Effects of insularity on life-history traits and on chemical communication in Hierophis viridiflavus: a widespread European colubrid (Reptilia: Serpentes).

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(1)Dipartimento di Biologia Corso di Dottorato in Biologia Evoluzionistica (curriculum animali) (PhD in Evolutionary Biology) VI ciclo; 2005-2007. Thesis. Effects of insularity on life-history traits and on chemical communication in Hierophis viridiflavus: a widespread European colubrid (Reptilia: Serpentes).. PhD Student: Sara Fornasiero (matr. No. 299233) Tutor: Prof. Fernando Dini.

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(3) Abstract The study of intraspecific variability offers a good opportunity to observe how different selective pressures can shape in different ways life-history attributes of organisms living under dissimilar conditions. However, the evolution of intraspecific variation may be counteracted by gene flow. Intraspecific divergence in biological, ecological and behavioural traits are most likely to evolve in a species in which local populations constitute genetically separate entities. In this scenario, islands may serve as natural laboratories, functioning essentially as closed systems. Insular populations of many animal species show ecology, general biology and behaviours often very different from those exhibited from their mainland conspecifics. This phenomenon is so common and widespread that is frequently named the ‘insular syndrome’. The overall aim of this study was to examine if the complete and long-lasting geographical isolation and the particular environmental conditions on the island of Montecristo (Tuscan Archipelago, Western Mediterranean) have influenced different life-history and behavioural attributes in the local population of a widespread European snake species, the European whip snake, Hierophis viridiflavus. This goal was achieved by comparing specific traits exhibited by the chosen model island population (island of Montecristo, Tuscan Archipelago, Italy) with the corresponding traits showed by at least one mainland population. The results of the present study show that individuals from the chosen insular population are characterised not only by reduced body size (a strong insular ‘dwarfism’, already known for the study species), but also by a series of demographic, reproductive and behavioural traits that strongly differ from their mainland relatives. Montecristo snakes are, on average, older and longer-living than their mainland conspecifics, with a lower absolute growth rate and a strong delay in sexual maturation. Moreover, reproductive investment and fecundity of insular. III.

(4) females are deeply reduced with respect to mainland females, while no differences were found in average egg size between the studied populations. This is in agreement with those theories predicting, under variable environmental conditions, a stronger selection in keeping a relatively constant egg size by varying the number of eggs (Optimal Egg Size theory). Among Montecristo reproductive females there is anyway the tendency to increase mean egg size as maternal body size increases. Moreover, insular females produce a high proportion of abortive eggs within the clutches; this proportion is negatively correlated with maternal body size and increases as clutch size increases. The isolation of Montecristo population also results in divergence of the chemical communication system. The crossed behavioural experiments conducted by testing insular and mainland males showed both the existence of intraspecific differences in the chemical composition of skin-derived pheromones and differences in the detection systems of males coming from the different populations. A certain degree of behavioural sexual isolation, with a strong asymmetric pattern, exists between mainland and insular snakes. In conclusion, Montecristo snakes show many traits that highlight the insularity of this population: body size variation, differences in mean age, growth pattern, sexual maturation, reproductive strategies and behaviour. Results are congruent with examples and theoretical models reported in literature. This work therefore represent an additional study-case for a better comprehension of general trends describing the response of organisms to variable environmental conditions.. IV.

(5) Acknowledgements Many, many sincere thanks to: ♦. Dr. Marco Zuffi (Museum of Natural History and Territory, University of Pisa) for having opened to me the doors of his ‘horror-room’ five years ago, for having taught me things that are usually supposed to be taught but also things that are usually not; for his patience in facing my frequent changes of mood, in giving me answers when I needed to, in giving me friendship every moment we spent together. For his trust in my capacities especially when I did not trust in me. Not enough words…thank you Marco!. ♦. Dr. Xavier Bonnet and Dr. Olivier Lourdais (CEBC, CNRS, France) for the many things I learned from them, for the time they dedicated me, since we first met in 2003 until my last visits this year, for their good, unique, incredible, contagious ‘madness’, their huge hospitality that made me feel at home when I was hundreds of kilometres far from home.. ♦. Elisa Bresciani and Federica Dendi, for the hours spent together with the snakes, with the maze and, most of all, with the pentane (!!!), for all the good time we had despite the strong effort and the tiredness, for all the discussions while preparing the next trial!!!. I would have never been able to do all this without you…part of this thesis is also yours. An additional thank to Federica, for her incredible help in the field (despite she used to frighten me during the night…)…did you finally understand how it works, Fé?!?.... ♦. Antonio Atzori, Elena Cecchinelli and Elena Foschi, for their help during the experiments, not because they had to, but ‘simply’ because they wanted to. Elena C. also helped me so many times in the field,. V.

(6) both with her hands in catching snakes, but, especially, with her incredible good mood, always…thank you elenina!!! ♦. Ispettore Eugenio Sereni, Corpo Forestale dello Stato, for the many snakes he catched and he helped me to catch in Montecristo…I will never forget our ambush-hunts…for his friendship…because whenever I needed him he was ready to help me.. ♦. Ministero dell’Ambiente, Servizio V, Roma, Corpo Forestale dello Stato and Parco Nazionale dell’Arcipelago Toscano for permission to enter to Montecristo island, permission to capture and study whip snake specimens and for transportation to and from Montecristo.. ♦. Prof. Jacques Castanet (Université Pierre et Marie Curie, Paris VII) for having patiently taught me how to use the skeletochronological method.. ♦. Prof. Anna Maria Pagni and Mr Antonio Masetti (Department of Biology, Unity of Botany, University of Pisa), for having let me use the instruments in their laboratory and for technical assistance.. ♦. Dr. Roberto Sacchi and Dr. Stefano Scali, for all the brainstorming and the long discussions around parts of this work…. ♦. Prof. Simonetta Citi (Department of Veterinary, University of Pisa), for helping with surgery equipment and with ultrasounds of gravid females.. ♦. Dr. Gunther Köler (Forschunginstitut und Naturmuseum Senckenberg, Senckenberg, Frankfurt/Main, Germany), for loaning all the specimens from Montecristo under his care at the Senckenberg Museum of Natural History.. VI.

(7) ♦. Riccardo Picchiotti, for having cleaned tons of dirty terraria during the experiments!. ♦. Prof. Paolo Joalé and Prof. Paolo Luschi, for their precious advices on a previous draft of the Thesis.. ♦. SHI (Societas Herpetologica Italica) for having granted part of this Thesis (Premio di studio Francesco Barbieri).. ♦. Prof. Fernando Dini, my supervisor, and the Department of Biology, University of Pisa, seat of my PhD Course.. In the end (but not in my heart)… …all my friends (special, additional thanks to Mirko, who thought and made all the drawings in this thesis)…the ones who are near…and the ones who are always too much far (playing with koalas)… …all the people I met during this adventure… …all the places I’ve been to… …my parents, who have always sustained me and my choiches, who gave me the possibility to live my dreams, who often faced the worst parts of me and tried to push them away…who always make me feel special to them… …Samuele, for being my strength when I was tired and hopeless, my positive thought when everything seemed to go in the wrong direction, my best friend and my lover every day since we met…. VII.

(8) Organisation of the Thesis This Thesis is organised into five main chapters. The first chapter is an overall introduction that considers the general overview of the issues at the basis of the present study, the aims of this Thesis, and presents the study species and the study site/population. Chapters 2, 3 and 4 deal with specific aspects of life-history and behaviour of the study species, each with a specific theory and applied methods. Hence, they are treated as separate sections for a better understanding. Chapter 5 presents a general conclusion and the main topics that need further investigation in future researches.. VIII.

