B&AH nº 25 [2011]

Revista EspaĂąola de HerpetologĂ­a

Journal of the Spanish Herpetological Society (AHE) Volume 25 (2011) http://bah.herpetologica.es bah@herpetologica.org

BASIC & APPLIED HERPETOLOGY REVISTA ESPAÑOLA DE HERPETOLOGÍA

Spanish Herpetological Society (AHE) President: Juan Manuel Pleguezuelos Gómez Vice-President: Jaime Bosch Pérez General Secretary: Miguel Ángel Carretero Fernández Vice-General Secretary: José Antonio Mateo Miras Treasurer: Javier Lluch Tarazona Vocals: Enrique Ayllón López (Management) César Ayres Fernández (Conservation) Víctor J. Colino Rabanal (Library) Francisco Javier Diego Rasilla (Web page and promotion) Andrés Egea Serrano (Editor, Boletín de la AHE) Marc Franch Quintana (Invasive species) Gustavo A. Llorente Cabrera (Atlas) Adolfo Marco Llorente (Marine turtles) Albert Montori Faura (Atlas) Manuel E. Ortiz Santaliestra (Editor, Basic & Applied Herpetology) Ana Perera Leg (Editor, Basic & Applied Herpetology) Alex Richter Boix (Editor, Boletín de la AHE) Xavier Santos Santiró (Editor, Boletín de la AHE)

Basic & Applied Herpetology (Editors) Manuel E. Ortiz Santaliestra (Amphibians) Instituto de Investigación en Recursos Cinegéticos (IREC). CSIC-UCLM-JCCM Ronda de Toledo, s/n.

13071 Ciudad Real (Spain) manuele.ortiz@uclm.es

Ana Perera Leg (Reptiles) CIBIO Campus Agrário de Vairão.

Rua Padre Armando Quintas-Castro 4485-661 Vairão (Portugal) perera@mail.icav.up.pt

Asociación Herpetológica Española Museo Nacional de Ciencias Naturales Cl. José Guitiérrez Abascal, 2 28006 Madrid http://www.herpetologica.es

ISSN 0213 - 6686 Impresión: igrafic. Url: www.igrafic.com

Depósito Legal: S. 633-1988 Maquetación: Marcos Pérez de Tudela. Url: www.marcos-pdt.com

BASIC & APPLIED HERPETOLOGY REVISTA ESPAÑOLA DE HERPETOLOGÍA

CONTENTS Volume 25 (2011)

Reviews GUEST CONTRIBUTION: Geometric morphometrics in herpetology: modern tools for enhancing the study of morphological variation in amphibians and reptiles A. Kaliontzopoulou

Pag. 5

Research papers Embryonic development of kidneys in viviparous Typhlonectes compressicauda (Amphibia, Gymnophiona) M. Bastit, J.-M. Exbrayat

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Population estimators and adult sex ratio for a population of Bolitoglossa altamazonica (Caudata: Plethodontidae) D.L. Gutiérrez-Lamus, J.D.Lynch, G.C. Martínez-Villate

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Behavioural responses of Iberian midwife toad tadpoles (Alytes cisternasii) to chemical stimulus of native (Natrix maura and Squalius pyrenaicus) and exotic (Procambarus clarkii) predators V. Gonçalves, S. Amaral, R. Rebelo

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Reproductive cycles in Bufo mauritanicus (Schlegel, 1841) in a wet area of Beni-Belaïd (Jijel, Algeria) O. Kisserli, S. Doumandji, J.-M. Exbrayat

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Age structure of Levant water frog, Pelophylax bedriagae, in Lake Sülüklü (Western Anatolia, Turkey) K. Çiçek, M. Kumaş, D. Ayaz, A. Mermer, Ş.D. Engin

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Population size, habitat use and movement patterns during the breeding season in a population of Perez’s frog (Pelophylax perezi) in central Spain G. Sánchez-Montes, I. Martínez-Solano

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A re-analysis of the molecular phylogeny of Lacertidae with currently available data P. Kapli, N. Poulakakis, P. Lymberakis, M. Mylonas

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Biometry and pholidosis of Thamnophis scaliger: an atypical example of sexual dimorphism in a natricine snake M. Feriche, S. Reguera, X. Santos, E. Mociño-Deloya, K. Setser, J.M. Pleguezuelos

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Cover illustration: Mauritanian toad (Bufo mauritanicus), Sidi Ifni, Morocco (see article by Kisserly et al. in this volume). Author: Luis García Cardenete.

BASIC & APPLIED HERPETOLOGY REVISTA ESPAÑOLA DE HERPETOLOGÍA

CONTENIDOS Volumen 25 (2011)

Revisiones ARTÍCULO INVITADO: Morfometría geométrica en herpetología: nuevas herramientas para promover el estudio de la variación morfológica en anfibios y reptiles A. Kaliontzopoulou

Pag. 5

Artículos de investigación Desarrollo embrionario de los riñones en Typhlonectes compressicauda vivíparas (Amphibia, Gymnophiona) M. Bastit, J.-M. Exbrayat

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Estimadores de población y razón de sexos en una población de Bolitoglossa altamazonica (Caudata: Plethodontidae) D.L. Gutiérrez-Lamus, J.D.Lynch, G.C. Martínez-Villate

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Respuestas conductuales de las larvas de sapo partero ibérico (Alytes cisternasii) a los estímulos químicos de depredadores nativos (Natrix maura y Squalius pyrenaicus) y exóticos (Procambarus clarkii) V. Gonçalves, S. Amaral, R. Rebelo

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El ciclo reproductor de Bufo mauritanicus (Schlegel, 1841) en el humedal de Beni-Belaïd (Jijel, Algeria) O. Kisserli, S. Doumandji, J.-M. Exbrayat

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Estructura de edad de la rana verde levantina, Pelophylax bedriagae, en el lago Sülüklü (Anatolia occidental, Turquía) K. Çiçek, M. Kumaş, D. Ayaz, A. Mermer, Ş.D. Engin

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Tamaño poblacional, uso del espacio y patrones de movimiento durante el periodo reproductor en una población de rana verde común (Pelophylax perezi) en España central G. Sánchez-Montes, I. Martínez-Solano

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Reanálisis de la filogenia molecular de los Lacertidae usando los datos disponibles en la actualidad P. Kapli, N. Poulakakis, P. Lymberakis, M. Mylonas

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Biometría y folidosis de Thamnophis scaliger: un ejemplo atípico de dimorfismo sexual en un colúbrido natricino M. Feriche, S. Reguera, X. Santos, E. Mociño-Deloya, K. Setser, J.M. Pleguezuelos

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Ilustración de portada: macho de sapo moruno (Bufo mauritanicus), Sidi Ifni, Marruecos (véase artículo de Kisserly et al. en este volumen). Autor: Luis García 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 2

CIBIO/UP, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrario de Vairão, 4485-661 Vairão, Portugal.

Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, Iowa 50011, USA.

* Correspondence: CIBIO/UP, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrario de Vairão, 4485-661 Vairão, 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 properties of the studied structures and thus improve our capacity for investigating patterns of morphological variation, and understanding their ecological and historical causes. Their application in herpetology has shed new light to the remarkable diversity observed among extant and extinct amphibians and reptiles. Here I first briefly consider the historical emergence of geometric morphometric methods, trying to provide a practical guide for herpetologists interested in implementing these tools to their investigation. 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 studied in reptiles, but less so in amphibians, while the contrary is true for phenotypic plasticity. The use of geometric morphometrics 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.

Morfometría geométrica en herpetología: nuevas herramientas para promover el estudio de la variación morfológica en anfibios y reptiles. El uso de la morfometría geométrica para estudiar la variación fenotípica en anfibios y reptiles ha aumentado notablemente durante la última década. Esta nueva herramienta proporciona un marco estadístico sólido para estudiar la forma de los organismos preservando las propiedades geométricas de las estructuras analizadas, mejorando así la comprensión de los factores ecológicos e históricos que explican los patrones de variación morfológica. Su aplicación en herpetología proporciona una nueva forma de explorar la diversidad morfológica de anfibios y reptiles tanto actuales como extintos. En esta revisión comienzo examinando la secuencia histórica que llevó a la aparición de la morfometría geométrica, tratando de ofrecer también una guía práctica para aquellos herpetólogos interesados en incorporar esta herramienta en su investigación. Después reviso un amplio muestrario de trabajos en los que la morfometría geométrica se usa para estudiar patrones morfológicos en anfibios y reptiles. Una pauta que emerge repetidamente es la existencia de similitudes generales, pero también de profundas diferencias, entre los miembros de los grupos taxonómicos de mayor rango. La existencia de una relación alométrica entre tamaño y forma es común en muchos grupos, pero también se observa una variabilidad considerable en las trayectorias alométricas entre taxones hermanados. El dimorfismo sexual se ha estudiado extensivamente en reptiles, pero no tanto en anfibios, mientras que con la plasticidad fenotípica ocurre lo contrario. El uso de la morfometría geométrica permite la detección de variaciones adaptativas en los patrones morfológicos y la investigación de sus causas. Finalmente, estos métodos tienen un valor incalculable para el estudio de organismos fósiles y proporcionan una mejor compresión de su paleobiología.

Key words: adaptación; alometría; dimorfismo sexual; morfometría geométrica; paleontología; plasticidad fenotípica.

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Morphology is undoubtedly one of the main components of an organism’s phenotype. As such, morphological traits have always been at the centre of attention of biological research, comprising important pieces of evidence for a wide variety of investigation fields. From traditional taxonomy and the modern school of systematics, through physiology, development, ecology, biogeography, and all the way to modern evolutionary 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 morphometric 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 understanding of the astonishing morphological variety that exists across amphibian and reptile 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 herpetofaunal groups, this is not meant to be an exhaustive account from a taxonomical perspective, but rather to give insight on how GM methods have advanced our understanding 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 nature 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 properties of a morphological trait of interest (ADAMS et al., 2004; SLICE, 2005). The application of multivariate statistics on the above biometric variables then allowed for the testing of specific biological hypotheses about an organism’s multivariate phenotype (i.e. “traditional morphometrics”; MARCUS, 1990).

However, morphometrics still suffered some shortcomings which troubled morphometricians 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 visualisation. With size and its effect on other morphological 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 mathematical 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 comparison), 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 regarded the selection of variables used for shape description; since the geometric relationships between linear measurements were not included in the dataset, one could neither predict

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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 captured, morphometric data could not be effectively 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 advances 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 techniques for capturing organismal form with a newly introduced, statistically robust shape theory led to the growth of a set of morphometric 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 threedimensional landmark-based GM methods are more frequently used today, I will concen-

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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 one’s explanations of biological processes are grounded” (BOOKSTEIN, 1991). It is through the definition of landmarks, based on biological intuition and previous 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 coordinates are first translated (b) by placing their centroid 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, orientation and position) and need to be mathematically processed in order to obtain shape variables. The dominant procedure today uses shape variables lying in Kendall’s shape space (or most frequently an approximation in a space tangent to the mean shape of the studied sample), Procrustes distance being the associated metric (Table 1). While several methods were proposed in the past for obtaining shape variables from landmark coordinates, methods using Kendall's space have been proven to be the most powerful and statistically 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), scaling (Fig. 1c) and rotation (Fig. 1d) of landmark coordinates. The specimen points aligned through this procedure can then be projected 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 Kendall’s space. One should be aware that, in addition to GPA, several other superimposition techniques exist, with potential benefits in particular datasets (SIEGEL & BENSON, 1982; BOOKSTEIN, 1991; ZELDITCH et al., 2004; SLICE, 2005).

Once non-shape variation has been removed, the superimposed landmark coordinates of specimens not differing in shape will per-

GEOMETRIC MORPHOMETRICS IN HERPETOLOGY

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

Term

Description

Allometry Centroid size

Shape change associated to size change.

The size measure used in GM. It is calculated as the square root of the sum of squared distances 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 distance over all sampled shapes by a least-squares fitting procedure.

Analysis (GPA) The geometric setting for analyses of Procrustes distances among arbitrary sets of landmarks.

Kendall's shape space 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 maximum 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 landmarks can be incorporated in GM analyses, but are not in fact considered true landmarks 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 (thinplate 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 vectors is approximately the squared Procrustes distance between the specimen and the consensus 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 Îą 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 translation, 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 landmark configurations. * Compiled and augmented from SLICE et al. (1996) and ZELDITCH et al. (2004).

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fectly coincide. In turn, specimens of different shape will present at least some differences in landmark positions. The largest the shape difference between specimens, the largest the difference in the positions of homologous landmarks after superimposition. Such shape difference 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. landmark coordinates after superimposition) can be used as shape variables to investigate shape variation. However, Procrustes residuals suffer the statistical adversities of not being a fullrank set of variables (due to superimposition) and of being non-Euclidean in nature, which frequently complicates their statistical treatment since they cannot be subjected to analysis 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 operations 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 infinitely 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 parsimoniously describing and modelling shape chan-

ge. The descriptors resulting from the application 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 according 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 statistical analyses (SLICE, 2005).

In addition to providing Euclidean shape variables for statistical hypothesis testing, the thin-plate spline is an essential tool for visualising shape variation in an integrated and intuitive manner (SLICE, 2005). Since the thinplate 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 software packages are available for conducting all the above GM analyses, performing statistical comparisons and visualising the results (see Appendix 1).

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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 caecilians. 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 extensively used for studying extinct amphibian and reptile taxa, with an important contribution to the total number of studies (Fig. 3) and remarkable results (see below).

Figure 2: How the thin-plate spline can be used to visualize shape differences found from landmarkbased GM methods. In this example, shapes A and B are being compared. Once landmark configurations have been aligned through superimposition (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 represent 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 considered here, a visible increase in the use of GM methods for studying extant or extinct amphibians and reptiles is observed after the

STUDYING PHENOTYPIC VARIATION: FIELDS OF APPLICATION

The integrated study of shape through GM motivated an explosion of morphological 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 general shape patterns observed across herpetofaunal taxa and discussing their potential causes as seen by herpetologists.

Allometric patterns: shape change due to size GM tools have been a cornerstone contribution to the study of development and ontogenetic shape change. By enhancing shape quantification and shape change visualisation, GM methods have provided the possibility not only

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Figure 3: Yearly evolution of the number of publications using GM methods to analyse morphological 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 changes throughout an organism’s development (LAWING & POLLY, 2010). Most studies investigating allometric relationships, both in an ontogenetic and static context of allometry (i.e.

GOULD, 1966), indicate that large part of the shape variation observed is attributable to sizeshape allometry (see MONTEIRO & ABE, 1997; BIRCH, 1999; BONNAN, 2007; SMITH & COLLYER, 2008; PIRAS et al., 2010; CHIARI & CLAUDE, 2011; IVANOVIĆ et al., 2011 for illustrative examples from different groups).

Caution is advised to avoid conceptual misunderstandings: while GM methods remove size variation through GPA or similar superimposition 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 association with size (either represented by centroid size or any other size variable) indicates deviation from isometry (ZELDITCH et al., 2004).

Extensive research about ontogenetic shape change at different developmental stages has

been carried out mainly in amphibians, revealing both similarities and profound differences among groups. Pre-metamorphic shape ontogeny 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 isometric 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 developmental 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 (IVANOVIĆ et al., 2007, 2008).

However, while general trends appear relatively uniform, allometric trajectories frequently vary among closely related species (LARSON, 2005; IVANOVIĆ et al., 2007, 2008), providing a potentially important mechanism of morphological 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; IVANOVIĆ et al., 2011), although further studies should investigate the generality of the results obtained for Triturus, since remarkable variation of allometric patterns 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 environmental variation (see below).

Allometric shape variation has been also extensively investigated in reptiles, again revealing 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; LJUBISAVLJEVIĆ et al., 2011; ZUFFI et al., 2011) context, includes a relative shortening of the anterior area and an enlargement of the posterior 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 caution, 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 principal components analysis). As for amphibians, allometric trajectories of the skull have been found to vary extensively among species

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(LJUBISAVLJEVIĆ et al., 2010), but intraspecific variation seems to be less common.

Regarding other reptile groups, size variation also seems to be a main determinant of shape variation in turtles, including ontogenetic, 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, evolutionary allometry of skull shape seems to be constrained, and similar to intraspecific allometry (CLAUDE et al., 2004), while extensive variation 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 modification of growth trajectories between two closely related lineages of Galápagos 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 variation 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 application of GM has importantly enhanced our understanding of the proximate and evolutio-

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nary causes of shape SD. Surprisingly, studies using GM methods to address SD in amphibians are very limited. Some studies investigated the effect of sex on shape and reported significant SD (IVANOVIĆ 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 general resemblance of global patterns, but also significant 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: LJUBISAVLJEVIĆ et al., 2011; Dalmatolacerta and Dinarolacerta: LJUBISAVLJEVIĆ et al., 2010; Lacerta: BRUNER et al., 2005; COSTANTINI et al. 2007; Podarcis: KALIONTZOPOULOU et al., 2007, 2008; LJUBISAVLJEVIĆ 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 disparate 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, investigation of the proximate causes of shape SD indicated that males and females follow common 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-independent 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 source of shape SD (BRUNER et al., 2005; LJUBISAVLJEVIĆ et al., 2010). This, together with the variation of allometric slopes observed among closely related species (Ljubisavljević 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, continuous 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 elucidation 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 relatively 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 discordance among authors regarding the comparison between traditional and geometric morphometrics for quantifying shape in turtles; while some authors use GM as a powerful method for capturing patterns invisible to traditional approaches, such as SD in hatchlings (VALENZUELA et al., 2004; LUBIANA & FERREIRA JĂšNIOR, 2009), others report a weaker 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 direction 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 number 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 signal (GENTILLI et al., 2009). Clearly, patterns of

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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 describing shape variation and associating it with both ecological factors and performance measures, thus providing evidence of the adaptive potential of certain shape traits. Extensive herpetological research has focused on the search for such variation, shedding new light on the way amphibians and reptiles respond morphologically 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 fundamental component of the turtle phenotype and it is recognised as one of the most remarkable 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 ecological niches, while preserving a basic body plan, thus constituting exceptional model organisms for studying the consequences of ecological diversification on morphological traits.

Several components of habitat use have been shown to directly influence turtle body shape.

The differentiation between aquatic and terrestrial life is undoubtedly one of the main causes of turtle phenotypic diversification, being reflected in the shape of the carapace (CLAUDE

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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 articulation (DEPECKER et al., 2006). Diet is an additional niche dimension involved in skull differentiation 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 differentiation, at least among emydines (ANGIELCZYK et al., 2011). By contrast, phylogenetic inertia seems to play a subsidiary role in turtle shape differentiation, at least in comparison 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 regimes are more domed (Fig. 5). Further supporting an adaptive explanation for the observed

patterns, the shape typical of fast-flowing regimes importantly reduced drag during swimming. Interestingly, a trade-off was also shown to exist between this hydrodynamic efficiency and mechanical strength. Flattened, hydrodynamic shells were more fragile (RIVERA & STAYTON, 2011), a pattern observed also between 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 inhabiting fast-flowing and slow-flowing water environments, 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 empirical 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 biomechanical (ADAMS & ROHLF, 2000) and behavioural (ADAMS, 2004) modifications.

Undoubtedly, head shape is a trait with strong evolutionary potential in this salamander group, an observation corroborated by the existence of an important heritable component (ADAMS, 2011). However, the detailed studies conducted also reveal that competitive interactions, while repeatable across space when the same pair of species is involved (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 variation in Plethodon, other factors are also involved. For example, MAERZ et al. (2006) reported significant intraspecific variation associated to trophic polymorphism. Diet also seems to be significantly associated to shape

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variation of the hyobranchial skeleton of anuran larvae (CANDIOTI, 2006). Other patterns of shape variation in salamanders concern potential adaptation to structural environment, where shape variation among closely related taxa also involves the modification of shape allometries. In a very interesting contrast of evolutionary patterns, JAEKEL & WAKE (2007) and ADAMS & NISTRI (2010) respectively described how divergence or convergence 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 morphological variation (foot webbing) and associate 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 ecomorphological 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 theoretical 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 hypothesis 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

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due to differences in diet, aggressive or antipredatory 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 affinity visibly dominates over feeding behaviour as a factor of skull shape differentiation.

Considering other factors, both habitat type (KALIONTZOPOULOU et al., 2010) and insularity (BĂNCILĂ 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 capturing shape variation, GM methods have been used for species discrimination and for describing morphological variation between closely related, and in many cases cryptic, taxa. For instance, LEACHÉ et al. (2009) used GM on cranial horn shape of the coast horned lizard species complex, combined with a large number 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 Galápagos tortoises and confirmed the morphological differentia-

tion between two genetically distinct lineages, which had been described to differ morphologically using linear methods. GM tools have also been used to analyse intraspecific geographic 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 relatedness and morphological similarity (IVANOVIĆ et al., 2008, 2009). From a purely taxonomical perspective, JAMNICZKY & RUSSELL (2004) used GM to investigate the “batagurine process”, a potential diagnostic character of the turtle family Bataguridae, while VIEIRA et al.

(2008) investigated the morphological differentiation between colour morphs of a toad population to examine the taxonomical implications 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 variables (either partial warps or their principal components) as cladistic characters (ADAMS et al., 2011). The main difficulty presented is that of transforming continuously varied, multivariate 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 importance 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 organismal shape as described by GM methods cannot 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 utility 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 estimating 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

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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 characterise morphological disparity patterns and investigate their temporal variation and to examine macroevolutionary trends (CANUDO & CUENCA-BESCĂ“S, 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 providing a deeper understanding of the paleobiology and paleoecology of archosaur taxa (BONNAN, 2004, 2007; PIERCE et al., 2009b; BONNAN et al., 2010). Finally, a very interesting 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 development of new methods The multivariate nature of GM data frequently challenges statistical methods and has stimulated morphometricians to extend existing methods for studying complex shapes.

Apart from addressing biological questions, several authors have used amphibians and reptiles as model organisms to develop new methodological approaches that utilise the toolbox of GM and further enhance our capacity 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 comprehensive review of how multivariate regression tech-

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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 processes involved in ontogenetic shape change. In a conceptually similar approach, COLLYER & ADAMS (2007) examined character displacement 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 multistate 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 univariate 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 patterns of plastron shape heritability in slider turtles. 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 crocodilian 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 traditional morphometric methods to GM and have reached the conclusion that GM techniques 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 implementation of such a sensitive tool, to keep with strong biological inference and avoid missing 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 tomography (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 future contributions. I hope the above review has provided an informed view of the questions that have been explored and the answers 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 anonymous 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 postdoctoral grant (SFRH/BPD/68493/2010) from Fundação para a Ciência e Tecnologia (FCT, Portugal).

REFERENCES ADAMS, D.C. (2004). Character displacement via aggressive interference in Appalachian salamanders. Ecology 85: 2664-2670.

ADAMS, D.C. (2010). Parallel evolution of character displacement driven by competitive selection in terrestrial salamanders.

BMC Evolutionary Biology 10: 72.

ADAMS, D.C. (2011). Quantitative genetics and evolution of head shape in Plethodon salamanders. Evolutionary Biology 38: 278-286.

ADAMS, D.C. & COLLYER, M.L. (2009). A general framework for the analysis of phe-

21

notypic trajectories in evolutionary studies. Evolution 63: 1143-1154.

ADAMS, D.C. & NISTRI, A. (2010).

Ontogenetic convergence and evolution of foot morphology in European cave salamanders (Family: Plethodontidae).

BMC Evolutionary Biology 10: 216.

ADAMS, D.C. & ROHLF, F.J. (2000).

Ecological character displacement in Plethodon: Biomechanical differences found from a geometric morphometric study. Proceedings of the National Academy of Sciences USA 97: 4106-4111.

ADAMS, D.C. & ROSENBERG, M.S. (1998).

Partial warps, phylogeny, and ontogeny: a comment on Fink and Zelditch (1995).

Systematic Biology 47: 168-173.

ADAMS, D.C.; ROHLF, F.J. & SLICE, D.E. (2004). Geometric morphometrics: ten years of progress following the ‘revolution’. Italian Journal of Zoology 71: 5-16.

ADAMS, D.C.; WEST, M.E. & COLLYER, M.L. (2007). Location-specific sympatric morphological divergence as a possible response to species interactions in West Virginia Plethodon salamander communities. Journal of Animal Ecology 76: 289-295.

ADAMS, D.C.; CARDINI, A.; MONTEIRO, L.R.; O’HIGGINS, P. & ROHLF, F.J. (2011). Morphometrics and phylogenetics: principal components of shape from cranial modules are neither appropriate nor effective cladistic characters. Journal of Human Evolution 60: 240-243.

ANGIELCZYK, K.D. (2007). How to be a miniature turtle: comparisons of ontogeny in the Emydinae using geometric morphometrics. Integrative and Comparative Biology 46 (supplement 1): e3.

22

KALIONTZOPOULOU

ANGIELCZYK, K.D. & SHEETS, H.D. (2007).

Investigation of simulated tectonic deformation in fossils using geometric morphometrics. Paleobiology 33: 125-148.

ANGIELCZYK, K.D.; FELDMAN, C.R. & MILLER, G.R. (2011). Adaptive evolution of plastron shape in emydine turtles.

