Acta Herpetologica 14(2): 117-122, 2019 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-7749 Ontogenetic and interspecific variation in skull morphology of two closely related species of toad, Bufo bufo and B. spinosus (Anura: Bufonidae) Giovanni Sanna Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands Department of Biological, Geological, and Environmental Sciences, University of Bologna, Via Selmi 3, 40126 Bologna, Italy Marine Research Department, Senckenberg am Meer, Südstrand 40, 26382 Wilhelmshaven, Germany E-mail: giovanni.sanna3@studio.unibo.it Submitted on: 2019, 31st January; Revised on: 2019, 6th June; Accepted on: 2019, 5th August Editor: Marcello Mezzasalma Abstract. Using micro-CT and 3D landmark-based geometric morphometrics, I investigated postmetamorphic shape variation in the skull of Bufo bufo and Bufo spinosus, two widespread European toad species with small phenotyp- ic differences. Two ontogenetic series were compared, for a total of 58 individuals. They exhibited similar allometric growth patterns, characterised by cranial widening with relative shortening and dorsoventral compression. However, some interspecific shape divergence was observed, particularly among adults: a relatively shorter skull and a more dorsally extended snout distinguished B. spinosus from B. bufo. This disparity, which gives further support to species separation, can probably be ascribed to changes in the allometric trajectories, and seen in light of the evolutionary history of the two lineages. Keywords. Allometry, Bufo bufo, Bufo spinosus, divergence, geometric morphometrics, landmark, ontogeny, skull shape. INTRODUCTION Divergent traits can be expected to be found in closely related species with a broad geographical distri- bution, relatively to ecological and climatic variation, but developmental constraints may also play an impor- tant role in restricting or channelling phenotypic evolu- tion (Cvijanović et al., 2014; Ivanović and Arntzen, 2018). The Common toad, Bufo bufo (Linnaeus, 1758), and the Spined toad, Bufo spinosus Daudin, 1803, are mem- bers of the Common toad species group of the western Palaearctic (Arntzen et al., 2013b). B. bufo has a wide Eurasian distribution that comprises northern and east- ern France, central and southern Europe (including Sar- dinia; Cossu et al., 2018), and stretches northwards into Scandinavia and eastwards deep into Russia; B. spinosus is found in the Iberian Peninsula, western and south- ern France, and North Africa, from Morocco to Tuni- sia (Arntzen et al., 2013a). Their lineages have diverged around 9 Ma (million years ago; Recuero et al., 2012), but in contrast to a deep genetic differentiation, B. bufo and B. spinosus appear phenotypically similar (Arntzen et al., 2013a). Whereas a few diagnostic characters were described for the external morphology, virtually nothing is known about interspecific osteological differences, nei- ther in the postcranial nor in the cranial skeleton. Such a complex structure as the skull is of particular interest in a wide range of studies, due to its fundamental biologi- cal functions and the fact that it often undergoes adaptive variation (Ivanović et al., 2012). Substantial changes are 118 Giovanni Sanna known to occur in the anuran skull after metamorphosis (Ponssa and Candioti, 2012). The purpose of this investigation was to highlight potential interspecific differences in the skull morphology of B. bufo and B. spinosus, in the context of postmeta- morphic development, using a geometric morphometrics approach. The analysis of ontogenetic shape variation in these two species is interesting not only for a taxonomic evaluation based on morphology; it can provide insights into their morphological evolution, to be interpreted in the context of their evolutionary history and past distri- bution. Moreover, this analysis could contribute to a bet- ter understanding of the interplay between ontogeny and morphological differentiation among anuran species. MATERIAL AND METHODS A total of 58 alcohol-stored specimens, including freshly metamorphosed, juvenile, and adult (male and female) toads, were analysed (Table S1). These toads had been collected from various populations in different localities of the Iberian Penin- sula (B. spinosus), France (B. spinosus, B. bufo), and the Neth- erlands (B. bufo). They were subdivided into two ontogenetic series, made up of 28 Bufo bufo and 30 Bufo spinosus individu- als, respectively. Body size in the whole sample ranged from 16.0 mm to 78.0 mm SUL (snout-urostyle length). Mature B. spinosus individuals were larger on average (mean SUL 71.5 mm) than B. bufo ones (mean SUL 60.4 mm), reflecting size disparity between western European Common toads and Spined toads (Cvetković et al., 2009). Three-dimensional imaging Skulls were CT-scanned with two x-ray machines: SkyScan 1172 (Bruker, Kontich, Belgium) and ZEISS Xradia 520 Versa (Carl Zeiss XRM, Pleasanton, CA, USA). The former was used for the smaller toads, with 0.5 mm Aluminium filter, 2K reso- lution (2000 × 1336), pixel size of 13.17 µm, voltage of 29-54 kV, exposure time of 420-750 ms, 0.4 rotation step, averaging of four frames. Voltage and exposure time were modified when scanning specimens of different sizes (which showed differences in skull density), in order to keep image quality consistent. Xra- dia scanner was employed for adult individuals, mainly due to its larger sample holder; resolution was set at 1K (1000 × 1024), with pixel size of 34.18 µm, and voltage of 80 kV. Scanning was followed by two-dimensional reconstruction of raw image data into stacks, which were processed with Avizo 9 software (FEI SAS, France): the segmentation editor was used to segregate homogeneous volumes (corresponding to skull bones), with manual adjustment of masking. Notable variation in the extent of cranial ossification, not merely restricted to small juveniles, was observed at this point. A 3D surface model of the skull was then generated, applying a variable degree of unconstrained smoothing according to ossification extent. Landmarks Thirty-one homologous landmark points were collected on each 3D surface model to describe overall skull shape (Fig. 1), using Landmark editor 3.6 (Institute for Data Analysis and Vis- ualization, University of California, Davis, 2007). Fifteen points were bilateral and symmetric, while one was median. Anatomi- cal description of points is provided in Table S2. Incomplete skull ossification of small juveniles made it challenging, at times, to perform an accurate placement of homologous land- marks (Zelditch et al., 2004). Geometric morphometrics Morphometric and statistical analyses based on the land- mark coordinates, and qualitative observation of the associated shape changes, were carried out with MorphoJ 1.06 software (Klingenberg, 2011). A generalized Procrustes analysis (GPA), consisting of a full Procrustes superimposition for object sym- metry, was applied: the symmetric components of shape varia- tion, as a measure of skull shape, and centroid size, as a meas- ure of skull size, were computed for each individual (Klingen- berg, 2016). The covariance matrix of shape variables was then generated and used to perform a principal component analysis (PCA), in order to explore overall patterns of shape variation. Shape changes in relation to growth were evaluated with a multivariate regression of shape (symmetric components, i.e. dependent variables) on size (log-transformed centroid size, i.e. independent variable), one for each ontogenetic series (Mon- teiro, 1999), in association with a permutation test against inde- pendence between dependent and independent variables (made up of 10⁴ randomization rounds). The angle between the two regression vectors was calculated for comparison, including a test against randomness of vector directions in shape tangent space. Interspecific morphological distinction was assessed by performing a discriminant function analysis (DFA) with leave- one-out cross-validation (Lachenbruch, 1967; Webster and Sheets, 2010) on shape data of the 25 largest individuals, 12 B. bufo and 13 B. spinosus toads comprising adults and subadults. Such a subsample was selected in order to maximize interspe- cific differences, since the PCA had previously shown shape divergence in late stages of growth (Fig. 2). Results of this anal- ysis were compared with those of a DFA on the remaining indi- viduals of the sample, namely juveniles. Both analyses included a parametric T-square test against the null hypothesis of equal group