(9) Table of contents ABSTRACT .................................................................................................... III ACKNOWLEDGEMENTS.............................................................................V ORGANISATION OF THE THESIS ....................................................... VIII TABLE OF CONTENTS............................................................................... IX LIST OF FIGURES ......................................................................................XII LIST OF TABLES ...................................................................................... XVI. 1.. INTRODUCTION.....................................................................................1. 1.1. General introduction and aims of the research..................................2. 1.2. Study species ..........................................................................................5. 1.3. Study site and study population...........................................................8. 2. 2.1. GROWTH, LONGEVITY AND AGE AT MATURITY ....................15 Introduction .........................................................................................16. 2.2 Materials and Methods .......................................................................20 2.2.1 Study sites .........................................................................................20 2.2.2 Sampling ...........................................................................................23 2.2.3 Skeletochronological analysis...........................................................24 2.2.3.1 Counting SGM...........................................................................24 2.2.3.2 Measure of “rapprochement”...................................................25 2.2.4 Data analysis .....................................................................................26 2.2.4.1 Body size ...................................................................................26 2.2.4.2 Age, longevity and population age structures...........................26 2.2.4.3 Relationship between age and body size...................................27 2.2.4.4 Growth ......................................................................................27 2.2.4.5 “Rapprochement”: age and size at maturity and potential reproductive lifespan.................................................................................28 2.3 Results ..................................................................................................28 2.3.1 Body size, age, longevity and population age structures ..................28 2.3.1.1 Chizé..........................................................................................28 IX.

(10) 2.3.1.2 Montecristo ...............................................................................30 2.3.1.3 Calimera....................................................................................32 2.3.1.4 Inter-population comparisons...................................................35 2.3.2 Relationship between age and body size...........................................38 2.3.2.1 Chizé..........................................................................................38 2.3.2.2 Montecristo ...............................................................................38 2.3.2.3 Calimera....................................................................................39 2.3.2.4 Inter-population comparisons...................................................39 2.3.3 Population growth curves..................................................................42 2.3.3.1 Chizé..........................................................................................42 2.3.3.2 Montecristo ...............................................................................42 2.3.3.3 Calimera....................................................................................42 2.3.3.4 Inter-population comparisons...................................................45 2.3.4 “Rapprochement”: age and size at maturity and potential reproductive lifespan.....................................................................................47 2.3.4.1 Chizé..........................................................................................47 2.3.4.2 Montecristo ...............................................................................48 2.3.4.3 Calimera....................................................................................48 2.3.4.4 Inter-population comparisons...................................................51 2.4 Discussion.............................................................................................53 2.4.1 General considerations ......................................................................53 2.4.2 Intraspecific variation in life-history traits........................................54. 3.. REPRODUCTIVE ECOLOGY.............................................................61. 3.1. Introduction .........................................................................................62. 3.2. Materials and Methods .......................................................................65. 3.3 Results ..................................................................................................68 3.3.1 Maternal body size and clutch size ...................................................68 3.3.1.1 Montecristo ...............................................................................68 3.3.1.2 Chizé..........................................................................................72 3.3.1.3 Inter-population comparisons...................................................74 3.3.2 Egg size .............................................................................................75 3.3.2.1 Montecristo ...............................................................................75 3.3.2.2 Chizé..........................................................................................79 3.3.2.3 Inter-population comparisons...................................................81 3.3.3 Egg mass, clutch mass and RCM......................................................81 3.3.3.1 Montecristo ...............................................................................81 3.3.3.2 Chizé..........................................................................................87 3.3.3.3 Inter-population comparisons...................................................92 3.3.4 Trade-off between number and quality of eggs ................................92 3.4. Discussion.............................................................................................95. X.

(11) 4. 4.1. INTRASPECIFIC CHEMICAL COMMUNICATION....................100 Introduction .......................................................................................101. 4.2 Materials and Methods .....................................................................104 4.2.1 Study populations and husbandry ...................................................104 4.2.2 Y-Maze design ................................................................................105 4.2.3 Pheromone collection......................................................................106 4.2.4 Experimental design........................................................................107 4.2.4.1 Intra-population experiments..................................................107 4.2.4.2 Inter-population experiments ..................................................107 4.2.5 General experimental conditions ....................................................108 4.2.6 Statistics ..........................................................................................110 4.3 Results ................................................................................................110 4.3.1 General biometric features ..............................................................110 4.3.2 Trailing experiments .......................................................................111 4.3.2.1 Intra-population trailing experiments.....................................112 4.3.2.2 Inter-population trailing experiments .....................................113 4.4. 5.. Discussion...........................................................................................118. GENERAL CONCLUSION AND FUTURE RESEARCHES..........125. LITERATURE CITED.................................................................................132. XI.

(12) List of figures Figure 1.1 Distribution of Hierophis viridiflavus. .............................................5 Figure 1.2 Different colour patterns in H. viridiflavus ......................................6 Figure 1.3 The island of Montecristo. ................................................................9 Figure 1.4 Montecristo island: the picture shows the only part of the island where abundant arboreal species can be found. ................................................10 Figure 1.5 An adult female whip snake from Montecristo. .............................11 Figure 1.6 An adult H. viridiflavus from Montecristo basking on the rocks. ..14 Figure 2.1 Climatic features of the study sites.. ...............................................22 Figure 2.2 External and internal view of a preserved H. viridiflavus after the removal of the bones of the right side of the head. ...........................................24 Figure 2.3 Parameters for the measure of "rapprochement". ...........................26 Figure 2.4 Examples of SGM reading on ectopterygoids, Chizé sample. .......29 Figure 2.5 Chizé population age structure.. .....................................................30 Figure 2.6 Examples of SGM reading on ectopterygoids, Montecristo sample.. ...........................................................................................................................31 Figure 2.7 Montecristo population age structure .............................................32 Figure 2.8 Examples of SGM reading on ectopterygoids, Calimera sample ...34 Figure 2.9 Calimera population age structure. .................................................34 Figure 2.10 Inter-population differences in adult body sizes...........................35 Figure 2.11 Inter-population differences in adult estimated ages. ...................36 Figure 2.12 Differences in age distributions among males and females of the three populations considered.............................................................................37 Figure 2.13 Relationship between age and body size in males and females of the three populations.. .......................................................................................41 Figure 2.14 Intraspecific differences in size-corrected age..............................41 Figure 2.15 Population growth curves for H. viridiflavus of the three populations ........................................................................................................44 Figure 2.16 Estimated cumulative Von Bertalanffy’s growth curves..............46 Figure 2.17 Relationship between Von Bertalanffy's parameters. ...................47 Figure 2.18 Annual bone growth in males and females Hierophis viridiflavus from Chizé.........................................................................................................49 Figure 2.19 Annual bone growth in males and females Hierophis viridiflavus from Montecristo...............................................................................................50 Figure 2.20 Annual bone growth in males and females Hierophis viridiflavus from Calimera. ..................................................................................................51 Figure 2.21 Relationship between age at maturity and longevity.. ..................52 Figure 2.22 Relationship between SVL at maturity and asymptotic SVL.. .....52 Figure 3.1 A clutch made of 3 separate eggs laid by a female from Montecristo island. ................................................................................................................68 Figure 3.2 A clutch made of 12 stuck eggs laid by a mainland female from the vicinity of Pisa (Italy). ......................................................................................68 Figure 3.3 Relationship between maternal body size and clutch size in reproductive females from Montecristo. ...........................................................69. XII.

