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.

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".

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

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.

Rapid colour shift by reproductive character displacement in Cupido butterflies.

Reproductive character displacement occurs when competition for successful breeding imposes a divergent selection on the interacting species, causing a divergence of reproductive traits. Here, we show that a disputed butterfly taxon is actually a case of male wing colour shift, apparently produced by reproductive character displacement. Using double digest restriction-site associated DNA sequencing and mitochondrial DNA sequencing we studied four butterfly taxa of the subgenus Cupido (Lepidoptera: Lycaenidae): Cupido minimus and the taxon carswelli, both characterized by brown males and females, plus C. lorquinii and C. osiris, both with blue males and brown females. Unexpectedly, taxa carswelli and C. lorquinii were close to indistinguishable based on our genomic and mitochondrial data, despite displaying strikingly different male coloration. In addition, we report and analysed a brown male within the C. lorquinii range, which demonstrates that the brown morph occurs at very low frequency in C. lorquinii. Such evidence strongly suggests that carswelli is conspecific with C. lorquinii and represents populations with a fixed male brown colour morph. Considering that these brown populations occur in sympatry with or very close to the blue C. osiris, and that the blue C. lorquinii populations never do, we propose that the taxon carswelli could have lost the blue colour due to reproductive character displacement with C. osiris. Since male colour is important for conspecific recognition during courtship, we hypothesize that the observed colour shift may eventually trigger incipient speciation between blue and brown populations. Male colour seems to be an evolutionarily labile character in the Polyommatinae, and the mechanism described here might be at work in the wide diversification of this subfamily of butterflies.

How long is 3 km for a butterfly? Ecological constraints and functional traits explain high mitochondrial genetic diversity between Sicily and the Italian Peninsula.

Populations inhabiting Mediterranean islands often show contrasting genetic lineages, even on islands that were connected to the mainland during glacial maxima. This pattern is generated by forces acting in historical and contemporary times. Understanding these phenomena requires comparative studies integrating genetic structure, functional traits and dispersal constraints. Using as a model the butterfly species living across the Messina strait (3 km wide) separating Sicily from the Italian Peninsula, we aimed to unravel the mechanisms limiting the dispersal of matrilines and generating genetic differentiation across a narrow sea strait. We analysed the mitochondrial COI gene of 84 butterfly species out of 90 documented in Sicily and compared them with populations from the neighbouring southern Italian Peninsula (1,398 sequences) and from the entire Palaearctic region (8,093 sequences). For each species, we regressed 13 functional traits and 2 ecological constraints to dispersal (winds experienced at the strait and climatic suitability) against genetic differentiation between Sicily and Italian Peninsula to understand the factors limiting dispersal. More than a third of the species showed different haplogroups across the strait and most of them also represented endemic haplogroups for this island. One fifth of Sicilian populations (and 32.3% of endemic lineages) had their closest relatives in distant areas, instead of the neighbouring Italian Peninsula, which suggests high relictuality. Haplotype diversity was significantly explained by the length of the flight period, an intrinsic phenology trait, while genetic differentiation was explained by both intrinsic traits (wingspan and degree of generalism) and contemporary local constraints (winds experienced at the strait and climatic suitability). A relatively narrow sea strait can produce considerable differentiation among butterfly matrilines and this phenomenon showed a largely deterministic fingerprint. Because of unfavourable winds, populations of the less dispersive Sicilian butterflies tended to differentiate into endemic variants or to maintain relict populations. Understanding these phenomena required the integration of DNA sequences, species traits and physical constraints for a large taxon at continental scale. Future studies may reveal if the patterns here shown for mitochondrial DNA are also reflected in the nuclear genome or, alternatively, are the product of limited female dispersal.

The conundrum of species delimitation: a genomic perspective on a mitogenetically super-variable butterfly.

