DNA条形码技术在河北保定、廊坊地区鳞翅目昆虫上的应用及小型区域数据库的构建

【目的】为了探究DNA条形码技术和小型区域数据库在蛾类鉴定上的可行性,本研究利用条形码通用引物扩增了采自河北保定、廊坊地区10种夜蛾82个样本的线粒体细胞色素c氧化酶亚基I(Mitochondrial cytochrome c oxidase subunit I,COⅠ)基因序列。【方法】基于进化树、距离、阈值和特征的方法。【结果】虽然整体分类效果较好,但基于进化树、距离、阈值的方法都无法将二点委夜蛾Athetis lepigone进行较好的分类;样本LF110802.008总是被分入标瑙夜蛾Maliattha signifera类群,与形态学分类结果发生分歧。基于特征的方法运用核基因28S进行分析,结果与形态分类一致。同时还探讨了基于特征方法得到的诊断特征数目与样本数量之间的关系,发现两者密切相关;基于特征的方法对小样本量的鉴定也比较有效。本研究建立了小型区域的DNA条形码数据库,使物种识别具有更强的针对性,有利于提高地区性蛾类病虫害防治效果。【结论】在蛾类鉴定中,DNA条形码有很好的分类效果,小型区域数据库很有实际应用价值。     

DNA barcoding should accompany taxonomy - the case of Cerebratulus spp (Nemertea)

Many issues in DNA barcoding need to be solved before it can reach its goal to become a general database for species identification. While species delimitations are more or less well established in several taxa, there are still many groups where this is not the case. Without the proper taxonomic background/knowledge and corroboration with other kinds of data, the DNA barcoding approach may fail to identify species accurately. The classification and taxonomy of phylum Nemertea (nemerteans, ribbon worms) are traditionally based on morphology, but are not corroborated by an increasing amount of genetic data when it comes to classification either into species or into higher taxa. The taxonomy of the phylum needs to be improved before the full potential of DNA barcoding can be utilized to make sure that valid Linnean names accompany the barcode sequences. We illustrate the problematic situation in the phylum Nemertea by a case study from the genus Cerebratulus.     

Identification of larval fish in mangrove areas of Peninsular Malaysia using morphology and DNA barcoding methods

The identification of larval fish has been an important morphological issue in marine biology due to the dramatic transformations that most species undergo from early larval stages to adulthood. Insufficient morphological diagnostic characters in larval fishes made it easy to misidentify them and a difficult process to key to genus and species level. The experiment aims to find out, by applying DNA barcoding, how consistent the morphological identifications can be among larval fish. Larval fish were mainly collected using plankton nets around mangrove areas in Pendas (Johor), Setiu (Terengganu), Pekan (Pahang) and Matang (Perak) Malaysia between April 2015 and October 2015. A total of 354 samples were morphologically identified, mostly to the family level and a few to the genus level. Larval fish ranged from 1.5 mm to 31 mm of total length, with the most abundant individuals being <3 mm. Among them, a total of 177 individuals were selected for DNA barcoding analyses. Molecular works involved polymerase chain reaction (PCR) and sequencing of mitochondrial Cytochrome c Oxidase I (COI) gene fragment (655 base pairs) methods. DNA barcoding enabled all samples to be identified down to species level. The overall genetic identities ranged from 91% to 100%. Morphological identification classified the specimens into 19 families and 11 genera while DNA barcoding identified them into 19 families 33 genera and 40 species. A comparison between the two methods showed a mismatched identification of 42.6% where the accuracy percentage for morphological identification was moderate for the family level (67.8%) but was low for genus level identification (30%). The DNA barcoding method also managed to successfully identify 86.4% of the samples up to their species level where morphological method has failed to do so. The most misidentified families in the study were Blenniidae, Sparidae, Apogonidae Ambassidae and Monachantidae while almost all samples from the family Gobiidae and Engraulidae were correctly identified to family level because of their distinct morphology. In conclusion, taxonomic studies of larval fish should continue using combination of both morphology and DNA barcoding methods. Morphological identification should be more conservative i.e., when in doubt, it is better to key only to family and not to the genus and species level. DNA barcoding is a better method for deeper taxonomic levels identification with the existence of robust sequence reference libraries and should be able to validate the accuracy of traditional larval fish identification.     

