The fine-scale recombination rate variation and associations with genomic features in a butterfly.

Recombination is a key molecular mechanism that has profound implications on both micro- and macroevolutionary processes. However, the determinants of recombination rate variation in holocentric organisms are poorly understood, in particular in Lepidoptera (moths and butterflies). The wood white butterfly (Leptidea sinapis) shows considerable intraspecific variation in chromosome numbers and is a suitable system for studying regional recombination rate variation and its potential molecular underpinnings. Here, we developed a large whole-genome resequencing data set from a population of wood whites to obtain high-resolution recombination maps using linkage disequilibrium information. The analyses revealed that larger chromosomes had a bimodal recombination landscape, potentially caused by interference between simultaneous chiasmata. The recombination rate was significantly lower in subtelomeric regions, with exceptions associated with segregating chromosome rearrangements, showing that fissions and fusions can have considerable effects on the recombination landscape. There was no association between the inferred recombination rate and base composition, supporting a limited influence of GC-biased gene conversion in butterflies. We found significant but variable associations between the recombination rate and the density of different classes of transposable elements, most notably a significant enrichment of short interspersed nucleotide elements in genomic regions with higher recombination rate. Finally, the analyses unveiled significant enrichment of genes involved in farnesyltranstransferase activity in recombination coldspots, potentially indicating that expression of transferases can inhibit formation of chiasmata during meiotic division. Our results provide novel information about recombination rate variation in holocentric organisms and have particular implications for forthcoming research in population genetics, molecular/genome evolution, and speciation.

Nascent evolution of recombination rate differences as a consequence of chromosomal rearrangements.

Reshuffling of genetic variation occurs both by independent assortment of chromosomes and by homologous recombination. Such reshuffling can generate novel allele combinations and break linkage between advantageous and deleterious variants which increases both the potential and the efficacy of natural selection. Here we used high-density linkage maps to characterize global and regional recombination rate variation in two populations of the wood white butterfly (Leptidea sinapis) that differ considerably in their karyotype as a consequence of at least 27 chromosome fissions and fusions. The recombination data were compared to estimates of genetic diversity and measures of selection to assess the relationship between chromosomal rearrangements, crossing over, maintenance of genetic diversity and adaptation. Our data show that the recombination rate is influenced by both chromosome size and number, but that the difference in the number of crossovers between karyotypes is reduced as a consequence of a higher frequency of double crossovers in larger chromosomes. As expected from effects of selection on linked sites, we observed an overall positive association between recombination rate and genetic diversity in both populations. Our results also revealed a significant effect of chromosomal rearrangements on the rate of intergenic diversity change between populations, but limited effects on polymorphisms in coding sequence. We conclude that chromosomal rearrangements can have considerable effects on the recombination landscape and consequently influence both maintenance of genetic diversity and efficiency of selection in natural populations.

The Afrotropical breeding grounds of the Palearctic-African migratory painted lady butterflies (Vanessa cardui).

Migratory insects are key players in ecosystem functioning and services, but their spatiotemporal distributions are typically poorly known. Ecological niche modeling (ENM) may be used to predict species seasonal distributions, but the resulting hypotheses should eventually be validated by field data. The painted lady butterfly (Vanessa cardui) performs multigenerational migrations between Europe and Africa and has become a model species for insect movement ecology. While the annual migration cycle of this species is well understood for Europe and northernmost Africa, it is still unknown where most individuals spend the winter. Through ENM, we previously predicted suitable breeding grounds in the subhumid regions near the tropics between November and February. In this work, we assess the suitability of these predictions through i) extensive field surveys and ii) two-year monitoring in six countries: a large-scale monitoring scheme to study butterfly migration in Africa. We document new breeding locations, year-round phenological information, and hostplant use. Field observations were nearly always predicted with high probability by the previous ENM, and monitoring demonstrated the influence of the precipitation seasonality regime on migratory phenology. Using the updated dataset, we built a refined ENM for the Palearctic-African range of V. cardui. We confirm the relevance of the Afrotropical region and document the missing natural history pieces of the longest migratory cycle described in butterflies.

