Historic speciation and recent colonization of Eurasian monkey gobies (Neogobius fluviatilis and N. pallasi) revealed by DNA sequences,microsatellites, and morphology

Aim Hidden diversity within an invasive ‘species’ can mask both invasion pathways and confound management goals. We assessed taxonomic status and population structure of the monkey goby Neogobius fluviatilis across Eurasia, comparing genetic variation across its native and invasive ranges. Location Native populations were analysed within the Black and Caspian Sea basins, including major river drainages (Dnieper, Dniester, Danube, Don and Volga rivers), along with introduced locations within the upper Danube and Vistula river systems. Methods DNA sequences and 10 nuclear microsatellite loci were analysed to test genetic diversity and divergence patterns of native and introduced populations; phylogenetic analysis of mtDNA cytochrome b and nuclear RAG‐1 sequences assessed taxonomic status of Black and Caspian Sea lineages. Multivariate analysis of morphology was used to corroborate phylogenetic patterns. Population genetic structure within each basin was evaluated with mtDNA and microsatellite data using F_ST analogues and Bayesian assignment tests. Results Phylogenetic analysis of mitochondrial and nuclear sequences discerned a pronounced genetic break between monkey gobies in the Black and Caspian Seas, indicating a long‐term species‐level separation dating to c. 3 million years. This pronounced separation further was confirmed from morphological and population genetic divergence. Bayesian inference showed congruent patterns of population structure within the Black Sea basin. Introduced populations in the Danube and Vistula River basins traced to north‐west Black Sea origins, a genetic expansion pattern matching that of other introduced Ponto‐Caspian gobiids. Main conclusions Both genetic and morphological data strongly supported two species of monkey gobies that were formerly identified as subspecies: N. fluviatilis in the Black Sea basin, Don and Volga Rivers, and the Kumo‐Manych Depression, and Neogobius pallasi in the Caspian Sea and Volga River delta. Genetic origins of introduced N. fluviatilis populations indicated a common invasion pathway shared with other introduced Ponto‐Caspian fishes and invertebrates.     

Discordance between morphological and molecular species boundaries among Caribbean species of the reef sponge Callyspongia

Sponges are among the most species‐rich and ecologically important taxa on coral reefs, yet documenting their diversity is difficult due to the simplicity and plasticity of their morphological characters. Genetic attempts to identify species are hampered by the slow rate of mitochondrial sequence evolution characteristic of sponges and some other basal metazoans. Here we determine species boundaries of the Caribbean coral reef sponge genus Callyspongia using a multilocus, model‐based approach. Based on sequence data from one mitochondrial (COI), one ribosomal (28S), and two single‐copy nuclear protein‐coding genes, we found evolutionarily distinct lineages were not concordant with current species designations in Callyspongia. While C. fallax, C. tenerrima, and C. plicifera were reciprocally monophyletic, four taxa with different morphologies (C. armigera, C. longissima, C. eschrichtii, and C. vaginalis) formed a monophyletic group and genetic distances among these taxa overlapped distances within them. A model‐based method of species delimitation supported collapsing these four into a single evolutionary lineage. Variation in spicule size among these four taxa was partitioned geographically, not by current species designations, indicating that in Callyspongia, these key taxonomic characters are poor indicators of genetic differentiation. Taken together, our results suggest a complex relationship between morphology and species boundaries in sponges.     

