Pizzigalli, C., Banfi, F., Ficetola, G.F., Falaschi, M., Mangiacotti, M., Sacchi, R., Zuffi, M.A.L., Scali, S

applyparastyle “fig//caption/p[1]” parastyle “FigCapt”

Biological Journal of the Linnean Society, 2020, 130, 345–358. With 6 figures.

Eco-geographical determinants of the evolution of Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020 ornamentation in vipers

CRISTIAN PIZZIGALLI1, FEDERICO BANFI2, GENTILE FRANCESCO FICETOLA3,4, MATTIA FALASCHI3, MARCO MANGIACOTTI5, ROBERTO SACCHI5, , MARCO A.L. ZUFFI6, and STEFANO SCALI7,*,

1CIBIO/InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto, Vairão, Portugal 2Laboratory of Functional Morphology, Department of Biology, University of Antwerp, Wilrijk, Belgium 3Dipartimento di Scienze e Politiche Ambientali, Università degli Studi di Milano, Milano, Italy 4Université Grenoble Alpes, CNRS, Université Savoie Mont Blanc, LECA, Laboratoire d’Ecologie Alpine, Grenoble, France 5Dipartimento di Scienze della Terra e dell’Ambiente, Università degli Studi di Pavia, Pavia, Italy 6Museo di Storia Naturale, Università di Pisa, Calci (Pisa), Italy 7Museo di Storia Naturale, Milano, Italy

Received 23 December 2019; revised 27 February 2020; accepted for publication 27 February 2020

Multiple hypotheses have been proposed to explain the variation of dorsal patterns observed in snakes, but no studies yet have tested them over broad taxonomic and geographical scales. The Viperidae offer a powerful model group to test eco-evolutionary processes that lead to disruptive and cryptic ornaments. We developed a database reporting dorsal ornamentation, ecological habitus, habitat features and climatic parameters for 257 out of 341 recognized species. Three patterns of dorsal ornamentation were considered: “zig-zag”, “blotchy” and “uniform” patterns. Phylogenetic comparative analyses were based on 11 mitochondrial and nuclear genes. Forty-eight species presented a zig-zag pattern type, 224 a blotchy pattern type and 32 a uniform pattern type. All the patterns showed a strong phylogenetic signal. Character phylogenetic reconstruction analyses suggested an ancestral state for blotchy ornamentation, with multiple independent evolutions of the other patterns. The blotchy pattern was more frequent in terrestrial species living in warm climates and sandy habitats, supporting the hypothesis of a disruptive function. The zig-zag pattern evolved independently in several isolated taxa, particularly in species living in cold climates and in dense vegetation or water-related habitats, supporting the hypothesis of disruptive and aposematic functions. Uniform coloration was particularly frequent in arboreal species, supporting the hypothesis of a cryptic function.

ADDITIONAL KEYWORDS: blotchy pattern – dorsal ornamentation – ecological correlates – phylogenetic- supported characterization – uniform pattern – Viperidae – zig-zag pattern.

INTRODUCTION (Pérez i de Lanuza & Font, 2007; Svensson et al., 2008). Furthermore, mimetic coloration, camouflage Coloration and patterns are among the most intriguing and disruptive patterns reduce the detectability of phenomena in biology, involving both plants and both predators and prey (De Bona et al., 2015; Morris animals. Flowering plants, for instance, show a great & Reader, 2016), whereas conspicuous coloration alerts variability of coloration, often associated with odours, to toxicity or unpalatability (aposematism) (Kraemer flavours and attractive chemicals for pollinator insects et al., 2015; Cuthill et al., 2017). Many studies have (Lev-Yadun & Ne’eman, 2012; Erbar et al., 2017). In evaluated correlations between ornamentations, animals, coloration can provide information about colorations, ecological variables and phylogeny across health status (Halliday et al., 2014; Trigo & Mota, taxa of terrestrial vertebrates (Poulton, 1890; Cott, 2015; Sepp et al., 2018) and reproductive condition 1966; Waage, 1981; Endler, 1990; Krebs, 1994). These *Corresponding author. E-mail: [email protected] studies highlighted that the evolution of coloration

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 130, 345–358 345 346 C. PIZZIGALLI ET AL. and patterns has been driven by the interplay exists, with some species exhibiting all of the zig-zag, between aposematism, crypsis, sexual selection, blotchy and uniform patterns (Fig. 1). The efficiency physiological constraints and social selection (Cuthill of the different patterns is expected to vary according et al., 2017), confirming that colour patterns and to the environmental circumstances. A uniform

colour polymorphism are associated with speciation coloration, for instance, can be more cryptic in Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020 dynamics (Arbuckle & Speed, 2015). canopies with a homogeneous background coloration, Vipers (family Viperidae) are an excellent model whereas a disruptive pattern can favour camouflage group to test evolutionary processes related to in ecotones with a mottled background. Eco- disruptive and cryptic patterns. Vipers are widespread geographical variables provide a broad-scale picture of and well known from a morphological, ecological, the habitat variation and species activity conditions, molecular and phylogenetic point of view and multiple which in turn, can affect the relative efficiency of mechanisms have been suggested for the evolution different pattern types. However, no analyses have of their coloration patterns. First, vipers are a clade yet tested the relationships between colour patterns of highly venomous snakes, thus recurrent highly of snakes and eco-geographical variables over broad detectable colours or patterns may be examples of taxonomic and geographical scales. In this study, we aposematic coloration or of Müllerian mimicry (Wüster performed extensive bibliographical research to collect et al., 2004; Valkonen et al., 2011a, b; Santos et al., distributional, ecological, behavioural and bioclimatic 2014). Harmful species, however, do not necessarily information for most of the recognised viper species. show bright colours in order to reduce the risk of being We then used exhaustive phylogenetic data (Alencar detected (Sherrat & Betty, 2003; Endler & Mappes, et al., 2016) to reconstruct the evolution of different 2004). In fact, some patterns (e.g. the zig-zag dorsal dorsal patterns and to identify the eco-geographical pattern of many vipers) can be examples of Müllerian factors related to the occurrence of dorsal patterns in mimicry that allow vipers to be identified as dangerous, vipers. without increasing their detectability (Wüster et al., 2004; Valkonen et al., 2011a, b). In addition, ambush hunting and predation avoidance have been proposed MATERIALS AND METHODS as drivers of the evolution of cryptic colorations and disruptive patterns (Cott, 1966; Ruxton et al., 2004; Data collection del Marmol et al., 2016). Disruptive patterns can be Data were acquired from both the literature and online achieved through colorations with complex (either sources (Supporting Information, Appendix S1) and regular or irregular) patterns. Such combinations then used to create a database with morphometric, decrease the detectability of an individual even if the ecological and zoogeographical information coloration of the body does not perfectly match the representing all the 341 recognised species of vipers environment (e.g. Bitis nasicornis, Bitis gabonica) (Uetz & Hošek, 2017). For each taxon, we collected (Stevens & Merilaita, 2009a, b). the following variables: dorsal patterns, ecology and Several studies observed a correlation between viper habitat, and climatic variables. colour, pattern and behaviour (Jackson et al., 1976; Allen et al., 2013). Many species displaying uniform and/or stripe coloration have limited defensive abilities Dorsal patterns and high escape capacity (Jackson et al., 1976), Dorsal patterns were classified into three main because a moving striped object can create either a categories: zig-zag, blotchy and uniform (Fig. 1). “Zig- “barber pole effect” or a “flicker-fusion effect”, giving zag” is a mostly continuous linear motif characterised the perception of a uniform pattern during motion by a sequence of small corners, roughly rounded, with that may confuse a potential predator and increase the variable inclinations. The “blotchy” category included escaping probability (Jackson et al., 1976; Lindell & species with regularly repeated motifs such as bars, Forsman, 1996; Allen et al., 2013). Conversely, snakes blotches, circles, ovals and transversal stripes. Lastly, with bright colours and/or blotched designs are usually “uniform” indicates patterns that do not show regular more inclined to fight (Jackson et al., 1976; Clark, 2006; motifs. In several cases, one single species can show Allen et al., 2013), even if it is unclear whether the multiple dorsal patterns (see Results). For instance, efficiency of a bar-like pattern may serve as an anti- Vipera aspis displays a high variability of patterns predatory diversion or not (Lindell & Forsman, 1996). among subspecies, and the three patterns are present Vipers present a striking variability of dorsal in this species (Fig. 1; Zuffi & Bonnet, 1999). Therefore, ornamentations. Some species show a motif that is for each species, we recorded the presence/absence regularly repeated several times on the body surface of the three distinct patterns, where every species (e.g. zig-zag or blotchy pattern) whereas others show a can have more than one character state. Melanistic uniform coloration. In addition, intraspecific variation individuals were not considered; due to possible motif