Rewinding the Ratchet: Rare Recombination Locally Rescues Neo-W Degeneration and Generates Plateaus of Sex-Chromosome Divergence.

Natural selection is less efficient in the absence of recombination. As a result, nonrecombining sequences, such as sex chromosomes, tend to degenerate over time. Although the outcomes of recombination arrest are typically observed after many millions of generations, recent neo-sex chromosomes can give insight into the early stages of this process. Here, we investigate the evolution of neo-sex chromosomes in the Spanish marbled white butterfly, Melanargia ines, where a Z-autosome fusion has turned the homologous autosome into a nonrecombining neo-W chromosome. We show that these neo-sex chromosomes are likely limited to the Iberian population of M. ines, and that they arose around the time when this population split from North-African populations, around 1.5 million years ago. Recombination arrest of the neo-W chromosome has led to an excess of premature stop-codons and frame-shift mutations, and reduced gene expression compared to the neo-Z chromosome. Surprisingly, we identified two regions of ∼1 Mb at one end of the neo-W that are both less diverged from the neo-Z and less degraded than the rest of the chromosome, suggesting a history of rare but repeated genetic exchange between the two neo-sex chromosomes. These plateaus of neo-sex chromosome divergence suggest that neo-W degradation can be locally reversed by rare recombination between neo-W and neo-Z chromosomes.

Meiotic drive against chromosome fusions in butterfly hybrids.

Species frequently differ in the number and structure of chromosomes they harbor, but individuals that are heterozygous for chromosomal rearrangements may suffer from reduced fitness. Chromosomal rearrangements like fissions and fusions can hence serve as a mechanism for speciation between incipient lineages, but their evolution poses a paradox. How can rearrangements get fixed between populations if heterozygotes have reduced fitness? One solution is that this process predominantly occurs in small and isolated populations, where genetic drift can override natural selection. However, fixation is also more likely if a novel rearrangement is favored by a transmission bias, such as meiotic drive. Here, we investigate chromosomal transmission distortion in hybrids between two wood white (Leptidea sinapis) butterfly populations with extensive karyotype differences. Using data from two different crossing experiments, we uncover that there is a transmission bias favoring the ancestral chromosomal state for derived fusions, a result that shows that chromosome fusions actually can fix in populations despite being counteracted by meiotic drive. This means that meiotic drive not only can promote runaway chromosome number evolution and speciation, but also that it can be a conservative force acting against karyotypic change and the evolution of reproductive isolation. Based on our results, we suggest a mechanistic model for why chromosome fusion mutations may be opposed by meiotic drive and discuss factors contributing to karyotype evolution in Lepidoptera.

Chromosome Fissions and Fusions Act as Barriers to Gene Flow between Brenthis Fritillary Butterflies.

Chromosome rearrangements are thought to promote reproductive isolation between incipient species. However, it is unclear how often, and under what conditions, fission and fusion rearrangements act as barriers to gene flow. Here we investigate speciation between two largely sympatric fritillary butterflies, Brenthis daphne and Brenthis ino. We use a composite likelihood approach to infer the demographic history of these species from whole-genome sequence data. We then compare chromosome-level genome assemblies of individuals from each species and identify a total of nine chromosome fissions and fusions. Finally, we fit a demographic model where effective population sizes and effective migration rate vary across the genome, allowing us to quantify the effects of chromosome rearrangements on reproductive isolation. We show that chromosomes involved in rearrangements experienced less effective migration since the onset of species divergence and that genomic regions near rearrangement points have a further reduction in effective migration rate. Our results suggest that the evolution of multiple rearrangements in the B. daphne and B. ino populations, including alternative fusions of the same chromosomes, have resulted in a reduction in gene flow. Although fission and fusion of chromosomes are unlikely to be the only processes that have led to speciation between these butterflies, this study shows that these rearrangements can directly promote reproductive isolation and may be involved in speciation when karyotypes evolve quickly.

