Range instability leads to cytonuclear discordance in a morphologically cryptic ground squirrel species complex

The processes responsible for cytonuclear discordance frequently remain unclear. Here, we employed an exon capture data set and demographic methods to test hypotheses generated by species distribution models to examine how contrasting histories of range stability vs. fluctuation have caused cytonuclear concordance and discordance in ground squirrel lineages from the Otospermophilus beecheyi species complex. Previous studies in O. beecheyi revealed three morphologically cryptic and highly divergent mitochondrial DNA lineages (named the Northern, Central and Southern lineages based on geography) with only the Northern lineage exhibiting concordant divergence for nuclear genes. Here, we showed that these mtDNA lineages likely formed in allopatry during the Pleistocene, but responded differentially to climatic changes that occurred since the last interglacial (~120,000 years ago). We find that the Northern lineage maintained a stable range throughout this period, correlating with genetic distinctiveness among all genetic markers and low migration rates with the other lineages. In contrast, our results suggested that the Southern lineage expanded from Baja California Sur during the Late Pleistocene to overlap and potentially swamp a contracting Central lineage. High rates of intraspecific gene flow between Southern lineage individuals among expansion origin and expansion edge populations largely eroded Central ancestry from autosomal markers. However, male‐biased dispersal in this system preserved signals of this past hybridization and introgression event in matrilineal‐biased X‐chromosome and mtDNA markers. Our results highlight the importance of range stability in maintaining the persistence of phylogeographic lineages, whereas unstable range dynamics can increase the tendency for lineages to merge upon secondary contact.     

Phylogeographic assessment of mtDNA paraphyly and the evolution of unisexuality in Calligrapha (Coleoptera: Chrysomelidae)

The leaf beetle genus Calligrapha is one of the few examples of animals with several obligate unisexual, female‐only species. Previous work showed that each one arose independently from interspecific hybridization events involving different species. However, all of them clustered in a single mtDNA clade together with some individuals of the parental bisexual species, which appeared as deeply polyphyletic in the mtDNA genealogy of the genus. The dating of these splits using a molecular clock placed them in the Quaternary and it was hypothesized that climatic change during this period may have favored range expansions and secondary contacts required for hybridization. In this work, we test this hypothesis and the origins of unisexuality in Calligrapha examining the diversity of mitochondrial (cox1) and nuclear (wingless, Wg) genes and the Bayesian continuous mtDNA phylogeography of a sample of more than 500 specimens of two bisexual species of Calligrapha at a continental scale and two unisexual species derived from them. Besides a major topological difference, whereby each bisexual species is monophyletic for Wg but paraphyletic for cox1, both gene datasets are consistent with a minimum of seven evolutionary lineages, coherent with geography and consistent with an ordered expansion to occupy their current ranges. The results also imply their survival in well‐established glacial refuges during the Last Glacial Maximum (LGM). Thus, for bisexual C. multipunctata there are two main, southern and northern lineages. The southern lineage expanded its range in two evolutionary branches, to the Rocky Mountains and to the northern Mississippi and Ohio River basins, respectively. The northern lineage has one branch in the Upper Mississippi and one that expanded west to the Pacific Northwest and east to the northeastern North Atlantic, finding refuge in both areas. These major lineages are parapatric in the Northern Great Plains, an area consistent with them having found refuge in the so‐called Driftless region during the LGM. For bisexual C. philadelphica, one northern lineage expanded west from the northern Appalachians and one east and southwest along the axis of the Appalachians, and the timing of events is consistent with their persistence in glacial refugia at both sides of the main Great Lakes lobe of the Laurentide Ice Sheet. There is evidence that the northeastern North Atlantic lineages of both species hybridized at the edge of their ranges after the LGM. The additional, divergent mtDNA lineage of these species shows evidence of range expansions of two lineages, one for each species, in an area south of the Laurentide Ice Sheet, and giving origin to the unisexual species by way of hybridization with other species in the Alleghanian region after the LGM. Interestingly, the individuals of supposedly bisexual species in this clade are all females. This suggests that unisexuality actually predates the origin of unisexual taxa in this system and that some bisexual species in Calligrapha may be species complexes instead, with cryptic species differing in their reproductive mode.     