Mitogenomic phylogeny of Acanthocephala reveals novel Class relationships

The Acanthocephala is a phylum of obligate endoparasitic animals comprising four classes (Archiacanthocephala, Palaeacanthocephala, Eoacanthocephala and Polyacanthocephala), although the phylogenetic interrelationships of these classes still remains unresolved. To investigate phylogenetic relationships of major acanthocephalan groups, we characterized the complete mitochondrial genome sequences of two palaeacanthocephalan species Centrorhynchus aluconis and Prosthorhynchus transversus (representing two different families of the order Polymorphida), and Polyacanthorhynchus caballeroi (the first mitogenomic representative of the class Polyacanthocephala) and used these new sequences for phylogenetic analyses, along with 32 platyzoan mtDNAs, including 10 additional acanthocephalans. Phylogenetic analyses using concatenated amino acid sequences for 12 protein‐coding genes with maximum likelihood and Bayesian inference methods supported monophyly of Acanthocephala. Within the phylum, Archiacanthocephala was positioned as the sister to the clade containing all three other acanthocephalan classes, with the polyacanthocephalan species P. caballeroi nested within Eoacanthocephala. This result contradicts morphology‐based classification systems that treated polyacanthorhynchids as one of the palaeacanthocephalan families, and instead suggests Polyacanthocephala is a member of Eoacanthocephala. Within the Palaeacanthocephala, Polymorphida monophyly was strongly supported and this is inconsistent with nuclear rDNA‐based molecular hypotheses that suggest non‐monophyly.     

Genetic and morphometric analysis of sixteenth century <Emphasis Type="Italic">Canis</Emphasis> skull fragments: implications for historic eastern and gray wolf distribution in North America

Resolving the taxonomy and historic ranges of species are essential to recovery plans for species at risk and conservation programs that aim to restore extirpated populations. In eastern North America, planning for wolf population restoration is complicated by the disputed historic distributions of two wolf species: the Old World-evolved gray wolf (Canis lupus) and the New World-evolved eastern wolf (C. lycaon). We used genetic and morphometric data from 4- to 500-year-old Canis samples excavated in London, Ontario, Canada to help clarify the historic range of these two wolf species in the eastern temperate forests of North America. We isolated DNA and sequenced the mitochondrial control region and found that none of the samples were of gray wolf origin. Two of the DNA sequences corresponded to those found in present day coyotes (C. latrans), but morphometric comparisons show an eastern wolf, not coyote, origin. The remaining two sequences matched ancient domestic dog haplotypes. These results suggest that the New World-evolved eastern wolf, not the gray wolf, occupied this region prior to the arrival of European settlers, although eastern-gray wolf hybrids cannot be ruled out. Furthermore, our data support the idea of a shared common ancestry between eastern wolves and western coyotes, and that the distribution of gray wolves at this time probably did not include the eastern temperate forests of North America.     

Are sympatrically speciating Midas cichlid fish special? Patterns of morphological and genetic variation in the closely related species Archocentrus centrarchus

Established empirical cases of sympatric speciation are scarce, although there is an increasing consensus that sympatric speciation might be more common than previously thought. Midas cichlid fish are one of the few substantiated cases of sympatric speciation, and they formed repeated radiations in crater lakes. In contrast, in the same environment, such radiation patterns have not been observed in other species of cichlids and other families of fish. We analyze morphological and genetic variation in a cichlid species (Archocentrus centrarchus) that co‐inhabits several crater lakes with the Midas species complex. In particular, we analyze variation in body and pharyngeal jaw shape (two ecologically important traits in sympatrically divergent Midas cichlids) and relate that to genetic variation in mitochondrial control region and microsatellites. Using these four datasets, we analyze variation between and within two Nicaraguan lakes: a crater lake where multiple Midas cichlids have been described and a lake where the source population lives. We do not observe any within‐lake clustering consistent across morphological traits and genetic markers, suggesting the absence of sympatric divergence in A. centrarchus. Genetic differentiation between lakes was low and morphological divergence absent. Such morphological similarity between lakes is found not only in average morphology, but also when analyzing covariation between traits and degree of morphospace occupation. A combined analysis of the mitochondrial control region in A. centrarchus and Midas cichlids suggests that a difference between lineages in the timing of crater lake colonization cannot be invoked as an explanation for the difference in their levels of diversification. In light of our results, A. centrarchus represents the ideal candidate to study the genomic differences between these two lineages that might explain why some lineages are more likely to speciate and diverge in sympatry than others.     

DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Complete mitochondrial genomes of prasinophyte algae Pyramimonas parkeae and Cymbomonas tetramitiformis

Mitochondria are archetypal eukaryotic organelles that were acquired by endosymbiosis of an ancient species of alpha‐proteobacteria by the last eukaryotic common ancestor. The genetic information contained within the mitochondrial genome has been an important source of information for resolving relationships among eukaryotic taxa. In this study, we utilized mitochondrial and chloroplast genomes to explore relationships among prasinophytes. Prasinophytes are represented by diverse early‐diverging green algae whose physical structures and genomes have the potential to elucidate the traits of the last common ancestor of the Viridiplantae (or Chloroplastida). We constructed de novo mitochondrial genomes for two prasinophyte algal species, Pyramimonas parkeae and Cymbomonas tetramitiformis, representing the prasinophyte clade. Comparisons of genome structure and gene order between these species and to those of other prasinophytes revealed that the mitochondrial genomes of P. parkeae and C. tetramitiformis are more similar to each other than to other prasinophytes, consistent with other molecular inferences of the close relationship between these two species. Phylogenetic analyses using the inferred amino acid sequences of mitochondrial and chloroplast protein‐coding genes resolved a clade consisting of P. parkeae and C. tetramitiformis; and this group (representing the prasinophyte clade I) branched with the clade II, consistent with previous studies based on the use of nuclear gene markers.     

Paraphyly and budding speciation in the hairy snail (Pulmonata,Hygromiidae)

Delimitation of species is often complicated by discordance of morphological and genetic data. This may be caused by the existence of cryptic or polymorphic species. The latter case is particularly true for certain snail species showing an exceptionally high intraspecific genetic diversity. The present investigation deals with the Trochulus hispidus complex, which has a complicated taxonomy. Our analyses of the COI sequence revealed that individuals showing a T. hispidus phenotype are distributed in nine highly differentiated mitochondrial clades (showing p‐distances up to 19%). The results of a parallel morphometric investigation did not reveal any differentiation between these clades, although the overall variability is quite high. The phylogenetic analyses based on 12S, 16S and COI sequences show that the T. hispidus complex is paraphyletic with respect to several other morphologically well‐defined Trochulus species (T. clandestinus, T. villosus, T. villosulus and T. striolatus) which form well‐supported monophyletic groups. The nc marker sequence (5.8S–ITS2–28S) shows only a clear separation of T. o. oreinos and T. o. scheerpeltzi, and a weakly supported separation of T. clandestinus, whereas all other species and the clades of the T. hispidus complex appear within one homogeneous group. The paraphyly of the T. hispidus complex reflects its complicated history, which was probably driven by geographic isolation in different glacial refugia and budding speciation. At our present state of knowledge, it cannot be excluded that several cryptic species are embedded within the T. hispidus complex. However, the lack of morphological differentiation of the T. hispidus mitochondrial clades does not provide any hints in this direction. Thus, we currently do not recommend any taxonomic changes. The results of the current investigation exemplify the limitations of barcoding attempts in highly diverse species such as T. hispidus.     

Non‐concordant phylogeographical patterns of three widely codistributed endemic Western Balkans lacertid lizards (Reptilia,Lacertidae) shaped by specific habitat requirements and different responses to Pleistocene climatic oscillations

The Balkan Peninsula is a hot spot for European herpetofaunal biodiversity and endemism. The rock climbing lizards Dalmatolacerta oxycephala and Dinarolacerta mosorensis and the ground‐dwelling Dalmatian wall lizard Podarcis melisellensis are endemic to the Western Balkans, and their ranges largely overlap. Here, we present a comparative phylogeographical study of these three species in the area of their codistribution in order to determine the level of concordance in their evolutionary patterns. Phylogenetic analyses were performed based on two mitochondrial genes (cytochrome b and 16S rRNA), and a molecular clock approach was used to date the most important events in their evolutionary histories. We also tested for correlations regarding genetic differentiation among populations and their geographical distances. For all three species, a significant correlation between genetic and geographical distances was found. Within D. oxycephala, two deeply separated clades (‘island’ and ‘mainland clade’), with further subdivision of the ‘mainland clade’ into two subclades (‘south‐eastern’ and ‘north‐western’), were found. High sequence divergences were observed between these groups. From our data, the time of separation of the two main clades of D. oxycephala can be estimated at about 5 mya and at about 0.8 mya for the two subclades of the mainland clade. Within D. mosorensis, coalescence time may be dated at about 1 mya, while D. mosorensis and D. montenegrina separated around 5 mya. The results imply the existence of complex palaeo‐biogeographical and geological factors that probably influenced the observed phylogeographical patterns in these lacertid species, and point to the presence of numerous glacial/interglacial refugia. Furthermore, the observed cryptic genetic diversity within the presently monotypic species D. oxycephala prompts for a revision of its taxonomic and conservation status.     