The opposed forces of differentiation and admixture across glacial cycles in the butterfly Aglais urticae.

Glacial cycles lead to periodic population interbreeding and isolation in warm-adapted species, which impact genetic structure and evolution. However, the effects of these processes on highly mobile and more cold-tolerant species are not well understood. This study aims to shed light on the phylogeographic history of Aglais urticae, a butterfly species with considerable dispersal ability, and a wide Palearctic distribution reaching the Arctic. Through the analysis of genomic data, four main genetic lineages are identified: European, Sierra Nevada, Sicily/Calabria/Peloponnese, and Eastern. The results indicate that the Sardo-Corsican endemic taxon ichnusa is a distinct species. The split between the relict lineages in southern Europe and the main European lineage is estimated to have happened 400-450 thousand years ago, with admixture observed during the Quaternary glacial cycles, and still ongoing, albeit to a much smaller extent. These results suggest that these lineages may be better treated as subspecific parapatric taxa. Ecological niche modelling supported the existence of both Mediterranean and extra-Mediterranean refugia during the glacial periods, with the main one located on the Atlantic coast. Nevertheless, gene flow between populations was possible, indicating that both differentiation and admixture have acted continuously across glacial cycles in this cold-tolerant butterfly, generally balancing each other but producing differentiated lineages in the southern peninsulas. We conclude that the population dynamics and the processes shaping the population genetic structure of cold-adapted species during the Quaternary ice ages may be different than those classically accepted for warm-adapted species.

A hydrogen isoscape for tracing the migration of herbivorous lepidopterans across the Afro-Palearctic range.

RATIONALE: Many insect species undertake multigenerational migrations in the Afro-tropical and Palearctic ranges, and understanding their migratory connectivity remains challenging due to their small size, short life span and large population sizes. Hydrogen isotopes (δ2 H) can be used to reconstruct the movement of dispersing or migrating insects, but applying δ2 H for provenance requires a robust isotope baseline map (i.e. isoscape) for the Afro-Palearctic. METHODS: We analyzed the δ2 H in the wings (δ2 Hwing ) of 142 resident butterflies from 56 sites across the Afro-Palearctic. The δ2 Hwing values were compared to the predicted local growing-season precipitation δ2 H values (δ2 HGSP ) using a linear regression model to develop an insect wing δ2 H isoscape. We used multivariate linear mixed models and high-resolution and time-specific remote sensing climate and environmental data to explore the controls of the residual δ2 Hwing variability. RESULTS: A strong linear relationship was found between δ2 Hwing and δ2 HGSP values (r2 = 0.53). The resulting isoscape showed strong patterns across the Palearctic but limited variation and high uncertainty for the Afro-tropics. Positive residuals of this relationship were correlated with dry conditions for the month preceding sampling whereas negative residuals were correlated with more wet days for the month preceding sampling. High intra-site δ2 Hwing variance was associated with lower relative humidity for the month preceding sampling and higher elevation. CONCLUSION: The δ2 Hwing isoscape is applicable for tracing herbivorous lepidopteran insects that migrate across the Afro-Palearctic range but has limited geolocation potential in the Afro-tropics. The spatial analysis of uncertainty using high-resolution climatic data demonstrated that many African regions with highly variable evaporation rates and relative humidity have δ2 Hwing values that are less related to δ2 HGSP values. Increasing geolocation precision will require new modeling approaches using more time-specific environmental data and/or independent geolocation tools.

Environmental stress during larval development induces DNA methylation shifts in the migratory painted lady butterfly (Vanessa cardui).