DNA barcoding of Canada's skates

DNA-based identifications have been employed across broad taxonomic ranges and provide an especially useful tool in cases where external identification may be problematic. This study explored the utility of DNA barcoding in resolving skate species found in Atlantic Canadian waters. Most species were clearly resolved, expanding the utility for such identification on a taxonomically problematic group. Notably, one genus (Amblyraja) contained three of four species whose distributions do not overlap that could not be readily identified with this method. On the other hand, two common and partially sympatric species (Little and Winter skates) were readily identifiable. There were several instances of inconsistency between the voucher identification and the DNA sequence data. In some cases, these were at the intrageneric level among species acknowledged to be prone to misidentification. However, several instances of intergeneric discrepancies were also identified, suggesting either evidence of past introgressive hybridization or misidentification of vouchered specimens across broader taxonomic ranges. Such occurrences highlight the importance of retaining vouchered specimens for subsequent re-examination in the light of conflicting DNA evidence.     

The unholy trinity: taxonomy, species delimitation and DNA barcoding

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this 'DNA barcoding' initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the 'DNA barcoding' initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the 'DNA barcoding' initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings--Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of 'DNA barcoding'.     

DNA barcoding of Murinae (Rodentia: Muridae) and Arvicolinae (Rodentia: Cricetidae) distributed in China

Identification of rodents is very difficult mainly due to high similarities in morphology and controversial taxonomy. In this study, mitochondrial cytochrome oxidase subunit I (COI) was used as DNA barcode to identify the Murinae and Arvicolinae species distributed in China and to facilitate the systematics studies of Rodentia. In total, 242 sequences (31 species, 11 genera) from Murinae and 130 sequences (23 species, 6 genera) from Arvicolinae were investigated, of which 90 individuals were novel. Genetic distance, threshold method, tree‐based method, online BLAST and BLOG were employed to analyse the data sets. There was no obvious barcode gap. The average K2P distance within species and genera was 2.10% and 12.61% in Murinae, and 2.86% and 11.80% in Arvicolinae, respectively. The optimal threshold was 5.62% for Murinae and 3.34% for Arvicolinae. All phylogenetic trees exhibited similar topology and could distinguish 90.32% of surveyed species in Murinae and 82.60% in Arvicolinae with high support values. BLAST analyses yielded similar results with identification success rates of 92.15% and 93.85% for Murinae and Arvicolinae, respectively. BLOG successfully authenticated 100% of detected species except Leopoldamys edwardsi based on the latest taxonomic revision. Our results support the species status of recently recognized Micromys erythrotis, Eothenomys tarquinius and E. hintoni and confirm the important roles of comprehensive taxonomy and accurate morphological identification in DNA barcoding studies. We believe that, when proper analytic methods are applied or combined, DNA barcoding could serve as an accurate and effective species identification approach for Murinae and Arvicolinae based on a proper taxonomic framework.     

Metabarcoding技术在植物鉴定和多样性研究中的应用

目前植物学研究已进入后植物志时代,iFlora的实现需要以传统植物分类学及相关研究为基础,整合基于高通量测序的DNA条形码（DNAbareoding）技术并开发便携式快速鉴定仪器及构建信息平台。传统植物鉴定多基于形态学分类,而近年来快速发展的DNA条形码快速鉴定技术被各界分类学家认可,在植物鉴定中也被广泛应用。但DNA条形码技术仍存在一些问题亟待解决,如种的鉴定需要多个条形码的解析、Sanger测序平台无法处理混合样品。本文介绍了传统植物分类技术和DNA条形码技术在植物研究中的应用和遇到的瓶颈;并重点介绍了基于高通量测序的metabarcoding技术在植物鉴定及多样性研究中的应用及前景,及其与iFlora的关系。     

Land plants and DNA barcodes: short-term and long-term goals

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.     

