Deceptive single‐locus taxonomy and phylogeography: Wolbachia‐associated divergence in mitochondrial DNA is not reflected in morphology and nuclear markers in a butterfly species

The satyrine butterfly Coenonympha tullia (Nymphalidae: Satyrinae) displays a deep split between two mitochondrial clades, one restricted to northern Alberta, Canada, and the other found throughout Alberta and across North America. We confirm this deep divide and test hypotheses explaining its phylogeographic structure. Neither genitalia morphology nor nuclear gene sequence supports cryptic species as an explanation, instead indicating differences between nuclear and mitochondrial genome histories. Sex‐biased dispersal is unlikely to cause such mito‐nuclear differences; however, selective sweeps by reproductive parasites could have led to this conflict. About half of the tested samples were infected by Wolbachia bacteria. Using multilocus strain typing for three Wolbachia genes, we show that the divergent mitochondrial clades are associated with two different Wolbachia strains, supporting the hypothesis that the mito‐nuclear differences resulted from selection on the mitochondrial genome due to selective sweeps by Wolbachia strains.     

Movements and flight morphology in the endangered Large Blue butterflies

Movements and flight morphology of the endangered Large Blue butterflies Phengaris teleius and P. nausithous in southern Poland were studied with mark-release-recapture surveys and GIS analyses. Most individuals moved relatively small distances (<40 metres) within their habitat patches. Distances covered by both species were positively related to season progression and wing length, and negatively related to body mass. P. teleius movement distances slightly exceeded those of P. nausithous. In addition, females moved longer distances than males, although the difference was significant only in P. teleius. Morphological traits appeared to be good indicators of the inter-specific and inter-sexual differences in mobility. While P. teleius individuals were heavier than P. nausithous ones, they had considerably longer wings, which may explain longer movements in the former species. Similarly, females were heavier than males in both species, but they invested more in wing size, which is likely to compensate for the negative impact of body mass on movement distances. Our results indicate that combination of GIS analysis of movement distances recorded with mark-release-recapture methods and morphometric measurements taken in field during non-lethal handling of captured individuals proved useful for studying the mobility potential of the endangered insect species.     

Phylogeny of Maculinea blues (Lepidoptera: Lycaenidae) based on morphological and ecological characters: evolution of parasitic myrmecophily

A phylogeny of blue butterflies of the genus Maculinea and related genera (Lycaenidae) is proposed, based on 91 morphological and ecological characters. The resulting tree shows that: (1) Phengaris is a derived group nested within Maculinea; (2) the Maculinea‐Phengaris clade is probably nested within Glaucopsyche; (3) there are three well supported groups within the Maculinea‐Phengaris clade: (alcon group ((teleius group) (arion‐Phengaris group))). Some species (M. alcon, M. arionides) appear to be non‐monophyletic and require reclassification. The two alternative strategies of parasitic myrmecophily in the Maculinea‐Phengaris clade, viz., “predatory” and “cuckoo”, seem to be derived characters of the alcon group, and of the teleius and arion‐Phengaris groups, respectively. The common ancestor of Maculinea used dorsal nectary organ secretions for ant attraction, while this trait was reduced in the ancestor of the alcon group and in M. nausithous (of the teleius group). The three recent Maculinea lineages utilize taxonomically diverse host plants, the asterid families Gentianaceae (alcon and arion‐Phengaris groups), Lamiaceae (arion‐Phengaris group), Campanulaceae (arion‐Phengaris group), and the rosid family Rosaceae (teleius group).     

Long-term monitoring of <Emphasis Type="Italic">Phengaris</Emphasis> (Lepidoptera: Lycaenidae) butterflies in the Přelouč surroundings (Czech Republic): is the waterway construction a serious threat?

The monitoring of sympatrically occurring Phengaris teleius and P. nausithous metapopulations in the surroundings of P?elou?, Czech Republic, was launched over a decade ago in connection with the planned waterway construction on the Elbe river. The mark-recapture surveys were initially restricted to 10 habitat patches on the right river bank, but in 2010 three new patches were discovered on the opposite bank. We use the monitoring results for 2011–2015 to assess how the discovery of additional populations alters the impact evaluation of the prospective construction. The overall abundance of P. teleius in the recently discovered populations was about twice as high as on the right bank (ca. 2,800 vs. 1,400 adults on average), while the numbers of P. nausithous on both banks proved to be balanced (at ca. 600 adults on average). Furthermore, we confirmed a substantial exchange of butterflies between population representing both banks, which indicates a well-integrated metapopulation in both species. Since the waterway would partly destroy the two largest populations on the right bank, the potential loss due to its construction is estimated at 40.1–64.3% P. teleius individuals and 20.2–47.4% P. nausithous individuals occurring there. Nevertheless, concerning the entire metapopulation, the predicted decline is considerably smaller, reaching 13.9–25.7% in P. teleius and 8.5–20.0% in P. nausithous. Consequently, a long-term survival of the species is likely even in the case of the waterway construction, as long as appropriate management is applied on the unaffected habitat patches.     

