Cryptic diversity in Indo-Pacific coral-reef fishes revealed by DNA-barcoding provides new support to the centre-of-overlap hypothesis

Diversity in coral reef fishes is not evenly distributed and tends to accumulate in the Indo-Malay-Philippines Archipelago (IMPA). The comprehension of the mechanisms that initiated this pattern is in its infancy despite its importance for the conservation of coral reefs. Considering the IMPA either as an area of overlap or a cradle of marine biodiversity, the hypotheses proposed to account for this pattern rely on extant knowledge about taxonomy and species range distribution. The recent large-scale use of standard molecular data (DNA barcoding), however, has revealed the importance of taking into account cryptic diversity when assessing tropical biodiversity. We DNA barcoded 2276 specimens belonging to 668 coral reef fish species through a collaborative effort conducted concomitantly in both Indian and Pacific oceans to appraise the importance of cryptic diversity in species with an Indo-Pacific distribution range. Of the 141 species sampled on each side of the IMPA, 62 presented no spatial structure whereas 67 exhibited divergent lineages on each side of the IMPA with K2P distances ranging between 1% and 12%, and 12 presented several lineages with K2P distances ranging between 3% and 22%. Thus, from this initial pool of 141 nominal species with Indo-Pacific distribution, 79 dissolved into 165 biological units among which 162 were found in a single ocean. This result is consistent with the view that the IMPA accumulates diversity as a consequence of its geological history, its location on the junction between the two main tropical oceans and the presence of a land bridge during glacial times in the IMPA that fostered allopatric divergence and secondary contacts between the Indian and Pacific oceans.     

Pay Attention to the Overlooked Cryptic Diversity in Existing Barcoding Data: the Case of Mollusca with Character-Based DNA Barcoding

With the global biodiversity crisis, DNA barcoding aims for fast species identification and cryptic species diversity revelation. For more than 10 years, large amounts of DNA barcode data have been accumulating in publicly available databases, most of which were conducted by distance or tree-building methods that have often been argued, especially for cryptic species revelation. In this context, overlooked cryptic diversity may exist in the available barcoding data. The character-based DNA barcoding, however, has a good chance for detecting the overlooked cryptic diversity. In this study, marine mollusk was as the ideal case for detecting the overlooked potential cryptic species from existing cytochrome c oxidase I (COI) sequences with character-based DNA barcode. A total of 1081 COI sequences of mollusks, belonging to 176 species of 25 families of Gastropoda, Cephalopoda, and Lamellibranchia, were conducted by character analysis. As a whole, the character-based barcoding results were consistent with previous distance and tree-building analysis for species discrimination. More importantly, quite a number of species analyzed were divided into distinct clades with unique diagnostical characters. Based on the concept of cryptic species revelation of character-based barcoding, these species divided into separate taxonomic groups might be potential cryptic species. The detection of the overlooked potential cryptic diversity proves that the character-based barcoding mode possesses more advantages of revealing cryptic biodiversity. With the development of DNA barcoding, making the best use of barcoding data is worthy of our attention for species conservation.     

Genetic analysis of an endemic archipelagic lizard reveals sympatric cryptic lineages and taxonomic discordance

The importance of genetic data in biodiversity conservation is well established, and knowledge of standing genetic variation within and between populations is important for designing conservation strategies. We investigated partitioning of genetic diversity in an endemic lizard (Leiocephalus psammodromus) distributed in the Turks and Caicos archipelago using mtDNA and AFLP data from 259 individuals sampled across 13 islands. Current taxonomy identifies six or more subspecies of L. psammodromus within the archipelago, several of which have undergone recent drastic reductions in range due to extirpation. However, our results indicate the presence of two independent lineages, one on each of the Turks and Caicos banks, and a third sympatric cryptic lineage on both banks. These lineages do not correspond to current taxonomy and alter our understanding of diversity and conservation of this species. Gross morphological data (mass and snout-vent length) indicate some variation in female size among lineages, indicating the possibility of cryptic morphological variation. Instead of initiating separate conservation measures for nominate subspecies, we recommend a more thorough investigation of the morphology and genetics of this group and a more inclusive conservation program. Our surprising results indicate that other endemic squamates in the Bahamas Archipelago might also exhibit sympatric cryptic diversity that does not correspond to current taxonomic understanding and could have significant impacts on our approach to conservation in this region.     

