Molecular divergence in the timeless and cpr genes among three sympatric cryptic species of the Anopheles triannulatus complex

Anopheles triannulatus s.l. is a malaria vector with a wide geographic distribution, ranging from Argentina-Nicaragua and Trinidad. Here we analysed sequences of two genes, timeless and cpr, to assess the genetic variability and divergence among three sympatric cryptic species of this complex from Salobra, central-western Brazil. The timeless gene sequences did not conclusively differentiate Anopheles halophylus and An. triannulatus species "C". However, a partial separation has been observed between these species and An. triannulatus s.s. Importantly, the analysis of the cpr gene sequences revealed fixed differences, no shared polymorphisms and considerable genetic differentiation among the three species of the An. triannulatus complex. The results confirm that An. triannulatus s.s., An. halophylus and An. triannulatus species C are distinct taxa, with the latter two likely representing a more recent speciation event.     

Did vicariance and adaptation drive cryptic speciation and evolution of brooding in Ophioderma longicauda (Echinodermata: Ophiuroidea), a common Atlanto‐Mediterranean ophiuroid?

Over the last decade, cryptic speciation has been discovered in an increasing number of taxa. Species complexes are useful models for the understanding of speciation processes. Motivated by the discovery of brooding specimens in the common Atlanto-Mediterranean broadcast spawning brittle star, Ophioderma longicauda, a recent study revealed the occurrence of divergent mitochondrial lineages. We analysed 218 specimens from 23 locations spread over the geographic range of the species with partial Cytochrome c Oxidase subunit I (COI) sequences. A subset of this sample was also surveyed with the internal transcribed spacer of the ribosomal DNA cluster (nuclear ITS-1). Our study revealed six highly divergent mitochondrial lineages, and the ITS-1 data confirmed that they most likely represent a species complex. Geographic ranges, abundances and genetic structures are contrasted among the putative cryptic species. Lineages in which brooding specimens have been found form a monophyletic group and are restricted to the Eastern Mediterranean basin, an oligotrophic zone. A phylogeny-trait association analysis revealed a phylogenetic signal for low 'chlorophyll a' values (our proxy for oligotrophy). An ecological shift related to the hyper oligotrophy of the Eastern Mediterranean region is therefore likely to have played a role in the evolution of brooding. This study revealed that a complex mixture of vicariance, population expansion, adaptive divergence and possibly high local diversification rates resulting from brooding has shaped the evolution of this species complex. The dating analysis showed that these events probably occurred in the Pleistocene epoch.     

Biogeographic barriers,Pleistocene refugia,and climatic gradients in the southeastern Nearctic drive diversification in cornsnakes (Pantherophis guttatus complex)

The southeastern Nearctic is a biodiversity hotspot that is also rich in cryptic species. Numerous hypotheses (e.g., vicariance, local adaptation, and Pleistocene speciation in glacial refugia) have been tested in an attempt to explain diversification and the observed pattern of extant biodiversity. However, previous phylogeographic studies have both supported and refuted these hypotheses. Therefore, while data support one or more of these diversification hypotheses, it is likely that taxa are forming within this region in species‐specific ways. Here, we generate a genomic data set for the cornsnakes (Pantherophis guttatus complex), which are widespread across this region, spanning both biogeographic barriers and climatic gradients. We use phylogeographic model selection combined with hindcast ecological niche models to determine regions of habitat stability through time. This combined approach suggests that numerous drivers of population differentiation explain the current diversity of this group of snakes. The Mississippi River caused initial speciation in this species complex, with more recent divergence events linked to adaptations to ecological heterogeneity and allopatric Pleistocene refugia. Lastly, we discuss the taxonomy of this group and suggest there may be additional cryptic species in need of formal recognition.     

Cryptic Genetic Diversity within the Anopheles nili group of Malaria Vectors in the Equatorial Forest Area of Cameroon (Central Africa)

Background

The Anopheles nili group of mosquitoes includes important vectors of human malaria in equatorial forest and humid savannah regions of sub-Saharan Africa. However, it remains largely understudied, and data on its populations’ bionomics and genetic structure are crucially lacking. Here, we used a combination of nuclear (i.e. microsatellite and ribosomal DNA) and mitochondrial DNA markers to explore and compare the level of genetic polymorphism and divergence among populations and species of the group in the savannah and forested areas of Cameroon, Central Africa.

