Unprecedented within-species chromosome number cline in the Wood White butterfly Leptidea sinapis and its significance for karyotype evolution and speciation

Background: Species generally have a fixed number of chromosomes in the cell nuclei while between-species differences are common and often pronounced. These differences could have evolved through multiple speciation events, each involving the fixation of a single chromosomal rearrangement. Alternatively, marked changes in the karyotype may be the consequence of within-species accumulation of multiple chromosomal fissions/fusions, resulting in highly polymorphic systems with the subsequent extinction of intermediate karyomorphs. Although this mechanism of chromosome number evolution is possible in theory, it has not been well documented. Results: We present the discovery of exceptional intraspecific variability in the karyotype of the widespread Eurasian butterfly Leptidea sinapis. We show that within this species the diploid chromosome number gradually decreases from 2n = 106 in Spain to 2n = 56 in eastern Kazakhstan, resulting in a 6000 km-wide cline that originated recently (8,500 to 31,000 years ago). Remarkably, intrapopulational chromosome number polymorphism exists, the chromosome number range overlaps between some populations separated by hundreds of kilometers, and chromosomal heterozygotes are abundant. We demonstrate that this karyotypic variability is intraspecific because in L. sinapis a broad geographical distribution is coupled with a homogenous morphological and genetic structure. Conclusions: The discovered system represents the first clearly documented case of explosive chromosome number evolution through intraspecific and intrapopulation accumulation of multiple chromosomal changes. Leptidea sinapis may be used as a model system for studying speciation by means of chromosomally-based suppressed recombination mechanisms, as well as clinal speciation, a process that is theoretically possible but difficult to document. The discovered cline seems to represent a narrow time-window of the very first steps of species formation linked to multiple chromosomal changes that have occurred explosively. This case offers a rare opportunity to study this process before drift, dispersal, selection, extinction and speciation erase the traces of microevolutionary events and just leave the final picture of a pronounced interspecific chromosomal difference

Biogéographie, écologie et conservation d’Erebia oeme (Hübner) dans les Carpathes (Lepidoptera : Nymphalidae : Satyrinae)

13 páginas, 8 figuras, 1 tabla.[EN]The European endemic Erebia oeme (Hübner [1804]) (Lepidoptera: Nymphalidae: Satyrinae) is discovered in the Carpathian Chain, from where it was considered to be absent. The single population found is situated in the southern part of the Romanian Carpathians (Retezat Mountains), where it fl ies sympatrically and synchronically with Erebia medusa ([Denis & Schiffermüller] 1775). The similar external morphology of these two species probably caused E. oeme to be overlooked in the Carpathians, leading to an unexpected information gap in the otherwise thoroughly studied European continent. The morphology of the Romanian specimens is compared to populations from the rest of the species’ range and to E. medusa. In addition, we tested DNA barcoding as a method to discriminate between these species and confi rmed that it represents an effective identifi cation tool for the taxa involved. The habitat of E. oeme, adults of both sexes and their genitalia are illustrated in comparison with E. medusa. Based on the study of several collections, we show that E. oeme is likely to be extremely local in the Carpathians and provide arguments to consider the species as vulnerable in Romania.[FR]L’espèce endémique Européenne, Erebia oeme (Hübner [1804]) (Lepidoptera: Nymphalidae: Satyrinae), a été découverte dans la Chaîne des Carpates d’où elle était considérée comme absente. La seule population trouvée se situe dans la partie méridionale des Carpates Roumaines (Massif du Retezat), où l’espèce est sympatrique et synchronique avec Erebia medusa ([Denis & Schiffermüller] 1775). La similarité morphologique externe entre ces deux espèces est probablement la raison pour laquelle E. oeme a été méconnu dans les Carpates ce qui a laissé un défi cit d’information inattendu au niveau du continent Européen ayant fait l’objet d’études approfondies. La morphologie des spécimens Roumains est comparée aux populations du reste de l’aire de répartition de cette espèce et avec E. medusa. D’autre part nous avons testé le codage à barres de l’ADN comme méthode pour identifi er ces deux espèces et avons confi rmé que ceci est en effet un outil effi cace d’identifi cation pour les taxons concernés. L’habitat d’ E. oeme, les adultes des deux sexes et leurs organes génitaux sont illustrés en comparaison à E. medusa. Basé sur l’étude de différentes collections, nous démontrons que E. oeme est probablement très local dans les Carpates et nous apportons des arguments pour considérer l’espèce comme vulnérable en Roumanie.Financiación para este proyecto de investigación: Ministerio de Ciencia e Innovación (CGL2007-60516/BOS) para Roger Vila y Vlad Dincă, beca predoctoral de la Universitat Autònoma de Barcelona para Vlad Dincă, y subvenciones de NSERC y Genome Canada para Paul D. N. Hebert.Peer reviewe