Mating trials validate the use of DNA barcoding to reveal cryptic speciation of a marine bryozoan taxon

Despite increasing threats to the marine environment, only a fraction of the biodiversity of the oceans has been described, owing in part to the widespread occurrence of cryptic species. DNA-based barcoding through screening of an orthologous reference gene has been proposed as a powerful tool to uncover biological diversity in the face of dwindling taxonomic expertise and the limitations of traditional species identification. Although DNA barcoding should be particularly useful in the sea, given the prevalence of marine cryptic species, the link between taxa identified through DNA barcodes and reproductively isolated taxa (biological species) has rarely been explicitly tested. Here, we use an integrated framework comparing breeding compatibility, morphology and mitochondrial (cytochrome c oxidase 1) and nuclear (elongation factor-1-alpha) DNA sequence variation among globally distributed samples of the cosmopolitan marine bryozoan Celleporella hyalina (L.). Our results reveal that C. hyalina comprises numerous deep, mostly allopatric, genetic lineages that are reproductively isolated, yet share very similar morphology, indicating rampant cryptic speciation. The close correspondence between genetic lineages and reproductively isolated taxa in the context of minimal morphological change suggests that DNA barcoding will play a leading role in uncovering the hidden biodiversity of the oceans and that the sole use of morphologically based taxonomy would grossly underestimate the number of marine species.     

DNA barcoding reveals extraordinary cryptic diversity in an amphipod genus: implications for desert spring conservation

DNA barcoding has revealed unrecognized species in several animal groups. In this study we have employed DNA barcoding to examine Hyalella, a taxonomically difficult genus of amphipod crustaceans, from sites in the southern Great Basin of California and Nevada, USA. We assessed the extent of species diversity using a species screening threshold (SST) set at 10 times the average intrapopulation cytochrome c oxidase subunit I (COI) haplotype divergence. Despite the fact that this threshold approach is more conservative in delineating provisional species than the phylogenetic species concept, our analyses revealed extraordinary levels of cryptic diversity and endemism. The SST discriminated two provisional species within Hyalella sandra, and 33 provisional species within Hyalella azteca. COI nucleotide divergences among these provisional species ranged from 4.4% to 29.9%. These results have important implications for the conservation of life in desert springs - habitats that are threatened as a result of groundwater over-exploitation.     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

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.     

Discovering Hidden Diversity of Characins (Teleostei: Characiformes) in Ecuador’s Yasuní National Park

Background

Management and conservation of biodiversity requires adequate species inventories. The Yasuní National Park is one of the most diverse regions on Earth and recent studies of terrestrial vertebrates, based on genetic evidence, have shown high levels of cryptic and undescribed diversity. Few genetic studies have been carried out in freshwater fishes from western Amazonia. Thus, in contrast with terrestrial vertebrates, their content of cryptic diversity remains unknown. In this study, we carried out genetic and morphological analyses on characin fishes at Yasuní National Park, in eastern Ecuador. Our goal was to identify cryptic diversity among one of the most speciose fish families in the Amazon region. This is the first time that genetic evidence has been used to assess the species content of the Napo Basin, one of the richest regions in vertebrate diversity.

Results

Phylogenetic analyses of partial mitochondrial 16S ribosomal RNA gene (∼600 pb) DNA sequences from 232 specimens of the family Characidae and its closest groups revealed eight candidate new species among 33 species sampled, representing a 24% increase in species number. Analyses of external morphology allowed us to confirm the species status of six of the candidate species.

Conclusions

Our results show high levels of cryptic diversity in Amazonian characins. If this group is representative of other Amazonian fish, our results would imply that the species richness of the Amazonian ichthyofauna is highly underestimated. Molecular methods are a necessary tool to obtain more realistic inventories of Neotropical freshwater fishes.     