(13) Figure 3.4 Relationship between maternal post-deposition body mass and clutch size in reproductive females from Montecristo......................................69 Figure 3.5 Relationship between maternal body size and the proportion of abortive eggs within the clutch in reproductive females from Montecristo......70 Figure 3.6 Relationship between clutch size and the proportion of abortive eggs within the clutch in reproductive females from Montecristo. Arrows indicate double points. ......................................................................................71 Figure 3.7 Relationship between maternal body condition and the proportion of abortive eggs within the clutch in reproductive females from Montecristo. 71 Figure 3.8 Relationship between maternal post-deposition body mass and the proportion of abortive eggs within the clutch in reproductive females from Montecristo. ......................................................................................................72 Figure 3.9 Relationship between maternal body size and clutch size in reproductive females from Chizé. .....................................................................73 Figure 3.10 Relationship between maternal body condition and clutch size in reproductive females from Chizé. .....................................................................74 Figure 3.11 Relationship between maternal post-deposition body mass and clutch size in reproductive females from Chizé................................................74 Figure 3.12 Relationship between maternal body size and mean egg length within the clutch in reproductive females from Montecristo. ...........................76 Figure 3.13 Relationship between maternal body size and mean egg volume within the clutch in reproductive females from Montecristo. ...........................76 Figure 3.14 Relationship between maternal post-deposition body condition and mean egg volume within the clutch in reproductive females from Montecristo. ...........................................................................................................................77 Figure 3.15 Relationship between maternal post-deposition body mass and mean egg volume within the clutch in reproductive females from Montecristo. ...........................................................................................................................77 Figure 3.16 Relationship between maternal body size and mean egg width within a clutch in reproductive females from Montecristo (only viable eggs). 79 Figure 3.17 Relationship between maternal post-deposition body condition and mean egg volume within a clutch in reproductive females from Montecristo (only viable eggs)..............................................................................................79 Figure 3.18 Relationship between maternal body size and mean egg length within the clutch in reproductive females from Chizé. .....................................80 Figure 3.19 Relationship between maternal post-deposition body condition and mean egg length within the clutch in reproductive females from Chizé...........80 Figure 3.20 Relationship between maternal post-deposition body mass and mean egg length within the clutch in reproductive females from Chizé...........81 Figure 3.21 Relationship between maternal body size and total clutch mass in reproductive females from Montecristo. ...........................................................82 Figure 3.22 Relationship between maternal post-deposition body condition and total clutch mass in reproductive females from Montecristo. ...........................82 Figure 3.23 Relationship between maternal post-deposition body mass and total clutch mass in reproductive females from Montecristo. ...........................83 Figure 3.24 Relationship between maternal body size and mean egg mass within the clutch in reproductive females from Montecristo. ...........................83 Figure 3.25 Relationship between maternal post-deposition body condition and mean egg mass within the clutch in reproductive females from Montecristo...84. XIII.

(14) Figure 3.26 Relationship between maternal post-deposition body mass and mean egg mass within the clutch in reproductive females from Montecristo...84 Figure 3.27 Relationship between maternal post-deposition body condition and mean egg mass within a clutch in reproductive females from Montecristo (only viable eggs). ......................................................................................................85 Figure 3.28 Relationship between maternal body size and RCM in reproductive females from Montecristo. ...........................................................86 Figure 3.29 Relationship between clutch size and RCM in reproductive females from Montecristo. ................................................................................86 Figure 3.30 Relationship between the proportion of abortive eggs within the clutch and RCM in reproductive females from Montecristo. ...........................86 Figure 3.31 Relationship between maternal body size and total clutch mass in reproductive females from Chizé. .....................................................................87 Figure 3.32 Relationship between maternal post-deposition body condition and total clutch mass in reproductive females from Chizé. .....................................87 Figure 3.33 Relationship between clutch size and total clutch mass in reproductive females from Chizé. .....................................................................88 Figure 3.34 Relationship between maternal body size and mean egg mass within the clutch in reproductive females from Chizé. .....................................88 Figure 3.35 Relationship between maternal post-deposition body condition and mean egg mass within the clutch in reproductive females from Chizé.............89 Figure 3.36 Relationship between maternal post-deposition body mass and mean egg mass within the clutch in reproductive females from Chizé.............89 Figure 3.37 Relationship between mean egg length and mean egg mass within the clutch in reproductive females from Chizé. ................................................89 Figure 3.38 Relationship between maternal body size and RCM in reproductive females from Chizé. .....................................................................90 Figure 3.39 Relationship between clutch size and RCM in reproductive females from Chizé. ..........................................................................................90 Figure 3.40 Relationship between mean egg length within the clutch and RCM in reproductive females from Chizé..................................................................91 Figure 3.41 Relationship between mean egg mass within the clutch and RCM in reproductive females from Chizé..................................................................91 Figure 3.42 Relationship between female post-deposition body condition and RCM in reproductive females from Chizé........................................................91 Figure 3.43 Relationship between clutch size and mean egg volume within the clutch in reproductive females from Montecristo. ............................................93 Figure 3.44 Relationship between clutch size and mean egg volume within the clutch in reproductive females from Montecristo (only viable eggs). ..............93 Figure 3.45 Relationship between clutch size and mean egg length within the clutch in reproductive females from Chizé. ......................................................94 Figure 3.46 Relationship between clutch size and mean egg mass within the clutch in reproductive females from Chizé. ......................................................94 Figure 4.1 Picture of the Y-maze system used to conduct trailing experiments in the study. .....................................................................................................106 Figure 4.2 One-stimulus trial. ........................................................................109 Figure 4.3 Two-stimuli trial. ..........................................................................109 Figure 4.4 Results of control trailing experiments.........................................111 Figure 4.5 Results of intra-population trailing experiments ..........................112 XIV.

(15) Figure 4.6 Results of trailing experimental group 2.......................................113 Figure 4.7 Results of trailing experimental group 3.......................................114 Figure 4.8 Results of trailing experimental group 4.......................................115 Figure 4.9 Results of trailing experimental group 5.......................................116 Figure 4.10 Typical substrate in S. Rossore Natural Park. ............................122 Figure 4.11 Typical substrate in Montecristo island. .....................................122. XV.

(16) List of tables Table 2-1 Climatic features of the study sites.. ................................................21 Table 2-2 Mean, minimum and maximum age values estimated for Chizé sample.. .............................................................................................................29 Table 2-3 Mean, minimum and maximum age values estimated for Montecristo sample. ..........................................................................................31 Table 2-4 Mean, minimum and maximum age values estimated for Calimera sample.. .............................................................................................................33 Table 2-5 Results of statistical comparisons of age distributions between the different populations. ........................................................................................38 Table 2-6 Coefficients of correlation and parameters of the Von Bertalanffy's estimated model for male and female H. viridiflavus from Chizé.. ..................43 Table 2-7 Coefficients of correlation and parameters of the Von Bertalanffy's estimated model for male and female H. viridiflavus from Montecristo island.. ...........................................................................................................................43 Table 2-8 Coefficients of correlation and parameters of the Von Bertalanffy's estimated model for male and female H. viridiflavus from Calimera...............43 Table 2-9 Coefficients of correlation and parameters of the Von Bertalanffy's estimated models for the three populations considered ....................................46 Table 3-1 Spearman correlation coefficients (ρ) between maternal parameters in H. viridiflavus reproductive females from Montecristo island.. ...................68 Table 3-2 Pearson correlation coefficients between maternal parameters in H. viridiflavus reproductive females from the forest of Chizé.. ............................72 Table 3-3 Mean values for egg length, egg width and egg volume for abortive and non-abortive eggs in Montecristo sample. .................................................78 Table 4-1 Biometrical features of H. viridiflavus specimens used in trailing experiments.. ...................................................................................................110 Table 4-2 Results of the experiments testing H. viridiflavus conspecific trailing behaviour.........................................................................................................117. XVI.

(17) 1. Introduction. 1.