A Novel Widespread Cryptic Species and Phylogeographic Patterns within Several Giant Clam Species (Cardiidae: Tridacna) from the Indo-Pacific Ocean

Giant clams (genus Tridacna) are iconic coral reef animals of the Indian and Pacific Oceans, easily recognizable by their massive shells and vibrantly colored mantle tissue. Most Tridacna species are listed by CITES and the IUCN Redlist, as their populations have been extensively harvested and depleted in many regions. Here, we survey Tridacna crocea and Tridacna maxima from the eastern Indian and western Pacific Oceans for mitochondrial (COI and 16S) and nuclear (ITS) sequence variation and consolidate these data with previous published results using phylogenetic analyses. We find deep intraspecific differentiation within both T. crocea and T. maxima. In T. crocea we describe a previously undocumented phylogeographic division to the east of Cenderawasih Bay (northwest New Guinea), whereas for T. maxima the previously described, distinctive lineage of Cenderawasih Bay can be seen to also typify western Pacific populations. Furthermore, we find an undescribed, monophyletic group that is evolutionarily distinct from named Tridacna species at both mitochondrial and nuclear loci. This cryptic taxon is geographically widespread with a range extent that minimally includes much of the central Indo-Pacific region. Our results reinforce the emerging paradigm that cryptic species are common among marine invertebrates, even for conspicuous and culturally significant taxa. Additionally, our results add to identified locations of genetic differentiation across the central Indo-Pacific and highlight how phylogeographic patterns may differ even between closely related and co-distributed species.     

Distribution and habitats of <Emphasis Type="Italic">Phengaris</Emphasis> (<Emphasis Type="Italic">Maculinea</Emphasis>) butterflies and population ecology of <Emphasis Type="Italic">Phengaris</Emphasis><Emphasis Type="Italic">teleius</Emphasis> in China

Many species of the butterfly genus Phengaris are regarded as endangered in many parts of their distribution. Several species are also widely distributed across northern China. Due to land use change and overgrazing, their habitats are declining and many patches have been lost. This paper investigates the distribution and habitats of the Chinese Phengaris species (of the subgenus Maculinea). Shrub-grassland near forests seem the most frequent habitat for Phengaris, while flat open grasslands are mostly over-grazed and thus survival for Phengaris butterflies there seems difficult. Throughout Europe, P. teleius is an endangered species, while there is still no information on its status in China. To improve the knowledge on the population ecology of P. teleius, its population structure, adult behaviour and movement were studied through mark–release–recapture methods in the Qinling Mountains of Taibai County. Eight grassland patches which were potentially suitable were found in the area in 2013. In total, 480 individuals (274 females) were marked, resulting in an overall recapture rate of 16 %. The average daily population size was 44 butterflies (±23 SD) during the adult flight period. Sixty-seven percent of the females and 38 % of the males moved less than 50 m, and 17 % of recaptured females and 38 % of males moved more than 200 m. The mean movement distance was 107 ± 177 m for males and 182 ± 122 m for females. The majority of the recaptures (86 %) were made within the patches, only a few individuals (14 %) moved between patches. Due to human disturbance and destruction, all of the eight potentially suitable patches are becoming smaller and increasingly isolated, thus these populations of P. teleius may face an increasing risk of extinction, which may well be a tip of the iceberg of habitat loss and fragmentation of P. teleius in Taibai County and possibly beyond. Hence we hope our initial study of P. teleius could have positive impacts on the conservation of Phengaris butterflies in China.     