DNA Barcoding of Neotropical Sand Flies (Diptera,Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.     

Neotropical bats: estimating species diversity with DNA barcodes

DNA barcoding using the cytochrome c oxidase subunit 1 gene (COI) is frequently employed as an efficient method of species identification in animal life and may also be used to estimate species richness, particularly in understudied faunas. Despite numerous past demonstrations of the efficiency of this technique, few studies have attempted to employ DNA barcoding methodologies on a large geographic scale, particularly within tropical regions. In this study we survey current and potential species diversity using DNA barcodes with a collection of more than 9000 individuals from 163 species of Neotropical bats (order Chiroptera). This represents one of the largest surveys to employ this strategy on any animal group and is certainly the largest to date for land vertebrates. Our analysis documents the utility of this tool over great geographic distances and across extraordinarily diverse habitats. Among the 163 included species 98.8% possessed distinct sets of COI haplotypes making them easily recognizable at this locus. We detected only a single case of shared haplotypes. Intraspecific diversity in the region was high among currently recognized species (mean of 1.38%, range 0-11.79%) with respect to birds, though comparable to other bat assemblages. In 44 of 163 cases, well-supported, distinct intraspecific lineages were identified which may suggest the presence of cryptic species though mean and maximum intraspecific divergence were not good predictors of their presence. In all cases, intraspecific lineages require additional investigation using complementary molecular techniques and additional characters such as morphology and acoustic data. Our analysis provides strong support for the continued assembly of DNA barcoding libraries and ongoing taxonomic investigation of bats.     

Molecular systematics of threadfin breams and relatives (Teleostei,Nemipteridae)

Threadfin breams and relatives of the family Nemipteridae comprise 69 currently recognized species in five genera. They are found in the tropical and subtropical Indo‐West Pacific and most are commercially important. Using recently developed molecule‐based approaches exploiting DNA sequence variation among species/specimens, this study reconstructed a comprehensive phylogeny of the Nemipteridae, examined the validity of species and explored the cryptic diversity of the family, and tested previous phylogenetic hypotheses. A combined data set (105 taxa from 41 morphospecies) with newly determined sequences from two nuclear genes (RAG1 and RH) and one mitochondrial gene (COI), and a data set with only COI gene sequences (329 newly obtained plus 328 from public databases from a total of 53 morphospecies) were used in the phylogenetic analysis. The latter was further used for species delimitation analyses with two different tools to explore species diversity. Our phylogenetic results showed that all the currently recognized genera were monophyletic. The monotypic genus Scaevius is the sister group of Pentapodus and they together are sister to Nemipterus. These three genera combined to form the sister group of the clade comprising Parascolopsis and Scolopsis. The validity of most of the examined species was confirmed except in some cases. The combined evidence from the results of different analyses revealed a gap in our existing knowledge of species diversity in the Nemipteridae. We found several currently recognized species contain multiple separately evolving metapopulation lineages within species; some lineages should be considered as new species for further assignment. Finally, some problematic sequences deposited in public databases (probably due to misidentification) were also revised in this study to improve the accuracy for prospective DNA barcoding work on nemipterid fishes.     

Conservation Genetics of the Philippine Tarsier: Cryptic Genetic Variation Restructures Conservation Priorities for an Island Archipelago Primate

Establishment of conservation priorities for primates is a particular concern in the island archipelagos of Southeast Asia, where rates of habitat destruction are among the highest in the world. Conservation programs require knowledge of taxonomic diversity to ensure success. The Philippine tarsier is a flagship species that promotes environmental awareness and a thriving ecotourism economy in the Philippines. However, assessment of its conservation status has been impeded by taxonomic uncertainty, a paucity of field studies, and a lack of vouchered specimens and genetic samples available for study in biodiversity repositories. Consequently, conservation priorities are unclear. In this study we use mitochondrial and nuclear DNA to empirically infer geographic partitioning of genetic variation and to identify evolutionarily distinct lineages for conservation action. The distribution of Philippine tarsier genetic diversity is neither congruent with expectations based on biogeographical patterns documented in other Philippine vertebrates, nor does it agree with the most recent Philippine tarsier taxonomic arrangement. We identify three principal evolutionary lineages that do not correspond to the currently recognized subspecies, highlight the discovery of a novel cryptic and range-restricted subcenter of genetic variation in an unanticipated part of the archipelago, and identify additional geographically structured genetic variation that should be the focus of future studies and conservation action. Conservation of this flagship species necessitates establishment of protected areas and targeted conservation programs within the range of each genetically distinct variant of the Philippine tarsier.     