One species hides many: Molecular and morphological evidence for cryptic speciation in a thread snake (Leptotyphlopidae: Leptotyphlops sylvicolus Broadley & Wallach, 1997)

We investigate the phylogeographic structure of a fossorial forest‐living snake species, the forest thread snake, Leptotyphlopssylvicolus Broadley & Wallach, 1997 by sampling specimens from the Eastern Cape and KwaZulu‐Natal provinces of South Africa. Phylogenetic results, using Bayesian inferences and maximum likelihood, from the combined mitochondrial sequence data (cyt b and ND4), along with population genetic analyses suggest the presence of phylogeographic breaks broadly congruent to those exhibited by other forest‐living taxa. Divergence‐time estimates indicate that cladogenesis within the study taxon occurred during the late Miocene climatic shifts, suggesting that cladogenesis was driven by habitat fragmentation. We further investigate the species‐level divergence within L. sylvicolus by including two partial nuclear loci (PRLR and RAG1). The three species delimitation methods (ABGD, bGMYC, and STACEY), retrieved 10–12 putative species nested within the L. sylvicolus species complex. These results were corroborated by iBPP implementing molecular and morphological data in an integrative Bayesian framework. The morphological analyses exhibit large overlap among putative species but indicate differences between grassland and forest species. Due to the narrow distributions of these putative species, the results of the present study have further implications for the conservation status of the L. sylvicolus species complex and suggest that forest and grassland habitats along the east coast of South Africa may harbor significantly higher levels of diversity than currently recognized.     

Species delimitation in the Aleochara fucicola species complex (Coleoptera: Staphylinidae: Aleocharinae) and its phylogenetic relationships

To assess the accuracy of species delimitation and phylogenetic relationships of the Aleochara fucicola species complex, we performed molecular phylogenetic analyses. Detailed micromorphological characters were also examined using scanning electron microscopy (SEM). The molecular data set included two mitochondrial (COI and COII) and three nuclear protein‐coding genes (CAD, EF1‐α and wg) for 19 populations of five species. Significant discordance was found between mitochondrial and nuclear gene trees. Two species, A. puetzi (Assing) and A. segregata Yamamoto and Maruyama, were not separated in the mitochondrial gene trees, but clearly were differentiated in the nuclear and concatenated gene trees. The SEM data also supported the separation of these two species, but an analysis of genetic divergence data did not. Adaptation to extremely harsh environments might reduce morphological variation in the A. fucicola species complex during the colonization of seashores. We present a multilocus phylogeny of the species complex. It suggests that the ancestor of the A. fucicola species complex occurred along the southern coasts of Northeast Asia, followed by dispersals to northern coasts.     

Integrating multiple lines of evidence to better understand the evolutionary divergence of humpback dolphins along their entire distribution range: a new dolphin species in Australian waters?