(18) Chapter 1 – Introduction. 1.1. General introduction and aims of the research. The study of intraspecific variability offers a good opportunity to observe how different selective pressures can shape in different ways life-history attributes of organisms living under dissimilar conditions. Performing comparisons of conspecific populations impedes to take into account eventual biological traits due to events occurred during ancient phylogenetic history of the organisms and to focus the attention exclusively on those patterns that have likely originated as a response to current environmental conditions (Pearson et al., 2002). Common, widespread species can therefore represent good study models (Parker & Plummer, 1987), and in this context several studies have been conducted on mammals (e.g. Dobson & Murie, 1987; Ebenhard, 1990), as well as on amphibians (e.g. Morrison & Hero, 2003) and on reptiles (e.g. Forsman & Shine, 1995). However, the evolution of intraspecific variation may be counteracted by gene flow. Populations that easily interbreed will tend to display similar gene frequencies over wide areas, even in presence of spatially heterogeneous selective forces (Shine, 1987; King, 1993a). Intraspecific divergence in biological, ecological and behavioural traits are most likely to evolve in a species in which local populations constitute genetically separate entities. In this scenario, islands may serve as natural laboratories, functioning essentially as closed systems (Adler & Levins, 1994). Islands have then often been of special interest to ecologists and evolutionary biologists because of the extremely rapid shifts possible in island taxa with small and discrete populations, living under conditions (and selective pressures) often very different from those experienced by their mainland conspecifics (Losos et al., 1997; Keogh et al., 2005; Forchler & Kalko, 2007). It is generally recognised that once a population becomes isolated on an island (or when populations are separated by inhabiting different isolated patches in heterogeneous habitats), population-level changes may take place, such that organisms on islands frequently exhibit unique features compared to their. 2.

(19) Chapter 1 – Introduction. mainland relatives. Different researches have suggested both adaptive and nonadaptive explanations for the evolution of these differences (Barton, 1996; Palkovacs, 2003; Harmon & Gibson, 2006). Adaptive divergence may be the response to the particular habitats and simplified community structures of islands, which typically result in changing of the available resources (both in quality and in quantity), changing in the level of competition (both intraspecific and interspecific) and relaxed predation pressure (Andrews, 1976; Case, 1978; Schwaner & Sarre, 1988; Adler & Levins, 1994; Lindell & Forsman, 1996; Losos, 1996; Palkovacs, 2003; Bonnet et al., 2005; Lomolino, 2005; Wu et al., 2006). Alternatively, random genetic drift in founder events may lead to the observed divergences (Barton, 1996; White & Searle, 2007). The overall pattern of morphological, ecological, physiological and behavioural changes that frequently differentiate island populations from their mainland conspecifics has been often named the ‘island syndrome’ (Thiollay, 1993; Adler & Levins, 1994; Blondel, 2000; Michaux et al., 2002; Goltsman et al., 2005; Magnanou et al., 2005; Matson, 2006; Bertolero et al., 2007; White & Searle, 2007). The first, evident ‘symptom’ of the syndrome is a drastic change in body size, towards dwarfism or gigantism, in island populations with respect to their mainland conspecifics (e.g. Case, 1978; Shine, 1987; King, 1989; Case & Schwaner, 1993; Adler & Levins, 1994; Michaux et al., 2002; Boback, 2003; Boback & Guyer, 2003; Keogh et al., 2005; Lomolino, 2005; Luiselli et al., 2005; Boback, 2006). As age at first reproduction, longevity and reproductive efficiency are all strictly tied to an organism’s size, variation in body size often reflect a cascade of variation in several other correlated life-history traits (Blondel, 2000; Boback, 2003 and refs therein; Palkovacs, 2003). In addition, arbitrary divergence due to genetic isolation, together with the plastic response or the genetic adaptation (or a combination of both) to local conditions (e.g. local habitat type, substrate, available prey type, abundance of predators), may determine a set of peculiar additional behaviours that differentiate insular organisms from their mainland relatives (e.g. antipredator tactics: Bonnet et al., 2005; Barnes et al., 2006; intraspecific communication: Forchler & Kalko, 3.

(20) Chapter 1 – Introduction. 2007; feeding preferences: Aubret et al., 2006; locomotor capacity: Aubret, 2004). Patterns that can be easily recognised as examples of insular syndrome are also known among snakes. Many snake species show intraspecific differences in body size, and ‘giant’ as well as ‘dwarf’ forms can be found among island populations (e.g. Shine, 1987; Schwaner & Sarre, 1988; King, 1989; Madsen & Shine, 1993; Keogh et al., 2005; Barnes et al., 2006; Fornasiero et al., 2007). Boback & Guyer (2003) analysed the largest body lengths of 618 different snake species and found that species smaller than 1.0 m tend to become larger on islands, while those larger than 1.0 m become smaller on islands. The previous studied insular snake populations also showed ecological, biological and behavioural traits often very different from mainland populations (Shine, 1987; Bonnet et al., 2005; Aubret, 2004; Aubret et al., 2006; Zuffi et al., in press). Due to their relatively simple structure and organisation, and to the absence of complex intraspecific interactions and behaviours (such as parental cares, social structures), snakes are furthermore optimal models to analyse patterns of intraspecific variation in size, shape, life-history traits and behaviour. The overall aim of this study was to examine if insularity have influenced different life-history and behavioural attributes in a widespread European snake species. This goal was achieved by comparing specific traits exhibited by the chosen model island population (island of Montecristo, Tuscan Archipelago, Italy) with the corresponding traits showed by at least one mainland population. Results were then discussed in the general framework of intraspecific variability studies and theories, that is with the final aim of understanding how dissimilar environmental conditions can determine intraspecific differences in life-histories and behaviour. More specifically, the insular and the mainland populations were compared by: −. Age structures, longevity, growth trajectories, age and size at maturity;. −. Reproductive ecology: number and size of eggs, reproductive investment; 4.

(21) Chapter 1 – Introduction. −. Intraspecific chemical communication: homotypic (within a population) and heterotypic (between different populations) pheromone trailing behaviour ability.. 1.2. Study species. The European whip snake, Hierophis viridiflavus (Lacépède, 1789), is a common, non venomous, colubrid snake, which inhabits a great variety of environments from sea level up to 2000 m a.s.l. The species is widely distributed throughout south-western mainland Europe, and is present also on most of western Mediterranean islands (Naulleau, 1997; Figure 1.1).. Figure 1.1 Distribution of Hierophis viridiflavus.. It is a sexually dimorphic species, with males attaining larger sizes than females (Heimes, 1993; Luiselli, 1995; Scali & Montonati, 2000; Fornasiero et al., 2007; Zuffi, 2007). Body size (total body length) is highly variable within the species, ranging from less than 100 cm in populations from small Mediterranean islands, up to nearly 150 cm in most of mainland populations (Heimes, 1993; Luiselli, 1995; Capula et al., 2000; Zuffi, 2001; Fornasiero et al., 2007; Zuffi, 2007). Ventral scale counts and general morphology also. 5.

(22) Chapter 1 – Introduction. strongly vary at the intraspecific level (Schätti & Vanni, 1986; Scali et al., 2002; Fornasiero et al., 2007; Zuffi, 2007). Normally coloured (black and yellow; Figure 1.2a) as well as melanic (totally black or greyish; Figure 1.2b) and partially melanic (mostly black, with light parts; Figure 1.2c) populations exist across the distribution range of the species (see Zuffi, 2007 for a review). a). b). c). Figure 1.2 Different colour patterns in H. viridiflavus: a) normally coloured; b) melanic (photo F. Pupin); c) partially melanic.. The species was originally subdivided in three different subspecies on the basis of external characteristics alone (Kramer, 1971): Hierophis viridiflavus viridiflavus Lacépède, 1789, representing the more common black-yellow populations; Hierophis viridiflavus carbonarius Bonaparte, 1883, representing the blackish phenotype, distributed in the south of Switzerland, south of Italy, north-east of Italy and northern Balcanic regions; Hierophis viridiflavus kratzeri Kramer, 1971, endemic of the island of Montecristo. However, the general tendency in the last 15 years has been to consider the species as monotypic and highly polymorphic (Schätti & Vanni, 1986). More recently, as a result of morphometric analyses on several Italian populations, Scali et al. (2002) proposed to re-validate the existence of subspecific taxonomic units: particularly, the Authors suggested to assign the Sardinian populations to the subspecies Hierophis viridiflavus sardus Suckow, 1798 and the Sicilian. 6.

