Journal of Biogeography (J. Biogeogr.) (2011) 38, 854–867

ORIGINAL Phylogenetic island disequilibrium: ARTICLE evidence for ongoing long-term population dynamics in two Mediterranean butterflies Leonardo Dapporto1*, Thomas Schmitt2, Roger Vila3, Stefano Scalercio4, Heinrich Biermann5, Vlad Dinca˘6,7, Severiano F. Gayubo8, Jose´ A. Gonza´lez8, Pietro Lo Cascio9 and Roger L. H. Dennis10,11

1Istituto Comprensivo Materna Elementere ABSTRACT Media Convenevole da Prato via 1 Maggio 40, Aim Our aims were to verify the existence of phylogenetic disequilibrium 59100 Prato, Italy, 2Department of Biogeography, Trier University, D-54296 Trier, between butterfly lineages at the subcontinental scale for islands and the nearest Germany, 3ICREA and Institute of mainland and to test the capacity of islands for hosting ancestral populations of Evolutionary Biology (CSIC-UPF), Passeig butterflies and the significance of such relict populations. Marı´tim de la Barceloneta 37-49, 08003 Location The western Mediterranean continental area of Europe and North 4 Barcelona, Spain, CRA Centro di Ricerca per Africa together with several large and small islands (Balearics, Tuscan l’Olivicoltura e l’Industria Olearia, I-87036 Archipelago, Aeolian Archipelago, Capri, Sardinia, Sicily, Corsica). Rende (Cosenza), Italy, 5Markusstrasse 17, D-3490, Bad Driburg, Germany, 6Institute of Methods Using geometric morphometrics, the shape of male genitalia was Evolutionary Biology (CSIC-UPF), Passeig analysed in two common butterflies (Pyronia cecilia and Pyronia tithonus), whose Marı´tim de la Barceloneta 37-49, 08003 spatial heterogeneity in the Mediterranean region has recently been described. Barcelona, Spain, 7Departament de Gene`tica i Observed patterns in genital shapes were compared with shapes predicted for Microbiologia, Universitat Auto`noma de islands and fossil islands to assess the contribution of historical and current events Barcelona, 08193 Bellaterra (Barcelona), in accounting for the transition from a refugial model to an equilibrium model. 8 Spain, A´rea de Zoologı´a, Facultad de Biologı´a, Measurements were taken for 473 specimens in 90 insular and mainland sites. Campus ‘Miguel de Unamuno’ Universidad de Salamanca, 37007 Salamanca, Spain, Results The shape of the genitalia of populations of most islands differed 9NESOS, Associazione pro Isole, Corso Vittorio substantially from that predicted by the equilibrium hypothesis while closely Emanuele, 24 98055 Lipari (ME), Italy, fitting the refugial hypothesis. The comparison between different models strongly 10School of Life Sciences, Oxford Brookes suggests that islands maintain ancestral lineages similar to those living in Spain University, Headington, Oxford OX3 0BP, UK, (P. cecilia) and France (P. tithonus). A high correlation between observed and 11Institute for Environment, Sustainability and predicted patterns on islands and fossil islands occurs during the first steps of Regeneration, Mellor Building, Staffordshire modelled introgressive hybridization while the following steps exposed a University, Stoke-on-Trent ST4 2DE, UK successively lower fit, suggesting that the process from a refugial to an equilibrium situation is highly skewed towards an earlier non-equilibrium. Main conclusions The observed non-equilibrium pattern supports the refugial hypothesis, suggesting that an ancestral lineage was originally distributed from Spain to Italy, and also occupied offshore islands. This lineage, replaced in Italy, has persisted on the islands owing to their isolation. A comparison of the distribution patterns for genetic and morphometric markers in several species indicates that the situation highlighted for Pyronia may represent a common

*Correspondence: Leonardo Dapporto, Istituto biogeographic feature for many Mediterranean butterflies. Comprensivo Materna Elementere Media Keywords Convenevole da Prato via 1 Maggio 40, 59100, Prato, Italy. Genitalia, geometric morphometrics, glaciations, hybridization, introgression, E-mail: [email protected] Pyronia, refugia, Rhopalocera, western Mediterranean.

854 http://wileyonlinelibrary.com/journal/jbi ª 2011 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2010.02452.x Phylogenetic disequilibrium in Mediterranean butterflies

