When the rule becomes the exception. no evidence of gene flow between two Zerynthia cryptic butterflies suggests the emergence of a new model group

Abstract

There is increasing evidence that most parapatric cryptic/sister taxa are reproductively compatible across their areas of contact. Consequently, the biological species concept, which assumes absence of interbreeding, is becoming a not so effective criterion in evolutionary ecology. Nevertheless, the few parapatric sister taxa showing complete reproductive barriers represent interesting models to study speciation processes and the evolution of reproductive isolation. In this study, we examined contact populations in northwestern Italy of two butterfly species, Zerynthia polyxena and Z. cassandra, characterized by different genitalic morphotypes. We studied levels of divergence among 21 populations distributed from Sicily to France using three genetic markers (the mitochondrial COI and ND1 genes and the nuclear wingless gene) and genitalic geometric morphometrics. Moreover, we performed species distribution modelling to estimate different climatic requirements of Z. polyxena and Z. cassandra. We projected climatic data into glacial maximum scenarios in order to verify if and to which extent glacial cycles could have contributed to speciation processes. Genetic and morphometric analyses identified two main groups. All specimens showed a concordant pattern of diversification, including those individuals sampled in the contact area. Haplotype distribution and climatic models showed that during glacial maxima both species experienced a strong range contraction and presumably remained separated into different microrefugia in southern France, in the Italian Peninsula and on the islands of Elba and Sicily. Long term separation was probably favoured by reduced dispersal ability and high phylopatry, while genitalic diversification probably favoured interbreeding avoidance. Conversely, the aposematic wing pattern remained almost identical. We compared our results with those obtained in other species and concluded that Z. polyxena and Z. cassandra represent a valuable model in the study of speciation.

Figure 1. Analysis of genitalic morphology.

a) Partitioning Around Medioids (PAM) analysis. The red arrow indicates the solution that assumes the existence of two morphotypes having the best fit (highest average silhouette width) among all possible partitions. b) Partition in two groups obtained by PAM analysis plotted for the first two relative warps (PC1 and PC2). A high concordance with geographic origin is shown for both Z. cassandra (c) and Z. polyxena (p) morphotypes with the only exception of the Mount Beigua population (black dots), which includes individuals belonging to both species. Deformation grids of male genitalia confirm previous results on morphological diversification between the studied species .

Figure 2

Median-joining haplotype network of mitochondrial DNA COI (a) and ND1 (b) sequences for Zerynthia cassandra and Z. polyxena. Eastern European haplotypes (C24–C29) are shown in the same bullet color. The lower map (c) shows distribution of haplotypes and genitalic morphotypes for each sampling location. Each pie is divided into a number of slices equal to the number of sampled butterflies for that location. Each slice is further divided into four sectors. Starting from the centre of the pie, the first two sectors show the COI and ND1 haplotype colors, respectively, found at that location (see networks reconstruction). The third and the outer sectors of the slice show assignment of wingless genotype and genitalic morphotype, respectively, to either Z. cassandra (black) or Z. polyxena (white). A grey sector indicates absence of the corresponding marker for that individual.

Figure 3. Majority rule (50%) consensus tree resulting from Bayesian analysis of the combined COI, ND1 and wingless gene datasets.

Node supports inferred from Bayesian posterior probability are shown above recovered branches.

Figure 4

Representation of the logistic output of the Maxent analyses for Zerynthia cassandra (a) and Z. polyxena (b). Values >0.5 indicate the likely presence of a species. Bullets show current distributions. In the lower map (c) blue and yellow areas show logistic output >0.5 for Z. cassandra and Z. polyxena, respectively. Areas where both species are predicted to occur are reported in green. Bullet and circles show current distribution of Z. cassandra and Z. polyxena, respectively.

Figure 5. Projection of the Maxent models (based on present species distribution and climate data) on climatic reconstruction for the last glacial maximum using MIROC and CCSM circulation models.

Dark areas show predicted species distribution (logistic output >0.5) during the last glacial age.