no more than 250
words (no more than 150 words for short notes).
Key words in Portuguese, French, German or Italian (optional): 3-6 key words arran-
ged in alphabetical order in the manuscript language, separated by semicolons, and
preceded by the term Key words followed by a colon.
Text, with the following sections for original research papers:
Introduction: without heading. Must be clear and concise, including a justification for
the study and a review of the state-of-the-art of the subject. Authors are encouraged to pre-
sent the objectives of the study at the end of the introduction.
Materials and Methods: authors must include all the information necessary to replicate
the study. When presenting the procedures for data analysis, authors are recommended to
state the probability values considered as significant (usually P < 0.05).
Results: must be clear and concise, without repetition of the results shown in tables and figures.
Discussion: do not repeat the results but explore their significance. Nevertheless, it is
recommendable to start the discussion with a brief summary of the more relevant results.
Acknowledgements (optional): limit wording, for example:
oJ. McAllister and C. Smith helped during the study instead of We thank to James
McAllister for his participation in the experimental design, and to Cathy Smith for her
help during field work
oFinanced by the Regional Government (ref ###) instead of Thanks to the Regional
Government for financing the study through the project ###
References (see below)
Introduction should begin on the first line of the first page of the text, without heading.
Write main headings for the rest of the sections, with the exception of acknowledgement, in
small caps (MATERIALS AND METHODS, RESULTS, DISCUSSION, REFERENCES) on a separate line
and keeping an empty line right before and after each heading. In addition to main headings,
authors can use subheadings that will be written in bold italics (e.g. Experimental design, Data
analysis), on a separate line and keeping an empty line right before and after each subheading.
Acknowledgements should be placed between the discussion and the references. The heading
(Acknowledgement) will be written in italics, on a separate line and keeping an empty line right
before and after.
Authors submitting reviews can replace the main headings corresponding to Materials and
Methods, Results and Discussion by different headings at their convenience, using small caps
for main headings and bold italics for subheadings.
Short notes will be structured and arranged as original research papers, although headings
of the sections Materials and Methods, Results and Discussion will be omitted.
Tables: should be numbered consecutively in Arabic numerals arranged as they are quoted
in the text. Each table should be typed on a separate page together with a clear descriptive
legend. Tables should not include vertical rules, and the main body of the table should not
contain horizontal rules. Keep tables as simple as possible and make them understandable
without reference to the text.
 119

Figure captions: should be typed (double-spaced) and grouped together on a page separa-
te from the figures. They should explain with clarity all the elements in the figure. Make figu-
res understandable without reference to the text.
Figures: illustrations, whether maps, drawings, diagrams or photographs, should be kept to
the minimum needed to clarify the text. They should be numbered consecutively in Arabic
numerals arranged as they are quoted in the text and submitted on separate pages (one figure
per page), each bearing the figure number and, if desired (e.g. in photographs), the name(s) of
the figure author(s). If good-quality versions of figures are necessary to be examined during the
review process, authors will include such version in the original submission, as separate files,
according to the instructions given in the next paragraph. In the text, Figure should be abbre-
viated (Fig. 2) except when beginning a sentence. Authors are encouraged to use a sans serif
font (Helvetica, Arial, Geneva) for all text associated with figures. The labelling must be cle-
arly legible and stand reduction to the final print size. Include a scale of distance or dimension
where appropriate.
Once the manuscript is accepted for publication, good-quality versions of figures should
be submitted in their original format with a minimum resolution of 300 dpi. For figures con-
sisting in a picture modified with symbols, text, etc., authors will be requested to send the ori-
ginal, clean, good-quality picture once the manuscript is accepted. For figures not originally
in a digital format (photographs, slides, drawings) authors are recommended to use a dedica-
ted scanner or send the original to the editors by postal mail. In these cases, the originals will
be returned to authors after digitalization. Given that B&AH assumes the costs of publishing
colour prints and slides, these will be accepted only when they are strictly necessary at the edi-
tors discretion. In some cases, colour figures can be accepted only for the digital version, being
displayed in greyscale format in the printed version.

REREFENCES
For references in the text give full surnames of the first author followed by the publica-
tion year and separated by a comma (Pleguezuelos, 1997). For papers with two authors, use
the term & to separate surnames (Semlitsch & Bodie, 2003). Papers with three or more
authors will be quoted with the surname of the first author followed by et al. (note italics)
(Stuart et al., 2004). To distinguish between two papers by the same author(s) in the same
year use lower-case letters (a,b) after the year, without space, arranged in alphabetical order
as the references are quoted in the text (Harris et al., 2004a,b). List multiple citations in
chronological order, using alphabetical order for citations within the same year. Separate
citations with semicolons (Tyler, 1991; Wake, 1991; Blaustein et al., 1994a,b; Stuart et al.,
2004). If the citation is part of the sentence, move the surname(s) of the author(s) out of
the brackets and delete the comma.
As pointed by Pleguezuelos (1997)
Blaustein et al. (1994a) reviewed the situation of amphibians
The reference list should include all and only the references mentioned in the text, tables
and figures. Cite references in the reference list in alphabetical order according to the authors'
surnames. Multiple citations for the same author should be organized as follows: single cita-
tions first (in chronological order), two-author citations second (in alphabetical order), three
or more authors third (in chronological order). Spell out (i.e. do not abbreviate) the names of
all journals. The references should conform to the following formats:
120

