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As cryptic diversity is being discovered, mostly thanks to advances in molecular techniques, it is becoming evident that many of these taxa display parapatric distributions in mainland and that they rarely coexist on islands. Genetic landscapes, haplotype networks and ecological niche modeling analyses were performed for two pairs of non-sister cryptic butterfly species, Aricia agestis-A. cramera and Polyommatus icarus—P. celina (Lycaenidae), to specifically assess non-coexistence on western Mediterranean islands, and to test potential causes producing such chequered distribution patterns. We show that the morphologically and ecologically equivalent pairs of species do not coexist on any of the studied islands, although nearly all islands are colonized by one of them. According to our models, the cryptic pairs displayed marked climatic preferences and ‘precipitation during the driest quarter’ was recovered as the most important climatic determinant. However, neither dispersal capacity, nor climatic or ecological factors fully explain the observed distributions across particular sea straits, and the existence of species interactions resulting in mutual exclusion is suggested as a necessary hypothesis. Given that the studied species are habitat generalists, feeding on virtually unlimited resources, we propose that reproductive interference, together with climatic preferences, sustain density-dependent mechanisms like “founder takes all” and impede coexistence on islands. Chequered distributions among cryptic taxa, both sister and non-sister, are common in butterflies, suggesting that the phenomenon revealed here could be important in determining biodiversity patterns.

The cryptic fraction of biodiversity is composed of morphologically similar species that are or have been overlooked by scientists. Although current research is increasingly documenting new cases, cryptic species are frequently ignored in large-scale studies and monitoring programs, either because they have not yet been discovered, or because of the practical difficulties involved in differentiating them. However, it is unknown if this could represent a bias extending beyond the number of missed species. By analyzing the butterfly fauna of the west Mediterranean (335 species), we defined cryptic species based on the current consensus of the scientific community, compared their properties to other congeneric species and investigated the consequences of their inclusion/exclusion in beta-diversity analyses. We show that, as defined, the cryptic fraction of butterfly diversity represents about 25% of the west Mediterranean fauna and is overwhelmingly composed by groups of species that are not sympatric. Our results show that co-occurrence among cryptic species is significantly lower than among congeneric non-cryptic species. Accordingly, albeit the frequency of cryptic species is homogenously distributed over the study area, their distribution pattern accounts for most beta-diversity turnover over sea (from 50 to 100%). Beta-diversity turnover, a direct measure of the frequency of species replacement from site to site, is recognized as a fundamental parameter in ecology and is widely used to detect biogeographic patterns. These findings represent a change of paradigm in showing that cryptic diversity comprises original qualitative aspects in addition to merely quantitative ones. This highlights the importance of differentiating cryptic species for various research fields and opens the door to the study of further potential particularities of cryptic diversity.

How common are cryptic species - those overlooked because of their morphological similarity? Despite its wide-ranging implications for biology and conservation, the answer remains open to debate. Butterflies constitute the best-studied invertebrates, playing a similar role as birds do in providing models for vertebrate biology. An accurate assessment of cryptic diversity in this emblematic group requires meticulous case-by-case assessments, but a preview to highlight cases of particular interest will help to direct future studies. We present a survey of mitochondrial genetic diversity for the butterfly fauna of the Iberian Peninsula with unprecedented resolution (3502 DNA barcodes for all 228 species), creating a reliable system for DNA-based identification and for the detection of overlooked diversity. After compiling available data for European butterflies (5782 sequences, 299 species), we applied the Generalized Mixed Yule-Coalescent model to explore potential cryptic diversity at a continental scale. The results indicate that 27.7% of these species include from two to four evolutionary significant units (ESUs), suggesting that cryptic biodiversity may be higher than expected for one of the best-studied invertebrate groups and regions. The ESUs represent important units for conservation, models for studies of evolutionary and speciation processes, and sentinels for future research to unveil hidden diversity.

[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.

Integrating genetic and/or phenotypic traits at population level is considered a fundamental approach in the study of evolutionary processes, systematics, biogeography and conservation. But combining the two types of data remain a complex task, mostly due to the high, and sometimes different, sample sizes required for reliable assessments of community traits. Data availability has been increasing in recent years, thanks to online resources, but it is uncommon that different types of markers are available for any given specimen. We provide new R functions aimed at directly correlating traits at population level, even if data sets only overlap partially. The new functions are based on a modified Procrustes algorithm that minimizes differences between bidimensional ordinations of two different markers, based on a subsample of specimens for which both characters are known. To test the new functions, we used a molecular and morphological data set comprising Mediterranean specimens of the butterfly Maniola jurtina. By using this method, we have been able to maximize similarities between genotypic and phenotypic configurations obtained after principal coordinate analysis for the model species and evaluated their degree of correlation at both individual and population level. The new recluster. Procrustes function retained the information of the relative importance of different morphological variables in determining the observed ordinations and preserved it in the transformed configurations. This allowed calculating the best combination of morphological variables mirroring genetic relationships among specimens and populations. Finally, it was possible to analyse the modality and variance of the phenotypic characters correlated with the genetic structure among populations. The genetic and phenotypic markers displayed high overall correlation in the study area except in the contact zone, where discrepancies for particular populations were detected. Interestingly, such discrepa