Three divergent lineages within an Australian marsupial (Petrogale penicillata) suggest multiple major refugia for mesic taxa in southeast Australia

Mesic southeastern Australia represents the continent's ancestral biome and is highly biodiverse, yet its phylogeographic history remains poorly understood. Here, we examine mitochondrial DNA (mtDNA) control region and microsatellite diversity in the brush‐tailed rock‐wallaby (Petrogale penicillata; n = 279 from 31 sites), to assess historic evolutionary and biogeographic processes in southeastern Australia. Our results (mtDNA, microsatellites) confirmed three geographically discrete and genetically divergent lineages within brush‐tailed rock‐wallabies, whose divergence appears to date to the mid‐Pleistocene. These three lineages had been hypothesized previously but data were limited. While the Northern and Central lineages were separated by a known biogeographic barrier (Hunter Valley), the boundary between the Central and Southern lineages was not. We propose that during particularly cool glacial cycles, the high peaks of the Great Dividing Range and the narrow adjacent coastal plain resulted in a more significant north–south barrier for mesic taxa in southeastern Australia than has been previously appreciated. Similarly, located phylogeographic breaks in codistributed species highlight the importance of these regions in shaping the distribution of biodiversity in southeastern Australia and suggest the existence of three major refuge areas during the Pleistocene. Substructuring within the northern lineage also suggests the occurrence of multiple local refugia during some glacial cycles. Within the three major lineages, most brush‐tailed rock‐wallaby populations were locally highly structured, indicating limited dispersal by both sexes. The three identified lineages represent evolutionarily significant units and should be managed to maximize the retention of genetic diversity within this threatened species.     

Molecular markers provide evidence for a broad‐fronted recolonisation of the widespread calcareous grassland species Sanguisorba minor from southern and cryptic northern refugia

• Calcareous grasslands belong to the most species‐rich and endangered habitats in Europe. However, little is known about the origin of the species typically occurring in these grasslands. In this study we analysed the glacial and post‐glacial history of Sanguisorba minor, a typical plant species frequently occurring in calcareous grasslands.
• The study comprised 38 populations throughout the whole distribution range of the species across Europe. We used molecular markers (AFLP) and applied Bayesian cluster analysis as well as spatial principal components analysis (sPCA) to identify glacial refugia and post‐glacial migration routes to Central Europe.
• Our study revealed significant differences in the level of genetic variation and the occurrence of rare fragments within populations of S. minor and a distinct separation of eastern and western lineages. The analyses uncovered traditional southern but also cryptic northern refugia and point towards a broad fronted post‐glacial recolonisation.
• Based on these results we postulate that incomplete lineage sorting may have contributed to the detected pattern of genetic variation and that S. minor recolonised Central Europe post‐glacially from Iberia and northern glacial refugia in France, Belgium or Germany. Our results highlight the importance of refugial areas for the conservation of intraspecific variation in calcareous grassland species.

     

Cryptic diversity in the pen shell Atrina pectinata (Bivalvia: Pinnidae): high divergence and hybridization revealed by molecular and morphological data

Cryptic species have been increasingly revealed in the marine realm through an analytical approach incorporating multiple lines of evidence (e.g., mtDNA, nuclear genes and morphology). Illustrations of cryptic taxa improve our understanding of species diversity and evolutionary histories within marine animals. The pen shell Atrina pectinata is known to exhibit extensive morphological variations that may harbour cryptic diversity. In this study, we investigated A. pectinata populations along the coast of China and one from Japan to explore possible cryptic diversity and hybridization using a combination of mitochondrial (cytochrome c oxidase subunit I, mtCOI) and nuclear (ribosomal internal transcribed spacer, nrITS) genes as well as morphology. Phylogenetic analyses of mtCOI ‘DNA barcoding gene’ sequences resolved six divergent lineages with intralineage divergences between 0.4% and 0.8%. Interlineage sequence differences ranged from 4.3% to 22.0%, suggesting that six candidate cryptic species are present. The nrITS gene revealed five deep lineages with Kimura 2‐parameter distances of 3.7–30.3%. The five nuclear lineages generally corresponded to mtCOI lineages 1–4 and (5 + 6), suggestive of five distinct evolutionary lineages. Multiple nrITS sequences of significant variance were found within an individual, clearly implying recent hybridization events between/among the evolutionary lineages, which contributed to cytonuclear discordance. Morphologically, five morphotypes matched the five genetic lineages, although the intermediates may well blur the boundaries of different morphotypes. This study demonstrates the importance of combining multiple lines of evidence to explore species cryptic diversity and past evolutionary histories.     