Principal Findings

All the markers provided support for the current classification within the An. nili group. However, they revealed high genetic heterogeneity within An. nili s.s. in deep equatorial forest environment. Nuclear markers showed the species to be composed of five highly divergent genetic lineages that differed by 1.8 to 12.9% of their Internal Transcribed Spacer 2 (ITS2) sequences, implying approximate divergence time of 0.82 to 5.86 million years. However, mitochondrial data only detected three major subdivisions, suggesting different evolutionary histories of the markers.

Conclusions/Significance

This study enlightened additional cryptic genetic diversity within An. nili s.s. in the deep equatorial forest environment of South Cameroon, reflecting a complex demographic history for this major vector of malaria in this environment. These preliminary results should be complemented by further studies which will shed light on the distribution, epidemiological importance and evolutionary history of this species group in the African rainforest, providing opportunities for in-depth comparative studies of local adaptation and speciation in major African malaria vectors.     

Assessing hybridization in natural populations of Penstemon (Scrophulariaceae) using hypervariable intersimple sequence repeat (ISSR) bands

Inferences regarding hybridization rely on genetic markers to differentiate parental taxa from one another. Intersimple sequence repeat (ISSR) markers are based on single-primer PCR reactions where the primer sequence is derived from di- and trinucleotide repeats. These markers have successfully been used to assay genetic variability among cultivated plants, but have not yet been tested in natural populations. We used genetic markers generated from eight ISSR primers to examine patterns of hybridization and purported examples of hybrid speciation in Penstemon (Scrophulariaceae) in a hybrid complex involving P. centranthifolius , P. grinnellii , P. spectabilis and P. clevelandii . This hybrid complex has previously been studied using three molecular data sets (allozymes, and restriction-site variation of nuclear rDNA and chloroplast DNA). These studies revealed patterns of introgression involving P. centranthifolius , but were unsuccessful in determining whether gene flow occurs among the other species, and support for hypotheses of diploid hybrid speciation was also lacking. In this study, we were able to fingerprint each DNA accession sampled with one to three ISSR primers and most accessions could be identified with a single primer. We found population- and species-specific markers for each taxon surveyed. Our results: (i) do not support the hybrid origin of P. spectabilis ; (ii) do support the hypothesis that P. clevelandii is a diploid hybrid species derived from P. centranthifolius and P. spectabilis ; and (iii) demonstrate that pollen-mediated gene flow via hummingbird vectors is prevalent in the hybrid complex.     

Molecular and morphological data reveal cryptic taxonomic diversity in the terrestrial slug complex Arion subfuscus/fuscus (Mollusca, Pulmonata, Arionidae) in continental north-west Europe

The importance and abundance of cryptic species among invertebrate taxa is well documented. Nowadays, taxonomic, phylogenetic and conservation biological studies frequently use molecular markers to delineate cryptic taxa. Such studies, however, often face the problem of the differential resolution of the molecular markers and techniques involved. This issue is explored in the present study of cryptic taxa within the terrestrial slug complex Arion subfuscus/fuscus in continental north-west Europe. To this end, morphological, allozyme and mitochondrial 16S rDNA sequence data have been jointly evaluated. Using allozyme data and gonad type, two distinct groups were consistently delineated, even under sympatric conditions. The 16S rDNA data strongly supported both those groups and even suggested the presence of three distinct taxa within one of them. However, in view of: (1) the allopatric distribution of three OTUs, (2) the lack of allozyme or morphological differentiation, and (3) the extremely high degree of intraspecific mtDNA variation reported in pulmonate gastropods, they are, for the time being, not regarded as valid species under the biological species concept. By means of 16S rDNA and allozyme data, the position of type and topotype material of A. subfuscus s.s. and A. fuscus relative to the newly defined OTUs was determined, thus clarifying the nomenclature of this species complex. Additionally, gonad type proved to be a useful character for distinguishing the two species in north-west Europe.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 23–38.     

On the scent of speciation: the chemosensory system and its role in premating isolation

Chemosensory speciation is characterized by the evolution of barriers to genetic exchange that involve chemosensory systems and chemical signals. Here, we review some representative studies documenting chemosensory speciation in an attempt to evaluate the importance and the different aspects of the process in nature and to gain insights into the genetic basis and the evolutionary mechanisms of chemosensory trait divergence. Although most studies of chemosensory speciation concern sexual isolation mediated by pheromone divergence, especially in Drosophila and moth species, other chemically based behaviours (habitat choice, pollinator attraction) can also play an important role in speciation and are likely to do so in a wide range of invertebrate and vertebrate species. Adaptive divergence of chemosensory traits in response to factors such as pollinators, hosts and conspecifics commonly drives the evolution of chemical prezygotic barriers. Although the genetic basis of chemosensory speciation remains largely unknown, genomic approaches to chemosensory gene families and to enzymes involved in biosynthetic pathways of signal compounds now provide new opportunities to dissect the genetic basis of these complex traits and of their divergence among taxa.     