Furthermore, it is well known that genital shape in Satyrinae INTRODUCTION is very highly correlated with genetic characteristics (e.g. MacArthur & Wilson’s (1967) equilibrium theory of island Cesaroni et al., 1994; Dapporto, 2010a, and literature therein) biogeography represents a milestone in understanding distri- and is perhaps also involved in female choice (Gilligan & bution patterns of species. Despite various criticisms, additions Wenzel, 2008). Consequently, there is strong evidence for the and revisions to their work, the central idea of island existence of many different lineages on islands compared with communities being shaped by complex dynamic interactions those occurring at their nearest mainland sources (Dapporto involving immigration, extinction and evolution is still the et al., 2009; Dapporto, 2010a), as has been demonstrated by basic paradigm of recent island biogeography (Losos & genetic studies (Thomson, 1987; Cesaroni et al., 1994). Ricklefs, 2010). Although most of the work confirming the MacArthur & Wilson (1967) applied their equilibrium equilibrium theory has focused on small and close-to-source theory to species numbers. Subsequently, several authors have islands (e.g. Diamond, 1969; Simberloff & Wilson, 1969; see indicated that processes driving island species richness may also Schoener, 2010, for a recent review), the main advances to also drive changes in the genotypic diversity of their popula- the MacArthur & Wilson model have been formulated with tions (reviewed by Vellend & Orrock, 2010). Mainland–island respect to oceanic islands. On oceanic islands, evolution, and systems that fail to fit equilibrium notions, maintained by consequently speciation, are expected to be more important immigration and extinction, are frequently observed; ‘island than immigration in generating community species diversity disequilibrium’ occurs. Disequilibria are mainly expected to (e.g. Emerson & Gillespie, 2008; Whittaker et al., 2008; Grant arise following natural and anthropogenic disturbance (e.g. & Grant, 2010). On islands located within the range of Bush & Whittaker, 1991; Schoener, 2010) and, interestingly, dispersal capabilities of most taxa, populations are usually disturbance and other changes creating mainland–island assumed to be similar to those on the neighbouring mainland; disequilibria are generally postulated to occur on islands. This ongoing immigration and gene flow is expected to prevent is primarily because mainland areas are assumed to be more island–mainland differentiation. This outcome should be more stable and thus more conservative than island communities evident for taxa with a high dispersal capability (Emerson & (see Fig. 1 of Emerson & Gillespie, 2008, p. 620). Even so, Gillespie, 2008; Lomolino et al., 2010). refugial islands are well known and house exceptional Accordingly, when analysed at the community level, the palaeoendemics [e.g. Lemuriformes on Madagascar (Karanth distributions of flying insects on Mediterranean and European et al., 2005) or Sphenodontia on islands off New Zealand offshore islands (none of which are oceanic) are demonstrated (Jones et al., 2009)]. In addition to such impressive examples, to be closely related to source populations (Dennis & Shreeve, many less prominent, but highly conservative, patterns have 1996; Dennis et al., 2000; Dapporto & Dennis, 2008, 2009; also been demonstrated to occur on Mediterranean islands (see Fattorini, 2009; Heiser & Schmitt, 2010). However, when the Me´dail & Diadema, 2009, for a recent review on plants; and distributions of individual species and infra-specific lineages Sto¨ck et al., 2008, for amphibians). It is also well known that are analysed, their patterns cannot always be explained by species richness, composition and species ranges on mainland means of recent dispersal events (Dapporto & Dennis, 2009, areas can change very rapidly (even over a few decades), 2010; Dapporto et al., 2009; Fattorini, 2009). For instance, on especially in response to human global influences (Fisher et al., west Mediterranean islands, all hitherto analysed Satyrinae 2010); thus, retrodiction of past climatic niche distribution is butterfly species have differently shaped genitalia from those an emerging challenge for macroecology (Nogue´s-Bravo, 2009; occurring on the nearest mainland (Dapporto, 2010a). Habel et al., 2010).

Figure 1 Map of the study area: 1, North Africa; 2, Spain; 3, France; 4, northern Italy; 5, southern Italy; 6, Corsica; 7, Sardinia; 8, Sicily; 9, Pianosa; 10, Giglio; 11, Capraia; 12; Elba; 13, Capri; 14, Lipari; 15, Mallorca; 16, Menorca; 17, Piombino; 18, M. Argentario, 19, Montenero; 20, M. Calvi.

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Butterfly species on Mediterranean islands could well be in possibilities explored ranged from the hypothesis that island disequilibrium with respect to the distribution of mainland populations were maintained for longer or shorter periods as lineages following rapid post-glacial climate changes respon- relicts from any neighbouring mainland influences (refugial sible for shaping most of the genetic structures in this region hypothesis), to the hypothesis that islands no longer host relict (Hewitt, 2001; Schmitt, 2007), especially in view of the rapidity populations but reflect the direct influence of the neighbouring with which these species can change their distributions in mainland (equilibrium hypothesis). The predictive power of relation to changes in climate (Parmesan et al., 1999). In a equilibrium and non-equilibrium models in explaining the review, Heaney (2007) suggested that large differences in island distributions of two closely related, yet ecologically different, distributions can also occur among phylogenetically similar butterfly species is compared with three main purposes: (1) to species as a function of small differences in ecological establish whether the islands show evidence for non-equilibrial specialization. In this respect, particular distribution patterns (relict) or equilibrial (mainland-like) populations; (2) to may emerge at a very subtle taxonomic level in response to determine if some mainland areas in the Mediterranean have species’ unique ecologies (Dapporto & Dennis, 2009; Lomo- maintained such ancestral populations; and (3) to reconstruct lino et al., 2010). a plausible history of colonization/introgression in the study To test these assumptions, we analysed the geographic area on the basis of our morphometric results and the recent distribution and phenotypic differentiation of genital mor- assessment of genetic markers for P. cecilia in Italy, Sicily and photypes for two congeneric and often sympatric butterflies, North Africa (Habel et al., 2010). Pyronia cecilia (Vallantin, 1894) and Pyronia tithonus (Linna- eus, 1767), for mainland areas and islands in the western MATERIALS AND METHODS Mediterranean. We excluded a third species of this genus occurring in the Mediterranean (Pyronia bathseba) owing to its Model species restricted distribution, but largely because of its absence from islands. A comprehensive genetic assessment aimed at recon- Pyronia cecilia and P. tithonus (see Fig. S1 in Appendix S1 in structing the phylogeography of these species over the whole the Supporting Information) often form large populations in Mediterranean region does not exist, but Dapporto (2010a) the Mediterranean region. Their adult dimensions and wing demonstrated that both species display different morphotypes shapes are very similar, the larvae of both species feed on in western Europe. Hitherto, no models have been applied to several grass species and both species have a univoltine flight reconstructing the biogeographic factors responsible for this period in summer. These similarities in life history suggest that variation. the species share the same ecological traits. However, P. cecilia Reproductively, many butterfly species are not completely is a thermophilic species living only in the circum-Mediter- isolated and tend to form hybrids with symmetrical or ranean region south of the Alps. Pyronia tithonus is a asymmetrical gene introgression (Mallet, 2008); this applies mesophilic species living in cooler and more humid, wooded even more so to infra-specific taxonomic units, which areas of the Mediterranean; its distribution extends to Britain generally inter-breed without major problems. Therefore, most and central Europe, while in north-western Africa it is morphotypes usually represent different conspecific genetic restricted to a few mountain localities in the Moroccan Rif. lineages that are completely inter-fertile and actually hybridize Pyronia cecilia inhabits most Mediterranean islands, forming to a greater or lesser degree in areas where they meet (Schmitt, large populations, while P. tithonus cannot be considered a 2007), often accompanied by the evolution of geographic common insular species because in the western Mediterranean clines (Dapporto, 2010a). From this perspective, the graded it inhabits only Sardinia, Corsica and Elba, where it is variation in genital shape shown by most Satyrinae, including restricted to scattered cool areas. the two Pyronia species, represents a useful and reliable tool for reconstructing colonization routes in this region and for Study sample and preparation of genitalia testing the occurrence of introgression among lineages (cf. for Mediterranean Satyrinae Thomson, 1987; Cesaroni et al., 1994; A total of 289 P. cecilia and 184 P. tithonus specimens (all Dapporto et al., 2009; Habel et al., 2009; and Dapporto, males) were examined from 90 different sites across five 2010a). Male genital structures are largely supposed to be mainland areas and 15 islands, some of which are fossil islands involved in female mate choice (Gilligan & Wenzel, 2008). As (i.e. former islands that are now directly connected to the with song and beak shape in Darwin’s finches (Grant & Grant, mainland). For P. cecilia the samples comprised the following 2010), genital shape not only represents a useful clue to specimens: mainland areas [North Africa (Tunisia and taxonomy and biogeography but can also be directly involved Morocco), n = 15; Spain, n = 19; France, n = 15; northern in the evolution of the observed biogeographic patterns (i.e. as Italy, n = 19; southern Italy, n = 15]; islands (Corsica, n = 21; a consequence of avoidance of inter-breeding). Sardinia, n = 19; Sicily, n = 21; Elba, n = 18; Capraia, n =6, Geometric morphometrics of male genitalia have been Pianosa, n = 12; Giglio, n = 14; Capri, n = 17; Lipari, n = 17; recorded at the sub-continental scale and then morphotype Mallorca, n = 20; Menorca, n = 17); Tuscan fossil islands distributions have been modelled. As a novel approach, a (Argentario, n = 15; Piombino, n = 12). For P. tithonus the model was constructed without any a priori assumptions. The samples comprised: mainland areas [North Africa (Morocco),