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 130, 345–358 ORNAMENTATION IN VIPERS 347 Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020

Figure 1. Example of intraspecific polymorphism in V. aspis, on the top a concolor individual displaying uniform pattern coloration, in the middle an example of blotchy pattern (bars in this case) and on the bottom a zig-zag pattern-like coloration. Credit to Matteo Di Nicola (http://www.matteodinicola.it/).

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 130, 345–358 348 C. PIZZIGALLI ET AL. changes during species’ ontogenesis (da Silva et al., mapping (Revell, 2013). For all the resulting trees, we 2017), we only considered the features of adults. showed character state probabilities on both nodes and along branches. In large phylogenetic trees, the rate of trait evolution can differ significantly among lineages Ecology and habitat (Beaulieu et al., 2013). We therefore used the Beaulieu Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020 The ecological habitus of each species was coded as et al. (2013) approach to compare a time-homogeneous a semi-quantitative variable (strictly ground living: model of trait evolution, with models assuming two 0; semi-arboreal: 0.5; arboreal: 1). Furthermore, we or more hidden rates. For the three considered traits, identified the habitat typologies where each species the time-homogeneous model always showed lower can be present [dummy variables: sandy areas, rocky Akaike’s Information Criterion corrected (AICc) for areas, open vegetated areas (e.g. grasslands, croplands, limited sample size than the models with hidden meadows, etc.), forests, water-associated (i.e. living in rates, therefore we assumed homogeneous evolution riparian or moist areas)]. across the tree. Stochastic reconstruction of character states was first performed for the three patterns separately (presence-absence of blotchy, zig-zag and Climatic variables uniform patterns). Furthermore, we used a Markov For each species, we calculated average values of model (i.e. a model of trait evolution and ancestral mean annual temperature and total precipitation. states reconstruction for discrete states) to analyse the Climatic parameters were calculated as the average three patterns in the same model. We used the make. value through the whole species range. Ranges were simmap function in phytools to perform stochastic obtained from Roll et al. (2017); climatic values were mapping analysis (1000 replicates; Revell, 2013). For obtained from the CRU TS v.4.01 (updated from polymorphic species, we assumed that the multiple Harris et al., 2014). Because no distribution map was states of the pattern have the same prior probability. available for Crotalus ornatus, we used the centroid We then reconstructed state evolution through the of the range as described in the Reptile Database to describe.simmap in phytools (Revell, 2013). extract climatic values (Uetz & Hošek, 2017). The We used D statistics (Fritz et al., 2010) to measure correlation between variables was generally weak (for the phylogenetic signal of dorsal patterns. D statistics all pairwise correlations, |r| ≤ 0.6), suggesting that is appropriate to measure phylogenetic signal collinearity between independent variables did not for discrete traits. The value D = 1 indicates no bias the results of the regression analyses (Dormann phylogenetic signal, whereas D values close to zero or et al., 2013). Although the average conditions across lower suggest very strong signal (Fritz et al., 2010). the range may not represent the full conditions We used 5000 random permutations to assess whether experienced by the species, they provide excellent D is significantly different from the values expected information on the ecogeographical factors driving the under no phylogenetic structure. evolution of species, when the climate of exact localities Subsequently, we used phylogenetic logistic is not available (e.g. Stark & Meiri, 2018). regression to identify the eco-geographical parameters related to the evolution of dorsal patterns (Ives & Garland, 2010) using the Alencar et al. (2016) tree to Data analysis consider the evolutionary history. We used a model- For phylogenetic comparative analyses, we used the selection approach, based on AICc to identify the calibrated tree based on 11 mitochondrial and nuclear combination of variables best explaining the occurrence genes by Alencar et al. (2016). The time-tree included all of the three dorsal patterns. First, we built regression the taxa for which we obtained pattern and ecological models including all the possible combinations of the variables, and was pruned to match the list of species considered variables and calculated the AICc of each with available data. We used stochastic reconstruction model. AICc trades off explanatory power vs. number of character states in order to assess the evolution of predictors; models explaining more variation with of dorsal patterns along the phylogeny. Stochastic a limited number of variables have the lowest AICc character mapping is a technique where possible values and are assumed to be the “best models” histories of characters are sampled in proportion (Symonds & Moussalli, 2011). We then calculated to their probability. Starting from the topology of Akaike’s weight (w) of each model, which infers the the Alencar et al. (2016) time-tree, we generated likelihood that a model is the best one given a set 1000 random simulations of a stochastic process of of candidate models (Burnham & Anderson, 2002; the character state, across the branches of the tree. Symonds & Moussalli, 2011). We also tested the The posterior probability of stochastically mapped possibility of non-linear relationships, assessing characters was plotted on the phylogeny, to provide quadratic terms of continuous variables included in a character state reconstruction via stochasticity highly supported models. Finally, we calculated the sum