Genetics and extreme confinement of three overlooked butterfly species in Romania call for immediate conservation actions

Abstract A good knowledge of species distributions and their genetic structure is essential for numerous types of research such as population genetics, phylogeography, or conservation genetics. We document the presence of extremely local populations of three butterfly species (Iolana iolas, Satyrus ferula and Melanargia larissa) in the Romanian fauna. Satyrus ferula and M. larissa are reported for the first time in the country, while I. iolas is rediscovered following presumed extinction. Based on mitochondrial DNA (cytochrome c oxidase subunit 1—COI sequences), we assessed the genetic structure of these populations and placed them into a broader context through comparisons with other populations from across the range of these species. Each of the three species had a single haplotype in Romania, suggesting low female effective population size possibly under genetic erosion. Two of the populations (S. ferula and M. larissa) are genetically unique, displaying endemic haplotypes in south-western Romania. The Romanian populations of the three species likely remained unnoticed due to their extremely limited extent of occurrence. Their restricted range, close to the northern limits of distribution in the Balkans, their apparent low female effective population size, the presence of endemic haplotypes, and habitat vulnerability (especially for I. iolas) highlight the need for monitoring and conservation measures for the safeguarding of these populations

Integrative analyses on Western Palearctic Lasiommata reveal a mosaic of nascent butterfly species

Abstract Satyrinae butterflies occurring in the Mediterranean apparently have reduced gene flow over sea straits, and for several species, recent wide-scale biodiversity surveys indicate the existence of divergent mitochondrial lineages. Here, we apply an integrative approach and examine the phylogeography of the genus Lasiommata in the Western Palearctic. Our research comprised molecular analyses (mitochondrial and nuclear DNA) and geometric morphometrics (wings and genitalia) for two main species groups, and a comparative GMYC analysis, based on COI, of all the tribes within Satyrinae from this region. The GMYC approach revealed a particularly fast coalescence rate in the Parargina subtribe. The Lasiommata group was divided into 12 evolutionary significant units: six clades for the L. maera species group, five for the L. megera species group, and one for L. petropolitana, with divergences of about 1%. The patterns of COI were mirrored by ITS2 in L. maera, but the two markers were generally inconsistent in L. megera. On the contrary, morphological differences were coherent with the results of COI for L. megera, but less clearly so for L. maera. L. paramegaera and L. meadewaldoi were considerably differentiated for all the analyzed markers and likely proceeded faster in the process of speciation because of geographic isolation and reduced effective population size, rendering the rest paraphyletic. Our study illustrates the continuous nature of speciation and the difficulties of delimiting species. In Lasiommata, the recognition of taxa as diverging lineages or distinct, possibly paraphyletic species, mostly depends on the criteria adopted by different species concepts

More hidden diversity in a cryptic species complex:a new subspecies of Leptidea sinapis (Lepidoptera, Pieridae) from Northern Iran

Abstract A new subspecies of Leptidea sinapis from Northern Iran, discovered by means of DNA barcoding, is described as Leptidea sinapis tabarestana ssp. nov. The new subspecies is allopatric with respect to other populations of L. sinapis and is genetically distinct, appearing as a well-supported sister clade to all other populations in COI-based phylogenetic reconstructions. Details on karyotype, genitalia, ecology and behaviour for the new subspecies are given and a biogeographical speciation scenario is proposed

Molecular phylogeny of the Palaearctic butterfly genus Pseudophilotes (Lepidoptera: Lycaenidae) with focus on the Sardinian endemic P. barbagiae