Discordant patterns of introgression across a narrow hybrid zone between two cryptic lineages of an Iberian endemic newt

The study of natural hybrid zones can illuminate aspects of lineage divergence and speciation in morphologically cryptic taxa. We studied a hybrid zone between two highly divergent but morphologically similar lineages (south‐western and south‐eastern) of the Iberian endemic Bosca's newt (Lissotriton boscai) in SW Iberia with a multilocus dataset (microsatellites, nuclear and mitochondrial genes). STRUCTURE and NEWHYBRIDS analyses retrieved few admixed individuals, which classified as backcrosses involving parental individuals of the south‐western lineage. Our results show asymmetric introgression of mtDNA beyond the contact from this lineage into the south‐eastern lineage. Analysis of nongeographic introgression patterns revealed asymmetries in the direction of introgression, but except for mtDNA, we did not find evidence for nonconcordant introgression patterns across nuclear loci. Analysis of a 150‐km transect across the hybrid zone showed broadly coincident cline widths (ca. 3.2–27.9 km), and concordant cline centres across all markers, except for mtDNA that is displaced ca. 60 km northward. Results from ecological niche modelling show that the hybrid zone is in a climatically homogenous area with suitable habitat for the species, suggesting that contact between the two lineages is unlikely to occur further south as their distributions are currently separated by an extensive area of unfavourable habitat. Taken together, our findings suggest the genetic structure of this hybrid zone results from the interplay of historical (biogeographic) and population‐level processes. The narrowness and coincidence of genetic clines can be explained by weak selection against hybrids and reflect a degree of reproductive isolation that is consistent with cryptic speciation.     

Detecting cryptic species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus) butterflies (Lepidoptera: Lycaenidae)

Modern multilocus molecular techniques are a powerful tool in the detection and analysis of cryptic taxa. However, its shortcoming is that with allopatric populations it reveals phylogenetic lineages, not biological species. The increasing power of coalescent multilocus analysis leads to the situation in which nearly every geographically isolated or semi‐isolated population can be identified as a lineage and therefore raised to species rank. It leads to artificial taxonomic inflation and as a consequence creates an unnecessary burden on the conservation of biodiversity. To solve this problem, we suggest combining modern lineage delimitation techniques with the biological species concept. We discuss several explicit principles on how genetic markers can be used to detect cryptic entities that have properties of biological species (i.e. of actually or potentially reproductively isolated taxa). Using these principles we rearranged the taxonomy of the butterfly species close to Polyommatus (Agrodiaetus) ripartii. The subgenus Agrodiaetus is a model system in evolutionary research, but its taxonomy is poorly elaborated because, as a rule, most of its species are morphologically poorly differentiated. The taxon P. (A.) valiabadi has been supposed to be one of the few exceptions from this rule due to its accurately distinguishable wing pattern. We discovered that in fact traditionally recognized P. valiabadi is a triplet of cryptic species, strongly differentiated by their karyotypes and mitochondrial haplotypes.     

Phylogeography and lineage‐specific patterns of genetic diversity and molecular evolution in a group of North American skinks

Geography influences the evolutionary trajectory of species by mediating opportunities for hybridization, gene flow, demographic shifts and adaptation. We sought to understand how geography and introgression can generate species‐specific patterns of genetic diversity by examining phylogeographical relationships in the North American skink species Plestiodon multivirgatus and P. tetragrammus (Squamata: Scincidae). Using a multilocus dataset (three mitochondrial genes, four nuclear genes; a total of 3455 bp) we discovered mito‐nuclear discordance, consistent with mtDNA introgression. We further tested for evidence of species‐wide mtDNA introgression by using comparisons of genetic diversity, selection tests and extended Bayesian skyline analyses. Our findings suggest that P. multivirgatus acquired its mitochondrial genome from P. tetragrammus after their initial divergence. This putative species‐wide mitochondrial capture was further evidenced by statistically indistinguishable substitution rates between mtDNA and nDNA in P. multivirgatus. This rate discrepancy was observed in P. multivirgatus but not P. tetragrammus, which has important implications for studies that combine mtDNA and nDNA sequences when inferring time since divergence between taxa. Our findings suggest that by facilitating opportunities for interspecific introgression, geography can alter the course of molecular evolution between recently diverged lineages.     