Radiation and divergence in the Rhagoletis pomonella species complex: inferences from DNA sequence data

Here, we investigate the evolutionary history and pattern of genetic divergence in the Rhagoletis pomonella (Diptera: Tephritidae) sibling species complex, a model for sympatric speciation via host plant shifting, using 11 anonymous nuclear genes and mtDNA. We report that DNA sequence results largely coincide with those of previous allozyme studies. Rhagoletis cornivora was basal in the complex, distinguished by fixed substitutions at all loci. Gene trees did not provide reciprocally monophyletic relationships among US populations of R. pomonella, R. mendax, R. zephyria and the undescribed flowering dogwood fly. However, private alleles were found for these taxa for certain loci. We discuss the implications of the results with respect to identifiable genetic signposts (stages) of speciation, the mosaic nature of genomic differentiation distinguishing formative species and a concept of speciation mode plurality involving a biogeographic contribution to sympatric speciation in the R. pomonella complex.     

Try Tri again? Resolving species boundaries in the Lomatium triternatum (Apiaceae) complex

Species boundaries have traditionally been delimited by applying phenotypic characters to a morphological species concept. With an increased understanding of the complexities of speciation as a process, species concepts have proliferated while at the same time, the ability to gather greater numbers and types of molecular characters has expanded the means by which species can be delimited. Phylogenetic studies of molecular data provide an opportunity to identify reciprocally monophyletic groupsand have led to the identification of cryptic or nearly cryptic species in which subtle differences in phenotypes or ecological niches can be uncovered only after monophyletic groups have been identified. Here, we investigate evolutionary relationships among a group of species in the Lomatium triternatum complex using molecular phylogenetic analyses for all samples, and ecological parameters for two of the 38 species included in this study. The results indicate that there are more reciprocally monophyletic groups in this complex than had been estimated using phenotypic data alone. The ecological data show a clear differentiation for the one pair of sister species where ecological sampling was available, implying that divergence within this group may have resulted from environmental selection for soil preferences that have been strong enough to result in speciation.     

The interplay of colour and bioacoustic traits in the differentiation of a Southeast Asian songbird complex

Morphological traits have served generations of biologists as a taxonomic indicator, and have been the main basis for defining and classifying species diversity for centuries. A quantitative integration of behavioural characters, such as vocalizations, in studies on biotic differentiation has arisen more recently, and the relative importance of these different traits in the diversification process remains poorly understood. To provide a framework within which to interpret the evolutionary interplay between morphological and behavioural traits, we generated a draft genome of a cryptic Southeast Asian songbird, the limestone wren‐babbler Napothera crispifrons. We resequenced whole genomes of multiple individuals of all three traditional subspecies and of a distinct leucistic population. We demonstrate strong genomic and mitochondrial divergence among all three taxa, pointing to the existence of three species‐level lineages. Despite its great phenotypic distinctness, the leucistic population was characterized by shallow genomic differentiation from its neighbour, with only a few localized regions emerging as highly diverged. Quantitative bioacoustic analysis across multiple traits revealed deep differences especially between the two taxa characterized by limited plumage differentiation. Our study demonstrates that differentiation in these furtive songbirds has resulted in a complex mosaic of colour‐based and bioacoustic differences among populations. Extreme colour differences can be anchored in few genomic loci and may therefore arise and subside rapidly.     

Species identification in the trematomid family using nuclear genetic markers

The Trematominae, a subfamily of the Nototheniidae, are typical of the high-Antarctic shelf waters. Within the Trematominae examples of phenotypic plasticity and possible cryptic speciation have been observed. Morphological identification of adult stages can be problematic in cases of high phenotypic plasticity or cryptic speciation. Additionally, postlarval and juvenile stages often have traits still under development and which lack distinction. A microsatellite DNA multiplex of six markers has been developed for Trematomus newnesi (Van Houdt et al. 2006). This multiplex was tested on five additional trematomid taxa: Pagothenia borchgrevinki, Trematomus bernacchii, Trematomus eulepidotus, Trematomus hansoni and Trematomus scotti. We used these six microsatellite loci to assess the genetic differentiation among species and the resolution power of these loci for individual-based assignment methods. The six species could be well discriminated by conventional methods such as principal component analysis and distance-based methods, and individual Bayesian assignment methods. This marker set can be used for a number of purposes, including the identification of eggs and larval and adult stages. It is also useful for the investigation of recent phylogenetic patterns, as well as the detection of cryptic speciation, which has been suggested for T. bernacchii and T. newnesi but never confirmed with high polymorphic genetic markers.     