The Palaearctic butterfly Melitaea didyma stands out as one of the most striking cases of intraspecific genetic differentiation detected in Lepidoptera: 11 partially sympatric mitochondrial lineages have been reported, displaying levels of divergence of up to 7.4%. To better understand the evolutionary processes underlying the diversity observed in mtDNA, we compared mtDNA and genome-wide SNP data using double-digest restriction site-associated DNA sequencing (ddRADseq) results from 93 specimens of M. didyma ranging from Morocco to eastern Kazakhstan. We found that, between ddRADseq and mtDNA results, there is a match only in populations that probably remained allopatric for long periods of time. Other mtDNA lineages may have resulted from introgression events and were probably affected by Wolbachia infection. The five main ddRADseq clades supported by STRUCTURE were parapatric or allopatric and showed high pairwise FST values, but some were also estimated to display various levels of gene flow. Melitaea didyma represents one of the first cases of deep mtDNA splits among European butterflies assessed by a genome-wide DNA analysis and reveals that the interpretation of patterns remains challenging even when a high amount of genomic data is available. These findings actualize the ongoing debate of species delimitation in allopatry, an issue probably of relevance to a significant proportion of global biodiversity.

Lack of gene flow: Narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species.

Genome scans in recently separated species can inform on molecular mechanisms and evolutionary processes driving divergence. Large-scale polymorphism data from multiple species pairs are also key to investigate the repeatability of divergence-whether radiations tend to show parallel responses to similar selection pressures and/or underlying molecular forces. Here, we used whole-genome resequencing data from six wood white (Leptidea sp.) butterfly populations, representing three closely related species with karyomorph variation, to infer the species' demographic history and characterize patterns of genomic diversity and differentiation. The analyses supported previously established species relationships, and there was no evidence for postdivergence gene flow. We identified significant intraspecific genetic structure, in particular between karyomorph extremes in the wood white (L. sinapis)-a species with a remarkable chromosome number cline across the distribution range. The genomic landscapes of differentiation were erratic, and outlier regions were narrow and dispersed. Highly differentiated (FST ) regions generally had low genetic diversity (θπ ), but increased absolute divergence (DXY ) and excess of rare frequency variants (low Tajima's D). A minority of differentiation peaks were shared across species and population comparisons. However, highly differentiated regions contained genes with overrepresented functions related to metabolism, response to stimulus and cellular processes, indicating recurrent directional selection on a specific set of traits in all comparisons. In contrast to the majority of genome scans in recently diverged lineages, our data suggest that divergence landscapes in Leptidea have been shaped by directional selection and genetic drift rather than stable recombination landscapes and/or introgression.

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

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.

Gene expression profiling across ontogenetic stages in the wood white (Leptidea sinapis) reveals pathways linked to butterfly diapause regulation.

In temperate latitudes, many insects enter diapause (dormancy) during the cold season, a period during which developmental processes come to a standstill. The wood white (Leptidea sinapis) is a butterfly species distributed across western Eurasia that shows photoperiod-induced diapause with variation in critical day-length across populations at different latitudes. We assembled transcriptomes and estimated gene expression levels at different developmental stages in experimentally induced directly developing and diapausing cohorts of a single Swedish population of L. sinapis to investigate the regulatory mechanisms underpinning diapause initiation. Different day lengths resulted in expression changes of developmental genes and affected the rate of accumulation of signal molecules, suggesting that diapause induction might be controlled by increased activity of monoamine neurotransmitters in larvae reared under short-day light conditions. Expression differences between light treatment groups of two monoamine regulator genes (DDC and ST) were observed already in instar III larvae. Once developmental pathways were irreversibly set at instar V, a handful of genes related to dopamine production were differentially expressed leading to a significant decrease in expression of global metabolic genes and increase in expression of genes related to fatty acid synthesis and sequestration. This is in line with a time-dependent (hour-glass) model of diapause regulation where a gradual shift in the concentration of monoamine neurotransmitters and their metabolites during development of larvae under short-day conditions leads to increased storage of fat, decreased energy expenditures, and ultimately developmental stasis at the pupal stage.

Use of genetic, climatic, and microbiological data to inform reintroduction of a regionally extinct butterfly.