means, and a permutation test for the T-square statistic with 10,000 randomization rounds. RESULTS The first two principal components (PC1, PC2) of the PCA accounted for 71.9% of total shape variation. PC1 alone grouped 65.4% of variation and was positive- ly correlated with size: therefore, the scatter plot of PC2 119Skull shape variation in Bufo toads vs. PC1 can be looked at as a morphospace that contains the ontogenetic shape trajectories of B. bufo and B. spi- nosus (Fig. 2). Cranial shape variation along PC1 was characterised, in the positive direction, by a broadening (at the level of the jaw joint), an overall shortening (at the level of both the exoccipital and the premaxilla), and dorsoventral compression. Some interspecific variation was comprised in PC2, especially for the largest toads; positive direction shape changes along this axis were an increase in skull length, dorsoventral compression, and a slight widening. Both regression analyses found a significant asso- ciation between shape and size (P < 0.0001 for both), indicating allometric growth: 63.8% of shape variation in B. bufo and 68.5% of shape variation in B. spinosus were predicted by regression on size (Fig. 3). The two vector directions formed an angle of 17.2° (whereas 0° denote complete correspondence, 90° maximum diver- gence) and were not random in the shape tangent space (P < 0.00001). The major shape changes associated with regression were corresponding between the two species and matched those related to the first principal compo- nent of the PCA, thus confirming the correlation of PC1 with skull size. Discriminant function analysis applied to the largest toads showed a clear distinction between the two species (Fig. 4): after cross-validation, 10/12 B. bufo individuals were reassigned to their true group and 2/12 were allo- cated to the Spined toad group, while 13/13 B. spinosus individuals were reassigned to their own group (Cohen’s K = 0.84). However, the T-square test for the difference between group means was not statistically significant (T² = 281, P = 0.82). Permutation produced a signifi- cant result instead (P < 0.0001). The major interspecific shape differences pointed out by the analysis concerned cranial length and height: B. spinosus exhibited a longer upper jaw (with a more posterior jaw joint), yet a shorter Fig. 1. Landmark positions on the digitized skulls, in dorsal (A), right lateral (B), and frontal (C) views. For the anatomical descrip- tion of landmarks, see Table S2. Fig. 2. Ontogenetic shape variation of B. bufo and B. spinosus, described by the scatter plot of the first two principal components (with 95% confidence ellipses). PC1 summarises allometric varia- tion, while PC2 displays species divergence in late development. 120 Giovanni Sanna skull (due to a shorter occipital region), with a dorsally expanded snout; skull width did not show noteworthy variation. DFA on juveniles yielded similar results, with a weaker interspecific distinction (Fig. 4): 13/16 B. bufo and 14/17 B. spinosus toads were reallocated to their group (K = 0.63); T-square test was not significant (T² = 790.5, P = 0.72), while permutation test was significant (P < 0.0001). DISCUSSION Most of the shape variation in the whole dataset was explained by differences in size, as shown by the con- cordant results of principal component and regression analyses. Therefore, I suggest that ontogenetic allometry (i.e., the association between morphological changes and ontogenetic growth; Klingenberg, 2016) characterises skull development of both Bufo bufo and Bufo spinosus, mainly in the form of a considerable widening (typi- cal of skull growth in frogs; Ponssa and Candioti, 2012), and a relative shortening and dorsoventral flattening. Cranial allometry has already been recognized in other anuran species, both in larval (e.g., in Rana sylvatica; Larson, 2002) and post-metamorphic development (e.g. in Rhinella marina and the Leptodactylus fuscus group; Birch, 1999; Ponssa and Candioti, 2012). Allometric con- straints are likely to limit phenotypic evolution of the skull, even in presence of selective pressure (Simon et al., 2016). This kind of scenario can be logically applied to the current study, which found shape disparities between Fig. 3. Shape changes of B. bufo (A) and B. spinosus (B) in relation to size, described by the scatter plot of regression scores against log- transformed centroid size. Fig. 4. Interspecific distinction, described by DFA histograms in which light grey bars represent B. bufo and dark grey bars represent B. spi- nosus. Species assignment and discriminant scores are shown before (A, C) and after (B, D) cross-validation, for the largest individuals (A, B) and juveniles (C, D). 