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 130, 345–358 ORNAMENTATION IN VIPERS 349 of weight of each variable, as the sum of the Akaike’s schweizeri, Atheris ceratophora, Atheris chlorechis, w where each variable appears. The sum of weights is Causus resimus, Tropidolaemus subannulatus, a measure of the relative importance of variables and Bothriechis guifarroi, Bothriechis lateralis, Bothriechis can be used when model selection reveals uncertainty schlegelii and Bothrops bilineatus. In each of these

in the identification of best model(s). The significance species, the character evolved independently (Fig. 4). Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020 of variables within the best AICc models was assessed The stochastic reconstruction of the three pattern using likelihood ratio tests. Analyses were performed states in the same model confirmed the blotchy pattern using the packages ape (Paradis et al., 2004), corHMM as the ancestral state, followed by multiple transitions (Beaulieu et al., 2013), phytools (Revell, 2012), maps (Fig. 5). The model suggested that the uniform pattern (Brownrigg, 2018), raster (Hijmas & van Etten, 2012), evolved from the blotchy pattern six times, whereas ggplot2 (Wickham, 2016), caper (Orme, 2013), MuMln the zig-zag pattern evolved from the blotchy pattern (Bartoń, 2015) and phylolm (Ho & Ane, 2014) in R five times. Furthermore, several reversals occurred, v.3.3 (R Core Team, 2017). particularly from the uniform to the blotchy pattern (14 transitions), whereas reversals from zig-zag to blotchy were rare (two transitions; Fig. 5). RESULTS Overall, we obtained complete information for 257 species. Forty-five species presented more than one ECO-GEOGRAPHICAL DETERMINANTS OF dorsal pattern (e.g. in several instances the blotchy DORSAL PATTERNS and zig-zag pattern occurred in individuals of the same species). All the patterns showed a strong Blotchy pattern phylogenetic signal, with a particularly strong signal The best AICc phylogenetic regression model suggested for the zig-zag and blotchy ornamentations (zig-zag: that the blotchy pattern was related to climate, D = -0.20; blotchy: D = 0.02; uniform: D = 0.28); in all species habitus and habitat (Table 1a). Blotches cases, the D values indicated a phylogenetic signal were particularly frequent in ground-living species 2 stronger than expected from random phylogenetic (χ 1 = 26.0, P < 0.0001) (Fig. 6b), in species living in 2 structure (all P < 0.0001). The blotchy pattern was sandy areas (χ 1 = 11.5, P = 0.0007) (Fig. 6c) and in warm 2 the most widespread pattern among vipers, being climates (χ 1 = 7.5, P = 0.006) (Fig. 6d). An alternative recorded in 224 species. The Trimeresurus and Vipera model, with very similar AICc values, included annual genera showed mainly the uniform coloration and the precipitation instead of sandy habitat and confirmed zig-zag ornamentation type, respectively. Character the high frequency of this pattern in species living in 2 phylogenetic reconstruction analyses suggested arid areas (χ 1 = 12.1, P = 0.0005). Ecological habitus and an ancestral state for blotchy ornamentation with association with mean temperature were the variables multiple independent evolution of both of the other with the highest relative importance (Table 2). two types of dorsal patterns (Figs. 2–4). The zig-zag pattern was present in 48 species. The character mainly occurred in the Montivipera- Zig-zag pattern Macrovipera-Daboia-Vipera clade, in the Mixcoatlus- The best AICc model suggested that the zig-zag pattern Ophryacus clade and in both the Atheris and was particularly frequent in species living in cold 2 Cerrophidion genera with a few exceptions (Vipera climates (χ 1 = 15.2, P < 0.0001) (Fig. 6a). The zig-zag transcaucasiana, Daboia deserti, Daboia siamensis, pattern tended to be more frequent in species living 2 Macrovipera schweizeri, Montivipera latifi, in water-related habitats (χ 1 = 2.5, P = 0.12) and was Montivipera bornmuelleri, Montivipera albizona, slightly less frequent in species living in open habitats 2 Atheris squamigera and Atheris chlorechis). According (χ 1 = 3.75, P = 0.053); however, these variables were to the character state reconstruction analysis, the not significant at the 5% level. The mean temperature trait evolved independently also in several isolated was the variable with the highest relative importance taxa throughout both the Viperinae and Crotalinae to explain the occurrence of this pattern (Table 2). sub-families: Pseudocerastes fieldi, Cerastes vipera, Echis pyramidum, Bothrops pictus, Atropoides occiduus, Atropoides nummifer, Trimeresurus gracilis, Uniform pattern Protobothrops elegans, Protobothrops sieversorum and According to the best AICc model, uniform coloration Protobothrops kaulbacki (Fig. 3). was particularly frequent in arboreal species 2 The uniform pattern was present in 32 species. This (χ 1 = 28.3, P < 0.0001) (Fig. 6e and Table 1c). None of pattern type mostly occurred in Asiatic taxa of the genus the remaining variables were included in models with Trimeresurus (Fig. 4). It also occurred in Macrovipera AICc weight > 0.01, indicating ecological habitus as

Por

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i Bothrops dipor i ajoensis

h Bothrops lutzi h yi

t t thidium lansbergii thidium arcosae thidium yucatanicum nni

r r Bothrops bar r er

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P r P baeus Por

Bothrops insular us Por Po arroi Atropoides picadoi vir Cerrophidion petlalcalensis Cerrophidion tz Cerrophidion godmani iechis schlegelii Cerrophidion sasai Atropoides me x Atropoides ol iechis supracilliar Bothrops ammodytoides Atropoides n iechis bicolor Atropoides occiduus iensis Atropoides indomitus iechis marchi BothropsBothropsBothrops alter f cotiara Ophr Mixcoatlus barbour iechis thalassinu Bothrocophias campbelli Mixcoatlus bro iechis ro Mixcoatlus melanur vicaudus Bothrocophias microphthalmus us Bothr iechis aur Bothr iechis later netti Bothr Bothr iechis guif lomhoffi Bothrocophias hy Bothr iechis nigro rmedius Bothrops pictus Bothr r onsecai Bothr Bothrops lojanus Bothr ydius ussur is Bothr o ydius bre Agkistrodon conto natus Both ydius b Gl ydius tsushimaensis Agkistrodon bilineatus Glo AgkistrodonAgkistrodon ho Glo ydius inte ensis iridis Glo ydius halys us gracilisav Glo ydius shedaoensis oprora Glo ydius saxatilis avov Glo ydius strauchi AgkistrodonAgkistrodon pisci t Glo nensis ru Gloimeresur wardglosseolusrtr Tr ophis okin ix Ov Crotalus polystictus Protobothrops tokarensis Crotalus ena ydi Protobothrops fl is Crotalus cerastesvoylo ri Protobothrops mucrosquamatusutus ru Protobothrops elegansrn s Protobothrops maolanensis m CrotalusCrotalus pusillus r yo Protobothrops dabiesharsoru Crotalus tr Protobothrops xiangchengensis av Protobothrops jerdonii Crotalusiser lepidusus Protobothrops co Crotalus aquilusiatus Protobothrops mangshanens Crotalus er Protobothrops sieve Protobothrops kaulbacki a Crotalus lannoicsmithi Ovophis tonkinensisyuensis Crotalus stejneger Ovophis monticola Crotalus horr mi Ovophis za Calloselasma rhodostom Crotalus or idusi Hypnale nepahypnale Crotalus totonacusnatus Hypnale a nulatus Crotalus basiliscus Hypnale zar Tropidolaemus subanwagleri Crotalus molossus Tropidolaemus Crotalus culminatus Deinagkistrodon acutus Crotalus du Garthius chaseni rissus Azemiops feae Crotalus simus Bitis xeropaga Crotalus tzcaban Bitis rubida Crotalus willardi Bitis corn Bitis ar uta Crotalus catalinensisx Bitis atroposmata Crotalus atrouber Bitis caudalis Crotalus r idis Bitis schneideri Crotalus vir us Bitis per us Bitis rhinocerosingu Crotalus oregan Bitis gabonicaeyi Crotalus cerber is Bitis nasicor Bitis par CrotalusCrotalus scutulatus tigr Bitis wo Bitis ar vioculanis Crotalus mitchelliiicei Proatherrthingtoni Causus ietansdefilippii CrotalusCrotalus adamanteus prersus Causus rhombeatus Causus resimis supercilia mediusius Causus lichtensteinii Crotalus tancitarensis Ather Crotalus transvus miliar Athe ris ur Ather is chlorechis CrotalusSistr interus catenatus ys Ather r us ur Ather is squamigera Sistr Ather is hispida Lachesis muta is Ather is desaixi Lachesis acrochorda Athe is nitschei bularum Montiviperais ce xanthina Montiviperais matildae w rus tibetanus Montiviperar albi Lachesis stenophrus ne Montiviperais bor barbouratophorri Montivipera us popeiorus fucatus Montivipera latifii Lachesisimeresu melanocephalausus sabahi baratiuniana Macro Tr ogeli Macr a imeresur i Daboia siamensis Tr us b Daboia palaestinae imeresurimeresur us v Daboia deser Tr imeresur Daboia maur agne Tr imeresur Vipe ovipevipera lebetin Tr Vipera transcaucasiana Tr Vipera latastei r z ri us sichuanensis is Vipera aspis addeinmuelleona imeresur uongsonensis Vipera anatolica is i Vipera renardi ra sch Tr imeresur Viper r us gumprechtius stejneger iensis Vipera dinniki a ammodytes r Vipera er Tr Vipera ursinii us tr u us Vipera or Vipera kazna us macrops Vipera seoanei imeresur i Vipera barani ri us yunnanensisus medoensis Viper w us venustusasciatus Er Tr Pseudocer Pseudoce ti eiz a Pseudocer a loti Cerastes vipera ei Cerastes ce itanica us insular Cerastes gasperetti Echis ocellatus Echis joger imeresur r Echis khosatzkii imeresur E er imeresurTr us f us cantorythrur ionalis isticophis macmahoni Tr us kanb us albolabr c i