The conservation of humpback dolphins, distributed in coastal waters of the Indo‐West Pacific and eastern Atlantic Oceans, has been hindered by a lack of understanding about the number of species in the genus (Sousa) and their population structure. To address this issue, we present a combined analysis of genetic and morphologic data collected from beach‐cast, remote‐biopsied and museum specimens from throughout the known Sousa range. We extracted genetic sequence data from 235 samples from extant populations and explored the mitochondrial control region and four nuclear introns through phylogenetic, population‐level and population aggregation frameworks. In addition, 180 cranial specimens from the same geographical regions allowed comparisons of 24 morphological characters through multivariate analyses. The genetic and morphological data showed significant and concordant patterns of geographical segregation, which are typical for the kind of demographic isolation displayed by species units, across the Sousa genus distribution range. Based on our combined genetic and morphological analyses, there is convincing evidence for at least four species within the genus (S. teuszii in the Atlantic off West Africa, S. plumbea in the central and western Indian Ocean, S. chinensis in the eastern Indian and West Pacific Oceans, and a new as‐yet‐unnamed species off northern Australia).     

Systematics,genetics, and biogeography of intertidal mites (Acari,Oribatida) from the Andaman Sea and Strait of Malacca

This study demonstrates for the first time the presence of marine‐associated mites in the Andaman Sea and Strait of Malacca and reveals a relatively high diversity of these taxa with six species from two different families: Selenoribatidae and Fortuyniidae. Indopacifica, a new genus of Selenoribatidae, is described from Thailand and Malaysia, with two new species, Indopacifica pantai n. sp. and Indopacifica parva n. sp. The genus is characterized by the unique combination of following characters: lacking lamellar ridges, incomplete dorsosejugal suture, fourteen pairs of notogastral setae, and presence of epimeral foveae. A phylogenetic reconstruction based on 18S ribosomal RNA sequences clearly confirms the distinctness of the new genus Indopacifica and places it close to the genus Rhizophobates. The lack of molecular genetic data of possible relatives impedes a clear assessment, and hence, we emphasize the need for further combined approaches using morphological and molecular genetic sequence data. All species show wide distribution areas within this geographic region suggesting that these taxa are good dispersers despite their minute size and wingless body. Molecular genetic data demonstrate recent gene flow between far distant populations of I. pantai n. sp. from the coasts of Thailand and two islands of Malaysia and hence confirm this assumption. The seasonally changing surface currents within this geographic area may favor hydrochorous dispersal and hence genetic exchange. Nevertheless, morphometric data show a slight trend to morphological divergence among the studied populations, whereas this variation is suggested to be a result of genetic drift but also of habitat differences in one population of Alismobates pseudoreticulatus.     

Species delimitation and digit number in a North African skink

Delimitation of species is an important and controversial area within evolutionary biology. Many species boundaries have been defined using morphological data. New genetic approaches now offer more objective evaluation and assessment of the reliability of morphological variation as an indicator that speciation has occurred. We examined geographic variation in morphology of the continuously distributed skink Chalcides mionecton from Morocco and used Bayesian analyses of nuclear and mitochondrial DNA (mtDNA) loci to examine: (i) their concordance with morphological patterns, (ii) support for species delimitation, (iii) timing of speciation, and (iv) levels of gene flow between species. Four digit individuals were found at sites between Cap Rhir (in the south) and the northern extreme of the range, whereas five‐digit individuals were found in two disjunct areas: (i) south of Cap Rhir and (ii) the north of the range where they were often syntopic with four‐digit individuals. The pattern of variation in generalized body dimensions was largely concordant with that in digit number, suggesting two general morphotypes. Bayesian analyses of population structure showed that individuals from sites south of Cap Rhir formed one genetic cluster, but that northern four‐ and five‐digit individuals clustered together. Statistical support for delimitation of these genetic clusters into two species was provided by a recent Bayesian method. Phylogenetic–coalescent dating with external time calibrations indicates that speciation was relatively recent, with a 95% posterior interval of 0.46–2.66 mya. This postdates equivalent phylogenetic dating estimates of sequence divergence by approximately 1 Ma. Statistical analyses of a small number of independent loci provide important insights into the history of the speciation process in C. mionecton and support delimitation of populations into two species with distributions that are spatially discordant with patterns of morphological variation.     