(23) Chapter 1 – Introduction. populations to Hierophis viridiflavus xanthurus Rafinesque Schmaltz, 1810. Moreover, preliminary genetic analyses on cytochrome b mtDNA revealed the existence of two well-identifiable genetic clades (Nagy et al., 2002): a western one including samples from France, Switzerland and Italy west of the Apennines and an eastern one found in Croatia, eastern and southern Italy. The latter one could be further subdivided into three subgroups, two of which occur in the south of Italy only (southern Calabria and Sicily). The Authors proposed therefore the recognition of two valid subspecies of the European whip snake: Coluber (Hierophis) viridiflavus viridiflavus Lacépède, 1789, to identify the western clade; Coluber (Hierophis) viridiflavus carbonarius Bonaparte, 1833 for the eastern one. The taxonomy of this species is however still a matter of discussion. Despite the European whip snake is very common within its distribution range, and often synanthropic (Vanni & Nistri, 2006), detailed information regarding its ecology, general biology and behaviour are still scarce and fragmentary. It is a diurnal species, capable of wide-ranging movements, especially during the reproductive season (Ciofi & Chelazzi, 1991, 1994; Bonnet et al., 1999), and inhabiting a wide variety of habitats (Heimes, 1993; Cortesogno, 1994; Capula et al., 1997). This European snake is highly opportunistic, with a very wide prey spectrum (Heimes, 1993; Luiselli & Angelici, 1996; Capula et al., 1997) and an ontogenetic shift in the diet composition (Heimes, 1993; Rugiero & Luiselli, 1995). Furthermore, diet composition strongly varies at the intraspecific level, following local prey availability (Zuffi, 2001, 2007). Reproduction in Hierophis viridiflavus appears to be one of the least studied aspects of this species biology. Mating takes place after winter latency, from early to mid spring (Ciofi & Chelazzi, 1994; Capula et al., 1995, 1997; Bonnet & Naulleau, 1996; Bonnet et al., 1999), with a high variability due to local climatic conditions (hence, determining the beginning of the active season), and eggs are laid around June and July (Capula & Luiselli, 1995; Luiselli, 1995; Capula et al., 1997; Filippi et al., 2007). Only a limited number of papers, however, addressed reproductive traits, all of which considered single populations from limited areas (Bonnet & Naulleau, 1994; Capula & Luiselli, 1995; Capula et al., 1995; Luiselli, 1995). Nevertheless, a recent, preliminary 7.

(24) Chapter 1 – Introduction. study highlighted the existence of strong intraspecific variability even in reproductive traits (Zuffi et al., in press).. 1.3. Study site and study population. Montecristo is a small (around 10 SqKm in surface, with a coastal perimeter of 16 Km), granitic island sited between the Western coast of peninsular Italy and the Eastern coast of the Island of Corsica, Western Mediterranean (42°20’N, 10°18’E). It is about 63 Km far from the nearest mainland coast (Mt. Argentario, Tuscany, Italy). It emerged from the sea approximately 7.5-7 million years ago (Innocenti et al., 1997) and has never been in contact with mainland (Bossio et al., 2000). Montecristo is now integral Natural Reserve and only few people (the guardian and his family, from two to three) permanently live on the island. Other people’s access is strictly regulated: maximum 1000 tourists are allowed to visit Montecristo each year, but only for a few hours each time and with the only possibility of following a marked path. Access is however authorized for scientific purposes.. 8.

(25) Chapter 1 – Introduction. Despite the relatively small surface, the island presents a quite considerable elevation: its highest peak reaches 645 m of altitude, making the entire island resembling a giant pyramid directly emerging from the sea (Figure 1.3).. Figure 1.3 The island of Montecristo (photo F. Dendi).. The overall landscape is dominated by huge rocky surfaces modelled principally by the action of the wind and, secondarily, by fresh water’s streaming. The vegetation on the Island of Montecristo suffered, during centuries, the consequences of human exploitation. Originally it was probably characterised by a high and rich Mediterranean maquis, dominated, at least at higher elevations, by little forests of holm oaks (Quercus ilex). Today only few specimens of this species are present on the island, and its natural expansion is prevented by the excessive number of wild rabbits (Oryctolagus cuniculus) and wild goats (Capra aegagrus hircus), which destroy and eat the acorns and the young plants. The tree coverage is therefore almost absent on Montecristo, except for the inhabited part of the island (Figure 1.4), where exotic and/or allochthonous species have been imported during centuries: just to mention some of them, stone pines (Pinus pinea), different species of palm-trees, eucalyptuses. (Eucalyptus. globulus),. evergreen. magnolias. (Magnolia. grandiflora) and the ailanthus (Ailanthus altissima). The spontaneous vegetation is characterised by a low and medium-height Mediterranean maquis with a strong prevalence of tree-heather (Erica arborea), rosemary (Rosmarinus officinalis), narrow-leaved cistus (Cistus monspeliensis), curry plant (Helichrysum italicum) and cat thyme (Teucrium marum). 9.

(26) Chapter 1 – Introduction. Figure 1.4 Montecristo island. The blue rectangle shows the only part of the island where abundant arboreal species can be found.. Many bird species can be found on the island, especially during spring migration: Montecristo is located along the Mediterranean migratory routes and therefore functions as ‘stop-over site’ for thousands of starving animals, which, often, dye, as soon as they touch the ground. Among nesting bird species, the yellow-legged gull (Larus michahellis) is by far the most abundant, but others reproduce on the island, such as some nesting pairs of kestrels (Falco tinnunculus), peregrine falcons (Falco peregrinus), scops owls (Otus scops) and many passerine birds, most belonging to the Sylvidae family. Mammalian vertebrate fauna on Montecristo includes the already mentioned wild rabbit, the wild goat and the black rat (Rattus rattus). The only amphibian species on the island is the Thyrrenian painted frog (Discoglossus sardus), a Mediterranean endemism, locally abundant and widespread. Among reptiles, in addition to the population of Hierophis viridiflavus object of the present study, Montecristo hosts an endemic subspecies of the Italian wall lizard (Podarcis sicula calabresiae), two species of geckos (the Turkish gecko, Hemidactylus turcicus and the European leaf-toed gecko, Euleptes europaea) and a population of Southern Italian asp viper (Vipera hugyi, Zuffi, 2002), introduced by man not more than 2000 years ago.. 10.