Abstract Background: The Palaearctic butterfly genus Pseudophilotes Beuret, 1958 (Lycaenidae: Polyommatinae), that today occurs in North Africa and in Eurasia, includes ten described species with various distribution ranges, including endemics such as the Sardinian P. barbagiae. Phylogenetic relationships among these species are largely unresolved. In the present study, we analysed 158 specimens, representing seven species out of ten described in the genus, from widely distributed sites throughout the western Palaearctic region, using nuclear markers (28S rRNA, wingless, Internal Transcribed Spacer 2 and Elongation Factor 1α) and the barcoding region of the mitochondrial cytochrome c oxidase subunit 1 gene to investigate if the current taxonomic entities match the phylogenetic pattern. Further, we attempt to infer the geographic origin of the genus Pseudophilotes and estimate the timing of its radiations, including the split of the Sardinian endemic P. barbagiae. Results: Maximum Likelihood and Bayesian inference analyses confirmed the monophyly of the genus Pseudophilotes and clearly supported the closer affinity of P. barbagiae to the species assemblage of P. baton, P. vicrama and P. panoptes as opposed to P. abencerragus. The currently accepted species P. baton, P. vicrama and P. panoptes turned out to be weakly differentiated from each other, while P. bavius and P. fatma emerged as highly distinct and formed a well supported clade. The split between the lineage comprising bavius and fatma (sometimes treated as a distinct genus, Rubrapterus) with Salvia species as larval host plants, and the remaining Pseudophilotes that utilize Thymus and other Lamiaceae (but not Salvia), dates back to about 4.9 million years ago (Mya). Conclusions: Our results show that the last common ancestor of the genus probably lived in the Messinian period (5.33–7.25 Mya). At species level, they support the current taxonomy of the genus, although P. panoptes, P. baton and P. vicrama display complex patterns based on phylogeographic relationships inferred from mtDNA. The Sardinian endemic P. barbagiae turned out to be a young endemic, but clearly with European instead of North African origin and evolved through allopatric isolation on the island of Sardinia only about 0.74 Mya

Versatility of multivalent orientation, inverted meiosis, and rescued fitness in holocentric chromosomal hybrids

Abstract Chromosomal rearrangements (e.g., fusions/fissions) have the potential to drive speciation. However, their accumulation in a population is generally viewed as unlikely, because chromosomal heterozygosity should lead to meiotic problems and aneuploid gametes. Canonical meiosis involves segregation of homologous chromosomes in meiosis I and sister chromatid segregation during meiosis II. In organisms with holocentric chromosomes, which are characterized by kinetic activity distributed along almost the entire chromosome length, this order may be inverted depending on their metaphase I orientation. Here we analyzed the evolutionary role of this intrinsic versatility of holocentric chromosomes, which is not available to monocentric ones, by studying F1 to F4 hybrids between two chromosomal races of the Wood White butterfly (Leptidea sinapis), separated by at least 24 chromosomal fusions/fissions. We found that these chromosomal rearrangements resulted in multiple meiotic multivalents, and, contrary to the theoretical prediction, the hybrids displayed relatively high reproductive fitness (42% of that of the control lines) and regular behavior of meiotic chromosomes. In the hybrids, we also discovered inverted meiosis, in which the first and critical stage of chromosome number reduction was replaced by the less risky stage of sister chromatid separation. We hypothesize that the ability to invert the order of the main meiotic events facilitates proper chromosome segregation and hence rescues fertility and viability in chromosomal hybrids, potentially promoting dynamic karyotype evolution and chromosomal speciation

Molecular evolution in introduced insect species:a mitochondrial perspective

Abstract Introduced species provide the opportunity to study evolutionary change on short time scales—a key first step to understand and manage the ecosystem-level impact of invasions. This study examined mitochondrial DNA sequence variation in cytochrome c oxidase subunit 1 (COI) for 26 insect species—Coleoptera (9), Hymenoptera (9), and Lepidoptera (8)—introduced to the Nearctic from the Palearctic. A total of 6,302 barcode records were retrieved from BOLD (boldsystems.org) to compare sequence diversity between the native and introduced range. As expected, genetic variation averaged nearly an order of magnitude lower in introduced populations (2.19 × 10⁻⁵ substitutions per nucleotide) than in the native range (1.48 ×10⁻⁴ substitutions per nucleotide). Nonsynonymous and synonymous changes had a similar incidence in the introduced populations (p-value = 0.83, averaging respectively 1.08 × 10⁻⁵ and 1.11 × 10⁻⁵ substitutions). By contrast, nonsynonymous changes were ten-fold less frequent than synonymous changes in the native populations (p-value < 0.001, averaging 1.74 × 10⁻⁵ and 1.3 × 10⁻⁴ substitutions, respectively). Patterns of sequence variation in the introduced range were largely congruent across the three insect orders which suggests that they are produced by general processes. This study explores the molecular evolution of introduced species, a fundamental aspect to improve understanding of their biology and manage their impact on ecosystems

A mirage of cryptic species:genomics uncover striking mitonuclear discordance in the butterfly Thymelicus sylvestris

Abstract Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double‐digest restriction‐site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex‐biased asymmetries, NUMTs, natural selection, introgression or Wolbachia‐mediated genetic sweeps. We suggest that this mitonuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events

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

Abstract 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”