Habitat requirements and niche selection of Maculinea nausithous and M. teleius (Lepidoptera: Lycaenidae) within a large sympatric metapopulation

Identifying the factors determining the distribution and abundance of species is a fundamental question in population ecology. Based on habitat characteristics, we sought to identify factors affecting the abundance of two vulnerable large blue butterflies, Maculinea teleius and Maculinea nausithous. Both species often occur in sympatry and thus seem to have very similar requirements with regard to their wetland habitats. Even though, M. nausithous is usually far more widespread and abundant than M. teleius in Central Europe. For understanding this difference despite a similar ecology, more data is needed on the niche selection of both species. We have recorded Maculinea numbers, vegetation structure, food-plant abundance and ant community for 120 patches within a large sympatric metapopulation of both species in the Westerwald area (Germany). Unexpectedly (based on previous data), both species were equally common in the study area, reaching high densities of up to 61 (M. teleius) and 58 (M. nausithous) individuals per 50 m2. In 116 out of 120 patches both species occurred sympatrically. The preferred habitat was extensively managed grassland, being mown or grazed twice a year. The abundance of both Maculinea species was positively related to vegetation height, food-plant and Myrmica rubra abundance. There was no association between M. teleius and its putative main host-ant Myrmica scabrinodis, indicating that M. rubra may serve as an important host in the Westerwald area. The positive effect of food-plant abundance was more pronounced for M. teleius, coinciding with a higher food-plant abundance at sites dominated by M. teleius compared to those dominated by M. nausithous. Thus, M. teleius seems to be slightly more demanding than M. nausithous in terms of food-plant availability.     

Choice behaviour of Myrmica rubra workers between ant larvae and larvae of their Phengaris (Maculinea) nausithous nest parasites

Larvae of Phengaris (Maculinea) butterflies are adopted by Myrmica workers and are obligate myrmecophiles. Brood recognition by Myrmica rubra workers was tested for concolonial larvae (M. rubra) versus allocolonial larvae (M. rubra and P. nausithous) to assay the mimetic efficiency of P. nausithous. In addition, we tested M. rubra ant colonies from different populations with and without the presence of Phengaris, to test for potential local adaptation in adoption behaviour. We show that M. rubra can distinguish between nest-mate and foreign larvae as well as between P. nausithous and their own larvae. Workers from the allopatric population inspected and rejected more P. nausithous larvae than workers from the sympatric population. This might reflect a local host adaptation in which the social parasite more efficiently mimes its sympatric host ants than allopatric ones.     

Microsatellite analysis supports the existence of three cryptic species within the bumble bee <Emphasis Type="Italic">Bombus lucorum sensu lato</Emphasis>

Mitochondrial cytochrome oxidase I (COI) partial sequences are widely used in taxonomy for species identification. Increasingly, these sequence identities are combined with modelling approaches to delineate species. Yet the validity of species delineation based on such DNA ‘barcodes’ is rarely tested and may be called into question by phenomena such as ancestral polymorphisms in DNA sequences, phylogeographic divergence, mitochondrial introgression and hybridization, or distortion of mitochondrial inheritance through such factors as Wolbachia infection. The common and widespread European bumble bee Bombus lucorum s. lato contains three distinct mitochondrial DNA lineages that are assumed to represent three cryptic species, namely Bombus cryptarum, B. lucorum s. str. and B. magnus. To test whether nuclear gene pools of the three putative species were differentiated, we genotyped 304 sympatric members of the lucorum complex (54 B. cryptarum females, 168 B. lucorum s. str. females and 82 B. magnus females, as defined using mtDNA COI haplotypes) from 11 localities spread across the island of Ireland at seven nuclear microsatellite loci. Multilocus genotypes clustered into three discrete groups that largely corresponded to the three mtDNA lineages: B. cryptarum, B. lucorum s. str. and B. magnus. The good fit of mitochondrial haplotype to nuclear (microsatellite) genotypic data supports the view that these three bumble bee taxa are reproductively isolated species, as well as providing a vindication of species identity using so-called DNA barcodes.     

Comparison of genetic population structure of the large blue butterflies Maculinea nausithous and M. teleius

We investigated the genetic population structure of two rare myrmecophilous lycaenid butterflies, Maculinea nausithous and M. teleius, which often live sympatrically and have similar biology. In Europe, both species occur in highly fragmented populations and are vulnerable to local extinction. The proportion of variable allozyme loci, average heterozygosity and genetic differentiation among populations was higher in M. nausithous than in sympatrically living M. teleius populations. We hypothesise that the differences in heterozygosity are mainly due to the known higher efficiency of typical host ant nests in rearing M. nausithous pupae compared to M. teleius pupae. This implies a larger probability of larval survival in M. nausithous, which buffers populations against environmental and demographic stochasticity. In contrast, the lower carrying capacity of ant nests in rearing M. teleius pupae requires higher nest-densities and makes M. teleius populations more prone to losing genetic variation through drift if this condition is not fulfilled. The single investigated Russian population of M. teleius showed much higher levels of heterozygosity than any of the Polish populations, suggesting a more viable and still intact metapopulation structure.     