(27) Chapter 1 – Introduction. Figure 1.5 An adult female whip snake from Montecristo.. The origin of whip snake population on Montecristo island is not known. Since this volcanic island emerged from the sea, nearly 7.5-7 million years ago, it has never been connected to the mainland. However, maybe even during the Mediterranean salinity crisis in late Miocene, but almost surely as a consequence of sea regressions occurred during Pleistocene glacial maxima, it was very close (less than 20 km, the actual distance between Montecristo and the Africhella islet) to the emerged peninsula connecting the Africhella islet to Pianosa island, to Elba and, finally, to coastal peninsular Italy (Bossio et al., 2000). This available land-bridge might therefore have been used by mainland Italian whip snakes to colonise Montecristo. It is indeed true that whip snakes from Corsica strongly resemble Montecristo ones, being small in size, blackish in colour and with high scale counts (Scali et al., 2002). This could therefore lead to hypothesise a possible alternative colonisation route from Corsica. However, no land-bridges were present between the two islands during the late Miocene salinity crisis or Quaternary glaciations (Bossio et al., 2000). In addition, the Miocene major tectonic movements in Western Mediterranean occurred only before and during Montecristo emergence (being, in fact, the main cause of the opening of the Thyrrenian basin and the formation of the volcanic and intrusive rocks of the so-called Tuscan Magmatic Province, to. 11.

(28) Chapter 1 – Introduction. which Montecristo and other island of the Tuscan Archipelago belong; Dini et al., 2002; Meulenkamp & Sissingh, 2003), while the subsequent Mediterranean tectonic change probably did not involve so much Thyrrenian regions (Meulenkamp & Sissingh, 2003). These considerations lead to presumably exclude a temporary proximity of Montecristo to Corsica, both during Miocene and during Pliocene, that could justify the second colonisation hypothesis. In their 2002 study, Nagy et al. did not analyse whip snake samples from Mediterranean islands. Such samples (some from Montecristo specimens) have been considered in an ongoing study which, using ND4 mtDNA marker, is basically confirming preliminary results by Nagy et al. (2002), that is the existence of two well-identifiable clades (see also section 1.2), separated at the intraspecific level. In this emerging scenario Montecristo samples fall entirely within the western clade, together with samples from France and from the Pyrenees (M. Carretero et al., unpublished data). The absence of any genetic differentiation suggests that the colonisation (and the subsequent isolation) of this insular population might be, under an evolutionary perspective, relatively recent, maybe occurred during the last Pleistocene glacial periods. However, in the absence of exhaustive genetic information and of a reliable molecular clock, it is only possible to make hypothesis about the colonisation routes and the phylogeographical history of this common European species (Nagy et al., 2002; Zuffi, 2007). Whip snakes from Montecristo are significantly smaller than conspecifics from most of other populations studied up to date, surely always smaller than relatives from any mainland population (Fornasiero, 2004; Fornasiero et al., 2007). Moreover, important differences exist in traits that in snakes show a high heritability (scale counts, Dohm & Garland, 1993; Fornasiero, 2004; Fornasiero et al., 2007). Whip snake population density on Montecristo is about 11.1-15.4 specimens/ha (M.A.L. Zuffi & S. Fornasiero, unpublished data). Considering that similar calculations on data from a mainland population in S. Rossore Natural Park (Pisa, Italy) conducted to an estimate of 6-8.8 specimens/ha (M.A.L. Zuffi, unpublished data) and that the only reliable estimation present in literature for this species reports values ranging from 2.3 12.

(29) Chapter 1 – Introduction. to 3.5 specimens/ha (Rugiero et al., 2002), then H.viridiflavus has to be considered locally abundant, with a high population density on the island. Due to the absence of micromammals, whip snakes on Montecristo feed almost exclusively on really small preys: lizards and on both adults and tadpoles of Discoglossus sardus (Zuffi, 2007). Other preys are occasionally represented by small passerine birds (e.g. adult pied and collared flycatchers, Ficedula hypoleuca and F. albicollis, pers. obs.) or, presumably, by their chicks. However, whip snakes seem to exploit this kind of prey only when small birds arrive exhausted and harmless during the spring migration season, rarely in other months during the snakes’ active season (Zuffi, 2007). Moreover, only bigger specimens are able to kill and ingest such kind of prey (pers. obs.). Adult vipers on Montecristo also feed on the same type of food (lizards and painted frogs), but resources are locally so abundant and viper population density so small (pers. obs.), that competition between the two species should be limited. No other competitors exist on the island. In addition, continuous predation pressure is likely to be almost absent on Montecristo: there are no terrestrial predators, while the usual ‘snake-eating’ aerial predators (e.g. the buzzard, Buteo buteo and the short-toed eagle, Circaetus gallicus) fly over the island almost exclusively during early spring and during autumn migrating seasons. Montecristo snakes exhibit a particularly ‘relaxed’ basking behaviour: blackish in colour, they can be often seen basking directly over light-coloured rocks, perfectly visible (Figure 1.6). Such a behaviour is highly improbable on the mainland, where snakes are among the most secretive animals, laying always partially hidden among rocks and vegetation. This observations support the statement of a reduced predation pressure on Montecristo island. Even nesting gulls do not seem to disturb whip snakes’ activity: some specimens were observed basking just nearby the nesting colonies. Furthermore, another great snake-predator is missing from the island: man!. 13.

(30) Chapter 1 – Introduction. Figure 1.6 An adult H. viridiflavus from Montecristo basking on the rocks (photo M.A.L. Zuffi).. By all these considerations, it emerges the utility and the importance of this population for the purposes of the present study: its long-lasting isolation, the unique conditions present on the island (type of prey, relaxed predation pressure, absence of human disturbance), the high population density and, moreover, the presence of at least one unambiguous sign that “something different” is going on, i.e. the strong dwarfism, lead to hypothesise that other strong peculiarities might have arose in life-history traits as well as in behavioural aspects. In the context of the study of intraspecific variation, Montecristo whip snakes are therefore ideal models.. 14.

(31) 2. Growth, longevity and age at maturity. 15.

(32) Chapter 2 – Growth, longevity and age at maturity. 2.1. Introduction. The study of life histories has always been a central theme in evolutionary biology. One of the crucial questions is how life history traits (such as growth, longevity, age and size at maturity and fecundity) may vary among different species and/or among different populations of the same species as a consequence of adaptive genetic differentiation, phenotypic plasticity in response to local environmental conditions, or a combination of both (e.g. Dunham, 1978; Berrigan & Charnov, 1994; Ford & Seigel, 1994; Adolph & Porter, 1996; Rohr, 1997; Bronikowski, 2000; Blouin-Demers et al., 2002; Cogălniceanu & Miaud, 2003). Among life history traits, age at maturity is a pivotal one because, especially in iteroparous species with indeterminate growth, fitness is often more sensitive to changes in this traits than to any other (Stearns, 1992): together with maximum longevity, age at maturity determines the overall potential reproductive lifespan, therefore influencing average lifetime fecundity. In stable populations, age at maturity should then evolve to maximise lifetime reproductive success (Bernardo, 1993; Wapstra et al., 2001). Under this assumption, and without any other constraint, organisms should experience fast juvenile growth and early maturation, in order to shorten generation time and increase individual potential reproductive lifespan (Ryser, 1996; Bronikowski & Arnold, 1999). Moreover, considering that among juveniles mortality is higher than in adults (Parker & Plummer, 1987; Bronikowski & Arnold, 1999; Webb et al., 2003), maturing earlier would enhance the probabilities to survive until first reproduction (Day & Taylor, 1997). However, many examples suggest that there may be costs associated with fast growth (Stamps et al., 1998; Bronikowski & Arnold, 1999; Bronikowski, 2000; Mangel & Stamps, 2001; Blouin-Demers et al., 2002; Yearsley et al., 2004), determining, among other consequences, shorter longevity among early-breeders. Moreover, other studies suggested that a delay in maturity would result in an improvement in the juvenile survival of the offspring produced (Stearns & Crandall, 1981; Stearns & Koella, 1986).. 16.