Lepidopteran genomes consist of small-sized holokinetic chromosomes with an ancestral chromosome number of n=31, and a prevalent WZ/ZZ sex chromosome system with female heterogamety. Although the holokinetic nature of lepidopteran chromosomes is supposed to facilitate karyotype evolution mainly due to chromosomal fusions and fissions, recent studies revealed a highly conserved synteny of genes between chromosomes of distantly related taxa and evolutionary stability of karyotypes. The high degree of conservation at the chromosomal level across the phylogenetic tree of Lepidoptera contrasts with exceptional diversity found in some taxa. A typical example is the butterfly genus Leptidea, which shows karyotype variability not only between but also within species. In this work we studied karyotypes of three cryptic Leptidea species (L. juvernica, L. sinapis, and L. reali) by means of standard and molecular cytogenetic techniques.

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. © 2013 European Society For Evolutionary Biology.

The generalized mixed Yule-coalescent (GMYC) model has become one of the most popular approaches for species delimitation based on single-locus data, and it is widely used in biodiversity assessments and phylogenetic community ecology. We here examine an array of factors affecting GMYC resolution (tree reconstruction method, taxon sampling coverage/taxon richness and geographic sampling intensity/geographic scale). We test GMYC performance based on empirical data (DNA barcoding of the Romanian butterflies) on a solid taxonomic framework (i.e. all species are thought to be described and can be determined with independent sources of evidence). The data set is comprehensive (176 species), and intensely and homogeneously sampled (1303 samples representing the main populations of butterflies in this country). Taxonomy was assessed based on morphology, including linear and geometric morphometry when needed. The number of GMYC entities obtained constantly exceeds the total number of morphospecies in the data set. We show that c. 80% of the species studied are recognized as entities by GMYC. Interestingly, we show that this percentage is practically the maximum that a single-threshold method can provide for this data set. Thus, the c. 20% of failures are attributable to intrinsic properties of the COI polymorphism: overlap in inter- and intraspecific divergences and non-monophyly of the species likely because of introgression or lack of independent lineage sorting. Our results demonstrate that this method is remarkably stable under a wide array of circumstances, including most phylogenetic reconstruction methods, high singleton presence (up to 95%), taxon richness (above five species) and the presence of gaps in intraspecific sampling coverage (removal of intermediate haplotypes). Hence, the method is useful to designate an optimal divergence threshold in an objective manner and to pinpoint potential cryptic species that are worth being studied in detail. However, the exist

[Aim] Adaptive trait continua are axes of covariation observed in multivariate trait data for a given taxonomic group. These continua quantify and summarize life-history variation at the inter-specific level in multi-specific assemblages. Here we examine whether trait continua can provide a useful framework to link life-history variation with demographic and evolutionary processes in species richness gradients. Taking an altitudinal species richness gradient for Mediterranean butterflies as a study case, we examined a suite of traits (larval diet breadth, adult phenology, dispersal capacity and wing length) and species-specific habitat measures (temperature and aridity breadth). We tested whether traits and species-specific habitat measures tend to co-vary, whether they are phylogenetically conserved, and whether they are able to explain species distributions and spatial genetic variation in a large number of butterfly assemblages., [Location] Catalonia, Spain., [Methods] We formulated predictions associated with species richness gradients and adaptive trait continua. We applied principal components analyses (PCAs), structural equation modelling and phylogenetic generalized least squares models. [Results] We found that traits and species-specific habitat measures covaried along a main PCA axis, ranging from multivoltine trophic generalists with high dispersal capacity to univoltine (i.e. one generation per year), trophic specialist species with low dispersal capacity. This trait continuum was closely associated with the observed distributions along the altitudinal gradient and predicted inter-specific differences in patterns of spatial genetic variability (FST and genetic distances), population responses to the impacts of global change and local turnover dynamics., [Main conclusions] The adaptive trait continuum of Mediterranean butterflies provides an integrative and mechanistic framework to: (1) analyse geographical gradients in species richness, (2) explain inter-specific differences in population abundances, spatial distributions and demographic trends, (3) explain inter-specific differences in patterns of genetic variation (FST and genetic distances), and (4) study specialist-generalist life-history transitions frequently involved in butterfly diversification processes. © 2012 Blackwell Publishing Ltd.

Polyommatus ripartii is a biogeographically and taxonomically poorly understood species of butterfly with a scattered distribution in Europe. Recently, it has been shown that this species includes several European endemic and localized taxa (galloi, exuberans, agenjoi) that were previously considered species and even protected, a result that poses further questions about the processes that led to its current distribution. We analysed mitochondrial DNA and the morphology of P.ripartii specimens to study the phylogeography of European populations. Three genetically differentiated but apparently synmorphic lineages occur in Europe that could be considered evolutionarily significant units for conservation. Their strongly fragmented and counterintuitive distribution seems to be the result of multiple range expansions and contractions along Pleistocene climatic oscillations. Remarkably, based on the 79 specimens studied, these genetic lineages do not seem to extensively coexist in the distributional mosaic, a phenomenon most evident in the Iberian Peninsula. One of the important gaps in the European distribution of P.ripartii is reduced by the discovery of new Croatian populations, which also facilitate a better understanding of the biogeography of the species. © 2013 The Linnean Society of London.