121Skull shape variation in Bufo toads B. bufo and B. spinosus that are moderate if compared to the common allometric variation. Nevertheless, there seems to be a shift in the ontoge- netic trajectories of the two species, highlighted by both the principal component analysis (Fig. 2) and the angle between regression vectors (17.2°), and reflected in the morphological distinction provided by the discrimi- nant function analyses (Fig. 4). Along with both latter analyses, T-square test against equal species mean shapes yielded significant results only after permutation, which probably compensated for the relatively small sample size. Interspecific divergence, however, resulted higher in the group of largest individuals: they showed greater agreement to true species membership, as described by a K coefficient of 0.84 against the 0.63 of juveniles. Con- sequently, it might be easier to discriminate between B. bufo and B. spinosus at mature stages, rather than among young individuals. This would be in contrast with the findings from other studies on amphibians, which point- ed out a decrease in interspecific disparity over ontogeny (Adams and Nistri, 2010; Ponssa and Candioti, 2012). DFA and, to some extent, PCA, indicate that B. bufo toads have on average a longer skull and a more dorsally compressed snout than B. spinosus toads. This morpho- logical divergence could theoretically be placed within either the non-allometric or the allometric regime. Vari- ation in non-allometric shape would imply some sort of relaxation of ontogenetic constraints, which seems very unlikely, as the present evidence suggests that these con- straints are strong in both species. Alternatively, inter- preting interspecific variation as comprised in the allo- metric framework should better reflect the results of the analyses. Allometric variation could have arisen in two ways. First, from changes in developmental timing of the ancestral shape trajectory, with a conserved shape- size relationship (ontogenetic scaling hypothesis; Strelin et al., 2016); however, this also seems unlikely, because the divergent skull shapes do not correspond to different stages of the same trajectory. Secondly – and more plau- sibly – divergent evolution of the ontogenetic programme in the two lineages may have occurred, with a conserved direction of early post-metamorphic shape trajectories. The latter hypothesis would imply that interspecific dif- ferences have arisen during the long-lasting separation of the lineages (about 9 Ma), and it could even be surprising that they are not more pronounced; as a possible expla- nation for this moderate divergence, a recent morpho- logical evolution, following rapid postglacial European expansion of B. bufo from its Balkan refugium, cannot be excluded (Recuero et al., 2012; Arntzen et al., 2016). Climate is thought to have an indirect influence on skull morphology, because it determines food type and availability (Simon et al., 2016), and changes in these eco- logical factors may lead to skull shape divergence – even in closely related species. Alternative climate-driven fac- tors could also induce skull differentiation, such as repro- duction sites (water pools) occurrence, and the ability of toads to detect them, which involves the olfactory cap- sules in the snout region (Trueb, 1993; Simon et al., 2016). Whether such factors are related to the interspecific dif- ferences found here – some of which concern the snout, more expanded in B. spinosus – it is not possible to say. Arntzen et al. (2013a) hypothesized that in European toads a larger body size, a more pronounced presence of keratinous spines on the cheek warts and a wider head shape might favour defence against predators, namely grass snakes. Although B. spinosus apparently meets these requirements more than B. bufo, relative