Evolution 65: 377-394.

ARENDT, J. (2010). Morphological correlates of sprint swimming speed in five species of spadefoot toad tadpoles: Comparison of morphometric methods. Journal of Morphology 271: 1044-1052.

ARIF, S.; ADAMS, D.C. & WICKNICK, J.A. (2007). Bioclimatic modelling, morphology, and behaviour reveal alternative mechanisms regulating the distributions of two parapatric salamander species.

Evolutionary Ecology Research 9: 843-854.

ASHLOCK, D.; ADAMS, D.C. & DOTY, D. (2003). Morphometric grayscale texture analysis using foot patterns. Proceedings of the 2003 Congress on Evolutionary Computation: 1575-1582.

BĂNCILĂ, R.; VAN GELDER, I.; ROTTEVEEL, E.; LOMAN, J. & ARNTZEN, J.W. (2010).

Fluctuating asymmetry is a function of population isolation in island lizards.

Journal of Zoology 282: 266-275.

BARDEN, H.E. & MAIDMENT, S.C.R. (2011).

Evidence for sexual dimorphism in the stegosaurian dinosaur Kentrosaurus aethiopicus from the Upper Jurassic of Tanzania. Journal of Vertebrate Paleontology 31: 641-651.

BASZIO, S. & WEBER, S. (2002). Potentials and limits of morphometry in the understanding of squamate osteological structures. Senckenbergiana lethaea 82: 13-22.

BIRCH, J.M. (1999). Skull allometry in the marine toad, Bufo marinus. Journal of

Morphology 241: 115-126.

BONNAN, M.F. (2004). Morphometric analysis of humerus and femur shape in Morrison sauropods: implications for functional morphology and paleobiology.

Paleobiology 30: 444-470.

BONNAN, M.F. (2007). Linear and geometric morphometric analysis of long bone scaling patterns in Jurassic neosauropod dinosaurs: their functional and paleobiological implications. The Anatomical Record 290: 1089-1111.

BONNAN, M.F.; FARLOW, J.O. & MASTERS, S.L. (2008). Using linear and geometric morphometrics to detect intraspecific variability and sexual dimorphism in femoral shape in Alligator mississippiensis and its implications for sexing fossil archosaurs. Journal of Vertebrate Paleontology 28: 422-431.

BONNAN, M.F.; SANDRIK, J.L.; NISHIWAKI, T.; WILHITE, D.R.; ELSEY, R.M. & VITTORE, C. (2010). Calcified cartilage shape in archosaur long bones reflects overlying joint shape in stress-bearing elements: Implications for nonavian dinosaur locomotion. The Anatomical Record 293: 2044-2055.

BOOKSTEIN, F.L. (1982). Foundations of Morphometrics. Annual Review of Ecology and Systematics 13: 451-470.

BOOKSTEIN, F.L. (1986). Size and shape spaces for landmark data in two dimensions.

Statistical Science 1: 181-222.

BOOKSTEIN, F.L. (1989a). “Size and shape”: a comment on semantics. Systematic Zoology 38: 173-180.

BOOKSTEIN, F.L. (1989b). Principal warps: thinplate splines and the decomposition of deformations. IEEE Transactions on Pattern Analysis and Machine Intelligence 11: 567-585.

BOOKSTEIN, F.L. (1991). Morphometric Tools for Landmark Data: Geometry and Biology.

GEOMETRIC MORPHOMETRICS IN HERPETOLOGY

Cambridge University Press, Cambridge, United Kingdom.

BOOKSTEIN, F.L. (1994). Can biometrical shape be a homologous character?, In B.K. Hall (ed.) Homology: The Hierarchical Basis of Comparative Biology. Academic Press, New York, pp. 197-227.

BOOKSTEIN, F.L. (1996). Biometrics, biomathematics and the morphometric synthesis.

Bulletin of Mathematical Biology 58: 313-365.

BOOKSTEIN, F.L. (1997). Landmark methods for forms without landmarks: localizing group differences in outline shape.

Medical Image Analysis 1: 225-243.

BRUNER, E. & COSTANTINI, D. (2007). Head morphological variation in Podarcis muralis and Podarcis sicula: a landmark-based approach. Amphibia-Reptilia 28: 566-573.

BRUNER, E.; COSTANTINI, D.; FANFANI, A. & DELL’OMO, G. (2005). Morphological variation and sexual dimorphism of the cephalic scales in Lacerta bilineata. Acta Zoologica 86: 245-254.

BURKE, A.C. (1989). Development of the turtle carapace: implications for the evolution of a novel Bauplan. Journal of Morphology 199: 363-378.

CANDIOTI, M.F.V. (2006). Ecomorphological guilds in anuran larvae: an application of geometric morphometric methods.

Herpetological Journal 16: 149-162.

CANDIOTI, M.F.V. (2008). Larval anatomy of Andean tadpoles of Telmatobius (Anura: Ceratophryidae) from Northwestern Argentina. Zootaxa 1938: 40-60.

CANUDO, J.I. & CUENCA-BESCÓS, G. (2004).

Morphometric approach to Titanosauriformes (Sauropoda, Dinosauria) femora: implications to the paleobiogeographic analysis, In A.M.T. Elewa (ed.) Morphometrics: Applications in

23

Biology and Paleontology. Springer-Verlag, Berlin, pp. 143-156.

CEBALLOS, C.P. & VALENZUELA, N. (2011).

The role of sex-specific plasticity in shaping sexual dimorphism in a long-lived vertebrate, the snapping turtle Chelydra serpentina.

Evolutionary Biology 38: 163-181.

CHIARI, Y. & CLAUDE, J. (2011). Study of the carapace shape and growth in two Galápagos tortoise lineages. Journal of Morphology 272: 379-386.

CLAUDE, J. (2008). Morphometrics with R. Springer, New York.

CLAUDE, J.; PARADIS, E.; TONG, H. & AUFFRAY, J.-C. (2003). A geometric morphometric assessment of the effects of environment and cladogenesis on the evolution of the turtle shell. Biological Journal of the Linnean Society 79: 485-501.

CLAUDE, J.; PRITCHARD, P..C.H.; TONG, H.; PARADIS, E. & AUFFRAY, J.-C. (2004).

Ecological correlates and evolutionary divergence in the skull of turtles: a geometric morphometric assessment. Systematic Biology 53: 933-948.

CLEMENTE-CARVALHO, R.B.G.; MONTEIRO, L.R.; BONATO, V.; ROCHA, H.S.; PEREIRA, G.R.; OLIVEIRA, D.F.; LOPES, R.T.; HADDAD, C.F.B.; MARTINS, E.G. & DOS REIS, S.F. (2008). Geographic variation in cranial shape in the pumpkin toadlet (Brachycephalus ephippium): a geometric analysis. Journal of Herpetology 42: 176-185.

CLEMENTE-CARVALHO, R.B.G.; ALVES, A.C.R.; PEREZ, S.I.; HADDAD, C.F.B. & DOS REIS, S.F. (2011). Morphological and molecular variation in the pumpkin toadlet, Brachycephalus ephippium (Anura: Brachycephalidae). Journal of Herpetology 45: 94-99.

24

KALIONTZOPOULOU

COLLYER, M.L. & ADAMS, D.C. (2007).

Analysis of two-state multivariate phenotypic change in ecological studies.

Ecology 88: 683-692.

COSTA, C.; ANGELINI, C.; SCARDI, M.; MENESATTI, P. & UTZERI, C. (2009).

Using image analysis on the ventral colour pattern in Salamandrina perspicillata (Amphibia: Salamandridae) to discriminate among populations. Biological Journal of the Linnean Society 96: 35-43.

COSTANTINI, D.; BRUNER, E.; FANFANI, A. & DELL’OMO, G. (2007). Male-biased predation of western green lizards by Eurasian kestrels. Naturwissenschaften 94: 1015-1020.

COSTANTINI, D., LAPRESA ALONSO, M., MOAZEN, M. & BRUNER, E. (2010). The relationship between cephalic scales and bones in lizards: a preliminary microtomographic survey on three lacertid species. The Anatomical Record 293: 183-194.

DAYTON, G.H.; SAENZ, D.; BAUM, K.A.; LANGERHANS, R.B. & DEWITT, T.J. (2005).

Body shape, burst speed and escape behavior of larval anurans. Oikos 111: 582-591.

DAZA, J.D.; HERRERA, A.; THOMAS, R. & CLAUDIO, H.J. (2009). Are you what you eat? A geometric morphometric analysis of gekkotan skull shape. Biological Journal of the Linnean Society 97: 677-707.

DEPECKER, M.; BERGE, C.; PENIN, X. & RENOUS, S. (2006). Geometric morphometrics of the shoulder girdle in extant turtles (Chelonii). Journal of Anatomy 208: 35-45.

DRYDEN, I.L. & MARDIA, K.V. (1998).

Statistical Shape Analysis. John Wiley & Sons, Chichester, United Kingdom.

ELEWA, A.M.T. (2004). Introduction, In A.M.T. Elewa (ed.) Morphometrics: Applications in Biology and Paleontology.

Springer-Verlag, Berlin, pp. 1-6.

EMERSON, S.B. & BRAMBLE, D.M. (1993).

Scaling, allometry, and skull design, In J. Hanken & B.K. Hall (eds.) The Skull.

Volume 3. Functional and Evolutionary Mechanisms. University of Chicago Press, Chicago, pp. 384-421.

FERSON, S.; ROHLF, F.J. & KOEHN, R.K. (1985). Measuring shape variation of two-dimensional outlines. Systematic Zoology 34: 59-68.

GARRIGA, N. & LLORENTE, G.A. (in press).

Chondrocranial ontogeny of Pelodytes punctatus (Anura: Pelodytidae). Response to competition: geometric morphometric and allometric change analysis. Acta Zoologica doi: 10.1111/j.1463-6395.2011.00520.x.

GENTILLI, A.; CARDINI, A.; FONTANETO, D. & ZUFFI, M.A.L. (2009). The phylogenetic signal in cranial morphology of Vipera aspis: a contribution from geometric morphometrics. Herpetological Journal 19: 69-77.

GOSNER, K.L. (1960). A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: 183-190.

GOULD, S.J. (1966). Allometry and size in ontogeny and phylogeny. Biological Reviews 41: 587-640.

HARMON, L.J.; KOLBE, J.J.; CHEVERUD, J.M. & LOSOS, J.B. (2005). Convergence and the multidimensional niche. Evolution 59: 409-421.

HEAD, J.J.; BLOCH, J.I.; HASTINGS, A.K.; BOURQUE, J.R.; CADENA, E.A.; HERRERA, F.A.; POLLY, P.D. & JARAMILLO, C.A. (2009).

Giant boid snake from the Palaeocene neotropics reveals hotter past equatorial temperatures. Nature 457: 715-717.

HERREL, A.; MCBRAYER, L.D. & LARSON, P.M. (2007). Functional basis for sexual

GEOMETRIC MORPHOMETRICS IN HERPETOLOGY

differences in bite force in the lizard Anolis carolinensis. Biological Journal of the Linnean Society 91: 111-119.

HOSSIE, T.J.; FERLAND-RAYMOND, B.; BURNESS, G. & MURRAY, D.L. (2010).

Morphological and behavioural responses of frog tadpoles to perceived predation risk: A possible role for corticosterone mediation? Ecoscience 17: 100-108.

HUYGHE, K.; HERREL, A.; ADRIAENS, D.; TADIĆ, Z. & VAN DAMME, R. (2009). It is all in the head: morphological basis for differences in bite force among colour morphs of the Dalmatian wall lizard. Biological Journal of the Linnean Society 96: 13-22.

IVANOVIĆ, A.; VUKOV, T.D.; DžUKIĆ, G.; TOMAŠEVIĆ, N. & KALEZIĆ, M.L. (2007).

Ontogeny of skull size and shape changes within a framework of biphasic lifestyle: a case study in six Triturus species (Amphibia, Salamandridae). Zoomorphology 126: 173-183.

IVANOVIĆ, A.; SOTIROPOULOS, K.; VUKOV, T.D.; ELEFTHERAKOS, K.; DžUKIĆ, G.; POLYMENI, R.M. & KALEZIĆ, M.L. (2008). Cranial shape variation and molecular phylogenetic structure of crested newts (Triturus cristatus superspecies: Caudata, Salamandridae) in the Balkans. Biological Journal of the Linnean Society 95: 348-360.

IVANOVIĆ, A.; SOTIROPOULOS, K.; DžUKIĆ, G. & KALEZIĆ, M.L. (2009). Skull size and shape variation versus molecular phylogeny: a case study of alpine newts (Mesotriton alpestris, Salamandridae) from the Balkan Peninsula. Zoomorphology 128: 157-167.

IVANOVIĆ, A.; CVIJANOVIĆ, M. & KALEZIĆ, M.L. (2011). Ontogeny of body form and metamorphosis: insights from the crested newts. Journal of Zoology 283: 153-161.

25

JAEKEL, M. & WAKE, D.B. (2007).

Developmental processes underlying the evolution of a derived foot morphology in salamanders. Proceedings of the National Academy of Sciences USA 104: 20437-20442.

JAMNICZKY, H.A. & RUSSELL, A.P. (2004). A geometric morphometric assessment of the ‘batagurine process’ of testudinoid turtles. Amphibia-Reptilia 25: 369-379.

JOHNSON, J.B.; BURT, D.B. & DEWITT, T.J. (2008). Form, function, and fitness: pathways to survival. Evolution 62: 1243-1251.

JONES, M.E.H. (2008). Skull shape and feeding strategy in Sphenodon and other Rhynchocephalia (Diapsida: Lepidosauria).

Journal of Morphology 269: 945-966.

JORGENSEN, M.E. & SHEIL C.A. (2008). Effects of temperature regime through premetamorphic ontogeny on shape of the chondrocranium in the American toad, Anaxyrus americanus. The Anatomical Record 291: 818-826.

KALIONTZOPOULOU, A.; CARRETERO, M.A. & LLORENTE, G.A. (2007). Multivariate and geometric morphometrics in the analysis of sexual dimorphism variation in Podarcis lizards. Journal of Morphology 268: 152-165.

KALIONTZOPOULOU, A.; CARRETERO, M.A. & LLORENTE, G.A. (2008). Head shape allometry and proximate causes of head sexual dimorphism in Podarcis lizards: joining linear and geometric morphometrics. Biological Journal of the Linnean Society 93: 111-124.

KALIONTZOPOULOU, A.; CARRETERO, M.A. & LLORENTE, G.A. (2010). Intraspecific ecomorphological variation: linear and geometric morphometrics reveal habitat-related patterns within Podarcis bocagei wall lizards. Journal of Evolutionary Biology 23: 1234-1244.

KALIONTZOPOULOU, A.; ADAMS, D.C.; VAN DER

26

KALIONTZOPOULOU

MEIJDEN, A.; PERERA, A. & CARRETERO, M.A. (in press). Relationships between head morphology, bite performance and ecology in two species of Podarcis wall lizards.

Evolutionary Ecology doi: 10.1007/s10682011-9539-y KENDALL, D.G. (1984). Shape-manifolds, Procrustean metrics, and complex projective spaces. Bulletin of the London Mathematical Society 16: 81-121.

KENDALL, D.G. (1985). Exact distributions for shapes of random triangles in convex sets.

Advances in Applied Probability 17: 308-329.

KLINGENBERG, C.P. (1996). Multivariate allometry, In L.F. Marcus, M. Corti, A. Loy, G.J.P. Naylor & D.E. Slice (eds.)

Advances in Morphometrics. Series: NATO ASI Series A: Life Sciences, vol. 284.

Plenum Press, New York, pp. 23-49.

KLINGENBERG, C.P. (2003). Quantitative genetics of geometric shape: heritability and the pitfalls of the univariate approach. Evolution 57: 191-195.

KLINGENBERG, C.P. (2010). There’ s something afoot in the evolution of ontogenies. BMC Evolutionary Biology 10: 221.

KLINGENBERG, C.P. (2011). MorphoJ: an integrated software package for geometric morphometrics. Molecular Ecology Resources 11: 353-357.

KLINGENBERG, C.P. & GIDASZEWSKI, N.A. (2010). Testing and quantifying phylogenetic signals and homoplasy in morphometric data. Systematic Biology 59: 245-261.

KLINGENBERG, C.P. & MONTEIRO, L.R. (2005).

Distances and directions in multidimensional shape spaces: implications for morphometric applications. Systematic Biology 54: 678-688.

LARSON, P.M. (2002). Chondrocranial development in larval Rana sylvatica (Anura:

Ranidae): morphometric analysis of cranial allometry and ontogenetic shape change.

Journal of Morphology 252: 131-144.

LARSON, P.M. (2004). Chondrocranial morphology and ontogenetic allometry in larval Bufo americanus (Anura, Bufonidae).

Zoomorphology 123: 95-106.

LARSON, P.M. (2005). Ontogeny, phylogeny, and morphology in anuran larvae: morphometric analysis of cranial development and evolution in Rana tadpoles (Anura: Ranidae).

Journal of Morphology 264: 34-52.

LAWING, A.M. & POLLY, P.D. (2010).

Geometric morphometrics: recent applications to the study of evolution and development. Journal of Zoology 280: 1-7.

LEACHÉ, A.D.; KOO, M.S.; SPENCER, C.L.; PAPENFUSS, T.J.; FISHER, R.N. & MCGUIRE, J.A. (2009). Quantifying ecological, morphological, and genetic variation to delimit species in the coast horned lizard species complex (Phrynosoma).

Proceedings of the National Academy of Sciences USA 106: 12418-12423.

LJUBISAVLJEVIĆ, K.; UROŠEVIĆ, A.; ALEKSIĆ, I. & IVANOVIĆ, A. (2010). Sexual dimorphism of skull shape in a lacertid lizard species (Podarcis spp., Dalmatolacerta sp., Dinarolacerta sp.) revealed by geometric morphometrics. Zoology 113: 168-174.

LJUBISAVLJEVIĆ, K.; POLOVIĆ, L.; UROŠEVIĆ, A. & IVANOVIĆ, A. (2011). Patterns of morphological variation in the skull and cephalic scales of the lacertid lizard Algyroides nigropunctatus. Herpetological Journal 21: 65-72.

LUBIANA, A. & FERREIRA JÚNIOR, P.D. (2009). Pivotal temperature and sexual dimorphism of Podocnemis expansa hatchlings (Testudines: Podocnemididae) from Bananal Island, Brazil. Zoologia 26: 527-533.

GEOMETRIC MORPHOMETRICS IN HERPETOLOGY

MAERZ, J.C.; MYERS, E.M. & ADAMS, D.C. (2006). Trophic polymorphism in a terrestrial salamander. Evolutionary Ecology Research 8: 23-35.

MAGWENE, P.M. (2001). Comparing ontogenetic trajectories using growth process data. Systematic Biology 50: 640-656.

MANIER, M.K. (2004). Geographic variation in the long-nosed snake Rhinocheilus lecontei (Colubridae): beyond the subspecies debate. Biological Journal of the Linnean Society 83: 65-85.

MARCUS, L.F. (1990). Traditional morphometrics, In F.J. Rohlf & F.L. Bookstein (eds.)

Proceedings of the Michigan Morphometrics Workshop. Series: Special Publications, vol. 2.

University of Michigan Museum of Zoology, Ann Arbor, Michigan, USA, pp. 77-122.

MITTEROECKER, P. & GUNZ, P. (2009).

Advances in geometric morphometrics.

Evolutionary Biology 36: 235-247.

MONTEIRO, L.R. (1999). Multivariate regression models and geometric morphometrics: the search for causal factors in the analysis of shape. Systematic Biology 48: 192-199.

MONTEIRO, L.R. (2000). Why morphometrics is special: the problem with using partial warps as characters for phylogenetic inference. Systematic Biology 49: 796-800.

MONTEIRO, L.R. & ABE, A.S. (1997).

Allometry and morphological integration in the skull of Tupinambis merianae (Lacertilia: Teiidae). Amphibia-Reptilia 18: 397-405.

MONTEIRO, L.R.; CAVALCANTI, M.J. & SOMMER III, H.J.S. (1997). Comparative ontogenetic shape changes in the skull of Caiman species (Crocodylia, Alligatoridae).

Journal of Morphology 231: 53-62.

MONTEIRO, L.R.; DINIZ-FILHO, J.A.F.; DOS REIS, S.F. & ARAĂšJO, E.D. (2002). Geometric esti-

27

mates of heritability in biological shape.

Evolution 56: 563-572.

MYERS, E.M. & ADAMS, D.C. (2008).

Morphology is decoupled from interspecific competition in Plethodon salamanders in the Shenandoah Mountains, USA. Herpetologica 64: 281-289.

MYERS, E.M.; JANZEN, F.J.; ADAMS, D.C. & TUCKER, J.K. (2006). Quantitative genetics of plastron shape in slider turtles (Trachemys scripta). Evolution 60: 563-572.

MYERS, E.M.; TUCKER, J.K. & CHANDLER, C.H. (2007). Experimental analysis of body size and shape during critical lifehistory events of hatchling slider turtles, Trachemys scripta elegans. Functional Ecology 21: 1106-1114.

NISHIZAWA, H.; ASAHARA, M.; KAMEZAKI, N. & ARAI, N. (2010). Differences in the skull morphology between juvenile and adult green turtles: implications for the ontogenetic diet shift. Current Herpetology 29: 97-101.

O'HIGGINS, P. & JONES, N. (2006). Tools for Statistical Shape Analysis. Hull York Medical School, York-Hull, United Kingdom. Available at http://sites.google.com/site/hymsfme/resources.

Retrieved on 08/20/2011.

PIERCE, S.E.; ANGIELCZYK, K.D. & RAYFIELD, E.J. (2008). Patterns of morphospace occupation and mechanical performance in extant crocodilian skulls: a combined geometric morphometric and finite element modeling approach. Journal of Morphology 269: 840-864.

PIERCE, S.E.; ANGIELCZYK, K.D. & RAYFIELD, E.J. (2009a). Morphospace occupation in thalattosuchian crocodylomorphs: skull shape variation, species delineation and temporal patterns. Palaeontology 52: 1057-1097.

28

KALIONTZOPOULOU

PIERCE, S.E.; ANGIELCZYK, K.D. & RAYFIELD, E.J. (2009b). Shape and mechanics in thalattosuchian (Crocodylomorpha) skulls: implications for feeding behaviour and niche partitioning. Journal of Anatomy 215: 555-576.

PIRAS, P.; COLANGELO, P.; ADAMS, D.C.; BUSCALIONI, A.; CUBO, J.; KOTSAKIS, T.; MELORO, C. & RAIA, P. (2010). The Gavialis-Tomistoma debate: the contribution of skull ontogenetic allometry and growth trajectories to the study of crocodylian relationships. Evolution & Development 12: 568-579.

POLLY, P.D. & HEAD, J.J. (2004). Maximumlikelihood identification of fossils: taxonomic identification of Quaternary marmots (Rodentia, Mammalia) and identification of vertebral position in the pipesnake Cylindrophis (Serpentes, Reptilia), In A.M.T. Elewa (ed.) Morphometrics: Applications in Biology and Paleontology.

Springer-Verlag, Berlin, pp. 197-222.

PRIETO-MARQUEZ, A.; GIGNAC, P.M. & JOSHI, S. (2007). Neontological evaluation of pelvic skeletal attributes purported to reflect sex in extinct non-avian archosaurs. Journal of Vertebrate Paleontology 27: 603-609.

PRITCHARD, P.C.H. (2008). Evolution and structure of the turtle shell, In J. Wyneken, M.H. Godfrey & V. Bels (eds.)

Biology of Turtles. CRC Press, Boca Raton, Florida, USA, pp. 45-83.

R DEVELOPMENT CORE TEAM (2010). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Available at http://www.r-project.org/. Retrieved on 08/20/2011.

RAIA, P.; GUARINO, F.M.; TURANO, M.; POLESE, G.; RIPPA, D.; CAROTENUTO, F.; MONTI,

D.M.; CARDI, M. & FULGIONE, D. (2010).

The blue lizard spandrel and the island syndrome. BMC Evolutionary Biology 10: 289.

RIVERA, G. (2008). Ecomorphological variation in shell shape of the freshwater turtle Pseudemys concinna inhabiting different aquatic flow regimes. Integrative and Comparative Biology 48: 769-787.

RIVERA, G. & CLAUDE, J. (2008).

Environmental media and shape asymmetry: a case study on turtle shells. Biological Journal of the Linnean Society 94: 483-489.

RIVERA, G. & STAYTON, C.T. (2011). Finite element modeling of shell shape in the freshwater turtle Pseudemys concinna reveals a tradeoff between mechanical strength and hydrodynamic efficiency. Journal of Morphology 272: 1192-1203.

RODRIGUES, L.A. & FARIA DOS SANTOS, V. (2004). Sauropod Tracks – a geometric morphometric study, In A.M.T. Elewa (ed.) Morphometrics: Applications in Biology and Paleontology. Springer-Verlag, Berlin, pp. 129-142.

ROHLF, F.J. (1986). Relationships among eigenshape analysis, Fourier analysis, and analysis of coordinates. Mathematical Geology 18: 845-854.