Allopatric origin of cryptic butterfly species that were discovered feeding on distinct host plants in sympatry

Surveys of tropical insects are increasingly uncovering cryptic species – morphologically similar yet reproductively isolated taxa once thought to comprise a single interbreeding entity. The vast majority of such species are described from a single location. This leaves us with little information on geographic range and intraspecific variation and limits our ability to infer the forces responsible for generating such diversity. For example, in herbivorous and parasitic insects, multiple specialists are often discovered within what were thought to be single more generalized species. Host shifts are likely to have contributed to speciation in these cases. But when and where did those shifts occur, and were they facilitated by geographic isolation? We attempted to answer these questions for two cryptic species within the butterfly Cymothoe egesta that were recently discovered on different host plants in central Cameroon. We first used mtDNA markers to separate individuals collected on the two hosts within Cameroon and then extended our analysis to incorporate individuals collected across the entire pan‐Afrotropical range of the original taxon. To our surprise, we found that the species are almost entirely allopatric, dividing the original range and overlapping only in the narrow zone of West‐Central Africa where they were first discovered in sympatry. This finding, combined with analyses of genetic variation within each butterfly species, strongly suggests that speciation occurred in allopatry, probably during the Pleistocene. We discuss the implications of our results for understanding speciation among other cryptic species recently discovered in the tropics and argue that more work is needed on geographic patterns and host usage in such taxa.     

Molecular systematics and phylogeography of the cryptic species complex Baetis rhodani (Ephemeroptera, Baetidae)

Genetic studies have highlighted cryptic diversity in many well-known taxa including aquatic insects, with the general implication that there are more species than are currently recognised. Baetis rhodani Pictet are among the most widespread, abundant and ecologically important of all European mayflies (Ephemeroptera), and used widely as biological indicators of stream quality. Traditional taxonomy and systematics have never fully resolved differences among suspected cryptic species in the B. rhodani complex because morphological characters alone do not allow reliable distinction. This is particularly true among larvae, the life-stage used most widely in monitoring. Here, we assess the molecular diversity of this complex in one of the largest such studies of cryptic species in the order Ephemeroptera to date. Phylogenies were constructed using data from the mitochondrial cytochrome oxidase subunit I (COI) gene. Two monophyletic groups were recovered consisting of one major haplogroup and a second clade of 6 smaller but distinct haplogroups. Haplogroup divergence ranged from 0.2-3% (within) to 8-19% (among) with the latter values surpassing maxima typically reported for other insects, and provides strong evidence for cryptic species in the B. rhodani complex. The taxonomic status of these seven haplogroups remains undefined. Their distributions across Western Europe reveal no obvious geographic pattern, suggesting widespread diffusion of genetic lineages since the last glacial maximum. The implications of these findings are far-reaching given the ecological and bioindicator significance of what now appears to be several taxa.     

Molecular, morphological and behavioural data reveal the presence of a cryptic species in the widely studied Drosophila serrata species complex

The Drosophila serrata species complex from Australia and New Guinea has been widely used in evolutionary studies of speciation and climatic adaptation. It is believed to consist of D. serrata, D. birchii and D. dominicana, although knowledge of the latter is limited. Here we present evidence for a previously undescribed cryptic member of the D. serrata species complex. This new cryptic species is widespread in far north Queensland, Australia and is likely to have been previously mistaken for D. serrata. It shows complete reproductive isolation when crossed with both D. serrata and D. birchii. The cryptic species can be easily distinguished from D. serrata and D. birchii using either microsatellite loci or visual techniques. Although it occurs sympatrically with both D. serrata and D. birchii, it differs from these species in development time, viability, wing size and wing morphology. Its discovery explains patterns of recently described mitochondrial DNA divergence within D. serrata, and may also help to clarify some ambiguities evident in early evolutionary literature on reproductive incompatibility within the D. serrata species complex.     