Species reintroductions are increasingly used as means of mitigating biodiversity loss. Besides habitat quality at the site targeted for reintroduction, the choice of source population can be critical for success. The butterfly Melanargia russiae (Esper´s marbled white) was extirpated from Hungary over 100 years ago, and a reintroduction program has recently been approved. We used museum specimens of this butterfly, mitochondrial DNA data (mtDNA), endosymbiont screening, and climatic-similarity analyses to determine which extant populations should be used for its reintroduction. The species displayed 2 main mtDNA lineages across its range: 1 restricted to Iberia and southern France (Iberian lineage) and another found throughout the rest of its range (Eurasian lineage). These 2 lineages possessed highly divergent wsp alleles of the bacterial endosymbiont Wolbachia. The century-old Hungarian specimens represented an endemic haplotype belonging to the Eurasian lineage, differing by one mutation from the Balkan and eastern European populations. The Hungarian populations of M. russiae occurred in areas with a colder and drier climate relative to most sites with extant known populations. Our results suggest the populations used for reintroduction to Hungary should belong to the Eurasian lineage, preferably from eastern Ukraine (genetically close and living in areas with the highest climatic similarity). Materials stored in museum collections can provide unique opportunities to document historical genetic diversity and help direct conservation.

Climatic niche evolution is faster in sympatric than allopatric lineages of the butterfly genus Pyrgus.

Understanding how speciation relates to ecological divergence has long fascinated biologists. It is assumed that ecological divergence is essential to sympatric speciation, as a mechanism to avoid competition and eventually lead to reproductive isolation, while divergence in allopatry is not necessarily associated with niche differentiation. The impact of the spatial context of divergence on the evolutionary rates of abiotic dimensions of the ecological niche has rarely been explored for an entire clade. Here, we compare the magnitude of climatic niche shifts between sympatric versus allopatric divergence of lineages in butterflies. By combining next-generation sequencing, parametric biogeography and ecological niche analyses applied to a genus-wide phylogeny of Palaearctic Pyrgus butterflies, we compare evolutionary rates along eight climatic dimensions across sister lineages that diverged in large-scale sympatry versus allopatry. In order to examine the possible effects of the spatial scale at which sympatry is defined, we considered three sets of biogeographic assignments, ranging from narrow to broad definition. Our findings suggest higher rates of niche evolution along all climatic dimensions for sister lineages that diverge in sympatry, when using a narrow delineation of biogeographic areas. This result contrasts with significantly lower rates of climatic niche evolution found in cases of allopatric speciation, despite the biogeographic regions defined here being characterized by significantly different climates. Higher rates in allopatry are retrieved when biogeographic areas are too widely defined-in such a case allopatric events may be recorded as sympatric. Our results reveal the macro-evolutionary significance of abiotic niche differentiation involved in speciation processes within biogeographic regions, and illustrate the importance of the spatial scale chosen to define areas when applying parametric biogeographic analyses.

Species-Level Para- and Polyphyly in DNA Barcode Gene Trees: Strong Operational Bias in European Lepidoptera.

The proliferation of DNA data is revolutionizing all fields of systematic research. DNA barcode sequences, now available for millions of specimens and several hundred thousand species, are increasingly used in algorithmic species delimitations. This is complicated by occasional incongruences between species and gene genealogies, as indicated by situations where conspecific individuals do not form a monophyletic cluster in a gene tree. In two previous reviews, non-monophyly has been reported as being common in mitochondrial DNA gene trees. We developed a novel web service "Monophylizer" to detect non-monophyly in phylogenetic trees and used it to ascertain the incidence of species non-monophyly in COI (a.k.a. cox1) barcode sequence data from 4977 species and 41,583 specimens of European Lepidoptera, the largest data set of DNA barcodes analyzed from this regard. Particular attention was paid to accurate species identification to ensure data integrity. We investigated the effects of tree-building method, sampling effort, and other methodological issues, all of which can influence estimates of non-monophyly. We found a 12% incidence of non-monophyly, a value significantly lower than that observed in previous studies. Neighbor joining (NJ) and maximum likelihood (ML) methods yielded almost equal numbers of non-monophyletic species, but 24.1% of these cases of non-monophyly were only found by one of these methods. Non-monophyletic species tend to show either low genetic distances to their nearest neighbors or exceptionally high levels of intraspecific variability. Cases of polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, as the detected cases reflected misidentifications or methodological errors. Taking into consideration variation in sampling effort, we estimate that the true incidence of non-monophyly is ∼23%, but with operational factors still being included. Within the operational factors, we separately assessed the frequency of taxonomic limitations (presence of overlooked cryptic and oversplit species) and identification uncertainties. We observed that operational factors are potentially present in more than half (58.6%) of the detected cases of non-monophyly. Furthermore, we observed that in about 20% of non-monophyletic species and entangled species, the lineages involved are either allopatric or parapatric-conditions where species delimitation is inherently subjective and particularly dependent on the species concept that has been adopted. These observations suggest that species-level non-monophyly in COI gene trees is less common than previously supposed, with many cases reflecting misidentifications, the subjectivity of species delimitation or other operational factors.