ROHLF, F.J. (1990). Fitting curves to outlines, In F.J. Rohlf & F.L. Bookstein (eds.) Proceedings of the Michigan Morphometrics Workshop.

Series: Special Publications, Vol. 2.

University of Michigan Museum of Zoology, Ann Arbor, Michigan, USA, pp. 167-177.

ROHLF, F.J. (1998). On applications of geometric morphometrics to studies of ontogeny and phylogeny. Systematic Biology 47: 147-158.

ROHLF, F.J. (1999). Shape statistics: Procrustes superimpositions and tangent spaces.

GEOMETRIC MORPHOMETRICS IN HERPETOLOGY

Journal of Classification 16: 197-223.

ROHLF, F.J. (2000a). On the use of shape spaces to compare morphometric methods.

Hystrix 11: 9-25.

ROHLF, F.J. (2000b). Statistical power comparisons among alternative morphometric methods. American Journal of Physical Anthropology 111: 463-478.

ROHLF, F.J. (2003). Bias and errors in estimates of mean shape in geometric morphometrics.

Journal of Human Evolution 44: 665-683.

ROHLF, F.J. (2011). Morphometrics at SUNY Stony Brook. Department of Ecology and Evolution, State University of New York, Stony Brook, New York, USA. Available at http://life.bio.sunysb.edu/morph/.

Retrieved on 11/16/2011.

ROHLF, F.J. & ARCHIE, J.W. (1984). A comparison of Fourier methods for the description of wing shape in mosquitoes (Diptera: Culicidae). Systematic Zoology 33: 302-317.

ROHLF, F.J. & BOOKSTEIN, F.L. (1987). A comment on shearing as a method for “size correction”. Systematic Zoology 36: 356-367.

ROHLF, F.J. & MARCUS, L.F. (1993). A revolution morphometrics. Trends in Ecology and Evolution 8: 129-132.

ROHLF, F.J. & SLICE, D. (1990). Extensions of the Procrustes method for the optimal superimposition of landmarks. Systematic Zoology 39: 40-59.

SHEETS, H.D. (2000). Integrated Morphometrics Package (IMP). Canisius College, Buffalo, New York, USA. Available at http://www2.canisius.edu/~sheets/.

Retrieved on 08/20/2011.

SIEGEL, A.F. & BENSON, R.H. (1982). A robust comparison of biological shapes.

Biometrics 38: 341-350.

SLICE, D.E. (2001). Landmark coordinates

29

aligned by Procrustes analysis do not lie in Kendall’s shape space. Systematic Biology 50: 141-149.

SLICE, D.E. (2005). Modern morphometrics, In D.E. Slice (ed.) Modern Morphometrics in Physical Anthropology. Series: Developments in Primatology: Progress and Prospects (R.H. Tuttle, ed.). Kluwer Academics / Plenum Publishers, New York, pp. 1-46.

SLICE, D.E.; BOOKSTEIN, F.L.; MARCUS, L.F. & ROHLF, F.J. (1996). Appendix I. A glossary for geometric morphometrics, In L.F. Marcus, M. Corti, A. Loy, G.J.P. Naylor & D.E. Slice (eds.) Advances in Morphometrics. Series: NATO ASI Series A: Life Sciences, vol. 284.

Plenum Press, New York, pp. 531-551.

SMITH, M.T. & COLLYER, M.L. (2008).

Regional variation and sexual dimorphism in head form of the prairie rattlesnake (Crotalus viridis viridis): comparisons using new analytical techniques and collection methods, In W.K. Hayes, K.R. Beaman, M.D. Cardwell & S.P. Bush (eds.) The Biology of Rattlesnakes.

Loma Linda University Press, Loma Linda, California, USA, pp. 79-90.

SOKAL, R.R. & ROHLF, F.J. (1995). Biometry: The Principles and Practice of Statistics in Biological Research, 3rd ed. W.H. Freeman, New York.

STAYTON, C.T. (2005). Morphological evolution of the lizard skull: A geometric morphometrics survey. Journal of Morphology 263: 47-59.

STAYTON, C.T. (2006). Testing hypotheses of convergence with multivariate data: morphological and functional convergence among herbivorous lizards. Evolution 60: 824-841.

STAYTON, C.T. (2009). Application of thinplate spline transformations to finite element models, or, how to turn a bog turtle into a spotted turtle to analyze both.

30

KALIONTZOPOULOU

Evolution 63: 1348-1355.

STAYTON, C.T. (2011). Biomechanics on the half shell: functional performance influences patterns of morphological variation in the emydid turtle carapace.

Zoology 114: 213-223.

STAYTON, C.T. & RUTA, M. (2006). Geometric morphometrics of the skull roof of stereospondyls (Amphibia: Temnospondyli).

Palaeontology 49: 307-337.

THOMPSON, D.W. (1917). On Growth and Form. Cambridge University Press, Cambridge, United Kingdom.

VALENZUELA, N.; ADAMS, D.C.; BOWDEN, R.M. & GAUGER, A.C. (2004). Geometric morphometric sex estimation for hatchling turtles: a powerful alternative for detecting subtle sexual shape dimorphism. Copeia 2004: 735-742.

VAN BUSKIRK, J. (2009). Natural variation in morphology of larval amphibians: Phenotypic plasticity in nature? Ecological Monographs 79: 681-705.

VIDAL, M.A.; ORTIZ, J.C.; RAMÍREZ, C.C. & LAMBOROT, M. (2005). Intraspecific variation in morphology and sexual dimorphism in Liolaemus tenuis (Tropiduridae).

Amphibia-Reptilia 26: 343-351.

VIDAL, M.A.; VELOSO, A. & MÉNDEZ, M.A. (2006). Insular morphological divergence in the lizard Liolaemus pictus (Liolaemidae).

Amphibia-Reptilia 27: 103-111.

VIEIRA, K.S.; ARZABE, C.; HERNÁNDEZ, M.I.M. & VIEIRA, W.L.S. (2008). An examination of morphometric variations in a

neotropical toad population (Proceratophrys cristiceps, Amphibia, Anura, Cycloramphidae).

PloS ONE 3: e3934.

WELLS, K.D. (2007). The Ecology and Behavior of Amphibians. The University of Chicago Press, Chicago.

WITZMANN, F.; SCHOLZ, H. & RUTA, M. (2009).

Morphospace occupation of temnospondyl growth series: a geometric morphometric approach. Alcheringa 33: 237-255.

YOUNG, M.T. & LARVAN, M.D. (2010).

Macroevolutionary trends in the skull of sauropodomorph dinosaurs – the largest terrestrial animals to have ever lived, In A.M.T. Elewa (ed.) Morphometrics for Nonmorpometricians. Series: Lecture Notes in Earth Sciences, vol. 124.

Springer-Verlag, Berlin, pp. 259-269.

YOUNG, M.T.; BRUSATTE, S.L.; RUTA, M. & DE ANDRADE, M.B. (2010). The evolution of Metriorhynchoidea (mesoeucrocodylia, thalattosuchia): an integrated approach using geometric morphometrics, analysis of disparity, and biomechanics. Zoological Journal of the Linnean Society 158: 801-859.

ZELDITCH, M.L.; SWIDERSKI, D.L.; SHEETS, H.D. & FINK, W.L. (2004). Geometric Morphometrics for Biologists: A Primer.

Elsevier Academic Press, San Diego, California, USA. ZUFFI, M.A.L.; SACCHI, R.; PUPIN, F. & CENCETTI, T. (2011). Sexual size and shape dimorphism in the Moorish gecko (Tarentola mauritanica, Gekkota, Phyllodactylidae).

North-Western Journal of Zoology 7: 189-197.

GEOMETRIC MORPHOMETRICS IN HERPETOLOGY

APPENDIX 1: SOFTWARE RESOURCES Several software packages have been developed for the application of GM methods, including 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 programs are freely available for download. Here I briefly provide an account of the main software packages more frequently used in recent publications. Apart from the software mentioned 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 programs, 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), modelling shape variation through the thin-plate spline and related methods (tpsRelW; tpsSplin) and a wide array of more specific

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exploratory and statistical procedures such as the visualization of thin-plate splines on trees (tpsTree), regression of shape onto independent variables and regression-related hypothesis testing (tpsRegr) and two-block partial least squares analysis (tpsPLS). Although some users might find the separation in different programs troublesome, this is a very question-driven software series, specifically designed for answering shape-related questions and accompanied by extremely thorough help pages that provide the user with both a conceptual and mathematical understanding of the operations involved. Both 2D and 3D analyses are supported. Designed for use in Windows, but will run without problems in Linux using Wine.

MorphoJ (KLINGENBERG, 2011): A userfriendly integrated software package that provides a platform for the most important types of analyses usually carried out with GM data.

These include 2D and 3D Procrustes superimposition with and without object symmetry, utility operations, generation of covariance matrices, matrix correlation, principal components analysis, two-block partial least squares, canonical variate and linear discriminant analyses, regression analysis, mapping 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 platform-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 generation of shape coordinates, principal components analysis, canonical variate analysis,

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shape regression and pair-wise multivariate shape comparisons. Both 2D and 3D analyses are supported. It is based on MATLAB, but will work without this software being installed on the computer. It is a Windowsbased 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 described by configurations of both 2D and 3D landmark 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 replaced by the EVAN toolbox (http://www.evansociety.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* Université de Lyon, Biologie Générale, Université Catholique de Lyon, EPHE Reproduction et Développement Comparé, Lyon, France. * Correspondence: Université de Lyon. UMRS 449. Biologie Générale, Université Catholique de Lyon, EPHE Reproduction et Développement Comparé, 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 stages 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 mesonephric 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 riñones en Typhlonectes compressicauda vivíparas (Amphibia, Gymnophiona).

La ontogénesis de los órganos en Gymnophiona es aún poco conocida. En la cecilia vivípara Typhlonectes compressicauda 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 embrión. En el estadio 23, se pueden apreciar claramente un par de riñones pronéfricos con varios nefrostomas que vierten a la cavidad celómica.

En el estadio 30, los pronefros que antes se podían apreciar al nivel de la segunda y quinta vértebras aparecen ahora entre las vértebras 24ª y 28ª. En este estadio, varios túbulos mesonéfricos aparecen entre las vértebras 29ª y 32ª, mientras que en la parte posterior del cuerpo se puede observar el tejido mesonéfrico. En el estadio 31, el pronefros comienza a desaparecer, mientras que el mesonefros presenta túbulos tanto proximales como distales. En los estadios 32-33, pueden apreciarse dos mesonefros con forma de capas alargadas, en lo que representa los riñones definitivos.

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

In vertebrates, the development of excretory organs is characterized by the formation of a first pair of primitive kidneys, the pronephroi, 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 fishes, and amphibians. The pronephros is characterized 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

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from excretory organs, appear in amphibians after the development of pronephroi.

Particularly in amphibians, both open and closed nephrons with or without glomeruli 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 studied the structure and development of urogenital system in Ichthyophis glutinosus, BRAUER (1900, 1902), who investigated kidney development 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 & SÜΒ (2000) the structure and ultrastructure of Ichthyophis kohtaoensis young larvae kidneys, and MØBJERG 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 mesonephros could form a unique kidney, called holonephros, which would be the supposed primitive situation in vertebrates.

We describe for the first time the development of both pronephroi and mesonephroi in the phylogenetically derived caecilian T. compressicauda in order to add data to the knowledge of kidney development in caecilians, to understand the relationships between

pronephroi and mesonephroi in basal and derived caecilians, and to understand the evolutionary relationships, based on kidney morphology 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 enabling embryo to grasp the uterine secretions and epithelial cells from the uterine wall (HRAOUI-BLOQUET et al., 1994; HRAOUIBLOQUET & 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 uterine wall, participating to the constitution of a placental structure (HRAOUI-BLOQUET & EXBRAYAT, 1994; EXBRAYAT & HRAOUIBLOQUET, 2006).

MATERIALS AND METHODS Embryos were obtained from gravid females belonging to a collection of ani-

KIDNEY DEVELOPMENT IN TYPHLONECTES COMPRESSICAUDA

mals retrieved from Kaw, a village situated 80 km southwest of Cayenne, French Guiana, in 1979, 1980 and 1982 (Table 1).

Animals were euthanized by immersion in MS 222; embryos at several stages of development were fixed in Bouin’s fluid, measured by placing them on graph paper, embedded in paraffin, then sectioned in 5 μm-thick pieces and stained with haemalum-eosin, Masson-Goldner’s trichrome or Romeis’s azan in order to obtain several views of morphological structures, according to EXBRAYAT (2001). To prepare the stains, haematoxylin and heamalum were purchased from RAL (Clichy, France), eosin from Acros Organics (Morris Plains, New Jersey, USA), iron alum, acid fuschin and azocarmine G from Sigma-Aldrich (Saint Louis, Missouri, USA), fast green from Edward Gurr (London) and aniline blue from Merck (Whitehouse Station, New Jersey, USA).

Table 1: Length and developmental stage ranges of Typhlonectes compressicauda embryos used for the study of kidney development, according to Sammouri et al. (1990).

Developmental stage

Length (mm)

N

18 19 21-22 23 24 25 26-27 28 29 30 31 32 33

Not measured Not measured 7 8-10 9-10 10-11 13-14 18-24 27-30 33-34 37-40 63-70 95-100

1 1 1 3 5 4 3 2 3 2 2 4 2

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RESULTS At early embryonic stages (18-19), at the level of the fifth somite, in a well anterior position, several undifferentiated cells disposed as a crown are the first indications of the Wolffian ducts (Fig. 1a).

At stages 22-23 (Fig. 1b) certain pronephric tubules are observed laterodorsally to each Wolffian duct. The lumen of the Wolffian duct is lined with a single layer of cubic cells.

Several nephrons are observed near the Wolffian ducts, with nephrostomes opening into the coelomic cavity. The lumen of the renal tubules is covered with a single layer of cubic cells without any apical specialization and nephrostomes are lined with a single layer of ciliated cubic cells. In the posterior part, the lumen of the Wolffian duct becomes circular, lined with a single layer of cubic cells. Several flexuous renal tubules develop from Wolffian ducts. Blood cells can agglomerate into a single glomerulus, and several blood vessels are observed near the tubules.

This posterior part corresponds to the beginning of mesonephros development (Fig. 1b).

At stage 24 (Figs. 1c, 1d), a pair of pronephroi is observed at the level of the second to the fifth vertebrae. They are composed of well differentiated renal tubules lined with cubic cells containing vacuoles. Two to four nephrostomes open into the coelomic cavity are also observed. Some tubules are wider than others.

The widest tubules, corresponding to proximal ones, are lined with cubic cells with abundant cytoplasm, some of which possess an apical brush border directed to the lumen. The narrowest tubules, corresponding to the intermediate and distal ones, are lined with cubic cells, with or without microvilli, whose cytoplasm is

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Figure 1: Sections of anterior kidney of early embryonic stages of Typhlonectes compressicauda. (a) Stage 19. Cross section stained with haemalum-eosin. (b) Stage 22. Longitudinal section stained with Masson-Goldner’s trichrome. The insert shows a cross section stained with haemalum-eosin. (c) (d) Stage 24. Cross sections stained with haemalum-eosin. (e) Stage 26.

Longitudinal section stained with haemalumeosin. (f ) Stage 26. Longitudinal section stained with Romeis’s azan. Gl: glomerulus, mesoneph: mesonephros, neph: nephrostome, NT: neural tube, proneph: pronephros, Tub: renal tubule, WD: Wolffian duct.

less developed than that of cells lining the widest tubules. Several capillaries extending between tubules can be observed in the pronephros. 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 single layer of columnar stereociliated cells.

At stages 27-28, nephrostomes are observed within the pronephroi. Each nephrostome 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

Wolffian duct. Renal tubules are lined with brush border cells.

At stage 29, each pronephros is well developed, 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 metamorphosis, all the organs seem to move towards the posterior part of the embryo’s 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 mesonephroi 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

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Figure 2: Sections of the anterior kidney of late embryonic stages of Typhlonectes compressicauda stained with Romeis’s azan. (a) Stage 27. Longitudinal section showing pronephros. (b) Stage 27. Longitudinal section showing mesonephros. (c) Stage 30.

Longitudinal section showing pronephros and mesonephros. (d) Stage 32. Longitudinal section showing mesonephros with a glomerulus. (e) Stage 32. Longitudinal section showing 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 separated 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 mesonephros 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 mesonephros still contains nephrostomes.

At stage 32 (Fig. 2d), mesonephroi containing voluminous glomeruli (Fig. 2e) appear partially segmented, but at this stage the pronephros 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 (mesonephroi) 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 Îźm 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 degeneration of pronephros.

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DISCUSSION For the first time, the development of kidneys has been described in T. compressicauda.

Kidneys have been described in several caecilian 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 & SÜΒ, 2000; MØBJERG et al., 2004). In T. compressicauda, the examination of kidney development shows the formation of a pair of pronephroi characterized by nephrostomes, large tubules, and small capillaries irrigating tubules. In Hypogeophis sp., BRAUER (1902) described the presence of very well differentiated nephrostomes throughout both pronephroi and mesonephroi. 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 & SÜΒ (2000), a large pronephros is observed caudally to the branchial 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 persistent 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 & SÜΒ, 2000). In I. kohtaoensis (WROBEL & SÜΒ, 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 several capillaries that occur between tubules, 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 & SÜΒ, 2000), pronephroi 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; MØBJERG et al., 2004). With the exception of T. compressicauda, no information is available on kidney structure during caecilian metamorphosis. In Hypogeophis sp., pronephroi degenerate when mesonephroi become 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 frontal position only (MØBJERG et al., 2004).

SAKAI et al. (1986, 1988a,b) gave structural and ultrastructural descriptions of adult kidneys 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 MØBJERG 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 caecilian species and did not see any nephrostomes open into the coelom.

Several comparative data about embryonic development of kidneys in amphibians have been published (BRACHET, 1935; GIPOULOUX, 1986; DITRICH & LAMETSCHWANDTNER, 1992; GIPOULOUX & CAMBAR, 1995; RICHTER, 1995; MØBJERG 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 between caecilians and other amphibians. In caecilians, kidneys are elongated like other organs, which is considered to be an adaptation to the burrowing habits of these animals (TAYLOR, 1968). Kidneys of caecilians also present a segmental disposition (WAKE, 1970) that does not exist in anurans 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 nephrostomes 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 contain osmiophilic granules that disappear at metamorphosis, indicating a change in kid-

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ney activity at that time (MØBJERG et al., 2000). The ablation of pronephroi in larvae of several anuran species provokes an important oedema followed by death (CAMBAR, 1947), which suggests that these first kidneys are functional at least during a part of larval development. In larval Ambystoma mexicanum, HAUGAN et al. (2010) proposed that pronephroi were important for modification of urine. In Bufo viridis, each pronephros is a single convoluted tubule open into the coelomic cavity through three nephrostomes (MØBJERG 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 & SÜΒ, 2000) and resembling a holonephros, the theoretical primitive 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 (MØBJERG et al., 2004) and T. compressicauda (this work), as well as in anurans and urodeles. In the caecilians whose kidney development has been studied, pronephroi 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 structure of T. compressicauda kidneys resemble that of other amphibians (BRACHET, 1935; GIPOULOUX

BASTIT & EXBRAYAT

40

& CAMBAR, 1995), with morphological differences related to burrowing habits. Within caecilians, 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. MarieThérèse Laurent for her technical assistance.

REFERENCES BALINSKY, B.I. (1975). Introduction to Embryology, 4th ed. Saunders, Philadelphia, Pennsylvania, USA. BRACHET, A. (1935). Traité d’Embryologie des Vertébrés. Nouvelle Édition Revue et Complétée par A. Dalcq et P. Gérard. Masson et Cie, Paris, France.

BRAUER, A. (1900). Zur kenntniss der entwicklung der excretionsorgane der gymnophionen. Zoologischer Anzeiger 23: 353-358.

BRAUER, A. (1902). Beiträge zur kenntnis der entwicklung und anatomie der gymnophionen. III. Die entwicklung der excretionsorgane. Zoologisches Jahrbuch für Anatomie 3: 1-176.

CAMBAR, R. (1947). Valeur fonctionnelle du pronéphros chez le très jeune tétard de grenouille. Comptes Rendus des

Scéances de la Société de Biologie et des ses Filiales 141: 754-756.

CARVALHO, E.T.C. & JUNQUEIRA, L.C.U. (1999). Histology of the kidney and urinary bladder of Siphonops annulatus (Amphibia-Gymnophiona). Archives of Histology and Cytology 62: 39-45.

CHATTERJEE, B.K. (1936) The anatomy of Uraeotyphlus menoni Annandale. Part I. The digestive, circulatory, respiratory, and urino-genital systems. Anatomische Anzeiger 81: 393-414.

DITRICH, H. & LAMETSCHWANDTNER, A. (1992). Glomerular development and growth of the renal blood vascular system in Xenopus laevis (Amphibia: Anura: Pipidae) during metamorphic climax.

Journal of Morphology 213: 335-340.

DRAWBRIDGE, J.; MEIGHAN, C.M.; LUMPKINS, R. & KITE, M.E. (2003). Pronephric duct extension in amphibian embryos: migration and other mechanisms. Developmental Dynamics 226: 1-11.

EXBRAYAT, J.-M. (1986). Quelques aspects de la biologie de la reproduction chez Typhlonectes compressicaudus (Duméril et Bibron, 1841), Amphibien Apode. D.Sci. Dissertation. Université Pierre et Marie Curie-Paris 6, Paris, France.

EXBRAYAT, J.-M. (2001). Genome Visualization

by Classic Methods in Light Microscopy. CRC Press, Boca Raton, Florida, USA. EXBRAYAT, J.-M. & HRAOUI-BLOQUET, S. (2006). Viviparity in Typhlonectes compressicauda, In J.-M. Exbrayat (ed.) Reproductive Biology and Phylogeny of Gymnophiona (Caecilians). Series: Reproductive Biology and Phylogeny, vol. 5 (B.G.M Jamieson, ed.) Science Publishers, Enfield, New Hampshire, USA, pp. 325-357.

KIDNEY DEVELOPMENT IN TYPHLONECTES COMPRESSICAUDA

EXBRAYAT, J.-M. & RAQUET, M. (2009).

Vertebrate evolution: the strange case of gymnophionan amphibians, In P. Pontarotti (ed.) Evolutionary Biology: Concept, Modeling and Application. Springer-Verlag, Berlin, Germany, pp. 71-89.

GARG, B.L. & PRASAD, J. (1962). Observations of the female urogenital organs of limbless amphibians Uraeotyphlus oxyurus. Journal of Animal Morphology and Physiology 9: 154-156.

GIPOULOUX, J.D. (1986). Prise de forme de l’embryon. Organogenèse. Aspects de physiologie embryonnaire, In P.P. Grassé & M. Delsol (eds.) Traité de Zoologie, Anatomie, Systématique, Biologie, Tome XIV, Fascicule I-B, Amphibiens. Masson, Paris, France, pp. 110-302.

GIPOULOUX, J.D. & CAMBAR, R. (1995).

L’appareil excréteur chez la larve et pendant la métamorphose. In P.P. Grassé & M. Delsol (eds.) Traité de Zoologie, Anatomie, Systématique, Biologie, Tome XIV, Fascicule I-A, Amphibiens. Masson, Paris, France, pp. 1051-1066.

HAUGAN, B.M.; HALBERG, K.A.; JESPERSEN, Å.; PREHN, L.R. & MØBJERG, N. (2010).

Functional characterization of the vertebrate primary ureter: structure and ion transport mechanisms of the pronephric duct in axolotl larvae (Amphibia). BMC Developmental Biology 10: 56.

HRAOUI-BLOQUET, S. & EXBRAYAT, J.-M. (1994). Développement des branchies chez les embryons de Typhlonectes compressicaudus, amphibien gymnophione vivipare.

Annales des Sciences Naturelles, Zoologie et Biologie Animale, 13ème Série 15: 33-46.

HRAOUI-BLOQUET, S. & EXBRAYAT, J.-M. (1996). Les dents de Typhlonectes compressicaudus (Amphibia, Gymnophiona) au cours

41

du développement. Annales des Sciences Naturelles, Zoologie, 13ème Série 17: 11-23.

HRAOUI-BLOQUET, S.; ESCUDIÉ, G. & EXBRAYAT, J.-M. (1994). Aspects ultrastructuraux de l’évolution de la muqueuse utérine au cours de la gestation chez Typhlonectes compressicaudus amphibien gymnophione vivipare. Bulletin de la Société Zoologique de France 119: 237-242.

LAMETSCHWANDTNER, A.; ALBRECHT, U. & ADAM, H. (1978). The vascularization of the kidneys in Bufo bufo (L.), Bombina variegata (L.), Rana ridibunda (L.) and Xenopus laevis (D.) (Amphibia, Anura) as revealed by scanning electron microscopy of vascular corrosion casts. Acta Zoologica 59: 11-23.

MØBJERG, N.; LARSEN, E.H. & JESPERSEN, Å.

(2000). Morphology of the kidney in larvae of Bufo viridis (Amphibia, Anura, Bufonidae).