Seasonal environmental fluctuations provide formidable challenges for living organisms, especially small ectotherms such as butterflies. A common strategy to cope with harsh environments is to enter diapause, but some species avoid unsuitable conditions by migrating. Despite a growing understanding of migration in the life cycles of some butterfly species, it remains unknown how individuals register and store environmental cues to determine whether and where to migrate. Here, we explored how competition and host plant availability during larval development affect patterns of DNA methylation in the migratory painted lady (Vanessa cardui) butterfly. We identify a set of potentially functional methylome shifts associated with differences in the environment, indicating that DNA methylation is involved in the response to different conditions during larval development. By analysing the transcriptome for the same samples used for methylation profiling, we also uncovered a non-monotonic relationship between gene body methylation and gene expression. Our results provide a starting point for understanding the interplay between DNA methylation and gene expression in butterflies in general and how differences in environmental conditions during development can trigger unique epigenetic marks that might be important for behavioural decisions in the adult stage.

Do chromosome rearrangements fix by genetic drift or natural selection? Insights from Brenthis butterflies.

Large-scale chromosome rearrangements, such as fissions and fusions, are a common feature of eukaryote evolution. They can have considerable influence on the evolution of populations, yet it remains unclear exactly how rearrangements become established and eventually fix. Rearrangements could fix by genetic drift if they are weakly deleterious or neutral, or they may instead be favoured by positive natural selection. Here, we compare genome assemblies of three closely related Brenthis butterfly species and characterize a complex history of fission and fusion rearrangements. An inferred demographic history of these species suggests that rearrangements became fixed in populations with large long-term effective size (Ne ), consistent with rearrangements being selectively neutral or only very weakly underdominant. Using a recently developed analytic framework for characterizing hard selective sweeps, we find that chromosome fusions are not enriched for evidence of past sweeps compared to other regions of the genome. Nonetheless, we do infer a strong and recent selective sweep around one chromosome fusion in the B. daphne genome. Our results suggest that rearrangements in these species likely have weak absolute fitness effects and fix by genetic drift. However, one putative selective sweep raises the possibility that natural selection may sometimes play a role in the fixation of chromosome fusions.

Diversification linked to larval host plant in the butterfly Eumedonia eumedon.

It is widely accepted that the relationship between phytophagous insects and their host plants influences insect diversification. However, studies addressed at documenting host-associated genetic differentiation (HAD) and the mechanisms that drive reproductive isolation in host-associated lineages (or host races) are still scarce relative to insect diversity. To uncover further evidence on the HAD processes in Lepidoptera, we investigated the genetic structure of the geranium argus butterfly (Eumedonia eumedon) and tested for isolation by ecology (IBE) vs. isolation by distance (IBD). Genomic data revealed an array of host races (three of them in the same mountain range, the Cantabrian Mountains, northern Iberia) at apparently distinct levels of reproductive isolation. We found a pattern of IBE mediated by HAD at both local and European scales, in which genetic differentiation between populations and individuals correlated significantly with the taxonomic relatedness of the host plants. IBD was significant only when considered at the wider European scale. We hypothesize that, locally, HAD between Geranium-feeding populations was caused (at least partially) by allochrony, that is via adaptation of adult flight time to the flowering period of each host plant species. Nevertheless, the potential reproductive isolation between populations using Erodium and populations using Geranium cannot be explained by allochrony or IBD, and other mechanisms are expected to be at play.

Migratory behaviour is positively associated with genetic diversity in butterflies.

Migration is typically associated with risk and uncertainty at the population level, but little is known about its cost-benefit trade-offs at the species level. Migratory insects in particular often exhibit strong demographic fluctuations due to local bottlenecks and outbreaks. Here, we use genomic data to investigate levels of heterozygosity and long-term population size dynamics in migratory insects, as an alternative to classical local and short-term approaches such as regional field monitoring. We analyse whole-genome sequences from 97 Lepidoptera species and show that individuals of migratory species have significantly higher levels of genome-wide heterozygosity, a proxy for effective population size, than do nonmigratory species. Also, we contribute whole-genome data for one of the most emblematic insect migratory species, the painted lady butterfly (Vanessa cardui), sampled across its worldwide distributional range. This species exhibits one of the highest levels of genomic heterozygosity described in Lepidoptera (2.95 ± 0.15%). Coalescent modelling (PSMC) shows historical demographic stability in V. cardui, and high effective population size estimates of 2-20 million individuals 10,000 years ago. The study reveals that the high risks associated with migration and local environmental fluctuations do not seem to decrease overall genetic diversity and demographic stability in migratory Lepidoptera. We propose a "compensatory" demographic model for migratory r-strategist organisms in which local bottlenecks are counterbalanced by reproductive success elsewhere within their typically large distributional ranges. Our findings highlight that the boundaries of populations are substantially different for sedentary and migratory insects, and that, in the latter, local and even regional field monitoring results may not reflect whole population dynamics. Genomic diversity patterns may elucidate key aspects of an insect's migratory nature and population dynamics at large spatiotemporal scales.