Articles in periodicals:
Stuart, S.N.; Chanson, J.S.; Cox, N.A.; Young, B.E.; Rodrigues, A.S.L.; Fishman, D.L.
& Waller, R.W. (2004). Status and trends of amphibian declines and extinctions worldwi-
de. Science 306: 1783-1786.
Wiens, J.J. & Penkrot, T.A. (2002). Delimiting species using DNA and morphological varia-
tion and discordant species limits in spiny lizards (Sceloporus). Systematic Biology 51: 69-91.
Books:
Dodd, Jr., C.K. (ed.) (2009). Amphibian Ecology and Conservation. A Handbook of Techniques.
Oxford University Press, Oxford.
Vitt, L.J. & Caldwell, J.P. (2009). Herpetology: An Introductory Biology of Amphibians and
Reptiles, 3rd ed. Academic Press, Burlington, Massachusetts.
Book chapters:
King, R.B. (2009). Population and conservation genetics, In S.J. Mullin & R.A. Seigel (eds.)
Snakes: Ecology and Conservation. Cornell University Press, Ithaca, New York, pp. 78-122.
Web pages (authors are recommended to keep the use of web pages to a minimum; use peer-
reviewed literature instead when possible):
IUCN (2010). The IUCN Red List of Threatened Species, v. 2010.3. International Union for
Nature Conservation and Natural Resources, Gland, Switzerland. Available at http://www.iucn-
redlist.org/. Retrieved on 10/31/2010.

SUPPORTING MATERIAL
Authors can submit with their manuscripts supporting material related to the work (additional
tables and figures, detailed protocols, data logs, audio and video recordings, etc.). The supporting
material will be uploaded to the online site of B&AH with a reference code that will be used to quote
such material in the final version of the article. In the initial version of the manuscript, supporting
material should be quoted as SM followed by a number according to the same format as for tables
and figures. Supporting material must be submitted as independent files. Name each file with the
code used in the initial version of the manuscript (SM1, SM2, etc.). Authors may also refer to sup-
porting material available from a different online site (e.g. GenBank, MorphoBank), in which case
the exact access reference will be indicated in the final version of the article.

BIOETHICAL CONSIDERATIONS
Because right animal use and care is an area of major concern to the AHE, authors must guaran-
tee that all animals used for research purposes are treated ethically and in accordance with the laws and
regulations established by governmental authorities and bioethics committees of each institution.
Therefore, authors are recommended to state in the Acknowledgement section that they have follo-
wed the corresponding regulation and legislation on animal care. Authors should cite in this section
the information regarding collection permits and experimental protocols approved by bioethics or ani-
mal care committees. Editors might request from authors as much information as they consider neces-
sary to confirm the fulfilment of such premises. Failure to comply with these bioethical principles will
suppose immediate rejection of the article, regardless of the reviewers recommendation.

Las normas de publicacin en castellano estn disponibles para su consulta

en la pgina web de Basic and Applied Herpetology
(http://bah.herpetologica.es/)
 BASIC & APPLIED HERPETOLOGY
REVISTA ESPAOLA DE HERPETOLOGA

On behalf of the Spanish Herpetological Society, the editorial board of Basic and Applied
Herpetology wants to acknowledge the work of the following experts who have acted as
manuscript reviewers for the elaboration of the present volume (in alphabetical order):

Telma Susana Alonso (Universidad Nacional del Sur-CONICET, Argentina)

Ines M. Araujo (University of Coimbra, Portugal)
Teresa Cristina Avila-Pires (Museu Paraense Emlio Goeldi, Brasil)
Carmen Blzquez (Centro Investigaciones Biolgicas Noroeste, Mexico)
Diva Maria Borges-Nojosa (CIBIO-Universidade de Porto, Portugal)
Stephen Busack (North Carolina Museum of Natural Sciences, USA)
Michael Collyer (Western Kentucky University, USA)
Andrs Egea-Serrano (Universidad de Murcia, Spain)
David James Harris (CIBIO-Universidade de Porto, Portugal)
Alexander Kupfer (Friedrich-Schiller-Universitt Jena, Germany)
Pasqualina Kyriakopoulou-Sklavounou (Aristotle University of Thessaloniki, Greece)
Claude Miaud (Universit de Savoie, France)
Nadja Mbjerg (University of Copenhaguen, Denmark)
Maria Ogielska (University of Wroclaw, Poland)
Vivan P. Pez (Universidad de Antioqua, Colombia)
Johannes Penner (Humboldt-Universitt zu Berlin, Germany)
Nuria Polo Cavia (Universidad Autnoma de Madrid, Spain)
Alex Richter Boix (Uppsala University, Sweden)
Mireille Rossel (Universit Montpellier 2, France)
Daniele Salvi (CIBIO-Universidade de Porto, Portugal)
Delfi Sanuy (Universitat de Lleida, Spain)
Vanessa Sarasola (Aranzadi Society of Sciences, Spain)
Ulrich Sinsch (Universitt Koblenz-Landau, Germany)
Josiah Townsend (University of Florida, USA)

AHE 2011