Journal of Morphology 245: 177-195.

MØBJERG, N.; JESPERSEN, Å. & WILKINSON, M. (2004). Morphology of the kidney in the West African caecilian, Geotrypetes seraphini (Amphibia Gymnophiona, Caeciliidae).

Journal of Morphology 262: 583-607.

PORTER, K.R. (1972). Herpetology. Saunders, Philadelphia, Pennsylvania, USA. RICHTER, S. (1995). The opisthonephros of Rana esculenta (Anura). I. Nephron development. Journal of Morphology 226: 173-187.

SAKAI, T.; BILLO, R. & KRIZ, W. (1986). The structural organization of the kidney of Typhlonectes compressicaudus (Amphibia, Gymnophiona).

Anatomy and Embryology 174: 243-252.

SAKAI, T.; BILLO, R.; NOBILING, R.; GORGAS, K. & KRIZ, W. (1988a). Ultrastructure of the kidney of a South American caecilian, Typhonectes compressicaudus (Amphibia, Gymnophiona). I. Renal corpuscle, neck segment, proximal tubule and intermediate seg-

42

BASTIT & EXBRAYAT

ment. Cell and Tissue Research 252: 589-600.

SAKAI, T.; BILLO, R. & KRIZ, W. (1988b).

Ultrastructure of the kidney of a South American caecilian, Typhonectes compressicaudus (Amphibia, Gymnophiona). II. Distal tubule, connecting tubule, collecting duct and Wolffian duct. Cell and Tissue Research 252: 601-610.

SAMMOURI, R.; RENOUS, S.; EXBRAYAT, J.-M. & LESCURE, J. (1990). Développement embryonnaire de Typhlonectes compressicaudus (Amphibia, Gymnophiona).

Annales des Sciences Naturelles, Zoologie, 13ème Série 11: 135-163.

SEMON, R. (1892). Studien über den bauplan des urogenitalsystems der wirbeltiere.

Dargelegt an der entwicklung dieses organsystems bei Ichthyophis glutinosus. Jenaische Zeitschrift für Naturwissenschaft 26: 89-203.

SPENGEL, J.W. (1876). Das urogenitalsystem der amphibien. I. Theil. Der anatomische bau des urogenitalsystems. Arbeit aus dem

Zoologisch-Zootomischen Institut in Würzburg 3: 1-114.

TAYLOR, E.H. (1968). The Caecilians of the World: A Taxonomic Review. University of Kansas Press, Lawrence, Kansas, USA. WAKE, M.H. (1970). Evolutionary morphology of the caecilian urogenital system.

Part II. The kidney and the urogenital ducts. Acta Anatomica 75: 321-358.

WIEDERSHEIM, R. (1879). Die anatomie der gymnophionen. Gustav Fisher, Jena, Germany.

WILKINSON, M. & NUSSBAUM, R.A. (2006).

Caecilian phylogeny and classification, In J.M. Exbrayat (ed.) Reproductive Biology and Phylogeny of Gymnophiona (Caecilians). Series: Reproductive Biology and Phylogeny, vol. 5 (B.G.M Jamieson, ed.). Science Publishers, Enfield, New Hampshire, USA, pp. 39-78.

WROBEL, K.-H. & SÜΒ, F. (2000). The significance of rudimentary nephrostomial tubules for the origin of the vertebrate gonad.

Anatomy and Embryology 201: 273-290.

Basic and Applied Herpetology 25 (2011): 43-54

Population estimators and adult sex ratio for a population of Bolitoglossa altamazonica (Caudata: Plethodontidae) Doris Laurinette Gutiérrez-Lamus1,*, John Douglas Lynch1, Germán C. Martínez-Villate2 1 2

Instituto de Ciencias Naturales, Amphibian's Laboratory, Universidad Nacional de Colombia, Bogotá, Colombia.

Faculty of Veterinary Medicine and Husbandry, Fundación Universitaria San Martín, Sede Bogotá, Colombia.

* Correspondence: Cra. 4 # 24-59 apt. 1504, Bogotá, 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 tropical 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 población y razón de sexos en una población de Bolitoglossa altamazonica (Caudata: Plethodontidae). Bolitoglossa altamazonica es la especie de pletodóntido con distribución más amplia en los trópicos. Sin embargo, mientras que los aspectos relacionados con el tamaño de la población, tasa de supervivencia, reclutamiento, razón de sexos y otras características de la historia vital están bien documentados en salamandras de zonas templadas, la información para especies tropicales es relativamente escasa. Llevamos a cabo un estudio intensivo de marcaje-recaptura en una población de B. altamazonica. Utilizamos el método de Jolly-Seber para estimar tres parámetros (tamaño, reclutamiento y supervivencia) tanto para el conjunto de la población como para machos, hembras y juveniles por separado. Todos los parámetros estimados experimentaron variaciones mensuales en cada una de las clases así como en el conjunto de la población. El reclutamiento de los juveniles sucedió entre noviembre y julio. La supervivencia de los juveniles se incrementó cuando no existía reclutamiento. La razón de sexos de la población adulta durante el periodo reproductor estuvo significativamente sesgada en favor de las hembras (hasta 1:3 dependiendo del mes).

Key words: razón de sexos; reclutamiento; salamandras; supervivencia; tamaño de población.

Reports on declining and disappearing 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 environmental perturbations (POUNDS et al., 1997),

and differentiating problematic declines from 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

GUTIÉRREZ-LAMUS ET AL.

alarming decline, known as the global amphibian 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 salamanders 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 consistent 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 & M’CLOSKEY, 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 reproductive health for a given population is, and survivorship often explains a large portion of an individual’s 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 understanding 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 behaviour, and population dynamics (KVARNEMO & AHNESJÖ, 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 relative 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 altamazonica occurs on the eastern slopes of the Andes from Venezuela and Colombia, through Ecuador, Peru and Bolivia, and as far east as eastern 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 examine three population parameters (size, survivorship 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 parameters of a population of B. altamazonica. This dataset provides the best opportunity to examine changes in salamander populations over

POPULATION ECOLOGY OF A NEOTROPICAL SALAMANDER

time, thus serving as a reference for comparison to populations in other localities from Colombia and along its distribution range.

MATERIALS AND METHODS The study site, Jardín Botánico de Villavicencio, is a local natural reserve since 1983 located in Villavicencio City, Meta, Colombia (04º 09' 09'' N, 73º 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, 25.9ºC and 76%, respectively. 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 Hidrología, Meteorología y Estudios Ambientales de Colombia).

At the study site, we established a 7300 m2plot. We captured salamanders within this plot by visual encounter (CRUMP & SCOTT, 1994; ANGULO et al., 2006). Two experienced researchers 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 surveys were conducted at night because the focal species is nocturnal and surface activity occurs at night. We marked animals using freeze branding with liquid nitrogen (DAUGHERTY, 1976).

Individual identification was achieved by combinations of marks in fifteen body locations (modified from NISHIKAWA & SERVICE, 1988), five on each side of the body and five mid-dorsally according to the following distribution: anterior and posterior to the fore limb, midbody, 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 salamanders 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 classified 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, survival 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 animals in the population at time i per animal in the population at time i - 1 (PRADEL, 1996), and it includes in situ reproduction and immigration. 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

GUTIÉRREZ-LAMUS ET AL.

46

mark-recapture data using Cormack JollySeber (CJS) and Pradel survival and recruitment extensions of Program MARK version 5.0 (WHITE & BURNHAM, 1999). We used an overall model selection procedure before parameter estimation. Ten a priori candidate models offered different biological representations of the role of capture probability among sexes and ages (adult or juvenile). Rather than including all possible permutations in the primary model selection procedure, we initially tested four models to evaluate sex differences in detectability and survival; then, we tested four additional models accounting for possible age differences in these variables. Parameters in the candidate models were either allowed to vary over time or to remain constant. Model selection was based on the small-sample Akaike’s information criterion (AICc; BURNHAM & ANDERSON, 2002). Some parameters were estimated after 15 000 simulations using Markov chain Monte Carlo (MCMC), keeping all the other parameters

that did not have standard errors extremely high or low fixed. We used the Jolly-Seber original model (JOLLY, 1965; SEBER, 1965) to estimate population size, since program Mark never reached numerical convergence. Adult sex ratio was expressed as the relative proportions of estimated mature males and females, and deviations from a 1:1 ratio were tested through a Chi-square test.

RESULTS We captured 880 individuals of B. altamazonica, 244 of which were recaptured for a total of 1124 captures in nine months.

Monthly captures regardless of sex or age ranged from 64 to 184 individuals, and fluctuated following the same pattern as rainfall, with a peak of captured salamanders in June (Fig. 1). Because of the intense sampling, frequency of captures of new individuals declined rapidly over time, in spite of which we 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 Akaike’s Information Criterion. g = gender/age-dependent, t = time-dependent.

Extension

Model

CJS (Sex)

CJS (Age)

Pradel

AICc

Delta AICc

AICc weight

Model likelihood

Deviance

Phi(t) p(t) Phi(g*t) p(g*t) Phi(g*t) p(t) Phi(t) p(g*t)

203.87 206.41 207.84 209.09

0.00 2.53 3.97 5.21

0.669 0.188 0.092 0.049

1.00 0.28 0.137 0.074

20.85 12.81 18.52 19.76

Phi(t) p(g*t) Phi(g*t) p(g*t) Phi(t) p(t) Phi(g*t) p(t)

1763.93 1768.23 1772.19 1777.36

0.00 4.299 8.263 13.426

0.882 0.103 0.014 0.001

1.00 0.116 0.016 0.001

239.94 229.49 264.82 253.37

Phi (t) p(g*t) f(g*t) Phi (t) p(g*t) f(t)

5444.219 5452.48

0.00 8.26

0.984 0.016

1.00 0.016

242.66 257.28

POPULATION ECOLOGY OF A NEOTROPICAL SALAMANDER

Figure 1: Monthly variation in the number of captured 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 capturerecapture 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 detectability, supported the time-varying detectability, 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 function of both factors (Table 1).

Population size estimates fluctuated between 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 salamanders 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 obtained 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 parameters, 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) estimates (± SE) for a population of Bolitoglossa altamazonica. No data from January were calculated because, due to the absence of sampling in December, they did not meet the Jolly-Seber method requirement of consecutive samplings.

Month

N

Phi

April May June July August September October November January

582 (± 253) 1348 (± 350) 1344 (± 280) 1267 (± 275) 776 (± 150) 2165 (± 896) 1298 (± 560) -

0.342 (± 0.107) 0.847 (± 0.140) 0.779 (± 0.125) 0.880 (± 0.162) 0.830 (± 0.171) 1.950 (± 0.196) 0.825 (± 0.362) 0.281 (± 17.995) -

GUTIÉRREZ-LAMUS ET AL.

48

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

Month

May June July August September October November January

Adults

Juveniles

f

p

f

p

1.145 (± 0.276) 0.825 (± 0.219) 0.122 (± 0.274) -0.008 (± 0.004) 0.268 (± 0.123) 0.407 (± 0.182) 0.327 (± 0.227) 0.027 (± 1.085)

0.174 (± 0.102) 0.152 (± 0.050) 0.136 (± 0.037) 0.127 (± 0.033) 0.200 (± 0.045) 0.120 (± 0.027) 0.082 (± 0.039) 0.104 (± 0.021)

7.558 (± 1.709) 0.061 (± 0.124) 0.320 (± 0.127) 0.011 (± 0.223) 0.006 (± 0.062) 0.006 (± 0.089) 0.024 (± 0.093) 0.011 (± 0.130)

0.242 (± 0.000) 0.238 (± 0.174) 0.105 (± 0.037) 0.111 (± 0.0321) 0.077 (± 0.017) 0.071 (± 0.016) 0.064 (± 0.015) 0.124 (± 0.055)

no new juveniles entered the population between September and October. We did not take into account juvenile recruitment estimates in May because of the high standard error.

We performed estimates for adults from 468 individuals, 175 of which were recaptured for a total of 643 captures. Adult recruitment was highest in June and dropped along with the rainfall; in August-September no new adults entered the population (Table 3).

Using the 100 males and 245 females captured during the breeding season, which runs from January to July (GUTIÉRREZ-LAMUS, 2009), we found a significantly female-biased sex ratio that ranged between months from 1:2 to 1:3 (Table 4).

DISCUSSION The results from this study are the first of its kind for B. altamazonica and contribute to the knowledge about this population in regards to its size, survivorship, recruitment and adult sex ratio.

The key estimates for many ecologists using the CJS model on capture-recapture data are the survival rates, with the capture probabilities often viewed as little important nuisance para-

meters (PLEDGER et al., 2003). Numerous authors have suggested that this assumption is met as capture probability is likely to vary among demographic groups over time (WILLSON et al., 2011). The mistake of assuming constant capture probability when variation actually exists can bias estimates of abundance or recruitment (POLLOCK et al., 1990; BAILEY et al., 2004). Imperfect detectability also extends to the estimation of survival (MAZEROLLE et al., 2007). Adjusted population estimates, which estimate the “true” population based on capture-recapture techniques, are labour-intensive but may yield a more accurate picture of the number of salamanders present in a population (JUNG et al., 2000).

Table 4: Chi-square test to analyse the sex ratio obtained in a population of Bolitoglossa altamazonica. The null hypothesis is a one-to-one sex ratio. P-values in bold face are significant.

Month N (males) N (females) Sex ratio χ2 (d.f. = 1) April May June July January Total

17 17 29 23 14 100

31 46 67 64 37 245

1:1.8 1:2.7 1:2.3 1:2.8 1:2.6 1:2.45

4.0833 13.3492 15.0417 19.3218 10.3726 60.9420

P 0.0433 0.0000 0.0003 0.0000 0.0013 0.0000

POPULATION ECOLOGY OF A NEOTROPICAL SALAMANDER

According to recruitment estimates, most juveniles enter the population in July, and recruitment gradually decreases during subsequent 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 individuals 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 captures. 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 fluctuations in our estimates; first, our inability to find and mark brooding females; second, sampling during rainy nights when activity notably decreased; third, the behavioural thermoregulation 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 litter, 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 capture 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 monitoring 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 conditions. We obtained the greatest values for captures, population size and recruitment when rainfall levels were high. It has been generally accepted 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 RĂ&#x153;E 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 densities 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

GUTIĂ&#x2030;RREZ-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 individuals / m2 in Plethodon glutinosus (SEMLITSCH, 1980), 0.4051-0.4989 individuals / m2 in A. lugubris (ANDERSON, 1960), 0.10-0.25 individuals / m2 in Aneides aeneus (GORDON, 1952) and 0.1482-0.1729 individuals / m2 in Ensatina eschscholtzii (STEBBINS, 1954). Unfortunately, most reports on population densities of plethodontids were calculated before herpetologists could deal with imperfect detection through more sophisticated 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 predators 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 progressive 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 attribute losses in the population to deaths or emigration accurately. Generally, adult survival is likely to be more important than recruitment for population persistence, because it determines how long a population can persist without recruiting new individuals (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 homogeneity 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 plethodontids 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, variations 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 conditions and are commonly used also by nonreproducing 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 salamander populations and environmental factors. The present study can be used as a starting point for comparison to other B. altamazonica 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 Jardín Botánico de Villavicencio and E. Gomez provided access to the study place. R. MorenoArias and two anonymous reviewers provided helpful comments that greatly improved this manuscript. This project complies with all laws of Colombia and was conducted under CORMACARENA’s scientific research permit (authorization No 000884), with funds provided by División de Investigaciones sede Bogotá - 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.

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REFERENCES AMPHIBIAWEB (2011). Information on Amphibian Biology and Conservation.

University of California, Berkeley, California, USA. Available at http://amphibiaweb.org/. Retrieved on 09/09/2011.

ANDERSON, P.K. (1960). Ecology and evolution in island populations of salamanders in the San Francisco Bay region. Ecological Monographs 30: 359-386.

ANGULO, A.; RUEDA-ALMONACID, J.V.; RODRÍGUEZ-MAHECHA, J.V. & LA MARCA, E. (2006). Técnicas de Inventario y Monitoreo para los Anfibios de la Región Tropical Andina. Series: Manuales de Campo, vol. 2 (J.V. RodríguezMahecha, J.V. Rueda-Almonacid & A. González-Hernández, eds.). Conservación Internacional, Bogotá, Colombia.

AUBERT DE LA RÜE, E. ; BOURLIÈRE, F. & HARROY, J.-P. (1957). The Tropics. Knopf, New York.

AZEVEDO-RAMOS, C.; REICHLE, S.; ALMANDÁRIZ, A & CASTRO, F. (2009).

Bolitoglossa altamazonica, In IUCN 2009. The IUCN Red List of Threatened Species. Version 2009.1. International Union for Nature Conservation and Natural Resources, Gland, Switzerland. Available at http:// www.iucnredlist.org. Retrieved on 08/04/2009.

BAYLEY, L.L.; SIMONS, T.R. & POLLOCK, K.H. (2004). Estimating detection probability parameters for plethodontid salamanders using the robust capture-recapture design. Journal of Wildlife Management 68: 1-13.

BLACKWELL, E.A.; CLINE, G.R. & MARION, K.R. (2004). Annual variation in population estimators for a southern population of Ambystoma maculatum. Herpetologica 60: 304-311.

52

GUTIÉRREZ-LAMUS ET AL.

BRUCE, R.C. (1988). An ecological life table for the salamander Eurycea wilderae.

Copeia 1988: 15-26.

BURNHAM, K.P. & ANDERSON, D.R. (2002).

Model Selection and Multimodel inference: A Practical Information-Theoretic Approach, 2nd ed. Springer, New York.

BURTON, T.M. & LIKENS, G.E. (1975).

Salamander populations and biomass in the Hubbard Brook Experimental Forest, New Hampshire. Copeia 1975: 541-546.

CRUMP, M.L. & SCOTT, JR., N.J. (1994). Visual encounter survey, In W.R. Heyer, M.A. Donelly, R.W. McDiarmid, L.-A.C Hayek & M.S. Foster (eds.) Measuring and Monitoring Biological Diversity. Standard Methods for Amphibians. Smithsonian Institution Press, Washington, D.C., USA, pp. 84-92.

DAUGHERTY, C.H. (1976). Freeze-branding as a technique for marking anurans.

Copeia 1976: 836-838.

DONELLY, M.A. & GUYER, C (1994).

Estimating population size: Mark-recapture, In W.R. Heyer, M.A. Donelly, R.W. McDiarmid, L.-A.C Hayek & M.S. Foster (eds.) Measuring and Monitoring Biological Diversity. Standard Methods for Amphibians.

Smithsonian Institution Press, Washington, D.C., USA, pp. 183-200.

DUELLMAN, W.E. & TRUEB, L. (1994). Biology of Amphibians. The Johns Hopkins University Press, Baltimore, Maryland, USA. DUMAS, P.C. (1956). The ecological relations of sympatry in Plethodon dunni and Plethodon vehiculum. Ecology 37: 484-495.

FEDER, M.E. (1982). Thermal ecology of neotropical lungless salamanders (Amphibia: Plethodontidae): environmental temperatures and behavioral responses. Ecology 63: 1665-1674.

FLATT, T.; DUMMERMUTH, S. & ANHOLT, B.R. (1997). Mark-recapture estimates of survival in populations on the asp viper, Vipera aspis aspis. Journal of Herpetology 31: 558-564.

GORDON, R.E. (1952). A contribution to the life history and ecology of the plethodontid salamander Aneides aeneus (Cope and Packard).

American Midland Naturalist 47: 666-701.

GORDON, R.E.; MACMAHON, J.A. & WAKE, D.B. (1962). Relative abundance, microhabitat, and behavior of some southern Appalachian salamanders. Zoologica 47: 9-14.

GUTIÉRREZ-LAMUS, D.L. (2009). Tamaño Poblacional, Reclutamiento, Microhábitat y Uso del Espacio en una Población de Bolitoglossa altamazonica (Caudata: Plethodontidae) Presente en el Jardín Botánico de Villavicencio.

M.Sc. Dissertation. Universidad Nacional de Colombia, Bogotá, Colombia.

HECNAR, S.J. & M’CLOSKEY, R.T. (1996).

Regional dynamics and the status of amphibians. Ecology 77: 2091-2097.

HENDRICKSON, J.R. (1954). Ecology and systematics of salamanders of the genus Batrachoseps. University of California Publications in Zoology 54: 1-46.

HOLDRIDGE, L.R.; GRENKE, W.C.; HATHEWAY, W.H.; LIANG, T & TOSI, JR., J.A. (1971).

Forest Environments in Tropical Life Zones: A Pilot Study. Pergamon Press, Oxford, UK. HUSTING, E.L. (1965). Survival and breeding structure in a population of Ambystoma maculatum. Copeia 1965: 352-362.

JOLLY, G.M. (1965). Explicit estimates from capture-recapture data with both death and immigration-stochastic model.

Biometrika 52: 225-247.

JUNG, R.E.; DROEGE, S.; SAUER, J.R. & LANDY, R.B. (2000). Evaluation of terrestrial and streamside salamander monitoring techniques at

POPULATION ECOLOGY OF A NEOTROPICAL SALAMANDER

Shenandoah National Park. Environmental Monitoring and Assessment 63: 65-79.

Krebs, C.J. (1999). Ecological Methodology, 2nd ed. Addison Wesley Longman, Menlo Park, California, USA. KVARNEMO, C. & AHNESJÖ, I. (2002).

Operational sex ratios and mating competition, In I.C.W. Hardy (ed.) Sex Ratios. Concepts and Research Methods. Cambridge University Press, Cambridge, UK, pp. 366-382.

LEBRETON, J.-D.; BURNHAM, K.P.; CLOBERT, J. & ANDERSON, D.R. (1992). Modeling survival and testing biological hypotheses using marked animals: a unified approach with case studies. Ecological Monographs 62: 67-118.

LIPS, K.R. (1998). Decline of a tropical montane amphibian fauna. Conservation Biology 12: 106-117.

MAZEROLLE, M.J.; BAILEY, L.L.; KENDALL, W.L.; ROYLE, J.A.; CONVERSE, S.J. & NICHOLS, J.D. (2007). Making great leaps forward: accounting for detectability in herpetological field studies. Journal of Herpetology 41: 672-689.

NISHIKAWA, K.C & SERVICE, P.M. (1988). A fluorescent marking technique for individual recognition of terrestrial salamanders. Journal of Herpetology 22: 351-353.

OLGUN, K.; MIAUD, C & GAUTIER, P. (2001).

Age, growth, and survivorship in the viviparous salamander Mertensiella luschani from southwestern Turkey. Canadian Journal of Zoology 79: 1559-1567.

ORGAN, J.A. (1961). Studies of the local distribution, life history, and population dynamics of the salamander genus Desmognathus in Virginia. Ecological Monographs 31: 189-220.

ORTEGA, J.E.; MONARES-RIAÑO, J.M & RAMÍREZ-PINILLA, M.P. (2009). Reproductive activity, diet, and microhabitat use in

53

Bolitoglossa nicefori (Caudata: Plethodontidae).

Journal of Herpetology 43: 1-10.

PARRA-OLEA, G.; GARCÍA-PARÍS, M. & WAKE, D.B. (1999). Status of some populations of Mexican salamanders (Amphibia: Plethodontidae). Revista de Biología Tropical 47: 217-223.

PECHMANN, J.H.K. &WILBUR, H.M. (1994).

Putting declining amphibian populations in perspective: natural fluctuations and human impacts. Herpetologica 50: 65-84.

PECHMANN, J.H.K.; SCOTT, D.E.; SEMLITSCH, R.D.; CALDWELL, J.P.; VITT, L.J. & GIBBONS, J.W. (1991). Declining amphibian populations: the problem of separating human impacts from natural fluctuations. Science 253: 892-895.

PLEDGER, S.; POLLOCK, K.H. & NORRIS, J.L. (2003). Open capture-recapture models with heterogeneity: I. Cormack-JollySeber model. Biometrics 59: 786-794.

POLLOCK, K.H; NICHOLS, J.D.; BROWNIE, C. & HINES, J.E. (1990). Statistical inference for capture-recapture experiments.

Wildlife Monographs 107: 1-97.

POPE, C.H. (1950). A statistical and ecological study of the salamander, Plethodon yonahlossee. Bulletin of the Chicago Academy of Sciences 9: 79-106.

POUNDS, J.A.; FOGDEN, M.P.L.; SAVAGE, J.M. & GORMAN, G.C. (1997). Test of null models for amphibian declines on a tropical mountain. Conservation Biology 11: 1307-1322.

PRADEL, R. (1996). Utilization of capturemark-recapture for the study of recruitment and population growth rate.

Biometrics 52: 703-709.

RANTA, E.; LUMMAA, V.; KAITALA, V. & MERILÄ, J. (2000). Spatial dynamics of adaptive sex ratios. Ecology Letters 3: 30-34.

54

GUTIÉRREZ-LAMUS ET AL.

RICHARDS, P.W. (1957). The Tropical Rain Forest: An Ecological Study. Cambridge University Press, Cambridge, UK. ROSENTHAL, G.M. (1957). The role of moisture and temperature in the local distribution of the plethodontid salamander Aneides lugubris. University of California Publications in Zoology 54: 371-420.