Tr h e imeresur a be imeresurimeresur icus i i w vi Tr imeresur s l Tr Tr us er ovi anensis neensis inatus

Tr imeresu us andersoniius malcolmi b pureomaculatus omaculatus us wiroti r us hageni ru Tr imeresurimeresur o us schultz ramineus imeresur av us mcgregor ramidum r rastes persicus astes fieldi s ko Tr imeresur k Tr Tr astes u us septentr us sumatranus us puniceus i py Tr n i vi us bor r astes us pur us fl us g i r imeresur r igonocephalus imeresu imeresur Tr us malabar Echis car imeresur Echis coloratus Tr Tr imeresur

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Figure 2. Ancestral character state reconstruction of the blotchy pattern along the branches of the phylogenetic tree of the Viperidae. Red indicates a high posterior probability of the occurrence of the blotchy pattern within a clade, blue indicates low probability of occurrence, and pink indicate incertitude. The phylogeny is from Alencar et al. (2016). the most important variable to explain the occurrence multiple factors can jointly determine the evolution of of this pattern (Table 2). a certain pattern, predominantly climatic conditions and lifestyle. We observed a strong phylogenetic signal, with highly conserved basal character (blotchy) from which different patterns arose independently multiple DISCUSSION times in different areas of the world. In most of the This study represents the first global scale cases, the evolution of the new patterns corresponded characterisation of dorsal ornamentations for the to a loss of the ancestral state (e.g. the green concolor family Viperidae. Our analyses clearly show that pattern in most of the Trimeresurus species and the

Por

thidium oph Bothrops chloromelas

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i Bothrops diporus i

h Bothrops lutzi h yi

t t r thidium lansbergii thidium arcosae

r azili r Bothrops bar rthidium yucatanicum er

rn o Bothrops itapetiningaeBothrops alcatraz ibbaeus o acus undulatus ythromelas moratus ra wle if Bothrops jararaca y alis idis

P rus P ei Por Bothrops insular Por Po arroi Atropoides picadoi vir Cerrophidion petlalcalensis Cerrophidion tz f Cerrophidion godmani iechis schlegelii Cerrophidion sasai Atropoides me x Atropoides ol iechis supracilliar Bothrops ammodytoides Atropoides n iechis bicolor Atropoides occiduus iensis Atropoides indomitus iechis marchi BothropsBothropsBothrops alter f cotiara Ophr Mixcoatlus barbour iechis thalassi Bothrocophias campbelli Mixcoatlus bro iechis ro Mixcoatlus melanur vicaudus Bothrocophias microphthalmus Bothr iechis aur Bothr iechis later netti Bothr Bothr iechis gui lomhoffi Bothrocophias Bothr iechis nigro rmedius Bothrops pictus Bothr onsecai Bothr Bothrops lojanus Bothr ydius ussur is Bothr o ydius bre Agkistrodon contor natus Bothr ydius b Gl ydius tsushimaensis Agkistrodon bilineatus Glo AgkistrodonAgkistrodon ho Glo ydius inte ensis ridis Glo ydius halys us gracilis vi hy Glo ydius shedaoensis vo oprora Glo ydius saxatilis Glo ydius strauchi AgkistrodonAgkistrodon pisciv t Glo nensis ru Gloimeresur wa sseolustr Tr ophis okinav rdglo ix Ov Crotalus polystictus Protobothrops tokarensis Crotalus enayloydi Protobothrops fla is Crotalus cerastes Protobothrops mucrosquamatusutus or ri Protobothrops elegans Crotalus ra us Protobothrops maolanensis rum Crotalus pusillus yo Protobothrops dabieshaerso Crotalus tr Protobothrops xiangchengensis vus Protobothrops jerdonii Crotalusiser lepidus Protobothrops corn Crotalus aquilusiatus Protobothrops mangshanens Crotalus er Protobothrops siev Protobothropsophis tonkinensis kaulbacki Crotalus lannoicsmithi Ov ayuensis Crotalus stejneger Ovophis monticola Crotalus horr mi Ovophis z Calloselasma rhodostoma Crotalus or idusi Hypnale nepaypnale Crotalus totonacusnatus Hypnale h a nulatus Crotalus basiliscus Hypnale zarmus suban Tropidolae wagleri Crotalus molossus Tropidolaemus Crotalus culminatus Deinagkistrodon acutus Crotalus du Garthius chaseni rissus Azemiops feae Crotalus simus Bitis xeropaga Crotalus tzcaban Bitis rubida Crotalus willardi Bitis corn Bitis ar uta Crotalus catalinensis Bitis atroposmata Crotalus atruberox Bitis caudalis Crotalus r dis Bitis schneide Crotalus viri Bitis per us Bitis rhinocerosingu ri Crotalus oreganus Bitis gabonicaeyi Crotalus cerber is Bitis nasico Bitis par CrotalusCrotalus scutulatus tigr Bitis rnis cei Bitis arwo viocula Crotalus mitchelliiri Proatherrthingtoni Causus ietansdefilippii CrotalusCrotalus adamanteus persus Causus rhombeatus Causus resimis superciliar mediusius Causus lichtensteinii Crotalus tancitarensis Ather Crotalus transvus miliar Athe is ur Ather is chlorechis CrotalusSistr interus catenatus ys Athe r us ur Athe is squamige Sistr Ather ri is hispida Lachesis muta ris Ather s desaixi Lachesis acrochorda Athe ris nitschei ularum Montiviperais ceratophor xanthin ra Montiviperis matildae us tibetanus Montiviperar albiz Lachesis stenophrus neb Montiviperais bor barbouri Montivipera raddei us popeious fucatus Montivipera latifii Lachesisimeresur melanocephalausus sabahi baratiuniana Macr Tr r ogeli Macr a imeresur i Daboia siamensis a Tr us b Daboia palaestinae w imeresurimeresur us v Daboia deser Tr imeresur Daboia maur ov agner Tr imeresu Vipe ovipera schweiz Tr Vipera transcaucasianaiper a Tr Viper us sichuanensis is Vipera aspis nmuellerona i imeresur uongsonensis Vipera anatolica a lebetina is Vipera renardi Tr imeresur ri Vipera loti r us gumprechtius stejneger nustusiensis r Vipera dinnik a ammodytes r Vipera Tr Viper us tr u us Vipera or a latastei Vipera kaznak us macrops Viper imeresur Viper us yunnanensisus medoensis Vipera be i us ve asciatus ri Er Tr Pseudocerastes fieldi Pseudocerastes persicus ti Pseudocer Cerastes vipera ei Cerastes ce itanica us insular Cerastes gasperettii Echis ocellatus Echis jogeri imeresur r Echis khosatzkii imeresur E a ursinii er imeresurTr us f us cantoythrur ionalis isticophis macmahoni Tr us kanb anus c e us albolab a seoanei i Tr imeresur h a ba r ev imeresurimeresur wiroti icus iw