Maternal genomic variability of the wild boar (Sus scrofa) reveals the uniqueness of East‐Caucasian and Central Italian populations

The phylogeography of the European wild boar was mainly determined by postglacial recolonization patterns from Mediterranean refugia after the last ice age. Here we present the first analysis of SNP polymorphism within the complete mtDNA genome of West Russian (n = 8), European (n = 64), and North African (n = 5) wild boar. Our analyses provided evidence of unique lineages in the East‐Caucasian (Dagestan) region and in Central Italy. A phylogenetic analysis revealed that these lineages are basal to the other European mtDNA sequences. We also show close connection between the Western Siberian and Eastern European populations. Also, the North African samples were clustered with the Iberian population. Phylogenetic trees and migration modeling revealed a high proximity of Dagestan sequences to those of Central Italy and suggested possible gene flow between Western Asia and Southern Europe which was not directly related to Northern and Central European lineages. Our results support the presence of old maternal lineages in two Southern glacial refugia (i.e., Caucasus and the Italian peninsula), as a legacy of an ancient wave of colonization of Southern Europe from an Eastern origin.     

Unravelling the evolutionary history and future prospects of endemic species restricted to former glacial refugia

The contemporary distribution and genetic composition of biodiversity bear a signature of species’ evolutionary histories and the effects of past climatic oscillations. For many European species, the Mediterranean peninsulas of Iberia, Italy and the Balkans acted as glacial refugia and the source of range recolonization, and as a result, they contain disproportionately high levels of diversity. As these areas are particularly threatened by future climate change, it is important to understand how past climatic changes affected their biodiversity. We use an integrated approach, combining markers with different evolutionary rates and combining phylogenetic analysis with approximate Bayesian computation and species distribution modelling across temporal scales. We relate phylogeographic processes to patterns of genetic variation in Myotis escalerai, a bat species endemic to the Iberian Peninsula. We found a distinct population structure at the mitochondrial level with a strong geographic signature, indicating lineage divergence into separate glacial refugia within the Iberian refugium. However, microsatellite markers suggest higher levels of gene flow resulting in more limited structure at recent time frames. The evolutionary history of M. escalerai was shaped by the effects of climatic oscillations and changes in forest cover and composition, while its future is threatened by climatically induced range contractions and the role of ecological barriers due to competition interactions in restricting its distribution. This study warns that Mediterranean peninsulas, which provided refuge for European biodiversity during past glaciation events, may become a trap for limited dispersal and ecologically limited endemic species under future climate change, resulting in loss of entire lineages.     

Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs

Subdivided Pleistocene glacial refugia, best known as “refugia within refugia”, provided opportunities for diverging populations to evolve into incipient species and/or to hybridize and merge following range shifts tracking the climatic fluctuations, potentially promoting extensive cytonuclear discordances and “ghost” mtDNA lineages. Here, we tested which of these opposing evolutionary outcomes prevails in northern Iberian areas hosting multiple historical refugia of common frogs (Rana cf. temporaria), based on a genomic phylogeography approach (mtDNA barcoding and RAD‐sequencing). We found evidence for both incipient speciation events and massive cytonuclear discordances. On the one hand, populations from northwestern Spain (Galicia and Asturias, assigned to the regional endemic R. parvipalmata), are deeply‐diverged at mitochondrial and nuclear genomes (~4 My of independent evolution), and barely admix with northeastern populations (assigned to R. temporaria sensu stricto) across a narrow hybrid zone (~25 km) located in the Cantabrian Mountains, suggesting that they represent distinct species. On the other hand, the most divergent mtDNA clade, widespread in Cantabria and the Basque country, shares its nuclear genome with other R. temporaria s. s. lineages. Patterns of population expansions and isolation‐by‐distance among these populations are consistent with past mitochondrial capture and/or drift in generating and maintaining this ghost mitochondrial lineage. This remarkable case study emphasizes the complex evolutionary history that shaped the present genetic diversity of refugial populations, and stresses the need to revisit their phylogeography by genomic approaches, in order to make informed taxonomic inferences.     