ROVITO, S.M.; PARRA-OLEA, G.; VÁSQUEZALMAZÁN, C.R.; PAPENFUSS, T.J. & WAKE, D.B. (2009). Dramatic declines in neotropical salamander populations are an important part of the global amphibian crisis. Proceedings of the National Academy of Sciences USA 106: 3231-3236.

SCHMIDT, B.R.; FELDMANN, R. & SCHAUB, M. (2005). Demographic processes underlying population growth and decline in Salamandra salamandra. Conservation Biology 19: 1149-1156.

SEBER, G.A.F. (1965). A note on the multiplerecapture census. Biometrika 52: 249-259.

SEMLITSCH, R.D. (1980). Geographic and local variation in population parameters of the slimy salamander Plethodon glutinosus. Herpetologica 36: 6-16.

SMYERS, S.D.; RUBBO,M.J.; TOWNSEND, JR., W.R. & SWART, C.C. (2002). Intra- and interspecific characterizations of burrow use and defense by juvenile ambystomatid salamanders. Herpetologica 58: 422-429.

SPOTILA, J.R. (1972). Role of temperature and water in the ecology of lungless salamanders. Ecological Monographs 42: 95-125.

STEBBINS, R.C. (1954). Natural history of the salamanders of the plethodontid genus Ensatina. University of California Publications in Zoology 54: 47-123.

STUART, S.N.; CHANSON, J.S.; COX, N.A.; YOUNG, B.E.; RODRIGUES, A.S.L.; FISCHMAN,

D.L. & WALLER, R.W. (2004). Status and trends of amphibian declines and extinctions worldwide. Science 306: 1783-1786.

TEST, F.H. & BINGHAM, B.A. (1948). Census of a population of the red-backed salamander (Plethodon cinereus). American Midland Naturalist 39: 362-372.

VIAL, J.L. (1968). The ecolology of the tropical salamander, Bolitoglossa subpalmata, in Costa Rica. Revista de Biología Tropical 15: 13-115.

WAKE, D.B. (1966). Comparative osteology and evolution of the lungless salamanders, family Plethodontidae. Memoirs of the Southern California Academy of Sciences 4: 1-111.

WHITE, G.C. & BURNHAM, K.P. (1999).

Program MARK: survival estimation from populations of marked animals. Bird Study 46: S120-S139.

WHITEMAN, H.H. & WISSINGER, S.A. (2005). Amphibian population cycles and long-term data sets, In M. Lannoo (ed.)

Amphibian Declines. The Conservation Status of United States Species. University of California Press, Berkeley, California, USA, pp. 177-184.

WHITFIELD, S.M.; BELL, K.E.; PHILIPPI, T.; SASA, M.; BOLAÑOS, F.; CHAVES, G.; SAVAGE, J.M. & DONNELLY, M.A. (2007). Amphibian and reptile declines over 35 years at La Selva, Costa Rica. Proceedings of the National Academy of Sciences USA 104: 8352-8356.

WILLSON, J.D.; WINNE, C.T. & TODD, B.D. (2011). Ecological and methodological factors affecting detectability and population estimation in elusive species. Journal of Wildlife Management 75: 36-45.

ZUG, G.R.; VITT, L.J. & CALDWELL, J.P. (2001). Herpetology. An Introductory Biology of Amphibians and Reptiles, 2nd ed. Academic Press, San Diego, California, USA.

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 Gonçalves1,*, Sandra Amaral2, Rui Rebelo2 1 2

Department of Evolutionary Biology “Leo Pardi”, University of Florence, Florence, Italy.

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 defences 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 tadpoles 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 ibérico (Alytes cisternasii) a los estímulos químicos de depredadores nativos (Natrix maura y Squalius pyrenaicus) y exóticos (Procambarus clarkii). La depredación es una importante presión selectiva que resulta en la evolución 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 estímulos químicos procedentes de un depredador exótico, Procambarus clarkii, con las respuestas a los estímulos químicos de dos de sus depredadores naturales, la culebra viperina (Natrix maura) y el cacho (Squalius pyrenaicus), los cuales utilizan estrategias de depredación diferentes. Las larvas reaccionaron de manera más intensa a N. maura, seguida de P. clarkii, sin que existieran respuestas significativas a S. pyrenaicus. La alteración en el uso de la columna de agua fue la conducta antipredatoria más utilizada ante los depredadores, ya fueran nativos o exóticos, adecuando las larvas su comportamiento al periodo de actividad y a la estrategia de cada depredador. Las larvas de A. cisternasii reaccionaron ante los estímulos procedentes de P. clarkii, un depredador introducido tan solo hace 20 años en el área de estudio. Estas reacciones podrían ser resultado de un rápido proceso micro-evolutivo, pero también ser una respuesta fortuita mostrada ante un estímulo novel para las larvas. Tampoco podemos descartar la posibilidad de aprendizaje por parte de las larvas ya que, dadas las características 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 acuático.

Key words: Alytes cisternasii; conducta antipredatoria; depredadores alóctonos; invasiones; Procambarus clarkii; quimiorrecepción.

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Native and alien predators can be an important force of selection in natural systems, resulting in the evolution or learning of antipredator defences by prey populations. Several studies have demonstrated that larval amphibians are able to innately recognize and respond 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 behaviour (SKELLY & WERNER, 1990; LARDNER, 2000). Antipredator behaviour may include the reduction of activity levels, alterations in the use of different microhabitats or an increased 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 predators differ in their predation strategies, prey frequently exhibit specific antipredator behaviour (RELYEA, 2001, 2004). Thus, when facing an introduced predator for the first time, naïve native prey may exhibit no antipredator behaviour (KIESECKER & BLAUSTEIN, 1997; NYSTRÖM 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 reproduction habitats (CRUZ et al., 2006). It is also known that Iberian midwife toad tadpoles,

Alytes cisternasii Boscá, 1879, show some behavioural modifications when faced with P. clarkii, namely by modifying their use of stream bed refuges and by fleeing to the margins during the night (GONÇALVES 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 (MÁRQUEZ, 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 observation). 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 disturbed, injured or consumed conspecifics (HETTYEY et al., 2010).

The objective of the present work is to compare 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 (Günther, 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 predation 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 antipredator 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 AprilMay 2005 (experiment 2). Part of our 2005 results were the subject of a previous paper (GONÇALVES et al., 2007), concerning differences between seasons. The present experiments 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 experiments were captured in the small streams of the Field Station of the CBA – the Herdade da Ribeira Abaixo (Serra de Grândola, Baixo Alentejo, SW Portugal; 38º06’28.57’’N; 8º34’14.56’’W). Tadpoles and fishes were captured with dip-nets; crayfishes were captured with baited funnel traps and snakes were captured by hand.

The Portuguese territory south of the Tagus River is a region where no native crayfish ever existed (ALMAÇA, 1991). Tadpoles for both experiments belonged to a population 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 station in PVC aquaria filled with spring water for one week, under a 12:12 light-dark photoperiod, and fed ad libitum with cooked lettuce and commercial fish food. The water temperature was kept at 10-14ºC (experiment 1) or 16-18ºC (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 pierced, 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 predator’ treatment), 10 replicates; cage with an individual of S. pyrenaicus (‘native predator’ treatment) 10 replicates; experiment 2, empty cage (control), 15 replicates; cage with an individual 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.

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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 preliminary tests that this did not affect tadpole behaviour (AMARAL, 2004). Each tadpole, fish and crayfish was used only once; however, 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; NYSTRÖM 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-

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 movement 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 variables 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 Fisher’s least significant difference (LSD) post-hoc tests.

Instead of using the time of the day as an additional factor in the analysis, the results of diurnal

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

Experiment 1 (Winter) Amaral (2004)

Experiment 2 (Spring) Present study

HL

Control

15.92 ± 1.89 (N = 75) 15.78 ± 1.85 (N = 75) –

N. maura

17.71 ± 1.81 (N = 45) 17.26 ± 1.77 (N = 50) 17.90 ± 1.51 (N = 50) –

Control P. clarkii S. pyrenaicus N. maura

25 25 27 –

41 41 – 37

P. clarkii S. pyrenaicus

Developmental stage

15.26 ± 2.13 (N = 75)

ANTIPREDATOR BEHAVIOUR OF A. CISTERNASII TADPOLES

and nocturnal trials were analysed separately because, as stated above, we used the same individuals for both trials. Therefore, the results of both trials were not independent from each other. All analyses were performed with the program 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 differences 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 subjected to P. clarkii cues decreased the use of refuge compared with tadpoles subjected to the control 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 involving 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 decreased the use of the substratum and increased their permanence at the surface of the experimental aquarium (LSD test, P < 0.05 for all mentioned variables and pair wise comparisons involving N. maura).

GONĂ&#x2021;ALVES ET AL.

60

Figure 2: Recorded behaviours of tadpole (a) activity, (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 (Âą 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

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 native 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 activity and fled from the water column towards the margins, whereas at night tadpoles remained more frequently at the surface. In the laboratory, N. maura presents both nocturnal

a

c

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 natural 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 Sum of squares

Refuge use Activity Water column Substratum Surface Margins

Treatment Error Treatment Error Treatment Error Treatment Error Treatment Error Treatment Error

34.53 412.67 4.04 21.07 4.58 25.2 76.84 710.26 86.58 824 12 10

Diurnal trials d.f.

F

2 42 2 42 2 42 2 42 2 42 2 42

ment. As the tadpoles of A. cisternasii approach metamorphosis, they tend to spend more time near the surface and margins of the aquaria (GONĂ&#x2021;ALVES et al., 2007), therefore reducing their presence at the substratum, where refuges were located.

According to our results, A. cisternasii tadpoles 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 compensate 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 ascribe this response as an antipredator behaviour adopted towards this alien species or simply as a reaction to a new, unknown cue. To clarify the meaning of this finding, future expe-

1.76

P

Sum of squares

0.18

6.53 264.27 0.31 14.93 1 3 88.31 378.93 98.8 449.2 21.38 363.6

4.03 <0.05 3.81 <0.05 2.27

0.11

2.21

0.12

4.31 <0.05

Noctural trials d.f.

F

2 42 2 42 2 42 2 42 2 42 2 42

P

0.52

0.60

0.44

0.65

0.69

0.51

4.89

<0.05

4.62

<0.05

1.23

0.30

riments will have to assess the effects of chemical 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-

GONÇALVES ET AL.

62

Figure 3: Recorded behaviours of tadpole (a) use of the substratum and (b) use of the surface during the nocturnal 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

a

nary time (about 10-15 generations, given the average lifespan of A. cisternasii (GARCÍA-PARÍS et al., 2004)). So, we may be witnessing the result from a learning process, which we cannot exclude, since it is not feasible to collect tadpoles of A. cisternasii before hatching and the individuals used in this experiment were thus all collected as tadpoles, with an unknown history of previous contact with P. clarkii. Using a completely different approach, a similar resistance of A. cisternasii to the effects of P. clarkii was also suggested by CRUZ et al. (2006).

Finally, it would be of interest to study the behavioural responses of tadpoles of A. cisternasii that have never been in contact with P. clarkii when faced with this species.

Acknowledgement This work was partially funded by project POCI/BIA-BDE/56100/2004 of Fundação para a Ciência e a Tecnologia (Portugal).

b

REFERENCES ALMAÇA, C. (1991). L’ecrevisse a pieds blancs, Astacus pallipes Lereboullet 1858, au Portugal.

L'Astaciculteur de France 28: 11-16.

ALTWEGG, R. (2002). Predator-induced lifehistory plasticity under time constraints in pool frogs. Ecology 83: 2542-2551.

AMARAL, S. (2004). Respostas comportamentais de girinos de Alytes cisternasii a estímulos químicos de dois predadores. Internship, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal.

BLANCO-GARRIDO, F.; SÁNCHEZ-POLAINA, F.J. & PRENDA, J. (2003). Summer diet of the Iberian chub (Squalius pyrenaicus) in a Mediterranean stream in Sierra Morena (Yeguas stream, Córdoba, Spain).

Limnetica 22: 99-106.

CRUZ, M.J. & REBELO, R. (2005). Vulnerability of Southwest Iberian amphibians to an introduced crayfish, Procambarus clarkii.

Amphibia-Reptilia 26: 293-303.

ANTIPREDATOR BEHAVIOUR OF A. CISTERNASII TADPOLES

CRUZ, M.J.; REBELO, R. & CRESPO, E.G. (2006). Effects of an introduced crayfish, Procambarus clarkii, on the distribution of south-western Iberian amphibians in their breeding habitats.

Ecography 29: 329-338.

GAMRADT, S.C. & KATS, L.B. (1996). Effects of introduced crayfish and mosquitofish on California newts. Conservation Biology 10: 1155-1162.

GARCÍA-PARÍS, M.; MONTORI, A. & HERRERO, P. (2004). Amphibia. Lissamphibia. Series: Fauna Ibérica, vol. 24 (M.A. Ramos, ed.).

Museo Nacional de Ciencias Naturales, Madrid, Spain.

GONÇALVES, V.; AMARAL, S. & REBELO, R. (2007). Iberian midwife toad (Alytes cisternasii) tadpoles show behavioural modifications when faced with a recently introduced predator, Procambarus clarkii. Munibe Suplemento-Gehigarria 25: 180-188.

GONZALO, A.; LÓPEZ, P. & MARTÍN, J. (2008).

Avoidance responses to scents of snakes that pose different risks of predation by adult natterjack toads, Bufo calamita.

Canadian Journal of Zoology 86: 928-932.

GOSNER, K.L. (1960). A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: 183-190.

HAILEY, A. & DAVIES, P.M.C. (1986). Diet and foraging behaviour of Natrix maura.

Herpetological Journal 1: 53-61.

HARPER, D.M.; SMART, A.C.; COLEY, S.; SCHMITZ, S.; GOUDER DE BEAUREGARD, A.C.; NORTH, R.; ADAMS, C.; OBADE, P. & KAMAU, M. (2002). Distribution and abundance of the Louisiana red swamp crayfish Procambarus clarkii Girard at Lake Naivasha, Kenya between 1987 and 1999. Hydrobiologia 488: 143-151.

63

HETTYEY, A.; ZSARNÓCZAI, S.; VINCZE, K.; HOI, H. & LAURILA, A. (2010). Interactions between the information content of different chemical cues affect induced defences in tadpoles. Oikos 119: 1814-1822.

KATS, L.B. & DILL, L.M. (1998). The scent of death: Chemosensory assessment of predation risk by prey animals. Ecoscience 5: 361-394.

KATS, L.B.; PETRANKA, J.W. & SIH, A. (1988).

Antipredator defenses and the persistence of amphibian larvae with fishes. Ecology 69: 1865-1870.

KIESECKER, J.M. & BLAUSTEIN, A.R. (1997).

Population differences in responses of redlegged frogs (Rana aurora) to introduced bullfrogs. Ecology 78: 1752-1760.

KIESECKER, J.M.; CHIVERS, D.P. & BLAUSTEIN, A.R. (1996). The use of chemical cues in predator recognition by western toad tadpoles. Animal Behaviour 52: 1237-1245.

LARDNER, B. (2000). Morphological and life history responses to predators in larvae of seven anurans. Oikos 88: 169-180.

MÁRQUEZ, R. (1992). Terrestrial paternal care and short breeding seasons: reproductive phenology of the midwife toads Alytes obstetricans and A. cisternasii.

Ecography 15: 279-288.

MOORE, R.D.; GRIFFITHS, R.A.; O’BRIEN, C.M.; MURPHY, A. & JAY, D. (2004). Induced defences in an endangered amphibian in response to an introduced snake predator.

Oecologia 141: 139-147.

NYSTRÖM, P.; SVENSSON, O.; LARDNER, B.; BRÖNMARK, C. & GRANÉLI, W. (2001).

The influence of multiple introduced predators on a littoral pond community.

Ecology 82: 1023-1039.

POLO-CAVIA, N.; GONZALO, A.; LÓPEZ, P. & MARTÍN, J. (2010). Predator recognition

64

GONĂ&#x2021;ALVES ET AL.

of native but not invasive turtle predators by naĂŻve anuran tadpoles. Animal Behaviour 80: 461-466.

RELYEA, R.A. (2001). The relationship between predation risk and antipredator responses in larval anurans. Ecology 82: 541-554.

RELYEA, R.A. (2004). Fine-tuned phenotypes: tadpole plasticity under 16 combinations of predators and competitors.

Ecology 85: 172-179.

RICHTER-BOIX, A.; LLORENTE, G.A. & MONTORI, A. (2007). A comparative study of predator-induced phenotype in tadpoles across a pond permanency gradient.

Hydrobiologia 583: 43-56.

SCHLAEPFER, M.A.; SHERMAN, P.W.; BLOSSEY, B. & RUNGE, M.C. (2005). Introduced species as evolutionary traps. Ecology Letters 8: 241-246.

SCHLEY, L. & GRIFFITHS, R.A. (1998). Midwife toads (Alytes muletensis) avoid chemical cues from snakes (Natrix maura). Journal of Herpetology 32: 572-574.

SIH, A. & KATS, L.B. (1994). Age, experience, and the response of streamside salamander hatchlings to chemical cues from predatory sunfish. Ethology 96: 253-259.

SKELLY, D.K. & WERNER, E.E. (1990).

Behavioral and life-historical responses of larval American toads to an odonate predator. Ecology 71: 2313-2322.

STRAUSS, S.Y.; LAU, J.A. & CARROLL, S.P. (2006). Evolutionary responses of natives to introduced species: what do introductions tell us about natural communities?

Ecology Letters 9: 357-374.

Basic and Applied Herpetology 25 (2011): 65-71

Reproductive cycles in Bufo mauritanicus (Schlegel, 1841) in a wet area of Beni-Belaïd (Jijel, Algeria) Omar Kisserli1, Salaheddine Doumandji2, Jean-Marie Exbrayat3,* Laboratoire de Pharmacologie et Phytochimie, Faculté des Sciences, Université de Jijel, Ouled Aissa, Jijel, Algeria.

Laboratoire de Zoologie Agricole et Forestière, Institut National Agronomique. El - Harrach, Alger, Algeria.

3 Université de Lyon, Laboratoire de Biologie Générale, Université Catholique de Lyon et Reproduction et Développement Comparé, Ecole Pratique des Hautes Etudes, Lyon, France.

1 2

* Correspondence: Université de Lyon. UMRS 449. Biologie Générale, Université Catholique de Lyon, Reproduction et Développement Comparé, 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 reproductive cycle in a population living in the wet area of Beni Belaïd, under a Mediterranean climate characterized 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-Belaïd (Jijel, Algeria).

Bufo mauritanicus es un anuro que habita en el Norte de África. Los ciclos reproductivos de esta especie han sido poco investigados, especialmente en lo que se refiere a las poblaciones de Argelia. Este estudio profundiza en el conocimiento del ciclo reproductor de una población de B. mauritanicus en la zona húmeda de Beni Belaïd, en Argelia, caracterizada por un clima mediterráneo con dos estaciones lluviosas, de enero a mayo y de septiembre a diciembre. El estudio de las gónadas 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; reproducción.

Reproductive cycles of amphibians have 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 species are rather little known. For example, some studies detected continuous annual cycles in several species (Bufo regularis, Ptychadena maccarthyensis, P. oxyrhynchus, Phrynobatrachus calcaratus, Xenopus laevis; DELSOL et al., 1980, 1981, 1995; GUEYDAN-BACONNIER, 1980;

GUEYDAN-BACONNIER et al., 1984a,b; PUJOL, 1985; PUJOL & EXBRAYAT, 1996, 2000, 2001; EXBRAYAT et al., 1998; DU PREEZ et al., 2005; VAN WYK et al., 2005). On the other side, discontinuous male and female cycles of reproduction have also been described in the viviparous Nectophrynoides occidentalis and other related species (LAMOTTE & TUCHMANDUPLESSIS, 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

KISSERLI ET AL.

66

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 reproductive cycles is still lacking. The aim of the present work was to analyse in detail the reproductive cycle and variations in the gonadal tissues of both male and female B. mauritanicus along the year and its relationships to environmental factors, namely precipitation. The analysis of hormones present during folliculogenesis was also investigated in order to understand hormonal regulation of female reproductive features.

MATERIALS AND METHODS The studied animals were randomly captured in the wet area of Beni-Belaid (Jijel, Algeria) throughout the year. This area is limited by the Mediterranean Sea in its northern part, by an agricultural area in its southern 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 collected across the year as follows: three males and 11 females during the wet season between January and May; four males and eight females 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 μm thick sections using a micrometer and stained with the Romeis’s azan according to Exbrayat (2001).

Histochemical stainings were used to characterize the chemical composition of tissues.

For that, frozen sections of gonads (14 μm 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 presence of neutral and acidic carbohydrates according to MARTOJA & MARTOJA (1967).

The detection of 17-β estradiol in ovaries was performed using immunohistochemical techniques. For that, sections were first incubated with H2O2 (Fluka, Buchs, Switzerland) in order to eliminate endogenous peroxidases, and then with bovine serum albumin (Sigma, St. Louis, Missouri, USA) to eliminate non-specific reactions. After this, a spe-

67

BUFO MAURITANICUS REPRODUCTIVE CYCLE

cific antibody directed against 17-β estradiol (Euromedex, Souffelweyersheim, France) was applied on the sections. After rinsing, immunoreactions were visualized with a streptavidin-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 deleting the first antibody. The observation of adjacent tissues was used as internal controls.

DISCUSSION

RESULTS

The histological study of both male and female reproductive organs of B. mauritanicus showed that spermatogenesis, oogenesis and folliculogenesis were continuous, and comparable to the ones observed in most African anurans. Like other amphibians, steroid 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 (stages Ia, Ib, II, III, IVa, IVb, V). Stages IVa an IVb were the vitellogenic stages at which the oocyte was increasingly filled with yolk platelets (Figs. 2a,b). Stage V was the final stage of maturation at which ovulation can occur. In addition, several atretic follicles were observed. 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 previtellogenic and vitellogenic oocytes (Fig. 2d).

The immunohistochemical detection of 17-β estradiol revealed the presence of this hormone in the follicular cells and connective theca in both previtellogenic and vitellogenic stages (Figs. 2e,f ).

Figure 1: Cross sections of Bufo mauritanicus testis. (a) All the spermatogenetic categories are present.

FS: Spermatozoa fascia, LTS: Lumen of the seminiferous tube, SP: Spermatid. Bar = 50 μm. (b) Detection of lipids on frozen sections stained with Sudan black. CL: Leydig-like cells, FS: Spermatozoa fascia. Bar = 50 μm.

68

KISSERLI ET AL.

Figure 2: Cross sections of Bufo mauritanicus ovary. (a) Cross section of B. mauritanicus ovary showing previtellogeic 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 vitellogenesis, 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 frozen sections stained with black Sudan. L: Lipids, VO: Vitellogenic oocyte. (e) Immunohistochemical detection of 17β-estradiol in follicles, during previtellogenesis. St. II: stage II follicle. The arrow indicates the presence of labelled follicle cells. (f) Immunohistochemical detection of 17β-estradiol in follicles during vitellogenesis. The arrow indicates the presence of labelled follicle cells. Bar = 50 μm.

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BUFO MAURITANICUS REPRODUCTIVE CYCLE

both males and females (DUMONT, 1972; DELSOL et al., 1980, 1981, 1995; GUEYDANBACONNIER, 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 continuous 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 discontinuous in males (GAVAUD, 1976, 1977) as well as in females (LAMOTTE & TUCHMANDUPLESSIS, 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 including 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 linked to rainfalls coupled with a brutal decrease 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 development. Reproduction of B. mauritanicus depends on local climate (SCHLEICH et al., 1996) and breeding is observed when rain falls down. When precipitations are insufficient, reproduction does not occur during several successive years (SCHLEICH et al.,

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 releasing 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.

REFERENCES BONS, J. & GENIEZ, P. (1996). Amphibiens et Reptiles du Maroc (Sahara Occidental Compris). Atlas Biogéographique. Asociación Herpetológica Española, Barcelona.

DELSOL, M.; GUEYDAN-BACONNIER, M.; NEYRAND DE LEFFEMBERG, F. & PUJOL, P. (1980). Cycle spermatogénétique continu chez des Batraciens tropicaux. Bulletin de la Société Zoologique de France 105: 232-233.

DELSOL, M.; FLATIN, J.; GUEYDAN-BACONNIER, M.; NEYRAND DE LEFFEMBERG, F. & PUJOL, P. (1981). Action des facteurs externes sur les cycles de reproduction chez les Batraciens. Bulletin de la Société Zoologique de France 106: 419-431.

DELSOL, M.; BLOND-FAYOLLE, C. & FLATIN, J. (1995). Appareil génital mâle, anatomie, histologie, déterminisme du cycle sexuel, In P.P. Grassé & M. Delsol (eds.) Traité de Zoologie tome XIV, fasc. 1-A, Masson, Paris, pp. 1187-1229.

DUMONT, J.N. (1972). Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.

Journal of Morphology 136: 153-180.