856 Journal of Biogeography 38, 854–867 ª 2011 Blackwell Publishing Ltd Phylogenetic disequilibrium in Mediterranean butterflies n = 24; Spain, n = 16; France, n = 22; northern Italy, n = 21; were regressed onto a third-order polynomial equation of southern Italy, n = 10]; islands (Corsica, n = 20; Sardinia, latitude and longitude with 10 potential terms. As suggested by n = 22; Elba, n = 23); Tuscan fossil islands (Montenero, Cardini et al. (2007), the terms were included in the multiple n = 15; Monte Calvi, n = 15) (see Appendix S2 for detailed regression as predictor variables and entered into the model by data and Fig. 1). The sample sizes (generally more than 10 a backward stepwise procedure. In order to reduce possible specimens per area/group) equal or exceed that of previous residual autocorrelation (Legendre, 1993), specimens belong- studies of geometric morphometrics in butterflies (Mutanen, ing to the same or to nearby localities (< 10 km apart) were 2005; Dapporto et al., 2009; Dapporto, 2010a) and can amalgamated by using the mean latitude, longitude and DF1 therefore be considered adequate. Genitalia were dissected values. This resulted in the identification of 27 mainland sites using standard procedures (Dapporto et al., 2009). The for both P. cecilia and P. tithonus (see Appendix S2 for the tegumen, left brachium and right valva were photographed location of sites and the number of specimens belonging to using a Nikon Coolpix 4500 camera mounted on a binocular each). The model was evaluated on the basis of mainland areas microscope. and the island DF1 values were predicted on the basis of their geographic location. In order to verify which islands and fossil islands showed a significantly different genital shape than Geometric morphometrics and statistical analyses predicted, a Wilcoxon sign test was carried out. The predicted A combination of landmarks and sliding semi-landmarks was values for DF1 were plotted on a map of the study area and applied (Bookstein, 1997). The tps (thin-plate spline) series of each island and fossil island was connected to the nearest point programs (available at http://life.bio.sunysb.edu/morph/) was on the map showing their observed values, in order to visualize used for these analyses. Lateral sections of the tegumen, the the ‘misplacement’ of the genital shape for each area examined. brachiae and the valvae were examined separately. Points on the outlines that could be precisely identified were considered The model as landmarks (type II and type III landmarks; Bookstein, 1997), whereas the other points were allowed to slide along the As for the previous analysis, centroid values for function 1 in outline trajectory to reduce uninformative variation (sliding discriminant analysis (i.e. DF1) described mean genital shape semi-landmarks) (see Figs S2 & S3 in Appendix S1). Digital for each source and island. On the basis of the suggestions data for landmarks on genital photographs were obtained made in previous papers (Dapporto et al., 2009; Dapporto, using tpsdig 2.16 (Rohlf, 2010a) and sliders were defined 2010a; Habel et al., 2010), the existence of an ancestral using tpsutil 1.46 (Rohlf, 2010b). Generalized Procrustes population was hypothesized to occur over the whole area of analysis (GPA) was applied to the landmark data in order to the western Mediterranean mainland and islands. Following remove non-shape variation in location, scale and orientation any change in the distribution of lineages over mainland areas, and to superimpose the objects in a common coordinate the emergence of a difference between populations on islands system. Partial warps were calculated using the shape residuals and their nearest mainland is expected. Populations on islands from GPA. By applying principal components analyses (PCA) are expected to change successively through introgressive to partial warps, relative warps (RWs) were obtained and used hybridization until they correspond to the nearest geographic as variables in discriminant analysis (Bookstein, 1997). RWs sources (Vellend & Orrock, 2010). The rapidity of this process are visualized by thin-plate spline deformation grids, which clearly depends on area and isolation. Immigration to nearby permit a visual comparison of shape differences. GPA, partial islands should be relatively fast and, if the island is also small, warp and RW calculations and thin-plate spline visualization the ancestral population should be readily replaced by were carried out using tpsrelw 1.49 (Rohlf, 2010c). The RW hybridization following colonization (Dapporto et al., 2009; scores were analysed by discriminant analysis on the groups of Vellend & Orrock, 2010). Ancestral populations on the largest specimens from continental areas, islands and fossil islands. islands – Sicily, Sardinia and Corsica – are expected to persist Wilks’ lambda was used to evaluate the significance and for longer. Consequently, the probability of immigration from validity of each discriminant function. As there are often large the mainland will correlate directly with an island’s perimeter numbers of RWs, only RWs explaining more than 1% of the and inversely with its distance from each source. This approach variance were included in the discriminant analysis (Dapporto assumes that ancestral populations persist on islands for longer et al., 2009). periods than on the mainland, once new lineages become The genital shape for islands and fossil islands was predicted established on neighbouring mainland areas. Conversely, if the based on a trend surface analysis (TSA) (Legendre & Legendre, islands do not host a ‘relict’ population, because the mainland 1998; Cardini et al., 2007). TSA is a generalized model population is stable over time or alternatively because the including both linear and nonlinear relationships between colonization–introgression process is too fast to permit an the geographic independent variables and the dependent ancestral island relict to persist, the characteristics of butterflies variable. Genital morphs can be identified from the first on islands should match those on the neighbouring mainland. discriminant function representing the combination of RWs For this reason our model starts with the assumption of the explaining most of the shape variance (Dapporto et al., 2009). occurrence of an ancestral population on islands (refugial Thus, the values obtained for discriminant function 1 (DF1) hypothesis), which over time is introgressed by individuals