i Tr imeresur s l i Tr Tr us er ovi anensis neensis inatus

Tr imeresu us andersoniius malcolmi b i pureomaculatus omaculatus rani r rus rus Tr imeresur o imeresur us schultz imeresur us mcgrego ramidum r

Tr imeresur r k Tr Tr astes u o us septentr us sumatr us puniceus i py Tr n vi us bor r astes us pur us flav us gramineus i imeresur r igonocephalus imeresur imeresu Tr us malabar Echis car imeresu Echis coloratus Tr Tr imeresurus hageni

imeresu imeresur Tr Echis omanensis r Tr Echis us tr arachnoides

Echis leucogaster imeresur Tr Tr imeresur imeresur imeresur Tr imeresu imeresur Tr Tr Tr imeresur 0PP(state=1)1 Tr Tr

Tr imeresur

imeresur length=24.835 Tr

Figure 3. Ancestral character state reconstruction of the zig-zag pattern along the branches of the phylogenetic tree of the Viperidae. Red indicates a high posterior probability of the occurrence of the blotchy pattern within a clade, blue indicates low probability of occurrence, and intermediate colours indicate incertitude. zig-zag pattern in Vipera). However, there are also by their dorsal patterns (Slowinski, 1995; Gutberlet multiple cases of intraspecific variability (species in & Harvey, 2004; Marques et al., 2013; Jowers et al., which a new pattern arose, but the blotchy remains, 2019): the monadal pattern (one black ring between e.g. V. aspis see Zuffi & Bonnet (1999)). Such complex two white or yellow annuli separated by red annuli), evolution of colour patterns along the phylogeny the triadal pattern (three black rings), and a bicolour has been observed in multiple snake lineages. For coloration. Jowers et al. (2019) confirmed that there instance, coral snakes (Micrurus), include distinct is a phylogeographic explanation behind the evolution phylogenetic lineages that can be also distinguished of the two predominant colour patterns in these

t Bothrops chloromelas

um Bothrops ja i Po

Bothrops bilineata i

Bothrops punctatus u Bothrops marajoensis Bothrops taeniata rt Porthidium dunni s

m Bothrops pulchra

Bothrops osbor hidium hespere a u er

r BothropsBothrops leucu moojeni s otzilor r Bothrops lanceolatus Bothrops brazili o