Deep divergence among mitochondrial lineages in African jackals

Recently, molecular analyses revealed that African and Eurasian golden jackals are distinct species. This finding suggests re‐investigation of the phylogenetic relationships and taxonomy of other African members of the Canidae. Here, we provide a study on the phylogenetic relationship between populations of African jackals Lupulella mesomelas and L. adusta inferred from 962 bp of the mitochondrial cytochrome b (cytb) gene. As expected from its disjunct distribution, with one population in eastern Africa and the other one in southern Africa, we found two mitochondrial lineages within L. mesomelas, which diverged about 2.5 million years ago (Ma). In contrast, in L. adusta with its more continuous distribution in sub‐Saharan Africa, we found only a shallower genetic diversification, with the exception of the West African population, which diverged around 1.4 Ma from the Central and East African populations. Both divergence ages are older than, for example the 1.1–0.9 million years between the grey wolf Canis lupus and the African golden wolf C. lupaster. One taxonomic implication of our findings might be that the two L. mesomelas populations warrant species status. However, genome‐wide data with adequate geographical sampling are needed to substantiate our results.     

Cytonuclear discordance and the species status of Myotis myotis and Myotis blythii (Chiroptera)

We used both highly variable mitochondrial and nuclear loci to investigate the large mouse‐eared bat species complex in the Western Palaearctic to clarify their systematic position. Although mitochondrial lineages show no species segregation and some haplotypes are shared between Myotis myotis and Myotis blythii sensu lato, Bayesian clustering methods based on multilocus genotypes indicate highly concordant nuclear and morphological species assignment. These multilocus, nuclear analyses detected only a single putative F₁ hybrid in the extensive areas of sympatry sampled, thus confirming the biological species status of M. myotis and M. blythii s.l. We propose that the strong cytonuclear discordance in these species complex results from a combination of prior spatial isolation of the two species in different glacial refugia, followed by a succession of mitochondrial introgression events that occurred during the eastward and westward expansions of M. myotis and of M. blythii, respectively. The nuclear markers further indicate the presence of a notable genetic discontinuity within M. myotis that broadly separates populations into an eastern and a western component with an overlap zone in the Balkans. This eastern and western discontinuity is also apparent in the mitochondrial lineages with the D haplogroup largely confined to samples found in Thrace and Asia Minor. None of these genetic discontinuities correspond to the distribution of the two commonly recognized M. myotis subspecies (myotis and macrocephalicus). We also show that distinct morphological subspecies within M. blythii (oxygnathus, omari, risorius and lesviacus) in Europe and the near‐East do not correlate with significant evolutionarily units, whether identified by mitochondrial or nuclear data and thus only represent local morphological variants with little taxonomic relevance.     

DNA barcoding supports reclassification of Japanese Chironomus species (Diptera: Chironomidae)

Non‐biting midges (Diptera: Chironomidae) adapt to species‐specific environmental conditions and hence are promising bioindicators for aquatic and ecotoxicological monitoring. Although their utility for these purposes was historically limited by difficulties in their morphological identification, DNA barcoding offers a possible solution. Here, eight Japanese species of the genus Chironomus, which is characterized by its worldwide distribution and abundance among Chironomidae, were subjected to DNA barcoding using cytochromec oxidase subunit I (COI). To examine whether this DNA barcode is a useful indicator for Japanese species of Chironomus, we calculated genetic distances within and between the COI sequences of Chironomus species both from this study and worldwide and constructed phylogenetic trees. Based on 415 bp COI sequences and the Kimura two‐parameter model, the average genetic distances within 37 species and between 72 species were 2.6% and 17.2%, respectively. Although the ranges of genetic distances within and between species overlapped from 0.8% to 17.3%, 99.7% of average genetic distances between species were >3.0%. Some of this overlap is attributable to distances within species that were “too large” as well as those between species that were “too small”. Of eight Japanese species examined, two showed genetic distances between species that were below a 3.0% threshold, and four had distances within species that were greater than 3.0%. These results suggest a possible reclassification of these species and the need for further sampling to unveil biogeographic variations among different countries and regions.