The Phylogenetic Signal in Cranial Morphology of <I>Vipera Aspis</I>
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HERPETOLOGICAL JOURNAL 19: 69–77, 2009 The phylogenetic signal in cranial morphology of Vipera aspis: a contribution from geometric morphometrics A. Gentilli1, A. Cardini2, D. Fontaneto3 & M.A.L. Zuffi4 1Dipartimento di Biologia Animale, Università di Pavia, Italy 2Dipartimento del Museo di Paleobiologia e dell’Orto Botanico, Università di Modena e Reggio Emilia, Italy & Hull York Medical School, The University of Hull, UK 3Imperial College London, Division of Biology, UK 4Museo di Storia Naturale e del Territorio, Università di Pisa, Italy Morphological variation in the frontal bone and cranial base of Vipera aspis was studied using geometric morphometrics. Significant differences in shape were found among samples from subspecies present in Italy (V. a. aspis, V. a. francisciredi, V. a. hugyi). Sexual dimorphism was negligible as well as allometry and size differences. The most divergent subspecies was V. a. aspis, possibly in relation to its recent history of geographic isolation in a glacial refugium. Shape clusters were in good agreement with clusters from studies of external morphology and completely congruent with results from molecular studies of mtDNA. Key words: asp viper, cranial shape and size, Italy, phylogeny, systematics INTRODUCTION those of other French populations. Garrigues et al. (2005) also made a preliminary comparison using mitochondrial he asp viper, Vipera aspis (Linnaeus, 1758), is among cytochrome b and ND2 DNA sequences. However, the Tthe best studied Palaearctic viperids (Mallow et al., limited number of samples analysed by Garrigues et al. 2003). It is a small to medium-sized snake, averaging 60– (2005) did not allow any robust reconstruction of relation- 70 cm total length (50–63 cm snout–vent length), up to 82 ships within V. aspis. In contrast, a recent study on cm maximum length (Saint Girons, 1978). It is only found in mtDNA variability investigating the phylogeography of western and southern Europe (Zuffi, 2002; Fig. 1) from sea the asp viper across its whole distribution range level to 3000 m (Saint Girons, 1997). Geographic variability (Ursenbacher et al., 2006) indicated that V. aspis can actu- in morphological features has been described (Phisalix, ally be separated into four distinct groups. Moreover, 1968; Kramer, 1980; Zuffi & Bonnet, 1999; Zuffi, 2002). For they considered V. atra, characterized by a relatively high instance, Zuffi (2002) studied characters of the external number of ventral scales and a marked black dorsal pat- morphology and copulatory apparatus, and found differ- tern, and distributed in northwestern Italy (Zuffi, 2002), as ences in the number of dorsal bars and of ventral and a synonym of V. a. aspis. Thus, the four well-supported subcaudal scales, together with a distinctive hemipenial molecular clades, corresponding to V. a. aspis, V. a. form. Thus, he suggested that Vipera aspis may indeed francisciredi, V. a. hugyi and V. a. zinnikeri, were consid- represent a species complex (sensu Hermann et al., 1999) ered as subspecies. including Vipera aspis, V. atra Meisner 1820, V. hugyi Whether populations that are recognized using soft- Schinz 1833 and V. zinnikeri Kramer 1958. A taxonomic tissue anatomy and molecular markers can also be revision was also carried out using immunological data by discriminated using bone morphology has never been ac- Pozio (1980), who showed that electrophoretic patterns of curately investigated. Moreover, incongruences between venom compositions were very different between V. aspis results from previous studies are present, requiring fur- atra, V. aspis francisciredi Laurenti, 1768, and V. aspis ther analyses and the clarification of independent hugyi, whereas the latter had the same electrophoretic characters. pattern as V. aspis montecristi Mertens, 1956. However, Cranial form provides potentially interesting traits for the reproductive isolation of populations within the V. investigating the systematic relationships of vipers (Guo aspis species group has been questioned by some au- & Zhao, 2006). In particular, the akinetic portion of the thors focusing on neurotoxins. For instance, Guillemin et skull has been considered more suitable for taxonomic al. (2003) found that interbreeding may have occurred comparisons in snakes (Kramer, 1980; Gloyd & Conant, even between two clearly separated species, V. aspis from 1990). This is because the neurocranium, which forms the southeastern France and the sand viper, Vipera akinetic portion of the skull, is less strongly influenced by ammodytes (Linnaeus 1758). On the other hand, De Haro selective pressures related to diet (prey swallowing) and et al. (2002) showed that in France at least one population it is not