Journal of Biogeography 38, 854–867 857 ª 2011 Blackwell Publishing Ltd L. Dapporto et al. from all neighbouring areas relative to island isolation and iteration of equation 3 tend asymptotically towards the island perimeter. This process gradually changes the island equilibrium values in which the ancestral population is population until the ancestral island morphotype is completely completely subsumed owing to immigration from sources. replaced by that of the mainland. Thus, as a first step the size The computation of the asymptotes is complex owing to the of the supposed ancestral island population is computed on inter-connections among equations for different islands shar- island area. As such, the centroid value (for genital shape) of ing similar nearest island sources. For this reason, we any supposed ancestral population is weighted by island performed a new run using 10,000 iterations and examined area: the shape of the curves to maximize the resolution of asymptotic values (see Fig. S4 in Appendix S1). Apop ¼ ACv0; ð1Þ The relationships between the observed and predicted Cv for where Apop is the weighted genitalia shape of the ancestral each iteration and for the equilibrium values were tested using population, A is island area and Cv0 is the ancestral shape Pearson correlations. However, the Pearson correlation coef- considered to inhabit the island. In the present study, no ficient is a non-dimensional measure and does not provide a priori predictions to identify the potential ancestral lineage absolute evaluation of dispersion between observed and were made, and instead a model was run five times, each time predicted values. Therefore, we also computed an absolute with a different ancestral centroid value (Cv0) relating to one index of dispersion for each iteration of the equation in the of the five mainland areas of North Africa, southern Italy, form of a standard deviate-like measure: northern Italy, southern France and Spain. At step zero all vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi populations from both islands and mainland sites are postu- u uPN u 2 lated to be the ancestral one. Following the establishment of t ðCvp CvoÞ the ancestral population on an island a perturbation is Dispersion ¼ i ; ð4Þ N supposed to have occurred, resulting in the currently observed distribution pattern of lineages on the mainland. At that stage where N is the number of islands tested, Cvp is the predicted the genital shape for each island is predicted to be in centroid value and Cvo is the observed centroid value. To make disequilibrium with that on the nearest continental area, but a direct comparison between P. cecilia and P. tithonus, the to change with immigration of individuals according to the observed centroid values in the model were standardized to formula have a variance equal to 1 in both species. Differences in

P6 dispersion between the two species could be a consequence of 1 Apop þ P ðCviðdiÞ Þ differences in number of islands and fossil islands (13 areas for ExCv ¼ i¼1 ; ð2Þ 1 P6 P. cecilia and 5 for P. tithonus). A potential bias of this kind 1 A þ P di was checked for by plotting the mean dispersion of groups for i¼1 an increasing number of areas. The data set was obtained by where ExCv1 is the expected shape at step 1, P is the island computing all possible combinations for groups composed by ) ) perimeter, Cvi is the centroid value for the ith source and di is n of k areas n!/[(n k)! k!] when the total number was less the minimal distance between the island and the ith source. than 100, and a random subset of 100 combinations when the To identify the predicted evolution of genital shape on the possible combinations were more numerous. Only the two islands and fossil islands studied, an iterative model was equilibrium formulae were used for this analysis because the applied in which ExCv1 was treated as the ancestral shape with refugial ones are numerous (10) and give different results for introgression established in successive steps for ExCv2, thus the 800 different steps. simulating ongoing colonization and hybridization. The pro- Island areas were taken from different official sources. cedure was iterated with 800 steps in Microsoft Excel: the Isolation from the four mainland areas was extracted from a Dapporto et al. (2009) model was implemented by including 1:1,000,000 atlas (Istituto Geografico de Agostini). Island among sources the nearest of the three large islands (Sicily, perimeters and Pleistocene island areas were measured by Corsica and Sardinia) and using as Cv for these sources the ImageJ (http://rsbweb.nih.gov/ij/) from 1:2,250,000 maps. spss Cv)1 value. This model takes inter-island faunal exchanges into Statistical analyses were performed using 13.0 and account: statistica 7 (Statsoft).