DNA barcodes as a tool in biodiversity research: testing pre-existing taxonomic hypotheses in Delphic Apollo butterflies (Lepidoptera,Papilionidae)

Numerous studies have demonstrated that DNA barcoding is an effective tool for detecting DNA clusters, which can be viewed as operational taxonomic units (OTUs), useful for biodiversity research. Frequently, the OTUs in these studies remained unnamed, not connected with pre-existing taxonomic hypotheses, and thus did not really contribute to feasible estimation of species number and adjustment of species boundaries. For the majority of organisms, taxonomy is very complicated with numerous, often contradictory interpretations of the same characters, which may result in several competing checklists using different specific and subspecific names to describe the same sets of populations. The highly species-rich genus Parnassius (Lepidoptera: Papilionidae) is but one example, such as several mutually exclusive taxonomic systems have been suggested to describe the phenotypic diversity found among its populations. Here we provide an explicit flow chart describing how the DNA barcodes can be combined with the existing knowledge of morphology-based taxonomy and geography (sympatry versus allopatry) of the studied populations in order to support, reject or modify the pre-existing taxonomic hypotheses. We then apply this flow chart to reorganize the taxa within the Parnassius delphius species group, solving long-standing taxonomic problems.     

The efficacy of DNA barcoding in the classification,genetic differentiation,and biodiversity assessment of benthic macroinvertebrates

Macroinvertebrates have been recognized as key ecological indicators of aquatic environment and are the most commonly used approaches for water quality assessment. However, species identification of macroinvertebrates (especially of aquatic insects) proves to be very difficult due to the lack of taxonomic expertise in some regions and can become time‐consuming. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in seven orders (Insecta: Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, and Odonata) from four large transboundary rivers of northwest China and further explored its potential application to biodiversity assessment. A total of 1,144 COI sequences, belonging to 176 species, 112 genera, and 53 families were obtained and analyzed. The barcoding gap analysis showed that COI gene fragment yielded significant intra‐ and interspecific divergences and obvious barcoding gaps. NJ phylogenetic trees showed that all species group into monophyletic species clusters whether from the same population or not, except two species (Polypedilum. laetum and Polypedilum. bullum). The distance‐based (ABGD) and tree‐based (PTP and MPTP) methods were utilized for grouping specimens into Operational Taxonomic Units (OTUs) and delimiting species. The ABGD, PTP, and MPTP analysis were divided into 177 (p = .0599), 197, and 195 OTUs, respectively. The BIN analysis generated 186 different BINs. Overall, our study showed that DNA barcoding offers an effective framework for macroinvertebrate species identification and sheds new light on the biodiversity assessment of local macroinvertebrates. Also, the construction of DNA barcode reference library of benthic macroinvertebrates in Eurasian transboundary rivers provides a solid backup for bioassessment studies of freshwater habitats using modern high‐throughput technologies in the near future.     

Taxon-specific PCR for DNA barcoding arthropod prey in bat faeces

The application of DNA barcoding to dietary studies allows prey taxa to be identified in the absence of morphological evidence and permits a greater resolution of prey identity than is possible through direct examination of faecal material. For insectivorous bats, which typically eat a great diversity of prey and which chew and digest their prey thoroughly, DNA-based approaches to diet analysis may provide the only means of assessing the range and diversity of prey within faeces. Here, we investigated the effectiveness of DNA barcoding in determining the diets of bat species that specialize in eating different taxa of arthropod prey. We designed and tested a novel taxon-specific primer set and examined the performance of short barcode sequences in resolving prey species. We recovered prey DNA from all faecal samples and subsequent cloning and sequencing of PCR products, followed by a comparison of sequences to a reference database, provided species-level identifications for 149/207 (72%) clones. We detected a phylogenetically broad range of prey while completely avoiding detection of nontarget groups. In total, 37 unique prey taxa were identified from 15 faecal samples. A comparison of DNA data with parallel morphological analyses revealed a close correlation between the two methods. However, the sensitivity and taxonomic resolution of the DNA method were far superior. The methodology developed here provides new opportunities for the study of bat diets and will be of great benefit to the conservation of these ecologically important predators.     

Evaluating DNA barcoding criteria using African duiker antelope (Cephalophinae) as a test case