DU PREEZ, L.H.; EVERSON, G.J.; HECKER, M.; CARR, J.A.; GIESY, J.P.; KENDALL, R.J.; SMITH, E.E.; VAN DER KRAAK, G. &

70

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SALOMON, K.R. (2005). Seasonal changes in testicular morphology in the african clawed frog, Xenopus laevis: a histometric analysis. 5th World Congress of Herpetology, Stellenbosch, South Africa, Abstracts: 42.

EXBRAYAT, J.-M. (2001). Genome Visualization

by Classic Methods in Light Microscopy.

CRC Press, Boca Raton, London, New York, Washington, D.C. EXBRAYAT, J.-M.; PUJOL, P. & LECLERCQ, B. (1998). Quelques aspects des cycles sexuels et nycthéméraux chez les amphibiens. Bulletin de la Société Zoologique de France 12: 113-124.

FERNANDEZ, S.N. & RAMOS, I. (2003).

Endocrinology of reproduction, In B.G.M. Jamieson (ed.) Reproductive Biology and Phylogeny of Anura. Science Publishers, Inc., Enfield, pp. 73-117.

GAVAUD, J. (1976). La gamétogenèse du mâle de Nectophrynoides occidentalis Angel (Amphibien Anoure vivipare). I. Etude quantitative au cours du cycle annuel chez l’adulte. Annales de Biologie Animale, Biochimie et Biophysique 16: 1-12.

GAVAUD, J. (1977). La gamétogenèse du mâle de Nectophrynoides occidentalis Angel (Amphibien Anoure vivipare). II. Etude expérimentale du rôle des facteurs externes sur la spermatogenèse de l’adulte, au cours du cycle annuel. Annales de Biologie Animale, Biochimie et Biophysique 17: 679-694.

GUEYDAN-BACONNIER, M. (1980). Le cycle sexuel chez les mâles et les femelles de Phrynobatrachus calcaratus (Peters, 1863) Batracien Anoure tropical. Ph.D. Dissertation, University of Paris VI, Paris.

GUEYDAN-BACONNIER, M.; NEYRAND DE LEFFEMBERG, F. & PUJOL, P. (1984a).

Comparaison de la vitesse spermatogéné-

tique entre trois batraciens tropicaux.

Bulletin de la Société Herpétologique de France 29: 69-70.

GUEYDAN-BACONNIER, M.; NEYRAND DE LEFFEMBERG, F.; PUJOL, P.; DELSOL, M. & FLATIN, J. (1984b). Etude comparative du dynamisme de la spermatogenèse chez trois Batraciens tropicaux par autoradiographie. Annales de Sciences Naturelles Zoologie, 13ème série 6: 191-196.

GUILLON, M.; LE LIARD, G. & SLIMANI, T. (2004). Nouvelles données sur la répartition et l’écologie de la reproduction de Bufo brongersmai, B. viridis et B. mauritanicus (Anura, Bufonidae) dans les Jbilets centrales (Maroc). Bulletin de la Société Herpétologique de France 111: 37-48.

HAUSEN, P. & RIEBESELL, M. (1991). The Early Development of Xenopus laevis. An Atlas of the Histology. Springer-Verlag, New York.

KISSERLI, O. & EXBRAYAT, J.M. (2006).

Premières données sur le cycle de reproduction des mâles de Bufo mauritanicus (Schlegel, 1841) dans la zone humide de Beni-Belaid (Jijel, Algérie). Bulletin de la Société Herpétologique de France 120: 5-13.

LAMOTTE, M. & TUCHMANN-DUPLESSIS, H. (1948). Structure et transformations gravidiques du tractus génital femelle chez un Anoure vivipare (Nectophrynoides occidentalis Angel). Comptes Rendus de l’Académie des Sciences de Paris 226: 597-599.

LAMOTTE, M.; REY, P. & VOGELI, M. (1964).

Recherches sur l’ovaire de Nectophrynoides occidentalis, Batracien anoure vivipare.

Archives d’Anatomie, de Microscopie et de Morphologie 5: 315-340.

MARTOJA, R. & MARTOJA, M. (1967).

Initiation aux Techniques de l’Histologie Animale. Masson, Paris.

BUFO MAURITANICUS REPRODUCTIVE CYCLE

MEASEY, G.J., SMITA, M., BEYO, R.S. & OOMMEN, O.V. (2008). Year-round spermatogenic activity in an oviparous subterranean caecilian, Boulengerula taitanus Loveridge 1935 (Amphibia Gymnophiona Caeciliidae). Tropical Zoology 21: 109-122.

NEYRAND DE LEFFEMBERG, F. & EXBRAYAT, J.M. (1995). Etude comparative du dynamisme de la spermatogenèse chez les Amphibiens par la méthode histoautoradiographique à la thymidine tritiée. Bulletin de la Société Linnéenne de Lyon 64: 356-372.

PUJOL, P. (1985). Quelques Aspects de la Reproduction du Crapaud Bufo regularis Reuss, 1834. Diplôme de l’E.P.H.E., Lyon.

PUJOL, P. & EXBRAYAT, J.M. (1996). Variations du tissu interstitiel du testicule et de l’hypophyse chez Bufo regularis mâle au cours du cycle sexuel. Bulletin de la Société Herpétologique de France 80: 27-37.

PUJOL, P. & EXBRAYAT, J.M. (2000). Mise en évidence de l’homogénéité des testicules multilobés de deux amphibiens par des méthodes morphométriques. Bulletin de la Société Herpétologique de France 95: 43-56.

71

PUJOL, P. & EXBRAYAT, J.M. (2001).

Quelques aspects de la biologie de la reproduction et des cycles sexuels chez Bufo regularis Reuss (1834), amphibien anoure. Bulletin Mensuel de la Société Linnéenne de Lyon 71: 12-52.

SANCHEZ, S.S. & VILLECCO, F.I. (2003).

Oogenesis. In B.G.M Jamieson (ed.)

Reproductive Biology and phylogeny of Anura.

Science Publishers, Inc, Enfield, pp. 27-71.

SCHLEICH, H., KÄSTLE, W. & KABISHI, K. (1996). Amphibians and reptiles of North Africa, Koltz Scientific Book, Keonigstein, Germany.

VAN WYK, J.H.; HURTER, E.; POOL.; E.J. & LESLIE, A.J. (2005). Seasonal variation in reproductive activity in natural Xenopus laevis populations in the Western Cape Province, South Africa. 5th World Congress of Herpetology, Stellenbosch, South Africa.

Abstracts: 103.

XAVIER, F. (1986). La reproduction des Nectophrynoides, In P.P. Grassé & M. Delsol (eds.) Traité de Zoologie tome XIV, fasc. 1-B, Masson, Paris, pp. 497-513.

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Basic and Applied Herpetology 25 (2011): 73-80

Age structure of Levant water frog, Pelophylax bedriagae, in Lake Sülüklü (Western Anatolia, Turkey) Kerim Çiçek*, Meltem Kumaş, Dinçer Ayaz, Ahmet Mermer, Ş. 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 Sülüklü (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 population 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 Sülüklü population were outlined.

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

Estructura de edad de la rana verde levantina, Pelophylax bedriagae, en el lago Sülüklü (Anatolia occidental, Turquía).

Durante el presente estudio obtuvimos datos, mediante un análisis esqueletocronológico, sobre la estructura de edades de la rana verde levantina, Pelophylax bedriagae, en el lago Sülüklü (Manisa, Turquía). 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 alcanzó tras la segunda hibernación, la edad modal fue dos años para los machos y tres años para las hembras.

La edad media en la población adulta fue 2,50 años (SD = 0,65, rango = 2-4) en los machos y 2,95 años (SD = 0,99, rango = 2-5) en las hembras. Por último, se indicaron algunas de las amenazas que afectan a la población estudiada.

Key words: occidental; edad; esqueletocronología; Pelophylax bedriagae; Turquía.

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 habitat 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 populations is essential in understanding the population dynamics and breeding of species.

Regarding amphibians and reptiles, skeletochronology is a widely used method for age estimation (CASTANET & SMIRINA, 1990;

ÇIÇEK ET AL.

74

CASTANET et al., 1993; SMIRINA, 1994).

Skeletochronology is based on the observation 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, skeletochronology is a non-destructive technique 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 skeletochronological study of phalanges, the age structure of P. bedriagae population inhabiting Lake Sülüklü in order to retrieve information about growth, size at maturity and longevity.

MATERIALS AND METHODS The study site, Lake Sülüklü, is located on the northeastern slope of Mount Spil (38.565035º N, 27.532617º 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 amphibian species other than P. bedriagae inhabit the lake: Pseudopidalea viridis, Lissotriton vulgaris 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 season, from Lake Sülüklü. The sex of all individuals was determined through the observation 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 sectioned 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 performed 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 samples 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 Ehrlich’s hematoxylin for 20 minutes. LAGs were counted by two observers who were blind to the identification of the individuals. All mount preparations were photographed with an Olympus LC20 Soft Imaging System (Olympus, Münster, 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 diameters of medullary cavity (MC), metamorphosis line (ML), resorption line (RL), each visible LAG, and periosteal outer margin were measured in each individual in order to estimate 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 & KYRIAKOUPOLOUSKLAVOUNOU, 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

TSIORA & KYRIAKOPOULOU-SKLAVOUNOU, 2002; YILMAZ et al., 2005; GUARINO et al., 2008; GÜL et al., 2011). Besides, the sexual maturity was confirmed when possible through the observation of the sexual secondary characters mentioned above. Longevity was determined as the age of the oldest individual of each sex in the population.

SVL and age structure were compared between sexes with a t-test and a Mann Whitney’s U-test, respectively. Growth was estimated by the equation of von BERTALANFFY (1983), which has been previously used in several studies on amphibians (ARNTZEN, 2000; MIAUD et al., 2001; COĞALNICEANU & MIAUD, 2003, SARASOLAPUENTE 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 metamorphosis), and K the growth rate coefficient. Units are given in growth increase per

75

year. SVLmax, K, tmet and their standard errors were estimated through the nonlinear leastsquare 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 individuals (Fig. 1a), barely perceptible in another nine, and different in thickness in the remaining 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 examined individuals.

The average age was 2.50 years (SD = 0.65; range 2-4) in males and 2.95 years (SD = 0.99;

Table 1: Snout-vent length mean values and ranges related to each sex and age, as estimated by skeletochronology, in Pelophylax bedriagae from Lake Sülüklü.

Age (years) N

Juveniles Mean (SD) Range

1 2 3 4 5

3 1

38.6 (1.3) 37.0-39.3 53.9

Total

4

42.4 (7.8) 37.0-53.9

N

Males Mean (SD) Range

N

Females Mean (SD) Range

8 5 1

52.6 (3.2) 50.4-60.0 58.2 (8.4) 50.7-72.2 73.2

8 10 1 3

59.8 (11.4) 45.8-83.1 64.0 (14.3) 52.2-86.2 64.2 78.6 (22.0) 53.2-92.4

14

56.1 (7.7) 50.4-73.2

22

64.5 (14.6) 45.8-92.4

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ÇIÇEK ET AL.

Figure 1: Phalangeal cross-sections of Pelophylax bedriagae from Lake Sülüklü (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 yearold 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 μm.

range 2-5) in females (Table 1). No statistical 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 metamorphosed individuals at the end of June from Lake Sülüklü (Fig. 1d). According to the von Bertalanffy equation, the SVLmax was calculated as 90.22 (SE = 31.89), K as 0.30 (SE = 0.33) and tmet as 0.03 (SE = 0.87) (Fig. 2).

DISCUSSION This study revealed that the estimated average age and longevity of P. bedriagae from Lake Sülüklü 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

SOCHA & OGIELSKA, 2010, GÜL et al., 2011). The first LAG of Lake Sülüklü population 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 starvation period, an exposure to cold temperatures during the activity period (SMIRINA et al., 1986) or other environmental factors (SOCHA & OGIELSKA, 2010).

Table 2 shows some age and growth parameters of other Pelophylax species. In this genus the maximum longevity reported was 12 years in Caucasian populations (namely for P. esculentus and P. lessonae) (SHALDYBIN, 1976; ALEXANDROVSKAYA & KOTOVA, 1986), while in European populations it was 11 years. Generally, longevity tends to increase in northern or mountain populations and it tends to decrease in southern or lowland populations (SMIRINA, 1994). In Lake

77

Figure 2: Growth curve of Pelopghylax bedriagae from Lake Sülüklü. SVLt= 90.2 - (90.2 - 26.0) e-0.30(t - 0.03).

Sülüklü, the maximum longevity was higher in females than in males, as previously reported by many other researchers working with green frogs (TSIORA & KYRIAKOPOULOUSKLAVOUNOU, 2002; ERIŞMIŞ, 2005; KYRIAKOPOULOU-SKLAVOUNOU et al., 2008; SOCHA & OGIELSKA, 2010; GÜL et al., 2011). Nevertheless, we must consider that sampling at Lake Sülüklü 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 P. ridibundus

Anatolia Russia

2-10 4-11

Georgia Anatolia Anatolia Anatolia Greece P. esculentus complex Romania Poland P. epeirotica Greece Anatolia P. bedriagae

2-7 1-7 2-8 4-11 1-5 4-10 2-7 1-5 2-5

ERIŞMIŞ (2005) SHALDYBIN, 1976; ALEXANDROVSKAYA & KOTOVA, 1986; SMIRINA, 1994 GOKHELASHVILI & TARKHNISHIVILI (1994) YILMAZ et al. (2005) GÜL et al. (2011) GÜL et al. (2011) KYRIAKOPOULOU-SKLAVOUNOU et al. (2008) COGĂLNICEANU & MIAUD (2003) SOCHA & OGIELSKA (2010) TSIORA & KYRIAKOPOULOU-SKLAVOUNOU (2002) Present study

2-3

2.78-4.03 3.90-3.72

2.96-3.73 5.0-6.7 4.1 2.82-3.22 2.50-2.95

2 2-4 2 3-4 1 4 2-3 1 2

0.22-0.28 0.76-0.59 0.88-0.49 0.30

ÇIÇEK ET AL.

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a single day and at the end of the breeding season, and older individuals might have already 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 Sülüklü (0.30) is lower than that of most European Pelophylax species (0.22-0.88; Table 2), which could be related to habitat quality, environmental conditions, and predator pressure. Lake Sülüklü 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 pressure 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 Sülüklü 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 longevity, survival and growth rate in comparison with other Pelophylax populations could be caused by impoverished habitat quality and environmental conditions, as well as by predator pressure.

Acknowledgement We thank Derek H. Ogle for your help in using R version 2.12.2 and Ivelin Mollow for reviewing English style.

REFERENCES ALEKSANDROVSKAYA, T.O. & KOTOVA, E.L. (1986). Preliminary data on age characteristics of Rana ridibunda Pallas from Armenia.

Proceedings of the Zoological Institute of the USSR Academy of Sciences 157: 177-181.

AMPHIBIAWEB (2011). AmphibiaWeb: Information on Amphibian Biology and Conservation.

University of California, Berkeley, California, USA. Available at http://www.amphibiaweb.org/. Retrieved on 05/30/2011.

ARNTZEN, J.W. (2000). A growth curve for the newt Triturus cristatus. Journal of Herpetology 34: 227-232.

BARAN, İ.; YILMAZ, İ., KUMLUTAŞ, Y. & KETE, R. (1992). Türkiye ova kurbağası (Rana ridibunda) stok tespiti (Anura, Ranidae).

Turkish Journal of Zoology 16: 289-299.

BEERLI, P. (1995). Rana bedriagae Camerano 1882. Version 20 December 1995, In D.R. Madison & K.-S. Schulz (eds.) The Tree of Life Web Project. The University of Arizona College of Agriculture and Life Sciences and The University of Arizona Library, Tucson, Arizona, USA. Available at http://tolweb.org/.

Retrieved on 05/30/2011.

CASTANET, J. & SMIRINA, E. (1990).

Introduction to the skeletochronological method in amphibians and reptiles. Annales des Sciences Naturelles, Zoologie et Biologie Animale, Séries 13 11: 191-196.

CASTANET, J.; FRANCILLON-VIEILLOT, H.; MEUNIER, J.F. & DE RICQLÉS, A. (1993).

Bone and individual aging, In B.K. Hall (ed.)

Bone, Volume 7: Bone Growth−B. CRC Press, Boca Raton, Florida, USA, pp. 245-283.

CASTANET, J.; MEUNIER, F. & DE RICQLÉS, A. (1977). L’enregistrement de la croissance cyclique par le tissue osseux chez

AGE OF PELOPHYLAX BEDRIAGAE FROM WESTERN ANATOLIA

les vertébrés poikilothermes: données comparatives et essai de synthèse.

Bulletin Biologique de la France et de la Belgique 111: 183-202.

COĞALNICEANU, D. & MIAUD, C. (2003).

Population age structure and growth in four syntopic amphibian species inhabiting a large river floodplain. Canadian Journal of Zoology 81: 1096-1106.

ERIŞMIŞ, U.C. (2005). Göller bölgesi Rana ridibunda (Anura: Ranidae) populasyonlarında yaş - boy, yaş – ağırlık ve boy-ağırlık ilişkilerinin araştırılması. Ph.D. dissertation, Ege University, Bornova-Izmir, Turkey.

FROST, D.R. (2011). Amphibian Species of the World 5.5, an Online Reference. American Museum of Natural History, New York, USA. Available at http://research.amnh. org/vz/herpetology/amphibia/. Retrieved on 05/30/2011.

GOKHELASHVILI, R.K. & TARKHNISHVILI D.N. (1994). Age structure of six Georgian anuran populations and its dynamics during two consecutive years (Anura). Herpetozoa 7: 11-18.

GUARINO, F.M., DI GIÀ, I. & SINDACO, R. (2008). Age structure in a declining population of Rana temporaria from northern Italy. Acta Zoologica Academiae Scientiarum Hungaricae 54: 99-112.

GÜL, S.; ÖZDEMIR, N.; ÜZÜM, N.; OLGUN, K. & KUTRUP, B. (2011). Body size and age structure of Pelophylax ridibundus populations from two different altitudes in Turkey.

Amphibia-Reptilia 32: 287-292.

HEMELAAR, A.S.M. & VAN GELDER, J.J. (1980).

Annual growth rings in phalanges of Bufo bufo (Anura, Amphibia) from the Netherlands and their use for age determination. Netherlands Journal of Zoology 30: 129-135.

79

HEMELAAR, A.S.M. (1985). An improved method to estimate the number of annular rings resorbed in phalanges of Bufo bufo (L.) and its application to populations from different latitudes and altitudes. Amphibia-Reptilia 6: 323-343.

HEMELAAR, A. (1988). Age, growth and other population characteristics of Bufo bufo from different latitudes and altitudes.

Journal of Herpetology 22: 369-388.

KLEINENBERG, S.E. & SMIRINA, E.M. (1969).

[A contribution to the method of age determination in amphibians]. Zoologicheskii Zhurnal 48: 1090-1094 (in Russian).

KYRIAKOPOULOU-SKLAVOUNOU, P.; STYLIANOU, P. & TSIORA, A. (2008). A skeletochronological study of age, growth and longevity in a population of the frog Rana ridibunda from southern Europe. Zoology 111: 30-36.

MIAUD, C.; ANDREONE, F.; RIBÉRON, A.; DE MICHELIS, S.; CLIMA, V.; CASTANET, J.; FRANCILLON-VIEILLOT, H. & GUYÉTANT, R. (2001). Variations in age, size at maturity and gestation duration among two neighbouring populations of the Alpine salamander (Salamandra lanzai). Journal of Zoology 254: 251-260.

PAPENFUSS, T.; KUZMIN, S.; DISI, A.M.M.; DEGANI, G.; UGURTAS, I.H.; SPARREBOOM, M.; ANDERSON, S.; SADEK, R.; HRAOUIBLOQUET, S.; GASITH, A.; ELRON, E.; GAFNY, S.; LYMBERAKIS, P.; BÖHME, W. & EL DIN, S.B. (2008). Pelophylax bedriagae, In IUCN (2010) The IUCN Red List of Threatened Species, v.

2010.4.

International Union for Nature Conservation and Natural Resources, Gland, Switzerland. Available at http://www.iucnredlist.org/. Retrieved on 05/30/2011.

80

ÇIÇEK ET AL.

R DEVELOPMENT CORE TEAM (2011). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Avaliable at http://www.r-project.org/. Retrieved on 05/30/2011.

ROZENBLUT, B. & OGIELSKA, M. (2005).

Development and growth of long bones in European water frogs (Amphibia: Anura: Ranidae), with remarks on age determination. Journal of Morphology 265: 304-317.

SARASOLA-PUENTE, V.; GOSÁ, A.; OROMÍ, N.; MADEIRA, M.J. & LIZANA, M. (2011).

Growth, size and age at maturity of the agile frog (Rana dalmatina) in an Iberian Peninsula population. Zoology 114: 150-154.

SHALDYBIN, S.L. (1976). [Age and sex structure of populations of anurans]. Prirodnye Resursy Volzhsko-Kamskogo Kraya 4: 112117 (in Russian).

SMIRINA, E.M. (1972). [Annual layers in bones of Rana temporaria]. Zoologicheskii Zhurnal 51: 1529-1534 (in Russian).

SMIRINA, E.M. (1994). Age determination and longevity in amphibians. Gerontology 40: 133-146.

SMIRINA, E.M.; KLEVEZAL, G.A. & BERGER, L. (1986). [Experimental investigation of the annual layer formation in bones of amphibians]. Zoologicheskii Zhurnal 65: 1526-1534 (in Russian).

SOCHA, M. & OGIELSKA, M. (2010). Age structure, size and growth rate of water frogs from central European natural Pelophylax ridibundus-Pelophylax esculentus mixed populations estimated by skeletochronology. Amphibia-Reptilia 31: 239-250.

TSIORA, A. & KYRIAKOPOULOUSKLAVOUNOU, P. (2002). A skeletochronological study of age and growth in relation to adult size in the water frog Rana epeirotica. Zoology 105: 55-60.

VON BERTALANFFY, L. (1983). A quantitative theory of organic growth (Inquiries on growth law. II). Human Biology 10: 181-213.

YILMAZ, N.; KUTRUP, B.; ÇOBANOĞLU, Ü. & ÖZORAN, Y. (2005). Age determination and some growth parameters of a Rana ridibunda population in Turkey. Acta Zoologica Academiae Scientiarum Hungaricae 51: 67-74.

Basic and Applied Herpetology 25 (2011): 81-96

Population size, habitat use and movement patterns during the breeding season in a population of Perez’s frog (Pelophylax perezi) in central Spain Gregorio Sánchez-Montes1, Iñigo Martínez-Solano2,* 1 2

Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain.

Instituto de Investigación en Recursos Cinegéticos, CSIC-UCLM-JCCM, Ciudad Real, Spain.

* Correspondence: Instituto de Investigación en Recursos Cinegéticos (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 common species thrive in heavily transformed habitats. A capture-mark-recapture study was performed on a population of Perez’s 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 polygons 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.

Tamaño poblacional, uso del espacio y patrones de movimiento durante el periodo reproductor en una población de rana verde común (Pelophylax perezi) en España central. Los estudios sobre demografía y uso del espacio resultan esenciales para la gestión 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 fuertemente alterados. En este trabajo presentamos resultados de un estudio de captura-marcaje-recaptura en una población de rana verde común (Pelophylax perezi) en España central. El área de estudio alberga tres puntos de reproducción, separados por distancias máximas de 700 metros en línea recta. El objetivo del estudio fue estimar parámetros demográficos y analizar patrones de uso del espacio y movilidad durante la temporada de reproducción en 2010. En uno de los puntos (Laguna de Valdemanco) se estimó una población de 173 ranas: 91 machos (intervalo de confianza al 95%: 51-130) y 82 hembras (IC 95%: 21-144). En otro punto de reproducción estimamos una población adulta de 62 machos (IC 95%: 42-83) y 17 hembras (IC 95%: 10-24). Se estimaron las áreas de actividad de 21 ejemplares en base al cálculo de los polígonos de área mínima 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, desviación estándar: 41 m, distancia máxima: 168 m), excepto un macho que se desplazó entre dos puntos de reproducción distantes entre sí 273 m. Estos resultados preliminares son aplicables al diseño de estrategias de gestión para ésta y otras especies codistribuidas.

Key words: anfibios; demografía; dispersión; España central; tamaño poblacional; uso del espacio.

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Several studies have described the spatial organization of different amphibian species as metapopulations (SJÖGREN-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 wellconnected network, including both aquatic breeding 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 dynamics cannot counteract local extinctions, compromising the long-term survival of populations (SJÖGREN GULVE, 1994; CARLSON & EDENHAMN, 2000). Thus, information about habitat use and demography is key for the effective management of amphibian populations (WELLS, 2007). For instance, based on these studies, areas that are important for the connectivity 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 populations is crucial, since they are more prone to extinction caused by purely stochastic processes (SJÖGREN GULVE, 1994; CUSHMAN, 2006).

Several studies have provided data on demographic parameters (ADAMA & BEAUCHER, 2006; KAYA et al., 2010), spatial movements (SJÖGREN-GULVE, 1998a,b) and habitat use (BLOMQUIST & HUNTER, 2009) in amphibian populations worldwide (see review

in WELLS, 2007), but data on Iberian amphibians are comparatively scarce (MALKMUS, 1982; DÍAZ PANIAGUA & RIVAS, 1987; LIZANA et al., 1989; GARCÍA-PARÍS et al., 2004).