DNA Barcodes Combined with Multilocus Data of Representative Taxa Can Generate Reliable Higher-Level Phylogenies.

Taxa are frequently labeled incertae sedis when their placement is debated at ranks above the species level, such as their subgeneric, generic, or subtribal placement. This is a pervasive problem in groups with complex systematics due to difficulties in identifying suitable synapomorphies. In this study, we propose combining DNA barcodes with a multilocus backbone phylogeny in order to assign taxa to genus or other higher-level categories. This sampling strategy generates molecular matrices containing large amounts of missing data that are not distributed randomly: barcodes are sampled for all representatives, and additional markers are sampled only for a small percentage. We investigate the effects of the degree and randomness of missing data on phylogenetic accuracy using simulations for up to 100 markers in 1000-tips trees, as well as a real case: the subtribe Polyommatina (Lepidoptera: Lycaenidae), a large group including numerous species with unresolved taxonomy. Our simulation tests show that when a strategic and representative selection of species for higher-level categories has been made for multigene sequencing (approximately one per simulated genus), the addition of this multigene backbone DNA data for as few as 5-10% of the specimens in the total data set can produce high-quality phylogenies, comparable to those resulting from 100% multigene sampling. In contrast, trees based exclusively on barcodes performed poorly. This approach was applied to a 1365-specimen data set of Polyommatina (including ca. 80% of described species), with nearly 8% of representative species included in the multigene backbone and the remaining 92% included only by mitochondrial COI barcodes, a phylogeny was generated that highlighted potential misplacements, unrecognized major clades, and placement for incertae sedis taxa. We use this information to make systematic rearrangements within Polyommatina, and to describe two new genera. Finally, we propose a systematic workflow to assess higher-level taxonomy in hyperdiverse groups. This research identifies an additional, enhanced value of DNA barcodes for improvements in higher-level systematics using large data sets. [Birabiro; DNA barcoding; incertae sedis; Kipepeo; Lycaenidae; missing data; phylogenomic; phylogeny; Polyommatina; supermatrix; systematics; taxonomy].

Linkage mapping and genome annotation give novel insights into gene family expansions and regional recombination rate variation in the painted lady (Vanessa cardui) butterfly.

Characterization of gene family expansions and crossing over is crucial for understanding how organisms adapt to the environment. Here, we develop a high-density linkage map and detailed genome annotation of the painted lady butterfly (Vanessa cardui) - a non-diapausing, highly polyphagous species famous for its long-distance migratory behavior and almost cosmopolitan distribution. Our results reveal a complex interplay between regional recombination rate variation, gene duplications and transposable element activity shaping the genome structure of the painted lady. We identify several lineage specific gene family expansions. Their functions are mainly associated with protein and fat metabolism, detoxification, and defense against infection - critical processes for the painted lady's unique life-history. Furthermore, the detailed recombination maps allow us to characterize the regional recombination landscape, data that reveal a strong effect of chromosome size on the recombination rate, a limited impact of GC-biased gene conversion and a positive association between recombination and short interspersed elements.

Linking large-scale genetic structure of three Argynnini butterfly species to geography and environment.