directly associated with the mechanics of enveno- of V. aspis had a specific venom profile distinct from mation. Such studies, however, either did not focus on Correspondence: M.A.L. Zuffi, Museo di Storia Naturale e del Territorio, Università di Pisa, via Roma 79, 56011 Calci (Pisa), Italy. E-mail: [email protected] 69 A. Gentilli et al. Fig. 1. Schematic distribution of Vipera aspis group (from Zuffi, 2002, modified, according to Ursenbacher et al., 2006). Small black dots indicate sampling localities of V. aspis subspecies (numbers indicate sample size >1); two large open dots indicate V. berus outgroups; the large dark grey dot indicates V. ammodytes outgroup. The small arrow at the black dot indicates the V. a. hugyi population of Montecristo Island. The large arrows at large grey dots indicate the transition forms, V. a. aspis*francisciredi in the north, and V. a. francisciredi*hugyi in the south (see Appendix 1 for references). European vipers or were limited by the paucity of avail- MATERIALS AND METHODS able information in terms of study specimens and number of metric characters. Morphometric tools are presently We considered only adult specimens (n=61) recognized available that allow accurate comparisons of the geomet- and classified following Zuffi & Bonnet (1999). Vipera a. ric shape of organisms or of their organs (Rohlf & aspis (4 males, 3 females), V. a. francisciredi (10 males, 8 Marcus, 1993; Adams et al., 2004). These methods, known females) and V. a. hugyi (14 males, 9 females) (sensu as geometric morphometrics, have been successfully ap- Ursenbacher et al., 2006) were analysed. Individuals from plied to detect subtle variation among closely related taxa contact areas between species, also showing intermedi- (Duarte et al., 2000; Cardini, 2003; Frost et al., 2003; ate numbers of dorsal bars and markings and numbers of Klingenberg et al., 2003; Nicola et al., 2003; Macholán, ventral scales, were considered separately, and indicated 2006). as V. a. aspis*francisciredi (5 males, 3 females) and V. a. Thus, we applied geometric morphometrics to explore francisciredi*hugyi (1 male). Specimens of V. berus (1 size and shape variation in the cranium of Italian male, 2 females) and V. ammodytes (1 female), closely re- populations of V. aspis that include three of the four sub- lated to V. aspis (Hermann et al., 1999), were used as species supported by the molecular analysis of outgroups. Museum catalogue numbers and localities of Ursenbacher et al. (2006). The analysis was performed by collection are given in Appendix 1. measuring crania using two-dimensional coordinates of Digital pictures of skulls were used in the analysis. Pic- anatomical landmarks in representatives of V. a. aspis, V. tures of the dorsal and ventral view of the akinetic portion a. francisciredi and V. a. hugyi. The main aims of the of the cranium were taken in standardized conditions us- study were to assess the magnitude of the variation and ing a Leica Digilux Zoom digital photocamera, with a 300 the significance of differences in cranial size and shape dpi resolution, mounted on a Leica MZ75 stereomicro- among these subspecies, and to reconstruct their similar- scope, and lit by optical fibres. A set of topographically ity relationships. corresponding anatomical landmarks (Marcus et al., 2000) 70 Cranial morphology in Vipera aspis Table 1. Anatomical description of landmarks of francisciredi and V. a. hugyi) by regressing shape vari- dorsal and ventral bones in viperid cranium. ables onto CS. Significance of regressions was tested with a permutation test for the generalized Goodall’s F Description (Goodall, 1991; Rohlf, 2005). Average shapes for the fron- Dorsal tal and cranial base were computed for each species with 1 Proximal margin of the suture between the pooled sexes. The two data sets were then combined frontals (Adams, 1999) and phenetic relationships among viper 2 Outermost point between prefrontals and taxa were summarized with multi-dimensional scaling frontals (MDS) and cluster analysis (performed on the matrix of 3 Point between the frontal and postfrontal Euclidean distances between mean shapes). A minimum- bones length spanning tree (MST; Rohlf, 1970) was 4 Suture point between the frontal, postfrontal superimposed on the MDS scatterplot to help detect local and parietal bones distortions. The matrix correlation (a Pearson correlation 5 Suture point between frontals and parietals on unfolded diagonal symmetric matrices) between the Ventral original matrix of Euclidean distances (all shape variables) 1-2-3-4 Higher points of the basisphenoid lateral and the one based on the position of the species in the crest three-dimensional MDS scatterplot was 0.960. Among dif- 5 Teeth of the basioccipital ferent clustering methods, the unweighted pair-group method using centroid (UPGMC)