P6 1 AExCv1 þ P ðCviðdiÞ Þ RESULTS ExCv ¼ i¼1 : ð3Þ 2 P6 1 For P. cecilia more than 1% of variance was explained by 12 A þ P ðdiÞ i¼1 tegumen RWs, 9 brachial RWs and 12 valval RWs (explaining a cumulative variance of 92.8%, 96.4% and 93.5%, respec- If the two species are at their population equilibrium, the tively). For P. tithonus more than 1% of variance was expected shape for each island can be considered as the average explained by 11 tegumen RWs, 8 brachial RWs and 12 valval shape of colonists from continental sources weighted for their RWs (explaining a cumulative variance of 94.7%, 97.2% and colonization potential (isolation). The values obtained by 93.5%, respectively).

858 Journal of Biogeography 38, 854–867 ª 2011 Blackwell Publishing Ltd Phylogenetic disequilibrium in Mediterranean butterflies

Discriminant analysis for P. cecilia highlighted a pattern and brachiae RW3 and RW5) are included in DF1 (see Fig. S5 largely reflecting recent geography; DF1 explained 66.5% of the in Appendix S1 for brachial variations). For P. tithonus the variance (Wilks’ lambda = 0.014, P < 0.001) and DF2 another overall mainland pattern was maintained in DF1 (explaining 19.0% (Wilks’ lambda = 0.113, P < 0.001) (Fig. 2a). For P. 40.6% of variance, Wilks’ lambda = 0.018, P < 0.001) and tithonus the pattern highlighted by DF1 (explaining 63.0% of DF2 (explaining 20.2% of variance, Wilks’ lambda = 0.054, variance, Wilks’ lambda = 0.033, P < 0.001) and DF2 P < 0.001), while the islands formed a more coherent pattern (explaining 15.5% of variance, Wilks’ lambda = 0.170, than for P. cecilia. Corsica and Sardinia are classified as near to P = 0.001) is congruent with continuous variation from France, and Elba to southern Italy (Fig. 2d). Relative warps Morocco (in the west) to Italy (in the east) (Fig. 2b). belonging to the tegumen and valva (tegumen RW1 and valva When islands were included in the analysis of data for RW5) are mostly related to the first function (see Fig. S6 in P. cecilia, the overall mainland pattern was maintained in DF1 Appendix S1 for tegumen variations). For both species, Tuscan (explaining 43.4% of variance, Wilks’ lambda = 0.007, fossil islands were classified as being closely similar to Tuscany. P < 0.001) and DF2 (explaining 15.5% of variance, Wilks’ TSA (d.f. = 26, R2 = 0.678, F = 10.513, P < 0.001) revealed lambda = 0.023, P < 0.001), but some islands were not that the best model for explaining the P. cecilia shape on the organized into a geographically coherent pattern. While mainland was Menorca, Mallorca, Sicily and Lipari are located near to their 2 2 geographically nearest sources (Fig. 2c), other islands appear DFI ¼ 0:254Lt 0:094Lg þ 0:127Lg 0:003LtLg 9:422; to have been misplaced, as in the case of Capri and more obviously the Tuscan islands (Capraia, Pianosa, Giglio and while for P. tithonus the best model (d.f = 26, R2 = 0.879, F = Elba). RWs belonging to the valvae and brachiae (valva RW4 19.968, P < 0.001) was

(a) (c)

(b) (d)

Figure 2 Discriminant function analyses displaying the relative positions of specimens of Pyronia cecilia and P. tithonus from the studied areas. Discriminant functions 1 and 2 are represented on the x- and y-axes, respectively. The analyses have been performed for mainland areas only for (a) P. cecilia and (b) P. tithonus and for the complete data set from mainland areas, islands and fossil islands for (c) P. cecilia and (d) P. tithonus.

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DFI ¼ 1:642Lg þ 0:051Lg2 0:004Lg3 0:0008Lt2Lg 1:289; Table 1 Wilcoxon Z and P values comparing the observed genital shape in the specimens of Pyronia cecilia and P. tithonus sampled where Lt and Lg are latitude and longitude, respectively. (discriminant function 1) with that predicted by trend surface The predicted values for DF1 are shown in Fig. 3(a,b). For analysis. both species, the nearest sampled area having the same genital shape as Corsica and Sardinia was located in the vicinity of the P. cecilia P. tithonus Gulf of Lion (southern France) and showed a significant Island ZP ZP difference (P < 0.05) between predicted and observed values (Fig. 3a,b, Table 1). For P. tithonus, Elba displayed a less severe Corsica )4.015 < 0.001 )3.472 0.001 but nevertheless significant misplacement (Fig. 3b, Table 1). Sardinia )3.622 < 0.001 )4.107 < 0.001 For P. cecilia the observed shapes for all the Tuscan Archipel- Sicily )0.643 0.520 ago islands (Elba, Capraia, Giglio and Pianosa) and Menorca Capraia )2.201 0.028 ) were significantly different from those predicted for the islands Pianosa 3.059 0.002 ) and also display the shape found for the area around the Gulf Giglio 3.296 0.001 Elba )3.823 < 0.001 )3.782 < 0.001 of Lion (Fig. 3a, Table 1). Pyronia cecilia from Sicily, Lipari Capri )1.018 0.309 and Capri, and both the species from fossil islands, have a Lipari )0.876 0.381 genital shape not significantly different from that predicted for Mallorca )0.635 0.526 their real positions (Table 1). Menorca )2.101 0.036 Predictions of genital shape, produced by the five models Piombino )1.779 0.075 based on the existence of an ancestral genital shape in turn for Argentario )1.854 0.064 each of the five mainland areas, revealed substantially different Montenero )1.063 0.288 results for the two species. For P. cecilia, the models for Spain, M. Calvi )0.202 0.840 France and North Africa produced an initial high Pearson Values for Z and P were obtained by Wilcoxon tests for islands and correlation (with a maximum for Spain of 0.87) which fossil islands. Significant results are highlighted in bold. decreased to the equilibrium value of 0.63. The northern Italy model showed very low initial Pearson values, reaching a maximum value of about 0.7 with iteration. The southern Italy value (Fig. 4a). For P. tithonus, the models for France, Spain model produced intermediate correlations in the first steps and North Africa also produced the highest Pearson values (maximum value 0.82), and then declined to the equilibrium initially (maximum 0.96), while the models for northern and