Capture-mark-recapture studies require continued, intensive sampling effort through many years, but they can provide very detailed information 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 complementary approach to the problem of amphibian declines may involve understanding the processes by which other species maintain positive demographic trends and thrive in the same environments where others disappear.

Perez’s 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 uniformly 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 (GOSÁ & ARIAS, 2009) and population age-structure as assessed by skeletochronology (PATÓN 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 (EGEASERRANO, 2009). In this paper, we present preliminary results about the demography, areas of activity and movement patterns

DEMOGRAPHY AND HABITAT USE IN PELOPHYLAX PEREZI

during the breeding season of a population of P. perezi in an area in the north of the province 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 individuals during the breeding season and to calculate their areas of activity.

3) To investigate possible differences in demographic parameters (population size, apparent survival, probability of recapture) and movement patterns between sexes and breeding sites.

4) To provide biometric (body mass and snoutvent length) data and analyze their relationship with demographic parameters (popula-

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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 delimited 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 corner 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.

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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 (MARTÍNEZ-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 Catálogo 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 vegetation is less abundant. In the east bank there are some willow trees (Salix sp.) and gum rockroses (Cistus ladanifer). During the course of the present 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 surveys, focused on breeding sites, with all the water surface and shores sampled homogeneously. Frogs were captured by hand or with the help of dip nets. We also surveyed terrestrial habitats, mostly along the trails delimiting the study area, but also covering the rest of the study area in search of adult frogs actively 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, starting 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 habitats. 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 recorded sex, snout-vent length (SVL) and body mass (measured with a Pesola MS 1000 scale (Pesola AG, Baar, Switzerland) with a precision of 0.2 g). When individuals were captured 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 inserted under the skin using a hypodermic needle (AVID Single Use Disposable Syringe monoject). Chips were read using an AVID Minitracker II RS232 reader device. All specimens 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 (MannWhitney’s U) to analyze differences in body mass between sites and sexes, whereas parame-

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tric tests (Student’s t) were used to test for differences 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 considered significant.

In recaptured frogs, we also calculated mass variation between the first and the last capture, taking into account the time elapsed between 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-Whitney’s U) tests, and between breeding sites using parametric (Student’s 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 populations, 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

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of variance explained, penalized by the number 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 assumptions of capture-mark-recapture (CMR) studies in open populations and is based on the estimation of four main parameters: 1) apparent survival of individuals between capture events (phi), 2) probability of capture in each survey (p), 3) rate of entrance of new individuals 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 different, albeit complementary ways, considering them as: a) constant, b) time-dependent, c) sexdependent 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 averaging the best models. The average of each parameter is calculated as the weighted average of the values estimated by the different models, where each model has a weight inversely proportional to its AICc. We also used MARK 6.0 to test for correlations between population parameters (phi and p) and biometric variables (body mass and SVL, separately).

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 distances 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 distance 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-Whitney’s 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 Spearman’s correlation coefficient to analyze linear relationships between movement patterns and biometry (body mass and SVL) in all observations pooled together.

Movement patterns and areas of activity

Sampling effort

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

Total sampling effort, measured as persons*hours, was 296 (150 at the mining pond, 83 at Laguna de Valdemanco and 63 in diurnal and nocturnal transects along trails). All captured frogs were found at or in the imme-

RESULTS

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 breeding 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 females: 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-Whitney’s 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-significantly lower than in adults (mean adult males = 0.63, SD = 0.87; mean adult females = 0.62, SD = 1.08) (Kruskal-Wallis’s K: H2 = 6.213; P = 0.469).

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Biometry Body mass and SVL were strongly correlated (Spearman’s Rho: Rho = 0.970; N = 124; P < 0.001). Very significant biometric differences were found between sexes, with females 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-Whitney’s 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; Student’s 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-Whitney’s 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; MannWhitney’s 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; Student’s t: t75 = -1.025; P = 0.309. Females: mean ± SD: L. Valdemanco = 81.8 ± 10.7 mm, mining pond = 82.5 ± 8.6 mm; Student’s 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 relative to initial body mass in individuals captured more than once revealed that females suffered in general stronger losses. However, gains of more than 20% with respect to body mass at the time of first capture were also recorded in individuals from both sexes (Fig. 2).

When differences relative to initial body mass

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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 between sexes were found (median / inter-quartile amplitude: males = 0.0017 / 0.0117 day-1, females = 0.0031 / 0.0131 day-1; MannWhitney’s U: U = 163.00; N (males) = 31, N (females) = 12; P = 0.547). However, whereas adult frogs in Laguna de Valdemanco experienced 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; Student’s 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 probability of entrance (pent) is time-dependent, whereas apparent survival (phi) and probability of capture (p) are either constant or sexdependent (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 = timedependent, and . = constant.

Pond

Model name

AICc

Laguna de phi(.)p(.)pent(t) 243.2289 Valdemanco phi(g)p(g)pent(t) 243.2908 phi(.)p(g)pent(t) 244.4822 phi(g)p(.)pent(t) 245.6365

AICc Num. of Weight params.

0.35544 0.34461 0.18994 0.10665

13 15 14 14

Mining pond phi(.)p(g)pent(t) 417.2786 0.68731 phi(g)p(g)pent(t) 420.4249 0.14254 phi(.)p(.)pent(t) 420.4340 0.14189

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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 relationship between demographic and biometric 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 relationship between apparent survival and SVL, whereas the probability of capture was independent of size (body mass and SVL). For females, however, there was no significant relationship between demographic and biometric 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 recapture 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 Valdemanco

Males Females

91 82

51-130 21-144

Mining pond

Males Females

62 17

42-83 10-24

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Movement patterns and areas of activity We recorded a total of 48 distances between captures, 19 in Laguna de Valdemanco (14 males and five females, including the longest distance recorded, an adult male that dispersed 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 minimum number of three locations are required to estimate the area of activity for a given individual, our dataset was reduced to samples 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 amplitude: 97.94 / 199 m2, Fig. 4).

We found no significant differences between 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-Whitney’s 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,

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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; MannWhitney’s 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-Whitney’s U: U = 52.00; N: males = 19, females = 8; P = 0.217). Timestandardized distances recorded in Laguna de Valdemanco were significantly larger than those recorded in the mining pond for males (Mann-Whitney’s U: U = 46.00; N: L. Valdemanco = 14, mining pond = 19; P = 0.001) but not for females (Mann-Whitney’s 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-Whitney’s 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: Spearman’s 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 population size estimates is also larger in the former. In spite of this, the difference in estimated population 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 differences 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).

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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 differences 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 summarized by EGEA-SERRANO (2009) and in contrast with REAL & ANTĂ&#x161;NEZ (1991). Size differences between sexes, with females being significantly 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 & ANTĂ&#x161;NEZ, 1991). However, these results have to be taken with caution, since our procedure overlooked a potential source of significant 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 thoroughly explored yet.

We did not detect any individual movement between the main breeding sites in our study area (with the single exception highlighted 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 dispersal 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 distributed and tend to occupy all the available space.

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European species of green waterfrogs (SJÖGREN 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 example, in our study area, Hyla arborea, I. Martínez-Solano, J. Gutiérrez & G. SánchezMontes, 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 individuals. Further monitoring of this population 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 individual 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 connected by superficial water currents or intermediate 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 relevance 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 permanent 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, resident frogs present longer within-pond distances 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 valuable 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 movement 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 between 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 preliminary, will be important in understanding finescale population dynamics in this species, including regional patterns of connectivity between breeding sites.

Acknowledgement The help of Garazi Rodríguez was key throughout the study. Thanks as well to Juan Zorrilla and José Serrano for their orientation and to every person who collaborated in the fieldwork: Esperanza and Jaime Iranzo, Ignacio Urbán, Fernando A. Fernández, Jorge Gutiérrez, Jaime Pérez, 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 Consejería de Medio Ambiente, Comunidad de Madrid. This study was funded by project CGL2008-04271-C02-01-BOS (Ministerio de Ciencia e Innovación, Spain, PI: I. Martínez-Solano). Legal regulations and ethical considerations related to work with live animals were strictly followed during the course of this study.

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REFERENCES ADAMA, D.B. & BEAUCHER, M.A. (2006).

Population Monitoring and Recovery of the Northern Leopard Frog (Rana pipiens) in Southeast British Columbia. Columbia Basin Fish and Wildlife Compensation Program, Nelson, British Columbia, Canada.

AKAIKE, H. (1974). A new look at the statistical model identification. IEEE Transactions on Automatic Control 19: 716-723.

BAUER, D.M.; PATON, P.W.C. & SWALLOW, S.K. (2010). Are wetland regulations cost effective for species protection? A case study of amphibian metapopulations.

Ecological Applications 20: 798-815.

BERVEN, K.A. & GILL, D.E. (1983).

Interpreting geographic variation in lifehistory traits. American Zoologist 23: 85-97.

BLACKWELL, E.A.; CLINE, G.R. & MARION, K.R. (2004). Annual variation in population estimators for a southern population of Ambystoma maculatum. Herpetologica 60: 304-311.

BLOMQUIST, S.M. & HUNTER, JR., M.L. (2009). A multi-scale assessment of habitat selection and movement patterns by Northern leopard frogs (Lithobates [Rana] pipiens) in a managed forest. Herpetological Conservation and Biology 4: 142-160.

BOSCH, J.; TEJEDO, M.; BEJA, P.; MARTÍNEZSOLANO, I.; SALVADOR, A.; GARCÍA-PARÍS, M.; RECUERO GIL, E. & BEEBEE, T.J.C. (2008). Pelophylax perezi, In IUCN (2010) The IUCN Red List of Threatened Species, v.

2010.4. International Union for Nature Conservation and Natural Resources, Gland, Switzerland. Available at http://www.iucnredlist.org/. Retrieved on 11/07/2011.

94

SÁNCHEZ-MONTES & MARTÍNEZ-SOLANO

BURNHAM, K.P. & ANDERSON, D.R. (2002).

Model Selection and Multimodel inference: A Practical Information-Theoretic Approach, 2nd ed. Springer, New York.

CARLSON, A. & EDENHAMN, P. (2000). Extinction dynamics and the regional persistence of a tree frog metapopulation. Proceedings of the Royal Society B 267: 1311-1313.

COMPTON, B.W.; MCGARIGAL, K.; CUSHMAN, S.A. & GAMBLE, L.R. (2007).

A resistant-kernel model of connectivity for amphibians that breed in vernal pools.

Conservation Biology 21: 788-799.

COMUNIDAD DE MADRID (2004). Revisión del Catálogo de Embalses y Humedales de la Comunidad de Madrid (Sección Humedales).

Consejería de Medio Ambiente y Ordenación del Territorio, Comunidad de Madrid, Madrid.

COMUNIDAD DE MADRID (2011). Visor de Cartografía Territorial Interactiva. Dirección General de Urbanismo y Estrategia Territorial, Consejería de Medio Ambiente, Vivienda y Ordenación del Territorio, Comunidad de Madrid, Madrid. Available at http://www.madrid.org/cartografia/planea/. Retrieved on 11/07/2011.

CONROY, S.D.S. & BROOK, B.W. (2003).

Demographic sensitivity and persistence of the threatened white- and orangebellied frogs of Western Australia.

Population Ecology 45: 105-114.

CUSHMAN, S.A. (2006). Effects of habitat loss and fragmentation on amphibians: A review and prospectus. Biological Conservation 128: 231-240.

DÍAZ PANIAGUA, C. & RIVAS, R. (1987).

Datos sobre actividad de anfibios y pequeños reptiles de Doñana (Huelva, España).

Mediterranea 9: 15-27.

EGEA-SERRANO, A. (2009). Rana común – Pelophylax perezi (López Seoane, 1885), In A. Salvador & I. Martínez-Solano (eds.)

Enciclopedia Virtual de los Vertebrados Españoles. Museo Nacional de Ciencias Naturales, Madrid. Available at http://www.vertebradosibericos.org/.

Retrieved on 11/07/2011.

EGEA-SERRANO, A.; OLIVA-PATERNA, F.J. & TORRALVA, M. (2005). Selección de hábitat reproductor por Rana perezi Seoane, 1885 en el N.O. de la Región de Murcia (S.E. Península Ibérica). Revista Española de Herpetología 19: 113-125.

ERISMIS, U.C. (2011). Abundance, demography and population structure of Pelophylax ridibundus (Anura: Ranidae) in 26-August National Park (Turkey). NorthWestern Journal of Zoology 7: 5-16.

ESTEBAN, M.; GARCÍA-PARÍS, M. & CASTANET, J. (1996). Use of bone histology in estimating the age of frogs (Rana perezi) from a warm temperate climate area. Canadian Journal of Zoology 74: 1914-1921.

FORTUNA, M.A.; GÓMEZ-RODRÍGUEZ, C. & BASCOMPTE, J. (2006). Spatial network structure and amphibian persistence in stochastic environments. Proceedings of the Royal Society B 273: 1429-1434.

GARCÍA-PARÍS, M.; MONTORI, A. & HERRERO, P. (2004). Amphibia. Lissamphibia. Series: Fauna Ibérica, vol. 24 (M.A. Ramos, coord.) Museo Nacional de Ciencias Naturales, Madrid.

GOSÁ, A. & ARIAS, A. (2009). Estado de las poblaciones de anfibios en un parque urbano de Pamplona. Munibe 57: 169-183.

GREEN, D.M. (2003). The ecology of extinction: population fluctuation and decline in amphibians. Biological Conservation 111: 331-343.

DEMOGRAPHY AND HABITAT USE IN PELOPHYLAX PEREZI

JEHLE, R.; WILSON, G.A.; ARNTZEN, J.W. & BURKE, T. (2005). Contemporary gene flow and the spatio-temporal genetic structure of subdivided newt populations (Triturus cristatus, T. marmoratus). Journal of Evolutionary Biology 18: 619-628.

JOLLY, G.M. (1965). Explicit estimates from capture-recapture data with both death and immigration-stochastic model. Biometrika 52: 225-247.

KAYA, U.; BAŞKALE, E.; ÇEVIK, I.E.; KUMLUTAŞ, Y. & OLGUN, K. (2010).

Population sizes of Taurus frog, Rana holtzi, in two different localities, Karagöl and Eğrigöl: new estimations, decline and a warning for their conservation. Russian Journal of Herpetology 17: 247-250.

KIE, J.G.; BALDWIN, J.A. & EVANS, C.J. (1996). CALHOME: a program for estimating animal home ranges. Wildlife Society Bulletin 24: 342-344.

LIZANA, M.; CIUDAD, M.J. & PÉREZMELLADO, V. (1989). Actividad, reproducción y uso del espacio en una comunidad de anfibios. Treballs de la Societat Catalana d´Ictiologia i Herpetología 2: 92-127.

LLORENTE, G.A.; MONTORI, A.; CARRETERO, M.A. & SANTOS, X. (2002). Rana perezi Seoane 1885. Rana común, In J.M. Pleguezuelos, R. Márquez & M. Lizana (eds.)

Atlas y Libro Rojo de los Anfibios y Reptiles de España. Dirección General de la Conservación de la Naturaleza - Asociación Herpetológica Española, Madrid, pp. 126-128.

MALKMUS, R. (1982). Beitrag zur verbreitung der amphibien und reptilien in Portugal.

Salamandra 18: 218-299.

MARSH, D.M. & TRENHAM, P.C. (2001).

Metapopulation dynamics and amphibian conservation. Conservation Biology 15: 40-49.

95

MARTÍNEZ-SOLANO, I. (2006). Atlas de distribución y estado de conservación de los anfibios de la Comunidad de Madrid. Graellsia 62 (número extraordinario): 253-291.

MAZEROLLE, M.J. (2001). Amphibian activity, movement patterns, and body size in fragmented peat bogs. Journal of Herpetology 35: 13-20.

PATÓN, D.; JUARRANZ, A.; SEQUEROS, E.; PÉREZ-CAMPO, R.; LÓPEZ-TORRES, M. & BARJA DE QUIROGA, G. (1991). Seasonal age and sex structure of Rana perezi assessed by skeletochronology. Journal of Herpetology 25: 389-394.

REAL, R. & ANTÚNEZ, A. (1991). Análisis e interpretación de las dimorfometrías en una población de Rana perezi. Anales de Biología 17: 63-69.

RICHTER, S.C. & SEIGEL, R.A. (2002). Annual variation in the population ecology of the endangered gopher frog, Rana sevosa Goin and Netting. Copeia 2002: 962-972.

RICHTER-BOIX, A.; LLORENTE, G.A. & MONTORI, A. (2006). Breeding phenology of an amphibian community in a Mediterranean area. Amphibia-Reptilia 27: 544-549.

SCHMIDT, B.R.; FELDMANN, R. & SCHAUB, M. (2005). Demographic processes underlying population growth and decline in Salamandra salamandra. Conservation Biology 19: 1149-1156.

SCHWARZ, C.J. & ARNASON, A.N. (1996). A general methodology for the analysis of capture-recapture experiments in open populations. Biometrics 52: 860-873.

SEBER, G.A.F. (1965). A note on the multiplerecapture census. Biometrika 52: 249-259.

SJÖGREN GULVE, P. (1994). Distribution and extinction patterns within a northern

96

SÁNCHEZ-MONTES & MARTÍNEZ-SOLANO

metapopulation of the pool frog, Rana lessonae. Ecology 75: 1357-1367.

SJÖGREN-GULVE, P. (1998a). Spatial movement patterns in frogs: Target-oriented dispersal in the pool frog, Rana lessonae.

Ecoscience 5: 31-38.

SJÖGREN-GULVE, P. (1998b). Spatial movement patterns in frogs: Differences between three Rana species. Ecoscience 5: 148-155.

SJÖGREN-GULVE, P. & RAY, C. (1996). Using logistic regression to model metapopulation dynamics: large-scale forestry extirpates the pool frog, In D.R. McCullough (ed.)

Metapopulations and Wildlife Conservation.

Island Press, Washington, D.C., pp. 111-139.

SMITH, M.A. & GREEN, D.M. (2005).

Dispersal and the metapopulation paradigm in amphibian ecology and conservation: are all amphibian populations metapopulations? Ecography 28: 110-128.

WELLS, K.D. (2007). The Ecology and Behaviour of Amphibians. The University of Chicago Press, Chicago.

WHITE, G.C. & BURNHAM, K.P. (1999).

Program MARK: survival estimation from populations of marked animals. Bird Study 46: S120-S139.

ZAMUDIO, K.R. & WIECZOREK, A.M. (2007).

Fine-scale spatial genetic structure and dispersal among spotted salamander (Ambystoma maculatum) breeding populations. Molecular Ecology 16: 257-274.

Basic and Applied Herpetology 25 (2011): 97-104

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 2

Natural History Museum of Crete, University of Crete, Irakleio, Crete, Greece.

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 molecular 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 analyses 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.

Reanálisis de la filogenia molecular de los Lacertidae usando los datos disponibles en la actualidad. La familia Lacertidae es una de las más diversas y ampliamente distribuidas en Eurasia y África. Varios estudios han intentado hasta ahora aclarar la filogenia de los Lacertidae usando datos morfológicos y moleculares. Sin embargo, las relaciones dentro del grupo permanecen poco claras. En un esfuerzo por explorar las relaciones filogenéticas dentro de la familia Lacertidae, se analizó una base de datos de 5727 pares de bases para 40 géneros diferentes de lacértidos obtenidos por la concatenación de seis genes (dos nucleares y cuatro mitocondriales), todos ellos disponibles en la base de datos de GenBank. Los análisis filogenéticos realizados usando métodos de máxima parsimonia (MP), inferencia bayesiana (BI) y máxima verosimilitud (ML), revelaron que el conjunto combinado de genes mitocondriales y nucleares utilizados no es capaz de resolver las relaciones filogenéticas de los lacértidos a un nivel taxonómico 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, considering both molecular and morphological characters. 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 establishment 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

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(1998, 2000) used mitochondrial DNA sequences to explore the relationships of lacertid lizards.

However, in all cases, the datasets were insufficient 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 ARNOLD’s (1989) “Primitive Palearctic and Oriental Lacertids”, while Eremiainae is equivalent to the “Ethiopian and Advanced Saharo-Eurasian forms” and to ARNOLD’s (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 subfamily Eremiainae into two clades, “Ethiopian” and “Saharo-Eurasian”. ARNOLD et al. (2007) downgraded Lacertinae and Eremiainae into tribes (i.e. Lacertini and Eremiadini, respectively) of the subfamily Lacertinae. PAVLICEV & MAYER (2009) argued that the use of the tribe as a taxonomic entity causes confusion while they concluded that the polytomy of Lacertini is more likely to be attributed to multiple cladogenesis in a geologically 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 phylogeny 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 molecular 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 corresponding 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, respectively. For the BI analysis in the cases where the model selected by jModeltest could not be implemented the closest more complica-

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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

Gallotia Psammodromus Acanthodactylus Adolfus Algyroides Anatololacerta Dalmatolacerta Darevskia Dinarolacerta Eremias Heliobolus Hellenolacerta Iberolacerta Latastia Meroles Mesalina Nucras Atlantolacerta Ophisops Parvilacerta Pedioplanis Poromera Tropidosaura Ichnotropis Phoenicolacerta Takydromus Teira Timon Zootoca Apathya Scelarcis Omanosaura Lacerta Podarcis Australolacerta Holaspis Iranolacerta Philochortus Pseuderemias Archaeolacerta Scincidae

GenBank accession numbers 12S

16s

Cyt b

CO1

c-mos

Rag1

AF206587 AF206588 AF206607 AF206615 AF206598 AJ238188 AF440601 AF206597 AF440600 AF206604 AF206608 AF440602 AF440598 AF206609 AF206610 AY035832 AF206612 AF206603 AF206605 AJ238187 AF206613 AF080368 AF206616 AF080365 NC_011606 AB080237 AJ004884 AF206595 AF206594 AF145444 AF206602 AF080347 AM176577 AF206601 FR751396 n/a GQ142088 n/a n/a AF206592 NC_000888

AF206587 AF206588 AF206607 AF206615 AF206598 GQ142107 AF440616 AF206193 AF440615 AF206604 AF206608 AF440617 AF440612 AF206609 AF206611 AF206606 AF206612 AF149945 AF206605 GQ142106 AF206613 AF080370 AF206616 DQ871149 NC_011606 AB080237 GQ142096 AF206595 AF206594 AF149946 AF206602 AF080352 AM176577 AF206601 FR751396 n/a GQ142111 n/a n/a AF206592 NC_000888

AF101224 AF206535 AF206536 AF206539 AF206529 DQ461765 AY278199 U88611.3 GQ142141 AF206549 AF206544 GQ142128 AY267242 AF206545 AF206540 FJ416173 AF206550 AF206537 AF206532 GQ142135 AF206546 AF080369 AF206541 AF080366 DQ461762 AB080237 GQ142121 DQ902142 AY714929 GQ142127 AF206538 AF080351 AM176577 AY234154 FR751398 n/a GQ142140 n/a n/a GQ142126 NC_000888

AF206562 AF206567 AF206568 AF206578 AF206557 n/a n/a AF206552 n/a AF206576 AF206583 n/a AF206571 AF206563 AF206581 AF206580 AF206565 AF206579 AF206556 n/a AF206566 n/a AF206582 n/a NC_011606 AF206558 AF372052 AF206569 AF206554 unpublished AF206570 n/a AF206551 AF206575 n/a n/a n/a n/a n/a n/a NC_000888

EF632260 EF632284 EF632252 EF632253 EF632255 DQ461743 EF632271 EF632257 EF632270 EF632259 EF632262 EF632269 EF632264 EF632272 EF632273 EF632274 EF632276 GQ142144 EF632278 EF632279 EF632280 EF632283 EF632291 EF632266 DQ461740 EF632288 EF632289 EF632290 EF632292 EF632268 GQ142145 EF632277 EF632267 EF632282 n/a EF632263 GQ142152 EF632281 EF632286 EF632256 AY217888

EF632215 n/a EF632207 EF632208 EF632210 EF632224 EF632228 EF632212 EF632227 EF632214 EF632217 EF632225 EF632219 EF632229 EF632230 EF632232 EF632233 GQ142154 EF632235 EF632236 EF632237 EF632240 EF632248 EF632221 EF632226 EF632245 EF632246 EF632247 EF632249 EF632223 GQ142155 EF632234 EF632222 EF632239 DQ871208 EF632218 GQ142162 EF632238 EF632243 EF632211 AY662634

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ted model was used (RONQUIST & HUELSENBECK, 2003).