a Bothrops caribbaeus o Bothrops marm p i xicanus is

n p

h Bothrops er e rus BothropsBothrops asper atro r y ummif

m Bothrops neuwiedi m r

o nu aracussu u u wni

i i Bothrops pubescens m

d d racilliar nus

i i Bothrops Bothropspauloensis dipo e p

h h Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020

t Bothrops lutzi t thidium lansbergii thidium arcosae thidium yucatanicum yi

r r a r

o o Bothrops bar s acus undulatus er Bothrops alcatraz ythromelas nei P y Bothrops itapetiningae r P wle if Po idis Bothrops jararaca Por Por us Atropoides picadoi oratus Cerrophidion petlalcalensis Cerrophidion tz Bothrops insula Cerrophidion godmani Cerrophidion sasai arroivir Atropoides m iechis schlegelii Atropoides olmec x Atropoides n iechis su Atropoides occiduus iechis bicolor Bothrops ammodytoides Atropoides indomitus Ophr iechis marchi iensis Bothrops cotiara Mixcoatlus barbour iechis thalassi BothropsBothrops alter f Mixcoatlus bro ensis ru Mixcoatlus mela iechis ro Bothrocophias campbelli Bothr iechis aur Bothr vicaudus Bothrocophias microphthalmus netti s Bothr iechis lateralis Bothr iechis guif Bothr iechis nigrolomhoffimedius Bothrocophias hy Bothr Bothrops pictus Bothr onsecai r Bothr ydius ussur Bothrops lojanus is Bothr ydius bre natus Bothr ydius b Agkistrodon contor Glo ydius tsushima Agkistrodon bilineatus Glo o Glo ydius inter idis AgkistrodonAgkistrodon h r Gl ydius halys ensis Glo ydius shedaoensisus gracilisav vir oprora Glo ydius saxatilis vo Glo ydius strauchi Glo AgkistrodonAgkistrodon pisciv ta imeresur ow usseolus Glo ophis okin trix Tr ardglo Ov abieshanensis Crotalus polystictus Protobothrops tokarensis Crotalus e ydi Protobothrops fla Crotalus cer ylor Protobothrops mucrosquamatus oru i Protobothrops elegansnutus Crotalus ny s Protobothrops maolanensis m Crotalus pusillus o Protobothrops d Crotalus t astes Protobothrops xiangchengensiseversoru ravus Protobothrops jerdonii Crotalusrise lepidus Protobothrops cor Crotalus aquilusriatus Protobothrops mangshanensis Crotalus er Protobothrops si Protobothropsophis tonkinensis kaulbacki Crotalus lannomiicsmithi Ov yuensis Crotalus stejneger Ovophis monticolaa Ovophis z Crotalus horri Calloselasma rhodostoma Crotalus or dusi Hypnale nepaypnale Crotalus totonacusnatus Hypnale h a Crotalus basiliscus Hypnale zar us subannulatus Tropidolaem agleri Crotalus molossus Tropidolaemus w Crotalus culminatus Deinagkistrodon acutus Crotalus dur Garthius chaseni issus Azemiops feae Crotalus simus Bitis xeropaga Crotalus tzcaban Bitis rubida Crotalus willardi Bitis cornuta Bitis a Crotalus catalinensisox Bitis atroposrmata Crotalus atruber Bitis caudalis Crotalus r ridis Bitis schneider Crotalus vi Bitis pe us Bitis rhinocerosringue i Crotalus oreganus Bitis gabonic yi Crotalus cerber ris Bitis nasicor Crotalus scutulatus Bitis pa a Crotalus tig Bitis nis Bitis arwortrviocula Crotalus mitchelliiicei Proather hingtoni Causus ietansdefilippii CrotalusCrotalus adamanteus prversus Causus rhombeatusis superciliaris Causus resimus rmediusius Causus lichtensteinii Crotalus tancitarensis Ather Crotalus urtransus miliar Athe Crotalus inte muta Athe is chlorechi Sistrurus catenatusr ys Ather ris squamiger Athe ris hispida Sistr us Ather is desaixi Lachesis is Ather r s Lachesis acrochordaular um Ather is nitschei Montiviperis ceratophor a us tibetan Montivipeis matildae Lachesis stenophus neb ati Montiviperais barbouralbiz Montivipera bo us popeiorus fucatus Montivipe Lachesisimeresu melanocephalarr us sabahi Montivipe Tr rus baruniana Macro a xanthina a imeresur ogeli Macro ra wagne Tr ri Daboia siamensis i imeresuimeresur us b Daboia palaestinae Tr Tr imeresur us v Daboia deser Tr imeresu Daboia maur vipe r Tr Vipera ammodytesvipe ra alatifi raddei Viper onar us sichuanensis Vipera latastei rn i imeresur uongsonensis is Vipera aspis ra lebetina muelle Tr r Vipera anatolica ra schweizer imeresuusr gumprechti is Vipera renardi r us stejnege iensis Vipera loti Tr Vipe i us tr Vipera er a transcaucasiana venustusur Vipera ursinii imeresur us macrops us Vipe ri us yunnanensisus medoensis r Viper Tr Vipera seoanei r us asciatus Vipera barani Vipera ber ru ri Er ti Pseudoce us insula Pseudocer r itanica imeresu Pseudoce ei Ce a dinniki imeresur Cerastes cer Cerastes gasperettii Echis ocellatus Echis jogeri imeresurTr us f z Echis khosatzkii

Tr us cantoythri ionalis E r us kanb r isticophis macmahoni a or i Tr r a kaznak

us albolabr c imeresur r imeresur rastes vipe evi imeresur h T imeresur cus iw Tr Tr i ri

s lo Tr us er ratus anensis imeresur neensis us andersoniius malcolmi inatus vi pureomaculatus omaculatus us wiroti b Tr imeresu us hageni imeresu imeresur o us av us mcgregous schult rastes field

Tr r imeresu rastes ur Tr astes persicus Tr k

yramidum o us septentr us puniceus

us sumatranus i Tr vi p us pur us bor n us fl astes r i imeresur us gramineus r igonocephalusr imeresur a Tr imeresur r us malaba imeresur Echis car Tr Tr imeresur Echis colo imeresur imeresur Tr Echis omanensis Tr Echis 0PP(state=1)1 a us t imeresur Echis leucogaster Tr Tr meresur r r imeresur r i imeresur achnoides T imeresu ri imeresur Tr r T Tr r imeresu T T

Tr imeresur

Tr length=24.835 imeresu Tr

Figure 4. Ancestral character state reconstruction of the uniform pattern along the branches of the phylogenetic tree of the Viperidae. Red indicates a high posterior probability of the occurrence of the blotchy pattern within a clade, blue indicates low probability of occurrence, and intermediate colours indicate incertitude. coral snakes, where from a basal triadal pattern the Avoiding detection by visual hunting predators is monadal form evolved in the Middle Miocene and more essential for ground-dwelling species. Previous studies recently also with a bicolour coloration. already assumed the disruptive function of the blotchy pattern, which mimics the dark shadows of the litter- free sand beneath the vegetation (Sherbrooke, 2002). Drivers of the evolution of blotchy patterns These properties of the blotchy pattern result in The evolution of different patterns in vipers was being a particularly effective anti-predator strategy strongly related to habitat and habitus. That is, of defence in ground-dwelling species (Brodie, 1992, each coloration is characteristic of species living 1993) because it allows them to confuse the outlines in specific climatic conditions, habitats or lifestyle. of their bodies with the substratum (e.g. B. gabonica), The blotchy ornamentation appears to be frequent with shrubs and grass stems (e.g. V. aspis) or disguise in ground-dwelling species living at low latitudes in them by blending their dorsal pattern with the environments with warm and arid climates (Fig. 6c-d). shadow created by bushes (Cott, 1966). Moreover,

● Zigzag

● Uniform P

t Bothrops chloromelas

Bothrops jararacussu i

Port ● Blotchy d rum

m Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020

i Bothrops bilineata Bothrops marajoensis Bothrops punctatus u

Bothrops taeniata u Porthidium dunni

m Bothrops osbor Bothrops pulch hidium hespere er

u Bothrops lanceolatusBothrops Bothrops leucur moojeni otzilo Bothrops brazili s Bothrops ma o Bothrops car i is

a p xicanus us

Bothrops er n

Bothrops neuwiediBothrops Bothrops asper atro r

o wni

Bothrops pubescens u nummif

i Bothrops pauloensis m

Bothrops diporus d

i Bothrops lutzi e

t thidium porrasi thidium arcosae eyi thidium yucatanicum

Bothrops bar r rthidium lansbergii r a er Bothrops itapetiningaeBothrops alcat rmoratusibbaeus r acus undulatus ythromelas o wl if idis o Bothrops jararaca nei ra s ir