(a)

(b)

Figure 3 Plot of predicted values from dis- criminant function 1 (DF1, grey scale) pro- duced using trend surface analysis. Island circles are marked with the grey tone matching the observed value and are con- nected by arrows to the nearest point on the map where the value is predicted to occur: (a) Pyronia cecilia; (b) P. tithonus. Dashed zones for Algeria, Tunisia, Sicily and eastern Italy indicate areas where P. tithonus is not recorded or not sampled in this study.

860 Journal of Biogeography 38, 854–867 ª 2011 Blackwell Publishing Ltd Phylogenetic disequilibrium in Mediterranean butterflies southern Italy had lower correlations. However, the equilib- values is quite large (0.55) (Fig. 4c), while it was much smaller rium values were also high, with a Pearson correlation of 0.95 for P. tithonus (0.16) (Fig. 4d). Moreover, the North African (Fig. 4b). Analysis of dispersion between observed and models for both species produced lower predictive power (not predicted shapes offered an even more precise picture. For detected by Pearson correlation). The model using northern P. cecilia, dispersion obtained by the equilibrium predicted and southern Italy as an ancestral population produced greater

(a) (b)

Figure 4 Trends in Pearson correlations (a, b) and dispersion (c, d) for each of the five models obtained after 800 iterations (1, North Africa; 2, Spain; 3, France; 4, northern Italy; 5, southern Italy; E, equilib- (c) (d) rium) for Pyronia cecilia (a, c) and P. tithonus (b, d).

(a)

(b)

Figure 5 Expected centroid value (ExCv) for island populations from the best fitting refugial equation (Ref.) [(a) Spain for Pyro- nia cecilia and (b) France for P. tithonus], for the equilibrium values (Eq.) and the observed values (Obs.). Ref. graphs show the evolution of the predicted centroid value representing most shape variation in genitalia over 200 iterations.

Journal of Biogeography 38, 854–867 861 ª 2011 Blackwell Publishing Ltd L. Dapporto et al. distances between observed and predicted values (Fig. 4c,d). Pyronia tithonus has a distribution that closely matches an The model results can be best appreciated by directly equilibrium model founded on recent island geography and on comparing the equilibrium and the observed values for each the present distributions of putative morphotypes at potential island for the most successful predictive model (Spain for mainland sources. Although island populations significantly P. cecilia, France for P. tithonus) (Fig. 5a,b). For both species, differ from those inhabiting the nearest Italian mainland areas, observed values for island populations are lower than predicted the Elba population phenotype resembles the Italian one. by the equilibrium hypothesis, as expected for the first steps of Furthermore, the second nearest source to Corsica is southern the refugial hypothesis (Fig. 5a,b). In particular, the extremely France. Sardinia, in turn, was predicted to be influenced by low values for Tuscan islands are responsible for the low Corsica and vice versa. The degree to which Sardinian Pearson correlation coefficients for P. cecilia. Differences in P. tithonus has diversified suggests evolution due to passive dispersion between the two species are unlikely, owing to drift (see Fig. 2d and Fig. S6 in Appendix S1). Immigration differences in the number of sampled areas. Indeed, plotting from France to Corsica might well have been favoured by the the mean dispersion values for combinations of subsets of prevailing wind direction, emanating from the north-west, areas, for both species, revealed that dispersion is not during the flight period of this species (June–August) (Pierini correlated with the number of areas comprising the combina- & Simioli, 1998). tions and the mean values for the two species remain The interpretation of the P. cecilia pattern is more compli- completely separated (see Fig. S7 in Appendix S1). cated; in contrast to P. tithonus, the equilibrium model failed to predict genital shapes on most islands. This failure is mainly owing to the strong differences between predicted and DISCUSSION observed values on Italian offshore islands and the generally Three main results emerge from our data. First, populations of lower than predicted values shown by all island populations. most islands do not resemble those occurring on the respective Although the similarity of populations on Sardinia and Corsica nearest mainland. Second, the models with the greatest to those in France can be explained, and the possibility that predictive power for the occurrence of ancestral populations these islands acted as stepping stones for wind dispersal cannot occurring on islands are those based on populations in Spain be completely ruled out, it is difficult to conceive that and France. Third, the strongest correlations between observed prevailing wind might have favoured colonization to Elba, and predicted genital shapes are obtained during the first Giglio and Pianosa from North Africa, France or Spain, which iterative steps of the refugial models. are located hundreds of kilometres to the north-west or south- west. Therefore, colonization from the nearby Italian mainland involving a bottleneck successively subjected to rapid modi- Patterns of island and mainland populations fication is a more likely explanation, as assumed above for and predictive power of different models P. tithonus on Sardinia. However, different islands should be Differences between observed and predicted genital shapes and expected to follow stochastic, and thus different, fates, but they geographic locations (latitude and longitude) can occur as a consistently resemble the typical genital shapes of more result of statistical bias when regressions have low resolution westerly populations, a feature shared with Maniola jurtina and predictive power; in such cases cross-validation samples and other Satyrinae (Dapporto, 2010a). Moreover, the model (islands and fossil islands in trend surface regressions) are on predicting the current North African, French and particularly average predicted to occur around the mean of the model Spanish populations to be the ancestral colonizers of western sample (i.e. France for longitude in our analysis). However, if Mediterranean islands produces a close fit during the initial this was the case for our data, then specimens from fossil steps (as found in P. tithonus and M. jurtina). Therefore, North islands having similar latitudes and longitudes to Elba, Giglio, African, Spanish and French populations may represent the Pianosa and Capraia should match this displacement. More- ancestral lineages of several butterfly species in many of the over, as fossil islands are virtually identical to Tuscan islands western Mediterranean islands. with respect to ecology, geology and geography, they represent a control against putative statistical biases and ecological Biogeographic interpretations of observed patterns influences on genital shape. For these reasons, we assume that the most parsimonious explanation is that several island Most biogeographic discussions about the Mediterranean are populations represent different lineages compared to nearest based on widespread evidence for the existence of some well- mainland areas. This proposition has also been argued for defined glacial refugia (Spain, Italy, the Balkans, Asia Minor, other case studies (Thomson, 1987; Cesaroni et al., 1994). North Africa) (Hewitt, 2001; Schmitt, 2007), and this Interestingly, the distribution patterns of P. cecilia and hypothesis of different lineages surviving in refugia and P. tithonus morphotypes are remarkably different from one repeatedly expanding (Dennis et al., 1991) is supported by a another. Although geographic shifts are apparently similar for large number of examples (Hewitt, 2001; Schmitt, 2007; the two species in Sardinia and Corsica (Fig. 3a,b), the Dapporto, 2010b). However, most genetic studies visualize solutions produced by the model have two very different lineages expanding and encountering each other to form outcomes. broader or narrower hybrid suture zones on the mainland, the