Species-specific distribution of two sympatric Maculinea butterflies across different meadow edges

An important consequence of habitat fragmentation is the increase of edge habitats. Environmental factors in the edges are different from those in the interiors, which causes changes in the distribution of plant and animal species. We aimed to study how edges affect the distribution of two butterfly species within meadow fragments. We therefore investigated the effect of distance from edge and edge type (road edge versus tree edge) on two sympatric large blue species (Maculinea teleius and M. nausithous). Our results showed that edge type had contrasting effects on the two species. M. teleius favoured both interiors and road edges, while M. nausithous preferred the tree edges. In the case of the latter species a strong positive edge effect was also found. This kind of within-habitat niche segregation is probably related to the different microenvironmental conditions at the edges. Foodplant density did not seem to limit the distribution of these species. Our results suggest that interiors of meadows are important for M. teleius, while tree edges maintain the habitats of the regionally rarer butterfly, M. nausithous.     

A striking lack of genetic diversity across the wide-ranging amphibian <Emphasis Type="Italic">Gastrophryne carolinensis</Emphasis> (Anura: Microhylidae)

We examine phylogeographic structure across a wide-ranging microhylid frog (Gastrophryne carolinensis) using both mitochondrial (mtDNA) and nuclear (AFLP) data. Species with similar ecological characteristics such as large range size, low vagility, or existence across known biogeographic barriers, often are comprised of multiple, cryptic lineages. Surprisingly, our analyses of both portions of the genome show very little phylogeographic or population genetic structure. The family Microhylidae is one of the largest families of anurans with over 60 genera and around 400 species distributed across much of the world (Americas, Asia, Africa, and Madagascar), but very few phylogeographic studies have assessed intraspecific genetic diversity across the mitochondrial and nuclear genomes. Our results suggest that G. carolinensis, one of only three species of microhylid native to the US, has experienced a severe population bottleneck with subsequent range expansion. Comparable molecular data from closely related microhylids, in addition to demographic and ecological analyses, will provide valuable insight into patterns of genetic diversity and the processes driving phylogeographic diversity in these wide-ranging frogs.     

Local host ant specificity of Phengaris (Maculinea) teleius butterfly,an obligatory social parasite of Myrmica ants

1. Phengaris butterflies are obligatory social parasites of Myrmica ants. Early research suggested that there is a different Myrmica host species for each of the five European Phengaris social parasites, but more recent studies have shown that this was an oversimplification. 2. The pattern of host ant specificity within a Phengaris teleius metapopulation from southern Poland is reported. A combination of studying the frequency distribution of Phengaris occurrence and morphometrics on adult butterflies were used to test whether use of different host species is reflected in larval development. 3. Phengaris teleius larvae were found to survive in colonies of four Myrmica species: M. scabrinodis, M. rubra, M. ruginodis, and M. rugulosa. Myrmica scabrinodis was the most abundant species under the host plant but the percentage of infested nests was similar to other host ant species at two sites and lower in comparison to nests of M. rubra and M. ruginodis at the other two sites. Morphometric measurements of adult butterflies reared by wild colonies of M. scabrinodis and M. ruginodis showed that wing size and number of wing spots were slightly greater for adults eclosing from nests of M. ruginodis. 4. Our results suggest that P. teleius in the populations studied is less specialised than previously suggested. The results are consistent with the hypothesis that P. teleius is expected to be the least specific of the European Phengaris species, as it has the largest and best defended fourth‐instar caterpillars and, as a predatory species, it spends less time in the central larval chambers of the host colonies. The fact that individuals reared by M. ruginodis had wider hind wings may suggest that P. teleius had better access to resources in M. ruginodis than in M. scabrinodis colonies.     

Phylogeographic patterns and cryptic speciation across oceanographic barriers in South African intertidal fishes

Biogeographic boundaries are the meeting zone of broadly distributed faunas, or the actual cause of a faunal break. In the latter case, closely related sister species should be found across such a boundary. To achieve such a situation, preliminary stages are expected, where phylogeographic breaks followed by genetic cryptic speciation would be observed. Biogeographic boundaries, in the Cape Point/Cape Agulhas region of southern Africa, offer an ideal system to test such predictions. Here, we studied two intertidal clinid fish species that are endemic to southern Africa, Clinus superciliosus (n = 127) and Muraenoclinus dorsalis (n = 114). Using mitochondrial control region, 16S rRNA, 12S rRNA and NADH2 genes and the nuclear rhodopsin and the first intron of the S7 ribosomal protein gene, we show both phylogeographic breaks and likely cryptic speciation in each species. Pairwise Φ(st) results suggest population genetic structuring for both species, with higher levels for M. dorsalis (Φ(st) = 0.34-0.93) than for C. superciliosus (Φ(st) = 0.1-0.74). Further, we recover two and three distinct lineages within M. dorsalis and C. superciliosus, respectively. Phylogenetic topologies, concordance between nuclear and mitochondrial markers and levels of sequence divergence, which are consistent with closely related sister species pairs, suggest the presence of cryptic species. Our results therefore meet the expectation for reduced gene flow at a biogeographic barrier, which translates into significant genetic breaks and cryptic sister species.     