cranial width did not show significant interspecific disparity in the current study. Thus, no link was found between widely divergent parotoid glands, a distinctive trait of B. spinosus, and a wider skull. Nevertheless, Spined toads could compensate by attaining a wider skull through their bigger size. For further assessments of drivers and extent of morphological divergence between B. bufo and B. spino- sus, it would be convenient to use a larger sample, espe- cially for adult individuals, in order to properly account for the role of sexual dimorphism and benefit from the highest disparity. Different populations should be consid- ered, since B. bufo exhibits remarkable variation across its wide range; for instance, Mediterranean Common toads appear to resemble Spined toads in body size – large – and skin texture – thick and warty (De Lange, 1973; Cvetković et al., 2009; Arntzen et al., 2013a). ACKNOWLEDGEMENTS This study was conducted in association with Natura- lis Biodiversity Center. I would like to thank Marta Cal- vo (Museo Nacional de Ciencias Naturales) and Esther Dondorp (Naturalis Biodiversity Center) for access to the material under their supervision, Ana Ivanović for precious suggestions on study design, Rob Langelaan for technical support with computed tomography, and Pim Arntzen and two anonymous reviewers for valuable feed- back and remarks on earlier versions of the manuscript. SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found at manuscript number 24709. 122 Giovanni Sanna REFERENCES Adams, D.C., Nistri, A. (2010): Ontogenetic convergence and evolution of foot morphology in European cave salamanders (Family: Plethodontidae). BMC Evol. Biol. 10: 216. Arntzen, J.W., McAtear, J., Recuero, E., Ziermann, J.M., Ohler, A., van Alphen, J., Martínez-Solano, I. (2013a): Morphological and genetic differentiation of Bufo toads: two cryptic species in Western Europe (Anura, Bufonidae). Contrib. Zool. 82: 147-169. Arntzen, J.W., Recuero, E., Canestrelli, D., Martínez- Solano, I. (2013b): How complex is the Bufo bufo spe- cies group? Mol. Phylogenet. Evol. 69: 1203-1208. Arntzen, J.W., Trujillo, T., Butôt, R., Vrieling, K., Schaap, O., Gutiérrez-Rodríguez, J., Martínez-Solano, I. (2016): Concordant morphological and molecular clines in a contact zone of the Common and Spined toad (Bufo bufo and B. spinosus) in the northwest of France. Front. Zool. 13: 52. Birch, J.M. (1999): Skull allometry in the marine toad, Bufo marinus. J. Morphol. 241: 115-126. Cossu, I.M., Frau, S., Delfino, M., Chiodi, A., Corti, C., Bellati, A. (2018): First report of Bufo bufo (Linnaeus, 1758) from Sardinia (Italy). Acta Herpetol. 13: 43-49. Cvetković, D., Tomašević, N., Ficetola, G.F., Crnobrnja- Isailović, J., Miaud, C. (2009): Bergmann’s rule in amphibians: combining demographic and ecological parameters to explain body size variation among pop- ulations in the common toad Bufo bufo. J. Zool. Syst. Evol. Res. 47: 171-180. Cvijanović, M., Ivanović, A., Kalezić, M.L., Zelditch, M.L. (2014): The ontogenetic origins of skull shape dispar- ity in the Triturus cristatus group. Evol. Dev. 16: 306- 317. De Lange, L. (1973): A contribution to the intraspecific systematics of Bufo bufo (Linnaeus, 1758) (Amphibia). Beaufortia 21: 99-116. Ivanović, A., Arntzen, J.W. (2018): Evolution of skull shape in the family Salamandridae (Amphibia: Cau- data). J. Anat. 232: 359-370. Ivanović, A., Sotiropoulos, K., Üzüm, N., Džukić, G., Olgun, K., Cogălniceanu, D., Kalezić, M.L. (2012): A phylogenetic view on skull size and shape variation in the smooth newt (Lissotriton vulgaris, Caudata, Sala- mandridae). J. Zool. Syst. Evol. Res. 50: 116-124. Klingenberg, C.P. (2011): MorphoJ: an integrated soft- ware package for geometric morphometrics. Mol. Ecol. Resour. 11: 353-357. Klingenberg, C.P. (2016): Size, shape, and form: concepts of allometry in geometric morphometrics. Dev. Genes Evol. 226: 113-137. Lachenbruch, P.A. (1967): An almost unbiased method of obtaining confidence intervals for the probability of misclassification in discriminant analysis. Biometrics 23: 639-645. Larson, P.M. (2002): Chondrocranial development in lar- val Rana sylvatica (Anura: Ranidae): morphometric analysis of cranial allometry and ontogenetic shape change. J. Morphol. 