Understanding which factors and processes are associated with genetic differentiation within and among species remains a major goal in evolutionary biology. To explore differences and similarities in genetic structure and its association with geographical and climatic factors in sympatric sister species, we conducted a large-scale (>32° latitude and >36° longitude) comparative phylogeographical study on three Argynnini butterfly species (Speyeria aglaja, Fabriciana adippe and F. niobe) that have similar life histories, but differ in ecological generalism and dispersal abilities. Analyses of nuclear (ddRAD-sequencing derived SNP markers) and mitochondrial (COI sequences) data revealed differences between species in genetic structure and how genetic differentiation was associated with climatic factors (temperature, solar radiation, precipitation, wind speed). Geographical proximity accounted for much of the variation in nuclear and mitochondrial structure and evolutionary relationships in F. adippe and F. niobe, but only explained the pattern observed in the nuclear data in S. aglaja, for which mitonuclear discordance was documented. In all species, Iberian and Balkan individuals formed genetic clusters, suggesting isolation in glacial refugia and limited postglacial expansion. Solar radiation and precipitation were associated with the genetic structure on a regional scale in all species, but the specific combinations of environmental and geographical factors linked to variation within species were unique, pointing to species-specific responses to common environments. Our findings show that the species share similar colonization histories, and that the same ecological factors, such as niche breadth and dispersal capacity, covary with genetic differentiation within these species to some extent, thereby highlighting the importance of comparative phylogeographical studies in sympatric sister species.

Hybridization fuelled diversification in Spialia butterflies.

The importance of hybridization and introgression is well documented in the evolution of plants but, in insects, their role is not fully understood. Given the fact that insects are the most diverse group of organisms, assessing the impact of reticulation events on their evolution may be key to comprehend the emergence of such remarkable diversity. Here, we used an insect model, the Spialia butterflies, to gather genomic evidence of hybridization as a promoter of novel diversity. By using double-digest RADseq (ddRADseq), we explored the phylogenetic relationships between Spialia orbifer, S. rosae and S. sertorius, and documented two independent events of interspecific gene flow. Our data support that the Iberian endemism S. rosae probably received genetic material from S. orbifer in both mitochondrial and nuclear DNA, which could have contributed to a shift in the ecological preferences of S. rosae. We also show that admixture between S. sertorius and S. orbifer probably occurred in Italy. As a result, the admixed Sicilian populations of S. orbifer are differentiated from the rest of populations both genetically and morphologically, and display signatures of reproductive character displacement in the male genitalia. Additionally, our analyses indicated that genetic material from S. orbifer is present in S. sertorius along the Italian Peninsula. Our findings add to the view that hybridization is a pervasive phenomenon in nature and in butterflies in particular, with important consequences for evolution due to the emergence of novel phenotypes.

Global invasion history of the agricultural pest butterfly Pieris rapae revealed with genomics and citizen science.

The small cabbage white butterfly, Pieris rapae, is a major agricultural pest of cruciferous crops and has been introduced to every continent except South America and Antarctica as a result of human activities. In an effort to reconstruct the near-global invasion history of P. rapae, we developed a citizen science project, the "Pieris Project," and successfully amassed thousands of specimens from 32 countries worldwide. We then generated and analyzed nuclear (double-digest restriction site-associated DNA fragment procedure [ddRAD]) and mitochondrial DNA sequence data for these samples to reconstruct and compare different global invasion history scenarios. Our results bolster historical accounts of the global spread and timing of P. rapae introductions. We provide molecular evidence supporting the hypothesis that the ongoing divergence of the European and Asian subspecies of P. rapae (∼1,200 y B.P.) coincides with the diversification of brassicaceous crops and the development of human trade routes such as the Silk Route (Silk Road). The further spread of P. rapae over the last ∼160 y was facilitated by human movement and trade, resulting in an almost linear series of at least 4 founding events, with each introduced population going through a severe bottleneck and serving as the source for the next introduction. Management efforts of this agricultural pest may need to consider the current existence of multiple genetically distinct populations. Finally, the international success of the Pieris Project demonstrates the power of the public to aid scientists in collections-based research addressing important questions in invasion biology, and in ecology and evolutionary biology more broadly.