P rPo Bothrops insula Po P arroi us f Po Atropoides picadoi Cerrophidion petlalcalensis Cerrophidion tz iechis schlegelii Cerrophidion godmani Bothrops ammodytoides Cerrophidion sasai iechis supracilliar x ●●● Atropoides me ●● ●●● Atropoides olmec iechis bicolor iensis ● Atropoides ● BothropsBothropsBothrops alter cotiara ●● ● Atropoides occiduus iechis marchi ● ● Atropoides indomitus Bothrocophias campbelli ●● ● Ophry iechis thalassinus ● Mixcoatlus barbour ● Mixcoatlus bro iechis ro Bothrocophias microphthalmus ● vicaudus ●● ● Mixcoatlus melanur iechis aur Bothr netti ●● ● Bothr iechis lateralis Bothrocophias ● ● ● ● Bothr iechis gui lomhoffimedius ● ● Bothr iechis nigrov Bothrops pictus raz ●● ● ● ● Bothr f ● ●● ● ● Bothr onsecai ● Bothrops lojanus ● ● ● Bothr r ● ●● ● Bothr ydius ussur is ● ● ● ydius bre Agkistrodon contor ● ● ● Bothr natus ● ● ● Bothr ydius b Agkistrodon bilineatus ●● ●● ● Glo ydius tsushimaensis AgkistrodonAgkistrodon ho russeolu ●●●● ●● ● ● ● Glo ydius inter ensis idis ● ● Glo ydius halys us gracilisv vir hy ●● ● ● ● ● Glo ydius shedaoensis vo oprora ● ● ● Glo ydius saxatilis ● ● ● ● Glo ● ● Gloo ydius strauchi AgkistrodonAgkistrodon pisci ta ● ● Gl ● ● ● ● ● Gloimeresur w tr ● ● ● Tr ophis okina ardglo ix ● ● ● ● ●●● Ov CrotalusCrotalus polystictus en ● ● ● ● Protobothrops tokarensis ydis ● ● ● ● ● Protobothrops fla Crotalus cer v ylor ● ● Protobothrops mucrosquamatusnutus orus i ● ● Protobothrops elegans Crotalus ra ● ● ● ● ●● ● Protobothrops maolanensis um Crotalus pusillus yo ●●● ● ● ● ● Protobothrops dabieshanensisversor Crotalus tr astes ● ● ● Protobothrops xiangchengensis vus ● ● ●● ● ● ● Protobothrops jerdonii Crotalusiser lepidus ● ● ● ● Protobothrops cor Crotalus aquilusiatus ● ●● ● ● ● Protobothrops mangshanensis Crotalus er ● ● ● ● Protobothrops sie ● Protobothrops kaulbacki Crotalus lannomiicsmithi ● ● ●●● Ovophis tonkinensisyuensis Crotalus stejneger ● ●● ● Ovophis monticola Crotalus hor ● ● ● Ovophis za ● ●●●● ● ● Calloselasma rhodostoma Crotalus or ridus●i ● ● Hypnale nepa Crotalus totonacusnatus● ● ● ● ● ● Hypnale hypnale Crotalus basiliscus● ● ● Hypnale zara ● ● ● ● Tropidolaemus subannulatuswagleri Crotalus molossus● ● Tropidolaemus Crotalus culminatus● ● ● ● ●● ● Deinagkistrodon acutus Crotalus dur ● ● ● ● ● Garthius chaseni issus● ●●● ● ● ● Azemiops feae Crotalus simus● ● ●● ● Bitis xeropaga Crotalus tzcaban●● ● ● ● Bitis rubida Crotalus willardi● ● ● ● ● Bitis co ● ● ● ● Bitis ar rnuta Crotalus catalinensis●x ● ●● ● Bitis atropomatas Crotalus atrouber● ● ● ● ●● Bitis caudalis Crotalus r idis● ● ●●● ● Bitis schneider Crotalus virnus● ● ● ●● ●●● Bitis pe us● ● ● ● Bitis rhinocerosringue i Crotalus orega ●●●● ● ● Bitis gabonica yi Crotalus cerber is● ● ● ● ●● Bitis nasico ● ● Bitis par CrotalusCrotalus scutulatus tigr ● ● ● ● Bitis ● ● ● Bitis ar viocularnis Crotalus mitchelliiicei● ● ● ●● ● Proatherworthingtoni ● ● ● Causus ietansdefilippii Crotalus prersus● ● ● Causus rhombeatus Crotalus adamanteus ● ● ● Causus resimusis superciliar mediusius● Causus lichtensteinii Crotalus tancitarensis ● ● ● Athe Crotalus transvus miliar ●● ● Ather is ur ● ● ● Ather ris chlorechis CrotalusSistr interus catenatusys ● ● Athe is squamiger ur ● Athe is hispida Sistr ●is Athe ri Lachesis muta r● ● ● ● Athe r s desaixi ulaum● ● ● ● Athe is nitschei Lachesis acrochorda ● ● ● ●● Montivipries ce us tibetanus ● ●●● Montiviperaris matildae w a ● ● ●● Montiviperaris barbour albi r Lachesis stenophrus neb ● ● Montivipera bo atopho ● ●Montivipera raddei us fucatus ● Montivipera● latifii us popeiorus sabahi ● ● ● ●● imeresur us baratiuniana ● Macrov ra xanthin LachesisTr melanocephala ogeli ● ● Macr● ra imeresur v ●ri ● Daboia siamensis Tr us b ● ● ● ● Daboia● palaestinae i Daboia● deser imeresurimeresur us ● Daboia maur agneri Tr Tr imeresur ●● ● ●● ● Vipera● ammodyteovipera schweiz imeresur ● ipera lebetina Tr ● Vipera● transcaucasiana ● ●● Vipera● latastei zo a Tr us sichuanensis ●is Vipe● rnmuelle r ● ● ● Vipera● anatolica na imeresur uongsonensis ●i ● ● Vipera renardi is ●● ● ●●● Vipe● Tr imeresurus gumprechti iensis ● Viper r us stejnege r ● ● ● Vipera● Tr Vipera●● ursini ● Vipera or us ● us tr ● Vipera ●kaznak● r ● ● Viper a aspis us macropsbu Vipera barani ● Viper ● ri imeresur us yunnanensisus medoensis ●ri E asciatus Pseudoce ● us venustus ● Pseudocerastes persicu r rur Pseudocerastes urar ● Ce Cerastes cerastes ● a loti ti Tr Cerastes gasperettii ei ● Echis ocellatus ● ● Echis joge us insula ● Echis khosatzkii ●● a dinniki itanica risticophis macmahoni r ●●● E ●● imeresuimeresur us f us cantoryth ionalis ●●● ●●● er ●●●c ●● Tr e imeresurTr us kan rastes vipera a seoanei us albolabr h i a be r ev Tr icus iw imeresur i

imeresurimeresur s s lo i Tr imeresur s wiroti anensis Tr Tr us er neensis inatus r b Tr imeresu us andersoniius malcolmiomaculatus vi pureomaculatus us hageni ru o i

amidum Tr imeresur rastes fieldi imeresur r us mcgregous schultz r s imeresur r k Tr imeresurTr ovi ri Tr us puniceus i py us septentr us sumatranus n Tr r us bor

i us pur us flav us gramineus imeresu igonocephalus imeresur imeresur us malabar Echis car Tr imeresuru Echis coloratus imeresur Tr Tr imeresu imeresur Tr Echis omanensis Echis Tr us tr r Echis leucogaster T Tr imeresur imeresu imeresur achnoide imeresur Tr imeresur imeresur Tr Tr r imeresur T Tr Tr s Tr imeresur

imeresur

Tr s

Figure 5. Ancestral character state reconstruction for the three patterns. Pies indicate the probability of the occurrence of each pattern within a clade. blotchy patterns create a “barber pole effect” when hours of the day) and open areas (to thermoregulate fleeing throughout the bushy and herbaceous or hunt). vegetation (Jackson et al., 1976; Lindell & Forsman, 1996). Shadow-like coloration has useful camouflage properties in sandy habitats (Serventy, 1971). Our Drivers of the evolution of zig-zag patterns results clearly show that blotchy coloration patterns Our phylogenetic reconstruction suggests that the are more frequent in species inhabiting sandy and arid zig-zag coloration pattern evolved multiple times environments, where most of the species move between (approximately 23 times) in vipers, mostly from an patches of vegetation (seeking shade in the warmest ancestor with a blotchy pattern (Figs 2-3). The zig-zag

Table 1. Candidate mixed models explaining variation in the occurrence of dorsal patterns among vipers. Results of phylogenetic logistic regression models. Models are ranked on the basis of corrected Akaike’s information criterion (AICc); only models with Akaike’s weight > 0.01 are reported.