Speciation history of three closely related oak gall wasps,Andricus mukaigawae,A. kashiwaphilus,and A. pseudoflos (Hymenoptera: Cynipidae) inferred from nuclear and mitochondrial DNA sequences

The Andricus mukaigawae complex of oak gall wasps is composed of cyclically parthenogenetic species: A. mukaigawae and Andricus kashiwaphilus, and a parthenogenetic species, Andricus pseudoflos. The component species differ in life history, host plant, karyotype, and asexual gall shape, although little difference is found in the external morphology of asexual adults. To understand the speciation history of this species complex, DNA sequences of one mitochondrial region and nine nuclear gene regions were investigated. The genetic relationship among the species suggested that a loss of sex occurred after host shift. Unexpectedly, two or three distinct groups in the parthenogenetic species, A. pseudoflos, were revealed by both mitochondrial and nuclear DNA data. Gene flow in nuclear genes from the species not infected by Wolbachia (A. kashiwaphilus) to the species infected by it (A. mukaigawae) was suggested by a method based on coalescent simulations. On the other hand, gene flow in mitochondrial genes was suggested to be in the opposite direction. These findings indicate possible involvement of Wolbachia infection in the speciation process of the A. mukaigawae complex.     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

DNA barcoding of bark and ambrosia beetles reveals excessive NUMTs and consistent east‐west divergence across Palearctic forests

A comprehensive DNA barcoding library is very useful for rapid identification and detection of invasive pest species. We tested the performance of species identification in the economically most damaging group of wood‐boring insects – the bark and ambrosia beetles – with particular focus on broad geographical sampling across the boreal Palearctic forests. Neighbour‐joining and Bayesian analyses of cytochrome oxidase I (COI) sequences from 151 species in 40 genera revealed high congruence between morphology‐based identification and sequence clusters. Inconsistencies with morphological identifications included the discovery of a likely cryptic Nearctic species of Dryocoetes autographus, the possible hybrid origin of shared mitochondrial haplotypes in Pityophthorus micrographus and P. pityographus, and a possible paraphyletic Xyleborinus saxeseni. The first record of Orthotomicus suturalis in North America was confirmed by DNA barcoding. The mitochondrial data also revealed consistent divergence across the Palearctic or Holarctic, confirmed in part by data from the large ribosomal subunit (28S). Some populations had considerable variation in the mitochondrial barcoding marker, but were invariant in the nuclear ribosomal marker. These findings must be viewed in light of the high number of nuclear insertions of mitochondrial DNA (NUMTs) detected in eight bark beetle species, suggesting the possible presence of additional cryptic NUMTs. The occurrence of paralogous COI copies, hybridization or cryptic speciation demands a stronger focus on data quality assessment in the construction of DNA barcoding databases.     

Population genetic structure of Penaeus merguiensis in Thailand based on nuclear DNA variation

Polymorphisms of nuclear DNA markers were used to study the genetic variation and differentiation among populations of Penaeus merguiensis in Thailand. The data consisted of three nuclear loci analysed for 163 individuals from five populations collected from the Gulf of Thailand (Trad, Surat Thani and Songkhla) and the Andaman Sea (Satun and Trang). The multilocus F_ST estimated at three nuclear loci revealed great and highly significant differentiation between the Gulf of Thailand and Andaman Sea (F_ST=0.203, P<0.001), mostly due to the polymorphism of locus PvAmy. In addition, significant population differentiation was also found within the Gulf of Thailand. These results were compared to that of a previous mitochondrial DNA survey spanning the same geographical range where two divergent mitochondrial clades were reported. The present study brings support to the fact that the existence of these two clades is not due to a mixture of cryptic species but reflects their phylogeographic origin. The strong genetic structure of P. merguiensis on each side of the Thailand peninsula for both mitochondrial and nuclear genes could thus be linked to the phylogeographic divide between Indian Ocean and Pacific forms on the west and east sides respectively.     

Cryptic species in putative ancient asexual darwinulids (Crustacea, Ostracoda)

Background

Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied.

Methodology/Principal Findings

We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies.

Conclusions/Significance

Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.     

Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex

Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter‐ and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.