Integrative analyses unveil speciation linked to host plant shift in Spialia butterflies.

Discovering cryptic species in well-studied areas and taxonomic groups can have profound implications in understanding eco-evolutionary processes and in nature conservation because such groups often involve research models and act as flagship taxa for nature management. In this study, we use an array of techniques to study the butterflies in the Spialia sertorius species group (Lepidoptera, Hesperiidae). The integration of genetic, chemical, cytogenetic, morphological, ecological and microbiological data indicates that the sertorius species complex includes at least five species that differentiated during the last three million years. As a result, we propose the restitution of the species status for two taxa often treated as subspecies, Spialia ali (Oberthür, 1881) stat. rest. and Spialia therapne (Rambur, 1832) stat. rest., and describe a new cryptic species Spialia rosae Hernández-Roldán, Dapporto, Dinca, Vicente & Vila sp. nov. Spialia sertorius (Hoffmannsegg, 1804) and S. rosae are sympatric and synmorphic, but show constant differences in mitochondrial DNA, chemical profiles and ecology, suggesting that S. rosae represents a case of ecological speciation involving larval host plant and altitudinal shift, and apparently associated with Wolbachia infection. This study exemplifies how a multidisciplinary approach can reveal elusive cases of hidden diversity.

Dynamic karyotype evolution and unique sex determination systems in Leptidea wood white butterflies.

BACKGROUND: Chromosomal rearrangements have the potential to limit the rate and pattern of gene flow within and between species and thus play a direct role in promoting and maintaining speciation. Wood white butterflies of the genus Leptidea are excellent models to study the role of chromosome rearrangements in speciation because they show karyotype variability not only among but also within species. In this work, we investigated genome architecture of three cryptic Leptidea species (L. juvernica, L. sinapis and L. reali) by standard and molecular cytogenetic techniques in order to reveal causes of the karyotype variability. RESULTS: Chromosome numbers ranged from 2n = 85 to 91 in L. juvernica and 2n = 69 to 73 in L. sinapis (both from Czech populations) to 2n = 51 to 55 in L. reali (Spanish population). We observed significant differences in chromosome numbers and localization of cytogenetic markers (rDNA and H3 histone genes) within the offspring of individual females. Using FISH with the (TTAGG) n telomeric probe we also documented the presence of multiple chromosome fusions and/or fissions and other complex rearrangements. Thus, the intraspecific karyotype variability is likely due to irregular chromosome segregation of multivalent meiotic configurations. The analysis of female meiotic chromosomes by GISH and CGH revealed multiple sex chromosomes: W1W2W3Z1Z2Z3Z4 in L. juvernica, W1W2W3Z1Z2Z3 in L. sinapis and W1W2W3W4Z1Z2Z3Z4 in L. reali. CONCLUSIONS: Our results suggest a dynamic karyotype evolution and point to the role of chromosomal rearrangements in the speciation of Leptidea butterflies. Moreover, our study revealed a curious sex determination system with 3-4 W and 3-4 Z chromosomes, which is unique in the Lepidoptera and which could also have played a role in the speciation process of the three Leptidea species.

A macroevolutionary role for chromosomal fusion and fission in Erebia butterflies.