(33) Chapter 2 – Growth, longevity and age at maturity. In organisms with indeterminate growth, such as Reptiles, sexual maturation implies the shift of resources from only growth during juvenile phase, to growth plus reproduction during adulthood (e.g. Shine & Charnov, 1992; Day & Taylor, 1997; Rohr, 1997; Wapstra et al., 2001; Stanford & King, 2004). As a result, growth rate slows down after the attainment of sexual maturity, and the variance in the body sizes of animals that have reached age at maturity will be maintained in later age-classes. This means that small first-breeding individuals will continue to be small for the rest of their lives (Halliday & Verrell, 1988) and that postponing sexual maturity would allow continuing to grow, hence attaining a larger size. In this context, early maturation means smaller adult body size. If fecundity is size-related (Halliday & Verrell, 1988), the over mentioned advantages for fecundity of early maturation are counterbalanced by a decrease in fecundity, as a consequence of smaller body size. Oppositely, the gain in body size derived from a delay in maturation would mean a related gain in future overall fecundity, together with the ecological and behavioural advantages of a larger body size (e.g. success in intra- and interspecific resource competition, less predation risk; Dunham, 1978; Stamps et al., 1998). On the other hand, maturing later increases generation length and decreases the probabilities to survive until the first reproductive season. By all these considerations, it is clear that size (which, in fact, is determined by juvenile growth rates and age at maturity, two traits that may be or may not be correlated, Rohr, 1997) and age at maturity represent a compromise among many different pressures (Stearns & Crandall, 1981; Stearns & Koella, 1986; Ford & Seigel, 1994). The result of this trade-off may anyway be different depending on genetic adaptation together with different environmental conditions. Studies that compare populations of the same species in different parts of its range can consequently be of particular value in understanding how environmental factors can shape age and size at maturity and related lifehistory attributes (age-size relationship, longevity, population age structures, fecundity; e.g. Grant & Dunham, 1990; Mateo & Castanet, 1994; Nobili & Accordi, 1997; Rohr, 1997; Bronikowski & Arnold, 1999; Lima et al., 2000; Miaud et al., 2001; Blouin-Demers et al., 2002; Miaud & Guillaume, 2005; 17.

(34) Chapter 2 – Growth, longevity and age at maturity. Sears, 2005). As intraspecific geographic variation in adult body size is often associated with differences in life history traits (Alcobendas & Castanet, 2000; Miaud et al., 2001; Kutrup et al. 2005), species that exhibit such variation throughout their distribution range should receive particular attention in life history studies. Considering that most of life history traits are age-dependent, knowledge of the age of adult specimens, population age structures and growth patterns is an important prerequisite when evaluating intraspecific differences in life history patterns (Halliday & Verrell, 1988). Many studies have been focused on determining longevity and population age structures. Many of these studies used the mark-recapture method, which is undoubtedly valid and complete, but usually requires a certain number of years of field effort to collect sufficient data. Skeletochronology has been proved to be another reliable method to obtain such kind of data (reviewed in Halliday & Verrel, 1988), with the advantage of a shorter time required to carry out the study. The skeletochronological method is based on counting the skeletal growth marks (SGM), which are naturally produced on the growing bone. Functionally SGM represent the expression of temporary variations in the rate of osteogenesis (Castanet et al., 1992). Three categories of SGM can be recognised: opaque layers (zones), translucent layers (annuli) and lines of arrested growth (LAG) (Castanet et al., 1993). Zones correspond to general active and fast osteogenesis and they are made of badly spatially structured bone matrix, rich in randomly distributed osteocytic lacunae. Due to their structure, zones are more opaque than other marks and appear therefore dark when observed under transmitted light (Castanet et al., 1993). Annuli alternate with zones and correspond to periods of slow osteogenesis. Bone matrix is well structured (often being made of lamellar bone) and usually poor in osteocytes. When observed under transmitted light, annuli appear narrower and more translucent than adjacent zones (Castanet et al., 1993). 18.

(35) Chapter 2 – Growth, longevity and age at maturity. LAGs functionally correspond to a temporary arrest of local osteogenesis. They always have a very low thickness and, when visible, they are typically bordering annuli or, sometimes, appearing inside them (Castanet et al., 1993). Skeletochronology is based on the assumption that growth marks are the histological expression of temporary and periodical variation in bone growth rate. Especially among temperate ectotherms, which experience the alternation of active and resting seasons, zones are the product of active bone growth occurred during spring-summer active life, whilst annuli and LAGs represent the result of winter latency. Together, a zone and an annulus/LAG, correspond therefore to one complete growth cycle (Castanet et al., 1993). Alternatively, in tropical species the presence of skeletal growth marks may depend more on factors other than temperature, such as humidity variations or food availability (e.g. Ortega-Rubio et al., 1993; Driscoll, 1999; Kumbar & Pancharatna, 2001). The annual periodicity of SGM formation has been demonstrated in several species of temperate and tropical Amphibians (e.g. Miaud, 1992; Guarino et al., 2003) and Reptiles (e.g. Castanet & Naulleau, 1974; Castanet, 1985; De Buffrénil & Castanet, 2000; Waye & Gregory, 1998). The current opinion is that SGMs originate from endogenous rhythms, and are reinforced and synchronised by external seasonality (Castanet & Naulleau, 1974; Castanet et al., 1993). Until sexual maturity, when body growth rate is high, annuli or LAGs are well separated by wide zones of fast-growing tissues, while the subsequent SGM appear narrower and more irregular. This pattern has been named “rapprochement” by Francillon-Vieillot et al. (1990). Rapprochement is interpreted as the result of a sudden decrease in growth rate, as a consequence of a shift in the allocation of resources among the competitive demands of growth and reproduction, after the attainment of sexual maturity (FrancillonVieillot et al., 1990; Castanet et al., 1993; Lagarde et al., 2001). Such rapprochement is, together with secondary bone remodelling, the cause of arising problems during the lecture of SGM for age estimation, especially in older individuals. For this reason the choice of the bones to use in skeletochronological studies is of primary importance. In snakes, despite a 19.

(36) Chapter 2 – Growth, longevity and age at maturity. recent study validated the use of caudal vertebrae for age estimation (Waye & Gregory, 1998), two flat skull bones, the supra-angular and the ectopterygoid, are mostly used for SGM counting. Among these, the ectopterygoid has been shown to provide the best sample for skeletochronology application (Hailey & Davies, 1987). The use of skull bones, unfortunately, limits the application of the method to dead specimens. The aims of this study were to: (1) determine and analyse, by means of skeletochronology, age structures, longevity, age-size relationships, growth curves, age and size at maturation and potential reproductive lifespan in three different populations of the European whip snake, Hierophis viridiflavus, inhabiting different parts of the distribution range of the species; (2) compare the over-mentioned life history traits among the three populations and (3) search for specific differences among the study sites that may account for the observed differences in life history patterns.. 2.2. Materials and Methods. 2.2.1 Study sites Specimens used in this study came from three distinct and disjoint populations in different parts of the distribution range of the species: (1) Forest of Chizé, situated in France, Deux-Sèvres department, in the vicinity of the CEBCCNRS (46°07’ N, 00°25’ W). The site is close to the northern border of the distribution range of the species; (2) Montecristo island, Tuscan Archipelago (Central Italy). Full description of this site is provided in section 1.3; and (3) surroundings of Calimera, Lecce (Apulia, Southern Italy; 40°15’ N, 18°16’ E), close to the southernmost limit of the distribution range of the species in peninsular Italy. Climatological data were obtained from the three metereological stations closer to the respective study sites: Niort (30 Km from the Forest of Chizé), Lecce (15 Km from Calimera) and Gorgona island (65 Km from Montecristo island; Elba island is closer to Montecristo, but Gorgona resembles much more Montecristo island in overall surface, vegetation and. 20.