862 Journal of Biogeography 38, 854–867 ª 2011 Blackwell Publishing Ltd Phylogenetic disequilibrium in Mediterranean butterflies line of contact effectively marking the initial geographic In some cases mainland and island populations display dislocation (Hewitt, 2001; Schmitt, 2007). In recent reviews, transitional (perhaps hybrid) genital shapes as suggested for Ricklefs (2008), Excoffier et al. (2009) and Fisher et al. (2010) the area between North Africa, Sicily, Lipari, southern Italy warn against postulating that local communities have temporal and Capri; in other cases, island populations seem to have integrity. For example, differential temperature tolerance maintained their ancestral integrity compared with those on probably maintains the parapatric species boundary in the the nearest mainland (Corsica and Tuscan islands). A similar Papilio glaucus–Papilio canadensis hybrid zone, but climate pattern is also evident in, and accounts for, the biogeographic change may well alter this equilibrium (Mercader & Scriber, distribution of several other species of Satyrinae (M. jurtina, 2008). Dapporto et al., 2009; Hipparchia aristaeus and Pararge From this perspective, it can be conceived that different aegeria, Dapporto, 2010a). conspecific lineages of butterflies may have changed their In a recent paper it was shown that the same allozyme distributions in the Mediterranean region in the past. In fact, pattern is shared among populations of Pyronia cecilia and there have been substantial range changes among European M. jurtina in North Africa, Sicily and the Italian peninsula butterflies during recent decades, probably as a consequence of (Habel et al., 2009, 2010). Our results coincide with those of climate changes (Parmesan et al., 1999; Hill et al., 2002). Habel et al. (2010) because we did not find any substantial However, distribution changes on the mainland are likely to be morphological shift coinciding with the Strait of Sicily faster than successful migration from mainland areas to (between North Africa and Sicily) for P. cecilia. However, islands, even in taxa usually considered to be good dispersers we observed that the Messina Strait (between Sicily and Italy) (Sparks et al., 2005). As shown in mark–recapture studies, coincided with a pronounced shift in genital morphology. most butterfly individuals do not undertake movements Habel et al. (2010) did not specifically test whether for greater than a few hundred metres (Junker & Schmitt, 2010, P. cecilia the Messina Strait represents a barrier to gene flow, and literature therein), and thus for non-migratory species but they suggested that a high similarity in allozymes occurs overseas dispersal of several kilometres will occur, but only between the Maghreb, Sicily and the Italian peninsula as rarely. Consequently, after a population (e.g. of Pyronia cecilia) shown in greater detail for M. jurtina (Habel et al., 2009). has been established on an island, its genetic structure is These data contrast with the observed pattern in genital expected to be largely fixed, even in the event of occasional shape, indicating a strong divergence between North Africa immigrants from different lineages. and Italy (this study; Dapporto et al., 2009; Dapporto, Island disequilibrium is usually considered to be generated 2010a). This apparent discrepancy between allozyme and by disturbance events such as hurricanes and human interfer- morphological data can be explained by recent observations ence; when the recovery to equilibrium takes longer than on several species of widespread gene introgression (reviewed disturbance frequency, communities are predicted never to be by Currat et al., 2008; and Excoffier et al., 2009). Allozymes in equilibrium (Schoener, 2010). There is, however, no reason are well known to convey adaptive characters such as to think that an island–mainland equilibrium cannot be resistance to climatic conditions (Rank & Dahloff, 2002; changed by the alternative mechanism following rapid changes Van Oosterhout et al., 2004). It is thus possible that changes to mainland communities and the persistence of insular ones. in allozyme alleles and frequencies could spread very rapidly If changes occur in the relative and absolute size of species by introgression over large areas, reflecting on processes populations comprising mainland communities, the island operating in ecological (very recent) time. However, the community will initially differ from the one predicted to occur opposite could be the case, and an ancestral set of allozymes, there on the basis of the new mainland species pool; isolation well adapted to a region, could be incorporated within the and area are key factors controlling this relationship (Dennis genome of any invading lineage by introgression. Based on et al., 2010). Our data on P. cecilia suggest that, in the past, the allozyme patterns in several butterflies, Habel et al. (2010) morphotypes different from the current Italian one were suggested a continuous link between North African and originally widely distributed from Spain to Italy as well as on Italian populations during the last major glaciation and thus, offshore islands. Subsequently, this lineage has been replaced implicitly, with those from islets. A clear signal for this in Italy by another that evolved in Italy or elsewhere; the connection between islands and the south-western mainland ancestral (western) lineage has persisted on some islands, even populations is still evident in the genital morphology of on small ones relatively close to the coast of Italy, thus creating P. cecilia and M. jurtina. From this perspective the different a phylogenetic disequilibrium between Italian islands and their patterns of morphology and allozymes would accord with the nearest potential mainland sources. Mediterranean islands are existence of ancient widespread populations in the western therefore, apparently, particularly suitable for maintaining Mediterranean area and islands subsequently being replaced ancient populations. Indeed, in the Mediterranean Sea extreme in Italy by an invading group (evolved in situ or elsewhere) weather events are rare and, among the islands sampled, other with a different genital morphology that has maintained catastrophic events such as volcanic activity occur only on the traces of this process in the introgression of an ancestral set very large island of Sicily; there, frequent eruptions of Mount of allozymes. The occurrence of both ancestral allozymes and Etna may destroy only inconsequential fractions of the ancestral genital shape is evident in Sicily (this study; Habel butterfly populations in the immediate vicinity. et al., 2010) where invasion has probably been partially