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. © 2012 Elsevier Inc.

[Aim] Distinct insular populations are generally considered important units for conservation. In island-mainland situations, unidirectional introgressive gene flow from the most abundant, typically continental, populations into the smaller island populations can erase native insular genetic units. As an indication of threat, the concept of phenetic slope is developed, a measure proportional to differentiation and to geographical proximity. [Location] The Western Mediterranean, including the following islands: Sardinia, Sicily, Corsica, Balearics, circum-Italian, circum-Sicilian and circum-Sardo-Corsican archipelagos. Eastern Europe is included for comparison. [Methods] Geometric morphometrics was applied to 2392 male genitalia of seven butterfly species groups. Geographic Information System techniques were used to depict the pattern in the distribution of morphotypes. The slope of variation in genital shape was computed to highlight geographical areas showing abrupt morphological changes. Correlation analyses were performed between the mean slope values across sea straits separating islands and nearest sources and ecological traits of the species that underlie their colonization and migration capacity. [Results] Phenetic slope analysis has revealed that the strait of Messina and the northern Tyrrhenian Sea support particularly contrasting populations. In these areas, mean slopes for species also correlated with certain ecological traits of the species. Sardinia emerges as the most stable refugium for ancestral mediterranean populations. [Main conclusions] There is strong support for the hypothesis that Italy has experienced invasion by populations from Eastern Europe with postglacial expansion of these populations across Italy. However, propagules are impeded from invading islands by the expanse of sea straits. Even so, sea straits are not invariably barriers. Our results suggest that wind direction in combination with habitat occupancy may have maintained ancestral

Based on material collected during 2007–2009, we report 225 species of macrolepidoptera from the steppes of Dobrogea (south-eastern Romania). Besides numerous rare species with particular ecological preferences, we report Tarachidia candefacta (Hübner, [1831]) (Noctuidae) and Rhodostrophia discopunctata Amsel, 1935 (Geometridae) for the first time in the Romanian entomofauna. Several taxa of high zoogeographical significance for the Romanian and European fauna are commented. The conservation status and interest of the steppe areas from Dobrogea is brought into discussion.

Pseudophilotes bavius (Eversmann, 1832) is a butterfly species of European conservation concern (listed in Annexes II and IV of the Habitats Directive 92/43/EEC) with a restricted and fragmented distribution. In Romania, where it reaches the north-western limit of its range, P. bavius has disjunct populations: Transylvania in the north-west and Dobrogea in the south-east, with a gap of about 400 km between the two. In this study we provide an overview on the distribution of P. bavius in Dobrogea, while reporting three new sites which double the number of known localities and extend the species’ distribution about 60 km north in this region. The known and potential distribution, habitat, phenology and conservation status of P. bavius in Dobrogea are discussed.

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.

[Aim] Our aims were to verify the existence of phylogenetic disequilibrium between butterfly lineages at the subcontinental scale for islands and the nearest mainland and to test the capacity of islands for hosting ancestral populations of butterflies and the significance of such relict populations., [Location] The western Mediterranean continental area of Europe and North Africa together with several large and small islands (Balearics, Tuscan Archipelago, Aeolian Archipelago, Capri, Sardinia, Sicily, Corsica)., [Methods] Using geometric morphometrics, the shape of male genitalia was analysed in two common butterflies (Pyronia cecilia and Pyronia tithonus), whose spatial heterogeneity in the Mediterranean region has recently been described. Observed patterns in genital shapes were compared with shapes predicted for islands and fossil islands to assess the contribution of historical and current events in accounting for the transition from a refugial model to an equilibrium model. Measurements were taken for 473 specimens in 90 insular and mainland sites., [Results] The shape of the genitalia of populations of most islands differed substantially from that predicted by the equilibrium hypothesis while closely fitting the refugial hypothesis. The comparison between different models strongly suggests that islands maintain ancestral lineages similar to those living in Spain (P. cecilia) and France (P. tithonus). A high correlation between observed and predicted patterns on islands and fossil islands occurs during the first steps of modelled introgressive hybridization while the following steps exposed a successively lower fit, suggesting that the process from a refugial to an equilibrium situation is highly skewed towards an earlier non-equilibrium., [Main conclusions] The observed non-equilibrium pattern supports the refugial hypothesis, suggesting that an ancestral lineage was originally distributed from Spain to Italy, and also occupied offshore islands. This lineage, replaced in Italy, has persisted on the islands owing to their isolation. A comparison of the distribution patterns for genetic and morphometric markers in several species indicates that the situation highlighted for Pyronia may represent a common biogeographic feature for many Mediterranean butterflies.

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 chr

[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.