Phylogenetic inference analyses were conducted 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 dataset 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, suboptimal trees. The percentage of samples recovering any particular clade in a BI analysis represents that clade’s posterior probability (HUELSENBECK & RONQUIST, 2001). A majority rule consensus tree (‘Bayesian’ tree) was then calculated from the posterior distribution of trees, and the posterior probabilities calculated as the percentage of samples recovering any particular clade (HUELSENBECK & RONQUIST, 2001), where probabilities ≥ 95% indicate significant 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 bisection-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 considered 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: α-shape parameters, GTR-rates, and base frequencies estimated and optimized for each partition (gen). Furthermore, gamma model of rate heterogeneity was assumed for all partitions while the invariable sites were estimated 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 differentiate 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 likelihood under the null hypothesis (branch has zero-length). Using the ‘describe trees’ command 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 χ2 distribution with one degree of freedom. Significance for the likelihood ratio test for each branch in the phylogeny was determined using the percentage point values under the GOLDMAN & WHELAN (2000) mixed model. We used α = 0.05 as significance level to account for possible Type I error.

PHYLOGENY OF THE LACERTIDAE

RESULTS Of the 5727 sites examined, 2557 were variable, 1860 of which were parsimony informative (2690 and 1945 respectively, when the outgroup was included). The MP analysis when the gap was considered as missing produced one tree with a length of 13 941 steps, while three equally parsimonious trees with a length of 14 361 steps were produced when the gap was treated as a fifth character. The topologies recovered were the same for most of the clades (differences are discussed below).

BI resulted in the topology shown in Fig.1 (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% bootstrap support). In the case of Eremiadini, MP (either when the gap was treated as missing or as a fifth character) could not resolve adequately the relationships of the taxa under study. On the contrary BI and ML resulted in the same topology with the exception that ML supported a basal position of Pedioplanis

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for the clade including Meroles, Ichnotropis, Tropidosaura and Australolacerta. Finally the unresolved relationships of the family, according to the likelihood ratio test (SLOWINSKI, 2001), could be considered as soft polytomies with the exception of the branch length separating Poromera (Îą > 0.05, Fig. 1).

DISCUSSION The genera comprised in the Eremiadini tribe (ARNOLD et al., 2007) form a subclade within Lacertinae in all analyses. This clade showed significant internal structure into

Figure 1: Phylogenetic relationships among the 40 genera included in the analyses as inferred by BI. Bayesian posterior probability values (> 0.95) are presented on the nodes followed by bootstrap values (> 50%) for ML, MP and MP* (considering the gap as a fifth character). Branch lengths statistically not significantly greater than zero are indicated with an asterisk (*). Eumeces anthracinus, E. ergegius and E. inexpectatus (Scincidae) were used as outgroup (not shown).

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three groups, two of which correspond to the division of MAYER & PAVLICEV’s (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 sister 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) propose 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 problem of reconstructing the phylogenetic relationships within Lacertini arose in all previous studies (HARRIS et al., 1998; FU, 2000; ARNOLD et al., 2007; MAYER & PAVLICEV, 2007, PAVLICEV & MAYER, 2009) with different 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 relationships 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 likelihood ratio test, conducted for the current dataset, suggest that the unresolved relationships 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 & MAYER’s (2009) view, two aspects could be further researched: 1) taxa sampling and 2) quantity/quality of genetic markers. As far as the first is concerned, even though Lacertidae has been the subject of several studies, the intra genera variation in most of the cases remains unexplored. Taking this under consideration it could be assumed that the unresolved relationships of the family could be attributed to inadequate data sampling. For the near future, 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’ polytomy 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 combination 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 restriction 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 primary 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 relationships 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.

REFERENCES ARNOLD, E.N. (1973). Relationships of the Palearctic lizards assigned to the genera Lacerta, Algyroides and Psammodromus (Reptilia: Lacertidae). Bulletin of the British Museum (Natural History), Zoology 25: 291-366.

ARNOLD, E.N. (1989). Towards a phylogeny and biogeography of the Lacertidae: Relationships within an Old-World family of lizards derived from morphology. Bulletin of the British Museum (Natural History), Zoology 55: 209-257.

ARNOLD, E.N.; ARRIBAS, O. & CARRANZA, S. (2007). Systematics of the Palaearctic and Oriental lizard tribe Lacertini (Squamata: Lacertidae: Lacertinae), with descriptions of eight new genera. Zootaxa 1430: 1-86.

BAIRD, N.A.; ETTER, P.D.; ATWOOD, T.S.; CURREY, M.C.; SHIVER, A.L.; LEWIS, Z.A.; SELKER, E.U.; CRESKO, W.A. & JOHNSON, E.A. (2008). Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS ONE 3: e3376.

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CASTRESANA, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis.

Molecular Biology and Evolution 17: 540-552.

EMERSON, K.J.; MERZ, C.R.; CATCHEN, J.M.; HOHENLOHE, P.A.; CRESKO, W.A.; BRADSHAW, W.E. & HOLZAPFEL, C.M. (2010). Resolving postglacial phylogeography using high-throughput sequencing. Proceedings of the National Academy of Sciences of the United States of America 107: 16196-16200.

FELSENSTEIN, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.

FU, J. (1998). Toward the phylogeny of the family Lacertidae: Implications from mitochondrial DNA 12S and 16S gene sequences (Reptilia: Squamata). Molecular Phylogenetics and Evolution 9: 118-130.

FU, J. (2000). Toward the phylogeny of the family Lacertidae â&#x20AC;&#x201C; Why 4708 base pairs of mtDNA sequences cannot draw the picture. Biological Journal of the Linnean Society 71: 203-217.

GOLDMAN, N. & WHELAN, S. (2000).

Statistical tests of gamma-distributed rate heterogeneity in models of sequence evolution in phylogenetics. Molecular Biology and Evolution 17: 975-978.

HARRIS, D.J.; ARNOLD, E.N. & THOMAS, R.H. (1998). Relationships of lacertid lizards (Reptilia: Lacertidae) estimated from mitochondrial DNA sequences and morphology. Proceedings of the Royal Society B 265: 1939-1948.

HUELSENBECK, J.P. & RONQUIST, F. (2001).

MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755.

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LUTZ, D. & MAYER, W. (1984). Albuminimmunologische und proteinelektrophoretische Untersuchugen zur systematischen Stellung von Lacerta lepida Daudin und Lacerta princeps Blanford (Sauria, Lacertidae).

Zoologischer Anzeiger 212: 95-104.

LUTZ, D. & MAYER, W. (1985). Albumin evolution and its phylogenetic implications in several lacertid lizards. AmphibiaReptilia 6: 53-61.

MAYER, W. & BENYR, G. (1995). Albuminevolution und phylogenese in der familie Lacertidae. Annalen des Naturhistorischen Museums in Wien 96 B: 621-648.

MAYER, W. &PAVLICEV, M. (2007). The phylogeny of the family Lacertidae (Reptilia) based on nuclear DNA sequences: convergent adaptations to arid habitats within the subfamily Eremiainae.

Molecular Phylogenetics and Evolution 44: 1155-1163.

PAVLICEV, M. & MAYER, W. (2009). Fast radiation of the subfamily Lacertinae (Reptilia: Lacertidae): History or methodical artefact? Molecular Phylogenetics and Evolution 52: 727-734.

POSADA, D. (2008). jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution 25: 1253-1256.

RONQUIST, F. & HUELSENBECK, J.P. (2003).

MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.

SALVI, D.; BOMBI, P. & VIGNOLI, L. (2011).

Phylogenetic position of the Southern Rock Lizard Australolacerta australis within the Lacertidae radiation. African Journal of Herpetology 60: 60-69.

SLOWINSKI, J.B. (2001) Molecular polytomies. Molecular Phylogenetics and Evolution 19: 114-120.

SPRINGER, M.S.; STANHOPE, M.J.; MADSEN, O.; WILFRIED, W. & DE JONG, W.W. (2004). Molecules consolidate the placental mammal tree. Trends in Ecology and Evolution 19: 430-438.

STAMATAKIS, A.; HOOVER, P., & ROUGEMONT, J. (2008). A rapid bootstrap algorithm for the RAxML Web servers. Systematic Biology 75: 758-771.

SWOFFORD, D.L. (2002). PAUP 4.0b10a: Phylogenetic Analysis Using Parsimony (and Other Methods). Sinauer, Sunderland, Massachusetts, USA. SWOFFORD, D.L.; OLSEN, G.J.; WADDEL, P.J. & HILLIS, D.M. (1996). Phylogenetic inference, In D.M. Hillis, C. Moritz & B.K. Mable (eds.) Molecular Systematics.

Sinauer, Sunderland, Massachusetts, USA, pp. 407-514.

Basic and Applied Herpetology 25 (2011): 105-113

Biometry and pholidosis of Thamnophis scaliger: an atypical example of sexual dimorphism in a natricine snake Mónica Feriche1,*, Senda Reguera1, Xavier Santos2,3, Estrella Mociño-Deloya1, Kirk Setser1, Juan M. Pleguezuelos1 Departamento de Zoología, Facultad de Ciencias, Universidad de Granada, Granada, Spain.

Departamento de Biología Animal, Universidad de Barcelona, Barcelona, Spain.

3 CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Vairão, Portugal.

1 2

* Correspondence: Departamento de Zoología, 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 subcaudal 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: México; sexual dimorphism; Thamnophis scalaris; Thamnophis scaliger; ventral and subcaudal scale number.

Biometría y folidosis de Thamnophis scaliger: un ejemplo atípico de dimorfismo sexual en un colúbrido natricino. La historia natural de Thamnophis scaliger (serpiente de jardín manchada de Mesa Central), endemismo mexicano recientemente separada de Thamnophis scalaris, es casi desconocida. Presentamos información sobre los rasgos biométricos y folidóticos en función del sexo de una amplia muestra de individuos, y comparamos estos datos con los disponibles para T. scalaris, con el objetivo de situar la especie en el contexto morfológico de un género con una elevada diversidad como Thamnophis. Además 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 también apuntan a que las hembras de T. scaliger superan a los machos en longitud hocico-cloaca, masa e índice de condición corporal (aspectos relacionados con la fecundidad), mientras que los machos superan a las hembras en número de escamas ventrales y subcaudales y longitudes absoluta y relativa de la cola (aspectos relacionados con la selección sexual). El número de escamas ventrales y subcaudales es un reflejo del número de vértebras (somitos).

Sugerimos que la razón por la que las hembras tienen un menor número de somitos que los machos aun siendo más largas y pesadas que éstos puede ser consecuencia de la necesidad de tener vértebras más grandes, y por tanto más robustas, para anclar unos músculos que tienen que mover un cuerpo más pesado.

Key words: Dimorfismo sexual; México; número de escamas ventrales y subcaudales; Thamnophis scalaris; Thamnophis scaliger.

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The genus Thamnophis (American garter snake), distributed throughout North and Central America between 10ºN and 60ºN and between 5ºW and 140ºW, 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 histories 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 requires knowledge of morphological parameters in all species (REEDER & WIENS, 1996).

The present study characterizes morphological 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 & LARAGÓNGORA, 1997). Its position within the genus Thamnophis, however, remains unclear.

In a mitochondrial DNA analysis of 29 species 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 agreement with morphological evidence (ROSSMAN & LARA-GÓNGORA, 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 polytypic, and indicates a need for additional data to quantify morphological variability. In view of this confusing situation, we compare morphological 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 morphological information for T. scaliger was provided only by ROSSMAN et al. (1996) and ROSSMAN & LARA-GÓNGORA (1997).

MATERIALS AND METHODS Thamnophis scaliger occurs in the Distrito Federal and the states of México, Aguascalientes and Michoacán (19-22°N, 98102°W; elevation 2230-3000 m above sea level; QUINTERO-DÍAZ et al., 1999; CANSECOMÁRQUEZ et al., 2007) in the northern portion of the Mexican Transverse Volcanic axis and the southern fringe of the Mesa Central.

The study area is situated near Atlacomulco, north-western México State (19.70ºN, 99.87ºW; 2500-2700 m above sea level), in the highlands of the Central Volcanic Belt of México, near and within the valley of the Río Lerma (Fig. 1). The study area for the sample considered here occupies 74 hectares of pasture 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 0.1-20.2ºC for January and 8.8-21.2ºC 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 portion of the year (January to May) averaging 3% of the annual total (Comisión 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 georeferenced by GPS in order to release individuals at the exact point of capture following proces-

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sing. Samples from two nearby areas, Lagunita (19.90ºN, 99.87°W, 2510 m above seal level) and La Estancia (19.86ºN, 99.80°W; 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 MOCIÑODELOYA et al. (2009), to obtain reliable biometric 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 MOCIÑO-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 parturition (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, respectively, ± 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 calculated from residuals of regression of log body mass on log SVL (BONNET & NAULLEAU, 1994) in mature individuals of each sex.

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Pholidotic variables measured included the numbers of ventral scales (V; following DOWLING, 1951), subcaudal scale pairs (SC), dorsal scale rows at mid-body (D), and the numbers of preocular scales (PRO), postocular scales (PTO), loreal scales (L), anterior temporal scales (AT), posterior temporal scales (PT), supralabial scales (SPL) and infralabial scales (IFL) on each side of the head (Table 1).

Because of high ontogenetic variation in snake body size, we included only mature individuals in our analysis to evaluate possible sexually-based differences in morphometric variables. Females mature at 267 mm SVL (SVL of the smallest gravid female; authorsâ&#x20AC;&#x2122; unpublisTable 1: Frequencies and percentage of the number of head (both sides) and dorsal scales in a population of Thamnophis scaliger in the Valle del RĂ­o Lerma, Atlacomulco, State of Mexico. Abbreviations are defined in the Materials and Methods section.

Head characters (N)

Possible arrangements

%

SPL (70)

7+7 6+7 6+6 9+9 8+9 8 + 10 1+1 3+3 2+2 2+3 2+1 1+1 2+1 1+1 1+2 2+2 2+3 3+3 1+2 19 17

91.4 7.1 1.4 84.3 14.3 1.4 100 47.1 29.4 19.1 4.4 98.6 1.4 97.1 2.9 81.2 11.6 5.8 1.4 70.8 29.2

IFL (70)

PRO (70) PTO (68)

L (70) AT (69) PT (69)

D (113)

hed data); we have no direct data, however, allowing estimation of size at maturity for male T. scaliger. PARKER & PLUMMER (1987), after reviewing 19 studies on 10 species of small viviparous colubrids (e.g. T. scaliger), found that, on average, males matured at 87% of the size of conspecific females. As males reach 84% of the maximum size of females (see Results), this approach is very similar to estimating sexual maturity in males by utilizing the difference in maximum size between both sexes (SHINE, 1990). Using the generalization calculated by PARKER & PLUMMER (1987), we estimated that male T. scaliger matured at 235 mm SVL. Variables not normally distributed (SVL of the top decile of each sex) were assessed by nonparametric statistical tests (Mann-Whitney U-test). Tail length, HW and HL varied with body size, and comparisons between sexes were made by analysis of covariance (ANCOVA) with SVL as the covariate. Although numbers of V, SC, and V + SC are not thought to vary over the life of a snake, larger body size has been associated with a greater number of ventral scales in Coronella girondica (SANTOS & PLEGUEZUELOS, 2003); in these variables, comparisons between sexes were also performed using ANCOVA with SVL as covariate.

Comparisons among dorsal scale numbers (D) were assessed using 2 x 2 contingency tables.

RESULTS Thamnophis scaliger typically exhibit 7 SPL, 9 IFL, 1 PRO, 3 PTO, 1 L, 1 AT, 2 PT, and 19 D (Table 1). The sample of T. scalaris was quite small, but in average, T. scalaris were larger and heavier than T. scaliger and females differed in number of SC: 51-56 in T. scalaris, 39-48 in T. scaliger (Table 2).

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Table 2: Pholidotic and biometric variables (range, mean ± σ: standard deviations) in populations of Thamnophis scaliger and T. scalaris in the Valle del Río 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

SVL (mm)

%

N Range Mean ± σ

TL (mm)

N Range Mean ± σ

HL (mm)

N Range Mean ± σ

HW (mm)

N Range Mean ± σ

Body mass (g)

N Range Mean ± σ

V

N Range Mean ± σ

SC

N Range Mean ± σ

V + SC

N Range Mean ± σ

T. scaliger Males

Females

68 87 127-369 126-439 278.4 ± 70.1 320.2 ± 72.0 F1,153 = 13.11; P = 0.0004 62 70 30-93 29-85 68.2 ± 19.9 64.8 ± 14.4 F1,130 = 178.51; P = 0.001 37 37 8.8-15.5 9.5-17.4 12.78 ± 1.769 13.53 ± 1.92 F1,72 = 2.62; P = 0.110 37 37 5.3-11.0 5.3-11.4 8.31 ± 1.317 8.77 ± 1.293 F1,72 = 0.67; P = 0.41 50 40 10.5-41.7 16.2-70.3 23.2 ± 8.7 36.4 ± 13.9 F1,88 = 7.63; P = 0.007 57 71 134-148 129-145 141.37 ± 3.2 135.3 ± 3.9 F1,126 = 771.7; P < 0.0001 55 63 48 - 59 39 - 48 53.2 ± 2.4 44.1 ± 2.4 F1,116 = 389.7; P < 0.0001 39 42 184 - 204 170 - 192 194.2 ± 4.9 179.5 ± 5.6 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:

Males 1 483

T. scalaris Females 3 360-500 431.0±70.0 3 99-125 111.7 ± 13.0 3 16.8-22.9 19.2 ± 3.2 3 10.7-16.9 12.8 ± 3.5

1 67 1 140

3 31.6-72.6 46.7 ± 22.5 3 138-142 139.7 ± 2.1 3 51 - 56 53.0 ± 2.6 3 190 - 195 192.7 ± 2.5

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

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relative scores, male tail was longer, representing 19.5% of the total length in this sex (N = 61), whereas in females the tail represented 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 demonstrated 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 correcting 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 (χ2 = 0, d.f. = 1, P = 0.96).

DISCUSSION Differences between T. scaliger and T. scalaris ROSSMAN et al. (1996), ROSSMAN & LARA-GÓNGORA (1997) and this study demonstrated broad overlap in morphological 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 relative 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 Río Lerma, Mexico; only individuals with complete tails considered. 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-GÓNGORA, 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 RAMÍREZ-BAUTISTA et al. (1995) near the study area far exceeded the largest snake recorded in our substantial T. scaliger sample, 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 biological 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 species 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, authorsâ&#x20AC;&#x2122; 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 higher number of subcaudal scales than females and, accordingly, 94.2% (90.6-98.5%, Îą = 0.05) of individuals were correctly sexed based solely on this character. Males also had a higher number 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

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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 success in mating or male-male rivalry (sexual selection; KING, 1989).

Body length and tail length are highly correlated 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 similar in males and females (SHINE, 2000). In species sexually dimorphic in size, this similar relationship 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 longer 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 larger and consequently more robust vertebrae to anchor muscles that have to move a heavier body.

We did not test this possibility by necropsy or

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X-rays, but the alternative hypothesis – i.e. female vertebrae are not larger but simply longer than male ones – is less plausible. Sexual dimorphism in body thickness has been found in other viviparous snakes and is interpreted as an adaptation to female reproductive function (AUBRET et al., 2002). Although we were unable to test our argument, 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. Martínez-Lombarry for generously offering access to his property, and also to P. Sánchez 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 SEMARNAT (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 Fundação para a Ciência e a Tecnologia (FCT, Portugal).

REFERENCES ALEXANDER, A.A. & GANS, C. (1966). The pattern of dermal-vertebral correlation in snakes and amphisbaenians. Zoologische Mededelingen 41: 171-190.

AUBRET, F.; BONNET, X.; SHINE, R. & LOURDAIS, O. (2002). Fat is sexy for females but not males: the influence of body reserves on reproduction in snakes (Vipera aspis). Hormones and Behavior 42: 135-147.

BONNET, X. & NAULLEAU, G. (1994). A body condition index (BCI) in snakes to study reproduction. Comptes Rendus de l'Académie des Sciences 317: 34-41.

BONNET, X.; NAULLEAU, G.; SHINE, R. & LOURDAIS, O. (2000). Reproductive versus ecological advantages to larger body size in female snakes, Vipera aspis. Oikos 89: 509-518.

L.; MENDOZACANSECO-MÁRQUEZ, QUIJANO, F. & QUINTERO-DÍAZ, G. (2007). Thamnophis scaliger, In IUCN 2010. IUCN Red List of Threatened Species, v. 2010.3. International Union for Nature Conservation and Natural Resources, Gland, Switzerland. Available at http://www.iucnredlist.org/. Retrieved on 10/15/2010.

DE QUEIROZ, A.; LAWSON, R. & LEMOSESPINAL, J.A. (2002). Phylogenetic relationships of North American garter snakes (Thamnophis) based on four mitochondrial genes: how much DNA sequence is enough? Molecular Phylogenetics and Evolution 22: 315-329.

DOWLING, H.G. (1951). A proposed standard system of counting ventrals in snakes.

British Journal of Herpetology 1: 97-99.

FITCH, H.S. (1981). Sexual size differences in reptiles. University of Kansas, Museum of Natural

BIOMETRY AND PHOLIDOSIS OF THAMNOPHIS SCALIGER

History. Miscellaneous Publication 70: 1-72.

KING, R.B. (1989). Sexual dimorphism in snake tail length: sexual selection, natural selection, or morphological constraint? Biological Journal of the Linnean Society 38: 133-154.

MOCIÑO-DELOYA, E.; SETSER, K.; PLEGUEZUELOS, J.M.; KARDON, A. & LAZCANO, D. (2009). Cannibalism of nonviable offspring by postparturient Mexican lance-geaded rattlesnake, Crotalus polystictus. Animal Behaviour 77: 145-150.

PARKER, W.S. & PLUMMER, M.V. (1987).

Population Ecology, In R.A. Seigel, J.T. Collins & S.S. Novak (eds.) Snakes: Ecology and Evolutionary Biology.

Macmillan Publishing Company, New York, USA, pp. 253-301.

QUINTERO-DÍAZ, G.; VÁZQUEZ-DÍAZ, J. & SMITH, H.M. (1999). Geographic distribution: Thamnophis scaliger. Herpetological Review 30: 237.

RAMÍREZ-BAUTISTA, A.; GUTIÉRREZ-MAYÉN, G. & GONZÁLEZ-ROMERO, A. (1995).

Clutch size in a community of snakes from the mountains of the valley of Mexico. Herpetological Review 26: 12-13.

REEDER, T.W. & WIENS, J.J. (1996). Evolution of the lizard family Phrynosomatidae as inferred from diverse types of data. Herpetological Monographs 10: 43-84.

REGUERA, S.; SANTOS, X.; FERICHE, M.; MOCIÑO-DELOYA, E.; SETSER, K. & PLEGUEZUELOS, J.M. (2011). Diet and energetic constraints of an earthworm specialist, the Mesa Central Blotched Garter Snake (Thamnophis scaliger). Canadian Journal of Zoology 89: 1178-1187.

ROSSMAN, D.A. & LARA-GÓNGORA, G. (1997). Variation in the Mexican garter snake Thamnophis scalaris Cope and the

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taxonomic status of T. scaliger (Jan).

Occasional Papers of the Museum of Natural History Science, Louisiana State University 74: 1-14.

ROSSMAN, D.A.; FORD, N.B. & SEIGEL, R.A. (1996). The Garter Snakes: Evolution and Ecology. University of Oklahoma Press, Norman, Oklahoma, USA. SANTOS, X. & PLEGUEZUELOS, J.M. (2003).

Variación morfológica en la culebra lisa meridional Coronella girondica (Daudin, 1803) en su área de distribución. Revista Española de Herpetología 17: 53-73.

SEIGEL, R.A. & FITCH, H.S. (1984).

Ecological patterns of relative clutch mass in snakes. Oecologia 61: 293-301.

SETSER, K. (2007). Use of anesthesia increases precision of snake length measurements.

Herpetological Review 38: 409-411.

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. McGrawHill, 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.

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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 worldwide. Science 306: 1783-1786.

• Wiens, J.J. & Penkrot, T.A. (2002). Delimiting species using DNA and morphological variation 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 peerreviewed 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.iucnredlist.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 supporting 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 guarantee 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 followed 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 animal care committees. Editors might request from authors as much information as they consider necessary 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 publicación en castellano están disponibles para su consulta en la página web de Basic and Applied Herpetology (http://bah.herpetologica.es/)

BASIC & APPLIED HERPETOLOGY REVISTA ESPAÑOLA DE HERPETOLOGÍA

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 Emílio Goeldi, Brasil) Carmen Blázquez (Centro Investigaciones Biológicas 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) Andrés Egea-Serrano (Universidad de Murcia, Spain) David James Harris (CIBIO-Universidade de Porto, Portugal) Alexander Kupfer (Friedrich-Schiller-Universität Jena, Germany) Pasqualina Kyriakopoulou-Sklavounou (Aristotle University of Thessaloniki, Greece) Claude Miaud (Université de Savoie, France) Nadja Møbjerg (University of Copenhaguen, Denmark) Maria Ogielska (University of Wroclaw, Poland) Vivan P. Páez (Universidad de Antioquía, Colombia) Johannes Penner (Humboldt-Universität zu Berlin, Germany) Nuria Polo Cavia (Universidad Autónoma de Madrid, Spain) Alex Richter Boix (Uppsala University, Sweden) Mireille Rossel (Université 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 (Universität Koblenz-Landau, Germany) Josiah Townsend (University of Florida, USA)

AHE

© 2011