Host plant diet affects growth and induces altered gene expression and microbiome composition in the wood white (Leptidea sinapis) butterfly.

In a time with decreasing biodiversity, especially among insects, a detailed understanding about specific resource utilization strategies is crucial. The physiological and behavioural responses to host switches in phytophagous insects are poorly understood. Earlier studies indicate that a host plant switch might be associated with distinctive molecular and physiological responses in different lineages. Expanding the assessment of such associations across Lepidoptera will reveal if there are general patterns in adaptive responses, or if each switch event is more of a unique character. We investigated host plant preference, fitness consequences, effects on expression profiles and gut microbiome composition in two common wood white (Leptidea sinapis) populations with different host plant preferences from the extremes of the species distribution area (Sweden and Catalonia). Our results show that female Catalonian wood whites lack preference for either host plant (Lotus corniculatus or L. dorycnium), while Swedish females laid significantly more eggs on L. corniculatus. Individuals from both populations reared on L. dorycnium had longer developmental times and smaller body size as adults. This indicates that both environmental and genetic factors determine the choice to use a specific host plant. Gene expression analysis revealed a more pronounced response to host plant in the Catalonian compared to the Swedish population. In addition, host plant treatment resulted in a significant shift in microbiome community structure in the Catalonian population. Together, this suggests that population specific plasticity associated with local conditions underlies host plant utilisation in wood whites.

Two ways to be endemic. Alps and Apennines are different functional refugia during climatic cycles.

Endemics co-occur because they evolved in situ and persist regionally or because they evolved ex situ and later dispersed to shared habitats, generating evolutionary or ecological endemicity centres, respectively. We investigate whether different endemicity centres can intertwine in the region ranging from Alps to Sicily, by studying their butterfly fauna. We gathered an extensive occurrence data set for butterflies of the study area (27,123 records, 269 species, in cells of 0.5 × 0.5 degrees of latitude-longitude). We applied molecular-based delimitation methods (GMYC model) to 26,557 cytochrome c oxidase subunit 1 (COI) sequences of Western Palearctic butterflies. We identified entities based on molecular delimitations and/or the checklist of European butterflies and objectively attributed occurrences to their most probable entity. We obtained a zoogeographic regionalisation based on the 69 endemics of the area. Using phylogenetic ANOVA we tested if endemics from different centres differ from each other and from nonendemics for key ecological traits and divergence time. Endemicity showed high incidence in the Alps and Southern Italy. The regionalisation separated the Alps from the Italian Peninsula and Sicily. The endemics of different centres showed a high turnover and differed in phylogenetic distances, phenology and distribution traits. Endemics are on average younger than nonendemics and the Peninsula-Sicily endemics also have lower variance in divergence than those from the Alps. The observed variation identifies Alpine endemics as paleoendemics, now occupying an ecological centre, and the Peninsula-Sicily ones as neoendemics, that diverged in the region since the Pleistocene. The results challenge the common view of the Alpine-Apennine area as a single "Italian refugium".

The Pleistocene species pump past its prime: Evidence from European butterfly sister species.

The Pleistocene glacial cycles had a profound impact on the ranges and genetic make-up of organisms. While it is clear that the contact zones that have been described for many sister taxa are secondary and have formed in the current interglacial, it is unclear when the taxa involved began to diverge. Previous estimates based on small numbers of loci are unreliable given the stochasticity of genetic drift and the contrasting effects of incomplete lineage sorting and gene flow on gene divergence. Here, we use genome-wide transcriptome data to estimate divergence for 18 sister species pairs of European butterflies showing either sympatric or contact zone distributions. We find that in most cases, species divergence predates the mid-Pleistocene transition or even the entire Pleistocene period. We also show that although post-divergence gene flow is restricted to contact zone pairs, they are not systematically younger than sympatric pairs. This suggests that contact zones are not limited to the initial stages of the speciation process, but can involve notably old taxa. Finally, we show that mitochondrial divergence and nuclear divergence are only weakly correlated and mitochondrial divergence is higher for contact zone pairs.