(37) Chapter 2 – Growth, longevity and age at maturity. climatic conditions). While it was possible to obtain data for Lecce only for the period between 1961 and 1990, data for France and Montecristo are referred to the same time span, between 1998 and 2006. Average monthly temperatures (°C) and average monthly rainfalls (mm) for the over cited time intervals are reported in Table 2-1 and Figure 2.1.. Table 2-1 Climatic features of the study sites. AR=Average Rainfall (mm); AT=Average Temperature (°C); AmT=Average minimum Temperature (°C); AMT=Average Maximum Temperature (°C). Station Niort. Gorgona. Lecce. J. F. M. A. M. J. J. A. S. O. N. D. AR. 64.5. 48.0. 54.7. 71.6. 65.1. 32.3. 63.0. 61.3. 61.9. 100.9. 88.8. 79.0. AT. 5.9. 6.3. 9.3. 10.9. 14.8. 18.6. 19.3. 20.4. 17.0. 13.3. 8.0. 6.3. AmT. 2.3. 1.8. 3.8. 5.3. 8.5. 11.6. 12.2. 13.2. 10.2. 8.2. 3.7. 2.7. AMT. 9.5. 10.9. 14.7. 16.5. 21.1. 25.6. 26.3. 27.5. 23.7. 18.4. 12.4. 9.9. AR. 31.3. 21.6. 48.4. 59.2. 21.8. 25.4. 15.5. 25.3. 74.1. 107.2. 82.6. 80.7. AT. 9.4. 9.9. 11.6. 12.9. 17.4. 21.4. 23. 24.1. 20.8. 17.8. 13.7. 10.8. AmT. 7.7. 8.1. 9.7. 10.7. 15.1. 18.8. 20.5. 21.8. 18.4. 15.9. 11.9. 9.1. AMT. 12.1. 12.2. 14.5. 15.7. 20.8. 25. 26.6. 27.7. 23.9. 20.6. 16.5. 13.5. AR. 63. 53.6. 67.6. 38.4. 27.5. 20.3. 18.2. 32.3. 53.5. 80.6. 91. 81.4. AT. 8.6. 9.1. 10.95. 13.8. 18.2. 22.25. 24.9. 24.95. 21.95. 17.5. 13.15. 9.9. AmT. 4.6. 4.9. 6.4. 8.6. 12.4. 16.2. 18.6. 19. 16.5. 12.9. 9. 6. AMT. 12.6. 13.3. 15.5. 19. 24. 28.3. 31.2. 30.9. 27.4. 22.1. 17.3. 13.8. 21.

(38) Chapter 2 – Growth, longevity and age at maturity. a) 120. 35. 110 100 90. 25. 80 20. 70 60. 15. 50 10. 40. Rainfall (mm). Temperature (°C). 30. 30. 5. 20 0. 10 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec. b) 120. 35. 110 100 90. 25. 80 20. 70 60. 15. 50 10. 40. Rainfall (mm). Temperature (°C). 30. 30. 5. 20 0. 10 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec. c) 120. 35. 110 100 90. 25. 80 20. 70 60. 15. 50 10. 40. Rainfall (mm). Temperature (°C). 30. 30. 5. 20 0. 10 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec. Figure 2.1 Climatic features of the study sites. a) Chizé b) Montecristo c) Calimera. ( Average min. Temperature); ( Average Max. Temperature); ( Average monthly Temperature); ( Average monthly Rainfall).. 22.

(39) Chapter 2 – Growth, longevity and age at maturity. 2.2.2 Sampling A total of 132 specimens were analysed during four years: 72 whip snakes came from the Forest of Chizé, 28 from Montecristo Island and 32 from Calimera. Some of the studied specimens were alcohol preserved and came from the herpetological collections of Florence (Museo Zoologico “La Specola”, Università di Firenze, Italy), Pisa (Museo di Storia Naturale e del Territorio, Università di Pisa, Italy) and Frankfurt (Senkemberg Museum Frankfurt a.M., Germany); other specimens were road-kills, while others were animals found dead in the wild or died in laboratory during captive maintenance. While older road-kills and freshly dead animals were collected and preserved in freezer at low temperature from 1998, the temporal origin of Montecristo Museum specimens covered, in some cases, the last sixty years. On the island it is rare to find dead animals in the wild. Considering the particular protection status of Montecristo island I decided to use as many samples as possible from herpetological collections, finally avoiding to intentionally kill any animal. Moreover, preliminary analyses of results showed no differences among “old” and “recent” specimens (no outliers depending on sampling date), showing the absence of time-dependent variations in the observed population patterns during the considered time-span. Each snake was sexed (sexing was not possible for newborns and juveniles), and measured (SVL, Snout-to-Vent Length) to the nearest mm by stretching it along a measuring tape. Montecristo sample included only one newborn specimen and adult specimens: snakes were considered to be adult on the basis of external body coloration and on the current knowledge of the biometrical features (e.g. adult mean SVL) of this population (Fornasiero et al., 2007). Samples from Chizé and from Calimera included newborns, juveniles and adults: in these samples snakes were considered to be adult on the basis of external body coloration and when SVL was greater than 500mm.. 23.

(40) Chapter 2 – Growth, longevity and age at maturity. 2.2.3 Skeletochronological analysis Skeletochronology was applied to ectopterygoid and supra-angular flat bones of each sample. In those specimens, like the road-kills, where the subsequent preservation of the entire animal was not necessary, bones for the skeletochronological analysis were obtained by cutting the head of the snake and leaving it in fresh water until the complete putrefaction of soft tissues. Then the ectopterygoid and the supra-angular of each side of the head were removed and stored in fresh water. In museum specimens, in order to preserve the external morphology and scalation of the head, the bones were removed from one side only of the head, by gently separating them from the outer skin (Figure 2.2). Then they were stored in fresh water and cleaned from tissues several times, until they were ready to be analysed. a). b). Figure 2.2 External (a) and internal (b) view of a preserved H. viridiflavus after the removal of the bones of the right side of the head (photo M.A.L. Zuffi).. 2.2.3.1 Counting SGM The analysis of SGM was made following the procedure reported by Castanet et al. (1993): bones were observed in toto with a binocular microscope, under transmitted light. During the reading of SGM, the bone was kept under water, in order to enhance the contrast between different growth marks. The analysis was made by 2 different people (or by the same observer on several separate occasions), always blind to sample identity. The results of the different readings on the same sample were then compared and, if counts differed among the occasions, the sample was inspected again in order to match the observations. If differences were not resolved, the sample was not included in. 24.

(41) Chapter 2 – Growth, longevity and age at maturity. the analyses. If the readings on the ectopterygoid and the supra-angular differed within the same sample, the ectopterygoid was chosen, because in previous studies it has been shown to provide the most reliable results (Hailey & Davies, 1987). If strong bone remodelling occurred, obviating any age estimation, the sample was again not included in the analyses. The last step of the study included a comparison between the preliminary skeletochronological results and the measured length of the animals: if the result looked inconsistent with the general pattern found, the sample was rejected as outlier. 2.2.3.2 Measure of “rapprochement” Good ectopterygoids of adults of each population were photographed using a LEICA. DC300F. camera. assembled. with. a. WILD. HEERBRUGG. MAKROSKOP M 420 1,25x. Using an image-editing computer software, the distance between the basis of the first visible zone (corresponding to the bone growth occurred during the first active season of the animal) and the basis of each subsequent zone was measured on each picture along a straight line that run across the bone flat surface where LAGs were visible (Figure 2.3). The thickness of each zone + annulus complex represents one year of bone growth, thus the annual bone growth (G) of each sample was estimated following the relation. Gn + 1 =. Rn + 1 − Rn R1. where Gn+1 represents therefore the increment of bone per year in relation to the increment of the first year, expressed in arbitrary units. The analysis was performed only on those pictures where the different zones were clearly visible and distinguishable one from the other.. 25.