A molecular phylogenetic approach to the phylum Cycliophora provides further evidence for cryptic speciation in Symbion americanus

Cryptic speciation is a phenomenon that has been reported in a wide range of marine invertebrates, including the American cycliophoran Symbion americanus . Although cryptic speciation is often proposed based on phylogeographical analyses, here we demonstrate the utility of multilocus molecular phylogenetic approaches in identifying cryptic lineages within the phylum Cycliophora. Thirteen individuals from three putative cryptic lineages of S. americanus , as well as two individuals each from the European species S. pandora and a new species of Symbion living on Homarus gammarus , were sequenced for up to 4.8 kb of genomic DNA over four loci (COI, 16S rRNA, 18S rRNA and 28S rRNA). Phylogenetic analyses of individual loci as well as combined data clearly support a division between American and European cycliophorans. Moreover, the American cycliophorans consistently form three well-supported clades, which is congruent with the presence of three putative reproductively isolated lineages in a species complex. Further studies are necessary to more precisely describe the evolution of reproductive isolation within S. americanus .     

Nested biological variation and speciation

The modes of speciation that are thought to have contributed most to the generation of biodiversity require population differentiation as the initial stage in the speciation process. Consequently, a complete understanding of the mechanisms of speciation requires that the process be examined not just after speciation is complete, or nearly so, but also much earlier. Because reproductive isolation defines biological species, and it evolves slowly, study of the process may require a prohibitive span of time. Even if speciation could be observed directly, selection of populations in the process of speciation is typically difficult or impossible, because those that will ultimately undergo speciation cannot be distinguished from those that will differentiate but never assume the status of new biological species. One means of circumventing this problem is to study speciation in taxa comprising several sibling species, at least one of which exhibits extensive population differentiation. We illustrate this approach by exploring patterns of population variation in the post-glacial radiation of the threespine stickleback, Gasterosteus aculeatus. We focus on lacustrine populations and species within this complex, demonstrating parallel axes of divergence within populations, among populations and among species. The pattern that emerges is one of parallel relationships between phenotype and fitness at all three hierarchical levels, a pattern that facilitates exploration of the causes and consequences of speciation and secondary contact. A second outcome of this exploration is the observation that speciation can be the consequence of a cascade of effects, beginning with selection on trophic or other characteristics that in turn force the evolution of other population characteristics that precipitate speciation. Neither of these conclusions could have been reached without comparative studies of wild populations at several hierarchical levels, a conclusion reinforced by a brief survey of similar efforts to elucidate the process of speciation. We address the issues most likely to be resolved using this approach, and suggest that comparisons of natural variation within taxa at several hierarchical levels may substantially increase our understanding of the speciation process.     

Hybridization, mitochondrial DNA phylogeography, and prediction of the early stages of reproductive isolation: lessons from New Zealand cicadas (genus Kikihia)

One of the major tenets of the modern synthesis is that genetic differentiation among subpopulations is translated over time into genetic differentiation among species. Phylogeographic exploration is therefore essential to the study of speciation because it can reveal the presence of subpopulations that may go on to become species or that may already represent cryptic species. Acoustic species-specific mating signals provide a significant advantage for the recognition of cryptic or incipient species. Because the majority of species do not have such easily recognized premating signals, data from acoustically signaling species can serve as a valuable heuristic tool. Acoustic signals are also convenient tools for recognizing hybridization events. Here, we demonstrate that evidence of hybridization in the form of intermediate song phenotypes is present in many contact zones between species of the New Zealand grass cicadas of the Kikihia muta species complex and that recurring mitochondrial DNA (mtDNA) introgression has created misleading patterns that make it difficult to identify certain taxa using song or mtDNA alone. In one case, introgression appears to have occurred between allopatric taxa by dispersal of introgressed populations of an intermediary species ("hybridization by proxy"). We also present a comparison of mtDNA-tree- and song-based taxonomies obtained for the K. muta complex. We find that 12 mtDNA candidate species are identified using shifts in phylogenetic branching rate found by a single-threshold mixed Yule-coalescent lineage model, while only 7 candidate species are identified using songs. Results from the Yule-coalescent model are dependent on factors such as the number of modeled thresholds and the inclusion of duplicate haplotypes. Genetic distances within song species reach a maximum at about 0.028 substitutions/site when likely cases of hybridization and introgression are excluded. Large genetic breaks or "gaps" are not observed between some northern (warmer climate) song clades, possibly because climate-induced bottlenecks have been less severe. These results support ongoing calls for multimarker genetic studies as well as "integrative taxonomy" that combines information from multiple character sources, including behavior, ecology, geography, and morphology.     