Glacial cycles drive rapid divergence of cryptic field vole species

Understanding the factors that contribute to the generation of reproductively isolated forms is a fundamental goal of evolutionary biology. Cryptic species are an especially interesting challenge to study in this context since they lack obvious morphological differentiation that provides clues to adaptive divergence that may drive reproductive isolation. Geographical isolation in refugial areas during glacial cycling is known to be important for generating genetically divergent populations, but its role in the origination of new species is still not fully understood and likely to be situation dependent. We combine analysis of 35,434 single‐nucleotide polymorphisms (SNPs) with environmental niche modeling (ENM) to investigate genomic and ecological divergence in three cryptic species formerly classified as the field vole (Microtus agrestis). The SNPs demonstrate high genomic divergence (pairwise F_ST values of 0.45–0.72) and little evidence of gene flow among the three field vole cryptic species, and we argue that genetic drift may have been a particularly important mechanism for divergence in the group. The ENM reveals three areas as potential glacial refugia for the cryptic species and differing climatic niches, although with spatial overlap between species pairs. This evidence underscores the role that glacial cycling has in promoting genetic differentiation and reproductive isolation by subdivision into disjunct distributions at glacial maxima in areas relatively close to ice sheets. Future investigation of the intrinsic barriers to gene flow between the field vole cryptic species is required to fully assess the mechanisms that contribute to reproductive isolation. In addition, the Portuguese field vole (M. rozianus) shows a high inbreeding coefficient and a restricted climatic niche, and warrants investigation into its conservation status.     

Early events in speciation: Cryptic species of Drosophila aldrichi

Understanding the earliest events in speciation remains a major challenge in evolutionary biology. Thus identifying species whose populations are beginning to diverge can provide useful systems to study the process of speciation. Drosophila aldrichi, a cactophilic fruit fly species with a broad distribution in North America, has long been assumed to be a single species owing to its morphological uniformity. While previous reports either of genetic divergence or reproductive isolation among different D. aldrichi strains have hinted at the existence of cryptic species, the evolutionary relationships of this species across its range have not been thoroughly investigated. Here we show that D. aldrichi actually is paraphyletic with respect to its closest relative, Drosophila wheeleri, and that divergent D. aldrichi lineages show complete hybrid male sterility when crossed. Our data support the interpretation that there are at least two species of D. aldrichi, making these flies particularly attractive for studies of speciation in an ecological and geographical context.     

Molecular evidence for ten species and Oligo-Miocene vicariance within a nominal Australian gecko species (<Emphasis Type="Italic">Crenadactylus ocellatus</Emphasis>, Diplodactylidae)

Background  

Molecular studies have revealed that many putative 'species' are actually complexes of multiple morphologically conservative, but genetically divergent 'cryptic species'. In extreme cases processes such as non-adaptive diversification (speciation without divergent selection) could mask the existence of ancient lineages as divergent as ecologically and morphologically diverse radiations recognised as genera or even families in related groups. The identification of such ancient, but cryptic, lineages has important ramifications for conservation, biogeography and evolutionary biology. Herein, we use an integrated multilocus genetic dataset (allozymes, mtDNA and nuclear DNA) to test whether disjunct populations of the widespread nominal Australian gecko species Crenadactylus ocellatus include distinct evolutionary lineages (species), and to examine the timing of diversification among these populations.     

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.     