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections

BackgroundHigh rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage.Conclusions/SignificanceThis study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale.     

Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo‐West Pacific uncovered by DNA barcoding

Identification of taxonomical units underpins most biological endeavours ranging from accurate biodiversity estimates to the effective management of sustainably harvested, protected or endangered species. Successful species identification is now frequently based on a combination of approaches including morphometrics and DNA markers. Sequencing of the mitochondrial COI gene is an established methodology with an international campaign directed at barcoding all fishes. We employed COI sequencing alongside traditional taxonomic identification methods and uncovered instances of deep intraspecific genetic divergences among flathead species. Sixty‐five operational taxonomic units (OTUs) were observed across the Indo‐West Pacific from just 48 currently recognized species. The most comprehensively sampled taxon, Platycephalus indicus, exhibited the highest levels of genetic diversity with eight lineages separated by up to 16.37% genetic distance. Our results clearly indicate a thorough reappraisal of the current taxonomy of P. indicus (and its three junior synonyms) is warranted in conjunction with detailed taxonomic work on the other additional Platycephalidae OTUs detected by DNA barcoding.     

Unexpectedly High Levels of Cryptic Diversity Uncovered by a Complete DNA Barcoding of Reptiles of the Socotra Archipelago

Few DNA barcoding studies of squamate reptiles have been conducted. Due to the significance of the Socotra Archipelago (a UNESCO Natural World Heritage site and a biodiversity hotspot) and the conservation interest of its reptile fauna (94% endemics), we performed the most comprehensive DNA barcoding study on an island group to date to test its applicability to specimen identification and species discovery. Reptiles constitute Socotra’s most important vertebrate fauna, yet their taxonomy remains under-studied. We successfully DNA-barcoded 380 individuals of all 31 presently recognized species. The specimen identification success rate is moderate to high, and almost all species presented local barcoding gaps. The unexpected high levels of intra-specific variability found within some species suggest cryptic diversity. Species richness may be under-estimated by 13.8–54.4%. This has implications in the species’ ranges and conservation status that should be considered for conservation planning. Other phylogenetic studies using mitochondrial and nuclear markers are congruent with our results. We conclude that, despite its reduced length (663 base pairs), cytochrome c oxidase 1, COI, is very useful for specimen identification and for detecting intra-specific diversity, and has a good phylogenetic signal. We recommend DNA barcoding to be applied to other biodiversity hotspots for quickly and cost-efficiently flagging species discovery, preferentially incorporated into an integrative taxonomic framework.     

Molecular phylogeny and DNA barcoding confirm cryptic species in the African freshwater oyster Etheria elliptica Lamarck, 1807 (Bivalvia: Etheriidae)

Recent molecular approaches to taxonomy have led to a steady increase in the identification of cryptic species. Within the Etheriidae, the species Etheria elliptica (freshwater oyster) is widespread and common and exists in most of the major African drainages. Within the African freshwater ecosystems, there are major threats to biodiversity and cryptic species complicate conservation strategies; unknown species exist and no conservation status has been assigned. Our objective here was to determine if E. elliptica from several locations in the Congo drainage are correctly classified as representing a single species. We analysed the genetic diversity at two mitochondrial loci (COI and 16S) and two nuclear loci (H3 and 28S), and estimated evolutionary relationships using phylogenetic and DNA barcoding techniques. Bayesian inference yielded three cryptic species of Etheria, and mismatch analysis revealed discrete differences between the cryptic species. We identified three cryptic species within these collections, and evidence indicates that the third species may resolve further with more sampling. In conclusion, the taxonomic history of E. elliptica makes finding cryptic species unsurprising. However, molecular studies such as this may finally help to resolve the number of species within this genus.     