252: 131-144. Monteiro, L.R. (1999): Multivariate regression models and geometric morphometrics: the search for causal factors in the analysis of shape. Syst. Biol. 48: 192-199. Ponssa, M.L., Candioti, F.V. (2012): Patterns of skull development in anurans: size and shape relationship during postmetamorphic cranial ontogeny in five spe- cies of the Leptodactylus fuscus Group (Anura: Lepto- dactylidae). Zoomorphology 131: 349-362. Recuero, E., Canestrelli, D., Vörös, J., Szabó, K., Poyark- ov, N.A., Arntzen, J.W., Crnobrnja-Isailovic, J., Kidov, A.A., Cogălniceanu, D., Caputo, F.P., Nascetti, G., Martínez-Solano, I. (2012): Multilocus species tree analyses resolve the radiation of the widespread Bufo bufo species group (Anura, Bufonidae). Mol. Phylo- genet. Evol. 62: 71-86. Simon, M.N., Machado, F.A., Marroig, G. (2016): High evolutionary constraints limited adaptive responses to past climate changes in toad skulls. Proc. R. Soc. B Biol. Sci. 283: 20161783. Strelin, M.M., Benitez-Vieyra, S., Fornoni, J., Klin- genberg, C.P., Cocucci, A.A. (2016): Exploring the ontogenetic scaling hypothesis during the diversifica- tion of pollination syndromes in Caiophora (Loasace- ae, subfam. Loasoideae). Ann. Bot. 117: 937-947. Trueb, L. (1993): Patterns of cranial diversity among the Lissamphibia. In: The skull: patterns of structural and systematic diversity, pp. 255-343. Hanken, J., Hall, B.K., Eds, University of Chicago Press, Chicago. Webster, M., Sheets, H.D. (2010): A practical introduc- tion to landmark-based geometric morphometrics. Paleontol. Soc. Pap. 16: 163-188. Zelditch, M.L., Swiderski, D.L., Sheets, H.D., Fink, W.L. (2004): Geometric morphometrics for biologists: a primer. Elsevier/Academic Press, Amsterdam. Acta Herpetologica Vol. 14, n. 2 - December 2019 Firenze University Press Podarcis siculus latastei (Bedriaga, 1879) of the Western Pontine Islands (Italy) raised to the species rank, and a brief taxonomic overview of Podarcis lizards Gabriele Senczuk1,2,*, Riccardo Castiglia2, Wolfgang Böhme3, Claudia Corti1 Substrate type has a limited impact on the sprint performance of a Mediterranean lizard Pantelis Savvides1,*, Eleni Georgiou1, Panayiotis Pafilis2,3, Spyros Sfenthourakis1 Coping with aliens: how a native gecko manages to persist on Mediterranean islands despite the Black rat? Michel-Jean Delaugerre1,*, Roberto Sacchi2, Marta Biaggini3, Pietro Lo Cascio4, Ridha Ouni5, Claudia Corti 3 PIT-Tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian Trogonophis wiegmanni Pablo Recio, Gonzalo Rodríguez-Ruiz, Jesús Ortega, José Martín* Occurrence of Batrachochytrium dendrobatidis in the Tensift region, with comments on its spreading in Morocco Ait El Cadi Redouane1, Laghzaoui El-Mustapha1, Angelica Crottini2, Slimani Tahar1, Bosch Jaime3,4, EL Mouden El Hassan1,* Hematological parameters of the Bolson tortoise Gopherus flavomarginatus in Mexico Cristina García-De la Peña1,*, Roger Iván Rodríguez-Vivas2, Jorge A. Zegbe-Domínguez3, Luis Manuel Valenzuela-Núñez1, César A. Meza Herrera4, Quetzaly Siller-Rodríguez1, Verónica Ávila-Rodríguez1 Ontogenetic and interspecific variation in skull morphology of two closely related species of toad, Bufo bufo and B. spinosus (Anura: Bufonidae) Giovanni Sanna Visible Implant Alphanumeric (VIA) as a marking method in the lesser snouted treefrog Scinax nasicus Andrea Caballero-Gini1,2,3,*, Diego Bueno Villafañe2,3, Lía Romero2, Marcela Ferreira2,3, Lucas Cañete4, Rafaela Laino2, Karim Musalem2,5 Morphological variation of the newly confirmed population of the Javelin sand boa, Eryx jaculus (Linnaeus, 1758) (Serpentes, Erycidae) in Sicily, Italy Francesco P. Faraone1,*, Salvatore Russotto2, Salvatore A. Barra3, Roberto Chiara3, Gabriele Giacalone4, Mario Lo Valvo3 Variability in the dorsal pattern of the Sardinian grass snake (Natrix natrix cetti) with notes on its ecology Enrico Lunghi1,2,3,4,*, Simone Giachello5, Manuela Mulargia6, Pier Paolo Dore7, Roberto Cogoni8, Claudia Corti1 Estimating abundance of the Stripeless tree-frog Hyla meridionalis by means of replicated call counts Federico Crovetto, Sebastiano Salvidio, Andrea Costa* AT-rich microsatellite loci development for Fejervarya multistriata by Illumina HiSeq sequencing Yan-Mei Wang, Jing-Yi Chen, Guo-Hua Ding*, Zhi-Hua Lin