The impact of large-scale chromosomal rearrangements, such as fusions and fissions, on speciation is a long-standing conundrum. We assessed whether bursts of change in chromosome numbers resulting from chromosomal fusion or fission are related to increased speciation rates in Erebia, one of the most species-rich and karyotypically variable butterfly groups. We established a genome-based phylogeny and used state-dependent birth-death models to infer trajectories of karyotype evolution. We demonstrated that rates of anagenetic chromosomal changes (i.e., along phylogenetic branches) exceed cladogenetic changes (i.e., at speciation events), but, when cladogenetic changes occur, they are mostly associated with chromosomal fissions rather than fusions. We found that the relative importance of fusion and fission differs among Erebia clades of different ages and that especially in younger, more karyotypically diverse clades, speciation is more frequently associated with cladogenetic chromosomal changes. Overall, our results imply that chromosomal fusions and fissions have contrasting macroevolutionary roles and that large-scale chromosomal rearrangements are associated with bursts of species diversification.

Biogeography and systematics of Aricia butterflies (Lepidoptera, Lycaenidae).

Butterflies of the Aricia species group represent a paradigm of unresolved taxonomy, both at the genus and species levels. We studied phylogenetic relationships, biogeography, and systematics based on genetic--nuclear and mitochondrial--and morphometric--external (wings) and internal (genitalia) - data. We show that Aricia is a monophyletic genus comprising the taxa Pseudoaricia, Ultraaricia and Umpria, which are here considered junior synonyms of Aricia. The taxa allous, inhonora, issekutzi, mandzhuriana, myrmecias and transalaica, which have often been raised to species rank, are shown to probably represent subspecies or synonyms. We show that montensis is likely a good species that is sister to all A. artaxerxes populations across the Palearctic region. The species A. anteros and A. morronensis are shown to display deep intraspecific divergences and they may harbor cryptic species. We also discovered that A. cramera and A. agestis exhibit a pattern of mutual exclusion on islands, and a parapatric distribution in mainland with a narrow contact zone where potential hybrids were detected. The lack of a prezygotic barrier that prevents their coexistence could explain this phenomenon. This study will hopefully contribute to the stability of the systematics of Aricia, a group with potential for the study of the link between speciation and biogeography.

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

A new subspecies of Leptideasinapis from Northern Iran, discovered by means of DNA barcoding, is described as Leptideasinapistabarestanassp. 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.

The genome sequence of the lesser marbled fritillary, Brenthis ino, and evidence for a segregating neo-Z chromosome.

The lesser marbled fritillary, Brenthis ino (Rottemburg, 1775), is a species of Palearctic butterfly. Male Brenthis ino individuals have been reported to have between 12 and 14 pairs of chromosomes, a much-reduced chromosome number than is typical in butterflies. Here, we present a chromosome-level genome assembly for Brenthis ino, as well as gene and transposable element annotations. The assembly is 411.8 Mb in length with a contig N50 of 9.6 Mb and a scaffold N50 of 29.5 Mb. We also show evidence that the male individual from which we generated HiC data was heterozygous for a neo-Z chromosome, consistent with inheriting 14 chromosomes from one parent and 13 from the other. This genome assembly will be a valuable resource for studying chromosome evolution in Lepidoptera, as well as for comparative and population genomics more generally.

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.

Complete DNA barcode reference library for a country's butterfly fauna reveals high performance for temperate Europe.

DNA barcoding aims to accelerate species identification and discovery, but performance tests have shown marked differences in identification success. As a consequence, there remains a great need for comprehensive studies which objectively test the method in groups with a solid taxonomic framework. This study focuses on the 180 species of butterflies in Romania, accounting for about one third of the European butterfly fauna. This country includes five eco-regions, the highest of any in the European Union, and is a good representative for temperate areas. Morphology and DNA barcodes of more than 1300 specimens were carefully studied and compared. Our results indicate that 90 per cent of the species form barcode clusters allowing their reliable identification. The remaining cases involve nine closely related species pairs, some whose taxonomic status is controversial or that hybridize regularly. Interestingly, DNA barcoding was found to be the most effective identification tool, outperforming external morphology, and being slightly better than male genitalia. Romania is now the first country to have a comprehensive DNA barcode reference database for butterflies. Similar barcoding efforts based on comprehensive sampling of specific geographical regions can act as functional modules that will foster the early application of DNA barcoding while a global system is under development.