DNA barcoding analysis of Coleoidea (Mollusca: Cephalopoda) from Chinese waters

Coleoids are part of the Cephalopoda class, which occupy an important position in most oceans both at an ecological level and at a commercial level. Nevertheless, some coleoid species are difficult to distinguish with traditional morphological identification in cases when specimens are heavily damaged during collection or when closely related taxa are existent. As a useful tool for rapid species assignment, DNA barcoding may offer significant potential for coleoid identification. Here, we used two mitochondrial fragments, cytochrome c oxidase I and the large ribosomal subunit (16S rRNA), to assess whether 34 coleoids accounting for about one-third of the Chinese coleoid fauna could be identified by DNA barcoding technique. The pairwise intra- and interspecific distances were assessed, and relationships among species were estimated by NJ and bayesian analyses. High levels of genetic differentiation within Loliolus beka led to an overlap between intra- and interspecific distances. All remaining species forming well-differentiated clades in the NJ and bayesian trees were identical for both fragments. Loliolus beka possessed two mitochondrial lineages with high levels of intraspecific distances, suggesting the occurrence of cryptic species. This study confirms the efficacy of DNA barcoding for identifying species as well as discovering cryptic diversity of Chinese coleoids. It also lays a foundation for other ecological and biological studies of Coleoidea.     

Allopatry as a Gordian Knot for Taxonomists: Patterns of DNA Barcode Divergence in Arctic-Alpine Lepidoptera

Many cold adapted species occur in both montane settings and in the subarctic. Their disjunct distributions create taxonomic complexity because there is no standardized method to establish whether their allopatric populations represent single or different species. This study employs DNA barcoding to gain new perspectives on the levels and patterns of sequence divergence among populations of 122 arctic-alpine species of Lepidoptera from the Alps, Fennoscandia and North America. It reveals intraspecific variability in the barcode region ranging from 0.00–10.08%. Eleven supposedly different species pairs or groups show close genetic similarity, suggesting possible synonymy in many cases. However, a total of 33 species show evidence of cryptic diversity as evidenced by the presence of lineages with over 2% maximum barcode divergence in Europe, in North America or between the two continents. Our study also reveals cases where taxonomic names have been used inconsistently between regions and exposes misidentifications. Overall, DNA barcodes have great potential to both increase taxonomic resolution and to make decisions concerning the taxonomic status of allopatric populations more objective.     

DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese‐breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour‐joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan‐specific cryptic species from trans‐Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans‐Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.     

Some ‘ant’swers: Application of a layered barcode approach to problems in ant taxonomy

DNA barcoding has emerged as a routine tool in modern taxonomy. Although straightforward, this approach faces new challenges, when applied to difficult situation such as defining cryptic biodiversity. Ants are prime examples for high degrees of cryptic biodiversity due to complex population differentiation, hybridization and speciation processes. Here, we test the DNA barcoding region, cytochrome c oxidase 1 and two supplementary markers, 28S ribosomal DNA and long‐wavelength rhodopsin, commonly used in ant taxonomy, for their potential in a layered, character‐based barcoding approach across different taxonomic levels. Furthermore, we assess performance of the character‐based barcoding approach to determine cryptic species diversity in ants. We found (i) that the barcode potential of a specific genetic marker varied widely among taxonomic levels in ants; (ii) that application of a layered, character‐based barcode for identification of specimens can be a solution to taxonomical challenging groups; (iii) that the character‐based barcoding approach allows us to differentiate specimens even within locations based on pure characters. In summary, (layered) character‐based barcoding offers a reliable alternative for problematic species identification in ants and can be used as a fast and cost‐efficient approach to estimate presence, absence or frequency of cryptic species.