A complex species complex: The controversial role of ecology and biogeography in the evolutionary history of Syllis gracilis Grube, 1840 (Annelida,Syllidae)

The cryptic diversity in the polychaete Syllis gracilis Grube, 1840, in the Mediterranean Sea was examined with an integrative morpho-molecular approach. Individuals of S. gracilis were collected at eleven Mediterranean localities to provide an insight into the role of brackish environments in inducing cryptic speciation. The examination of morphological features combined with a molecular genetic analysis based on a partial sequence of the 16S rRNA gene highlighted discrepancies between morphological and molecular diversity. Morphological data allowed to identify a morphotype with short appendages occurring in coralline algae communities and another one with long appendages observed in brackish-water environments and Sabellaria reefs. Multivariate analyses showed that sampling localities were the greatest source of morphological divergence, suggesting that phenotypic plasticity may play a role in local adaptations of S. gracilis populations. Molecular data showed the occurrence of four divergent lineages not corresponding to morphological clusters. Different species delimitation tests gave conflicting results, retrieving, however, at least four separated entities. Some lineages occurred in sympatry and were equally distributed in marine and brackish-water environments, excluding a biogeographic or ecological explanation of the observed pattern and suggesting instead ancient separation between lineages and secondary contact. The co-occurrence of different lineages hindered the identification of the lineage corresponding to S. gracilis sensu stricto. The discrepancy between morphological and molecular diversity suggests that different environmental and biogeographic features may interact in a complex and unpredictable way in shaping diversity patterns. An integrative approach is needed to provide a satisfactory insight on evolutionary processes in marine invertebrates.     

The hidden history of the snowshoe hare,Lepus americanus: extensive mitochondrial DNA introgression inferred from multilocus genetic variation

Hybridization drives the evolutionary trajectory of many species or local populations, and assessing the geographic extent and genetic impact of interspecific gene flow may provide invaluable clues to understand population divergence or the adaptive relevance of admixture. In North America, hares (Lepus spp.) are key species for ecosystem dynamics and their evolutionary history may have been affected by hybridization. Here we reconstructed the speciation history of the three most widespread hares in North America – the snowshoe hare (Lepus americanus), the white‐tailed jackrabbit (L. townsendii) and the black‐tailed jackrabbit (L. californicus) – by analysing sequence variation at eight nuclear markers and one mitochondrial DNA (mtDNA) locus (6240 bp; 94 specimens). A multilocus–multispecies coalescent‐based phylogeny suggests that L. americanus diverged ~2.7 Ma and that L. californicus and L. townsendii split more recently (~1.2 Ma). Within L. americanus, a deep history of cryptic divergence (~2.0 Ma) was inferred, which coincides with major speciation events in other North American species. While the isolation‐with‐migration model suggested that nuclear gene flow was generally rare or absent among species or major genetic groups, coalescent simulations of mtDNA divergence revealed historical mtDNA introgression from L. californicus into the Pacific Northwest populations of L. americanus. This finding marks a history of past reticulation between these species, which may have affected other parts of the genome and influence the adaptive potential of hares during climate change.     

Molecular evidence for the presence of cryptic evolutionary lineages in the freshwater copepod genus <Emphasis Type="Italic">Hemidiaptomus</Emphasis> G.O. Sars, 1903 (Calanoida,Diaptomidae)

The pattern of morphological and mtDNA cytochrome b diversity of three calanoid copepod species belonging to the diaptomid genus Hemidiaptomus has been investigated with the aim of checking the reliability of the morphological characters currently used for species identification, and the possible presence of cryptic taxa. A sharply different molecular structuring has been observed in the studied species: while Hemidiaptomus amblyodon exhibits a remarkable constancy throughout the European range of its distribution area (maximum inter-populations cytochrome b divergence of 3%), observed distances between presumed conspecific lineages of Hemidiaptomus gurneyi (maximum divergence of 21.5%) and Hemidiaptomus ingens (maximum 19.1%) suggest that under these binomens are in fact included complexes of cryptic, or currently just unrecognized, independent evolutionary lineages. The application of the “4x rule” shows that the two lineages singled out within H. ingens are in fact independent evolutionary units, while the complex molecular structure observed in H. gurneyi s.l. could not be resolved based on the currently available data. Applying standard crustacean mtDNA evolutionary rates to the observed divergence values, the separation of the main lineages within both H. ingens and H. gurneyi might dates back to the Miocene; however, it has also to be considered that the rate of mtDNA evolution might be accelerated in copepods, as already observed in other arthropod taxa. Present results gives further evidences of the high potential for copepod speciation with no or little morphological changes, and stress the need of a revision of the most controversial Palaearctic diaptomid genera.