Rank Independent variables AICc ΔAICc* w† Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020 a) Dependent: occurrence of blotchy pattern 1 Ecological habitus (-), mean temperature (+), sandy habitat (+) 134.04 0.00 0.48 2 Ecological habitus (-), mean temperature (+), precipitation (-) 134.67 0.63 0.35 3 Ecological habitus (-), forest habitat (-) 137.09 3.06 0.11 4 Ecological habitus (-) 138.41 4.38 0.05 b) Dependent: occurrence of zig-zag pattern 1 Mean temperature (-), water habitat (+), open habitat (-) 169.26 0.00 0.45 3 Mean temperature (-) 169.43 0.18 0.41 4 Water habitat (+), sandy habitat (+) 175.39 6.13 0.02 5 Rock habitat (+), forest habitat (-) 175.58 6.32 0.02 6 Precipitation (-), sandy habitat (+) 175.64 6.39 0.02 c) Dependent: occurrence of uniform pattern 3 Ecological habitus (+) 140.79 0.00 > 0.99

*ΔAICc = AICc difference with the best model. †w = Akaike’s weight pattern presents a very strong phylogenetic signal likely improves crypsis in the canopy. Cases of uniform and is more common in species living in cold climates coloration (or concolor form) have also been reported in (Fig. 6c). In these areas, more time is needed for individuals within the genus Vipera (e.g. former Viper thermoregulation and consequently snakes are more aspis atra, Viper aspis aspis and Viper berus bosniensis), exposed to predation. The zig-zag pattern has been especially at high altitudes in rocky and open areas proposed to have a disruptive effect (from afar) but can (Colombo & Di Nicola, 2012; Tessa, 2016; Nikolić & also represent a case of Müllerian mimicry (Valkonen Simović, 2017). In this genus, individuals with uniform et al., 2011b). The hypothesis of Müllerian mimicry is pattern display a greyish coloration, which can have supported by a strong phylogenetic signal. The zig-zag the same cryptic function of the green coloration in pattern is particularly clustered within the phylogeny arboreal or semi-arboreal Asian pit-vipers. However, (Fig. 3), and in closely related species which often live our model does not completely explain the evolution in nearby geographical regions (Warren et al., 2014). of all the uniform coloration patterns. In some species, For instance, all the species of the genus Vipera display individuals often are uniformly dark (melanism), and this colour pattern and are mostly distributed in the multiple hypotheses have been proposed to explain same geographic region, Europe. This has probably these colorations, such as thermoregulation (Kettlewell, allowed potential predators to learn from more than 1973; Kingsolver & Wiernasz, 1991; Trullas et al., 2007), one species-model that animals showing zig-zag crypticity (Kettlewell, 1973; Endler, 1984), aposematism coloration patterns are most likely a danger. Further (Turner, 1977), protection from UV radiation (Gunn, support to this hypothesis is the presence of several 1998) and sexual selection (Wiernasz, 1989); however, cases of Batesian mimicry from innocuous species further investigation is needed to corroborate or confute emulating this coloration pattern, e.g. the viperine these assumptions. water snake, Natrix maura, which is harmless and belongs to the Colubridae family (Santos et al., 2017). Limitations Our study provides one of the most complete Drivers of the evolution of uniform patterns evaluations of colour evolution in snakes, still it cannot Repeated evolutions have also been observed for the be regarded as exhaustive since we have not explored uniform dorsal pattern, which evolved from both blotchy all possible drivers for colour patterns. First, we and zig-zag patterns (Figs 2-3). Uniform coloration has adopted a macroevolutionary perspective and focused proved to be particularly frequent in Asian clades and on eco-geographical drivers; however, additional specifically in species with arboreal habits (Fig. 6a) processes were certainly at work. For instance, sexual for hunting, thermoregulation, roosting or all of three selection is one of the most frequent drivers of colour activities combined. Accordingly, many species with patterns (Cuthill et al., 2017). In our study, we did not uniform pattern exhibit a green coloration, which consider sexual selection as a factor because of the

© 2020 The Linnean Society of London, Biological Journal of the Linnean Society, 2020, 130, 345–358 ORNAMENTATION IN VIPERS 355 Downloaded from https://academic.oup.com/biolinnean/article-abstract/130/2/345/5815721 by Divisione Coord. Bib. UNI Milano user on 04 June 2020

Figure 6. Occurrence of dorsal patterns in relation to the variables, predicted on the basis of the best phylogenetic logistic regression models. Boxplots indicate: the occurrence of the zig-zag pattern in relation to (a) mean annual temperature; the occurrence of blotchy pattern predicted by (b) ecological habitus, (c) occurrence in sandy habitat and (d) mean annual temperature; the occurrence of uniform pattern type predicted by (e) ecological habitus. The categorical variables ecological habitus (b) and (e) and occurrence in sandy habitat (c), express the probability that a species has a given pattern based on the habitat where it lives. limited information on sexual dimorphism in patterns pattern). The possible bias determined by incomplete in vipers; however, this hypothesis requires future information hampered the analysis of intraspecific attention. Another process that we did not consider variability; however, improving the completeness is the change in coloration during the ontogenesis. of information could allow analysing intraspecific Unfortunately, detailed information on the coloration of variability. Finally, our analysis at a broad phylogenetic juveniles and sub-adults is only available for a subset scale used a coarse definition of patterns. For instance, of species. Nevertheless, the mortality of juveniles is the “blotchy” patterns group includes a broad range not consistently higher than that of adults (Pike et al., of patterns (blotches, transversal lines, ellipses, bars, 2008), suggesting that our conclusions are not biased by etc.). Furthermore, both green, grey and black vipers difference in mortality among age classes. Additional are “uniform”, but the role of these colorations is hypotheses that can be tested in the future include probably different. Future analyses could consider the the role of fine-scale interactions between individuals fine-scale variation within the different patterns. and their micro-habitat (relating colour patterns to vegetation cover), diet and hunting strategies. Our results could be partially affected by the uneven CONCLUSIONS distribution of information. First, some taxa are less known, for instance because they live in inaccessible/ Our study revealed the complexity of factors poorly studied areas. For these taxa, it is possible that determining the evolution of colour patterns in vipers, intraspecific variation exists (i.e. more than one single suggesting that multiple processes, ranging from

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Appendix S1. Sources of data used in the study.