Gene flow between chromosomal forms of the malaria vector Anopheles funestus in Cameroon, Central Africa, and its relevance in malaria fighting

Knowledge of population structure in a major vector species is fundamental to an understanding of malaria epidemiology and becomes crucial in the context of genetic control strategies that are being developed. Despite its epidemiological importance, the major African malaria vector Anopheles funestus has received far less attention than members of the Anopheles gambiae complex. Previous chromosomal data have shown a high degree of structuring within populations from West Africa and have led to the characterization of two chromosomal forms, "Kiribina" and "Folonzo." In Central Africa, few data were available. We thus undertook assessment of genetic structure of An. funestus populations from Cameroon using chromosomal inversions and microsatellite markers. Microsatellite markers revealed no particular departure from panmixia within each local population and a genetic structure consistent with isolation by distance. However, cytogenetic studies demonstrated high levels of chromosomal heterogeneity, both within and between populations. Distribution of chromosomal inversions was not random and a cline of frequency was observed, according to ecotypic conditions. Strong deficiency of heterokaryotypes was found in certain localities in the transition area, indicating a subdivision of An. funestus in chromosomal forms. An. funestus microsatellite genetic markers located within the breakpoints of inversions are not differentiated in populations, whereas in An. gambiae inversions can affect gene flow at marker loci. These results are relevant to strategies for control of malaria by introduction of transgenes into populations of vectors.     

A standard photomap of ovarian nurse cell chromosomes in the European malaria vector Anopheles atroparvus

Anopheles atroparvus (Diptera: Culicidae) is one of the main malaria vectors of the Maculipennis group in Europe. Cytogenetic analysis based on salivary gland chromosomes has been used in taxonomic and population genetic studies of mosquitoes from this group. However, a high‐resolution cytogenetic map that could be used in physical genome mapping in An. atroparvus is still lacking. In the present study, a high‐quality photomap of the polytene chromosomes from ovarian nurse cells of An. atroparvus was developed. Using fluorescent in situ hybridization, 10 genes from the five largest genomic supercontigs on the polytene chromosome were localized and 28% of the genome was anchored to the cytogenetic map. The study established chromosome arm homology between An. atroparvus and the major African malaria vector Anopheles gambiae, suggesting a whole‐arm translocation between autosomes of these two species. The standard photomap constructed for ovarian nurse cell chromosomes of An. atroparvus will be useful for routine physical mapping. This map will assist in the development of a fine‐scale chromosome‐based genome assembly for this species and will also facilitate comparative and evolutionary genomics studies in the genus Anopheles.     

Genome‐wide single‐nucleotide polymorphism data reveal cryptic species within cryptic freshwater snail species—The case of the Ancylus fluviatilis species complex

DNA barcoding utilizes short standardized DNA sequences to identify species and is increasingly used in biodiversity assessments. The technique has unveiled an unforeseeably high number of morphologically cryptic species. However, if speciation has occurred relatively recently and rapidly, the use of single gene markers, and especially the exclusive use of mitochondrial markers, will presumably fail in delimitating species. Therefore, the true number of biological species might be even higher. One mechanism that can result in rapid speciation is hybridization of different species in combination with polyploidization, that is, allopolyploid speciation. In this study, we analyzed the population genetic structure of the polyploid freshwater snail Ancylus fluviatilis, for which allopolyploidization was postulated as a speciation mechanism. DNA barcoding has already revealed four cryptic species within A. fluviatilis (i.e., A. fluviatilis s. str., Ancylus sp. A–C), but early allozyme data even hint at the presence of additional cryptic lineages in Central Europe. We combined COI sequencing with high‐resolution genome‐wide SNP data (ddRAD data) to analyze the genetic structure of A. fluviatilis populations in a Central German low mountain range (Sauerland). The ddRAD data results indicate the presence of three cryptic species within A. fluviatilis s. str. occurring in sympatry and even syntopy, whereas mitochondrial sequence data only support the existence of one species, with shared haplotypes between species. Our study hence points to the limitations of DNA barcoding when dealing with organismal groups where speciation is assumed to have occurred rapidly, for example, through the process of allopolyploidization. We therefore emphasize that single marker DNA barcoding can underestimate the true species diversity and argue in strong favor of using genome‐wide data for species delimitation in such groups.