Journal of Biogeography 38, 854–867 863 ª 2011 Blackwell Publishing Ltd L. Dapporto et al. impeded by isolation. The same phenomenon seems to have surrogates for fossil data. Persistence on islands of the ancestral occurred on most Italian islands. lineages probably generated the phylogenetic disequilibrium observed in most of the west Mediterranean islands. The patterns in Pyronia are not unique, but seem to represent the Stage of the disequilibrium phenomenon rule for many Tyrrhenian butterflies (e.g. Pontia daplidice, Our model fit for P. cecilia, and that developed for M. jurtina Aricia cramera, Pararge aegeria, Coenonympha lyllus, M. jurtina, (Dapporto et al., 2009), suggests that we are observing an Hipparchia aristaeus revised by Dapporto, 2010a), thus rein- initial stage in this proposed phenomenon. We might actually forcing the present hypothesis. Modelling past distributions of be witnessing the beginning of the disequilibrium processes in species still lacks robust retrodictive procedures and more which most island populations are still very different from prominent patterns obtained by theoretical models need continental ones owing to recent distribution changes or empirical validation (Nogue´s-Bravo, 2009). From this per- impacts of disturbance events on the mainland (such as spective, we suggest that comparative biogeographic analyses invading lineages following environmental changes). However, between islands and mainland at the subcontinental scale at the beginning of the 20th century, Roger Verity collected represent a unique opportunity for macroecology to expose many specimens of Satyrinae in Tuscany and on Elba, but their population dynamics operating over different time-scales from genital shapes do not apparently differ from those collected glaciations to the present day. about 100 years later (L.D., unpublished data). The high correlation with the first steps of the model could be a false ACKNOWLEDGEMENTS signal for a recent invasion of the mainland. Indeed, persis- tence of ancestral lineages on islands might have been This study was conducted in collaboration with the Tuscan particularly favoured, thus producing a misleading signal for Archipelago National Park and partially funded by the ENEL an initial stage in hybridization. The following processes might and Legambiente project ‘Insieme per la Biodiversita`:un apply here: (1) P. cecilia forms sufficiently large populations on santuario per le farfalle nel Parco Nazionale dell’Arcipelago islands so that the contribution and the replacement proba- Toscano’ and by the project ‘Definizione dello status di bilities of occasional mainland immigrants are of little conservazione delle falene e della malacofauna terrestre importance (Vellend & Orrock, 2010); and (2) islands are dell’Arcipelago Toscano’. Support was also provided by the often characterized by microevolution creating stochastic drift Spanish Ministerio de Ciencia e Innovacio´n (CGL2007-60516/ or populations adapted to the particular island habitats (Grant BOS) to R.V. and V.D., and by a pre-doctoral fellowship from & Grant, 2010). Such drifting populations may enhance some Universitat Auto`noma de Barcelona to V.D. Thanks are also due tendency to mate avoidance with respect to ancestral lineages to Luca Bartolozzi (Museo Zoologico ‘La Specola’, Firenze), or, more likely, to more genetically distant new immigrant Marco Dellacasa (Museo di Storia Naturale e del Territorio, lineages, generating reduced survival of hybrids (see Biermann Pisa), G. Govi, M. Romano, F. Strumia, R. Eastwood, J. Estela, & Eitschberger, 1996, for Mediterranean butterflies) or mate E. Garcı´a-Barros, J. Herna´ndez-Rolda´n, M. Munguira, D. Carr- avoidance (Grant & Grant, 2010). eras, S. Estrade´ and O. Garcia for the collection and the loan of several specimens. We also thank Melodie McGeoch and three anonymous referees for their helpful comments. CONCLUSIONS

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866 Journal of Biogeography 38, 854–867 ª 2011 Blackwell Publishing Ltd Phylogenetic disequilibrium in Mediterranean butterflies

BIOSKETCH

Leonardo Dapporto is a naturalist. His research work is mainly focused on unravelling historical and present-day factors determining insect distributions on Mediterranean islands. He has also worked on chemical communication in lemurs and social wasps.

Author contributions: L.D., T.S., R.D. and R.V. produced the text. L.D. carried out the geometric morphometrics and analysed the data. All the authors contributed to data interpretation. L.D., R.V., V.D., P.L.C., S.S., H.B., F.G. and J.A. collected field data.

Editor: Melodie McGeoch

Journal of Biogeography 38, 854–867 867 ª 2011 Blackwell Publishing Ltd