African duikers in the subfamily Cephalophinae (genera Cephalophus, Philantomba and Sylvicapra) constitute an important target for DNA barcoding efforts because of their importance to the bushmeat trade and protection under the Convention for International Trade in Endangered Species (CITES). Duikers also make a challenging test case of barcoding methods due to their recent diversification, substantial intra-specific genetic variation and high species richness. However, no study to date has evaluated how well DNA barcoding methods can be used to delineate all of the taxa within this group. To address this question, cytochrome c oxidase subunit 1 (COX1) sequences from all eighteen species within this subfamily and an outgroup taxon (genus Tragelaphus) were used to build a neighbor-joining tree, identify species-specific diagnostic synapomorphies, and determine whether species exceed a given pair-wise genetic distance threshold commonly employed in DNA barcoding studies. Tree-based analyses of the data indicate that several species within two clusters of closely related taxa consistently failed to form reciprocally monophyletic clades and similarly lack species-specific synapomorphies. Furthermore, one additional taxon failed to constitute a diagnosable clade and another occupied an unresolved position in the tree. Of the two genetic distance criteria evaluated, the 3% threshold was far more effective in delimiting species than a threshold level based on the ratio of inter- to intra-specific distances. However, neither approach could effectively delineate all sister species. While the taxonomy of this group might be open to question, the fact that barcodes consistently failed to differentiate several currently recognized sister taxa challenges the routine application of this approach in forensic studies of duiker species. Future barcoding work of this group should always include a complete taxonomic sampling and strive to include a broader geographic sampling of sequence diversity than has been carried out to date. Lastly, this work highlights the need to re-examine the taxonomy of this group, which may illuminate why some barcoding criteria fail to reliably differentiate species.     

Intragenomic variation in nuclear ribosomal markers and its implication in species delimitation,identification and barcoding in fungi

Intragenomic variation is the molecular variation within the genome among repetitive DNA. As a multigene family, nuclear ribosomal DNA (rDNA) has been widely used in fungal taxonomy for their ease in amplification and suitable variability to attain various levels of taxonomic resolution. At the intraspecific level, rDNA is believed to be under concerted evolution and the internal transcribed spacers (ITS) region is actually accepted as a universal barcoding marker for fungi. However, documentation of intragenomic variation of rDNA indicated that it can be problematic in species delimitation and identification. Fungal taxonomic studies have not generally taken into account the intragenomic variation of rDNA in a systematic manner. In this review, our objective is to address the definition, the origin and the mechanisms for maintenance of intragenomic variation, as well as its implication in the domain of fungal molecular taxonomy, particularly for species delimitation, identification and DNA barcoding. With advanced sequencing technologies (second and third generations), we also addressed how these technologies can be used to study the intragenomic variation of rDNA and also how the intragenomic variation will impact on DNA barcoding via high-throughput sequencing.     

Testing the utility of mitochondrial cytochrome oxidase subunit 1 sequences for phylogenetic estimates of relationships between crane (Grus) species

Morphology and biogeography are widely used in animal taxonomy. Recent study has suggested that a DNA-based identification system, using a 648-bp portion of the mitochondrial gene cytochrome oxidase subunit 1 (CO1), also known as the barcoding gene, can aid in the resolution of inferences concerning phylogenetic relationships and for identification of species. However, the effectiveness of DNA barcoding for identifying crane species is unknown. We amplified and sequenced 894-bp DNA fragments of CO1 from Grus japonensis (Japanese crane), G. grus (Eurasian crane), G. monacha (hooded crane), G. canadensis (sandhill crane), G. leucogeranus (Siberian crane), and Balearica pavonina (crowned crane), along with those of 15 species obtained from GenBank and DNA barcoding, to construct four algorithms using Tringa stagnatilis, Scolopax rusticola, and T. erythropus as outgroups. The four phylum profiles showed good resolution of the major taxonomic groups. We concluded that reconstruction of the molecular phylogenetic tree can be helpful for classification and that CO1 sequences are suitable for studying the molecular evolution of cranes. Although support for several deeper branches was limited, CO1 data gave remarkably good separations, especially considering that our analysis was based on just a fragment of the gene and that CO1 has generally been viewed as useful only for resolving shallow divergences.     

Alpine lichen diversity in an isolated sky island in the Colorado Plateau,USA—Insight from an integrative biodiversity inventory

Lichens are major components of high altitude/latitude ecosystems. However, accurately characterizing their biodiversity is challenging because these regions and habitats are often underexplored, there are numerous poorly known taxonomic groups, and morphological variation in extreme environments can yield conflicting interpretations. Using an iterative taxonomic approach based on over 800 specimens and incorporating both traditional morphology‐based identifications and information from the standard fungal DNA barcoding marker, we compiled a voucher‐based inventory of biodiversity of lichen‐forming fungi in a geographically limited and vulnerable alpine community in an isolated sky island in the Colorado Plateau, USA—the La Sal Mountains. We used the newly proposed Assemble Species by Automatic Partitioning (ASAP) approach to empirically delimit candidate species‐level lineages from family‐level multiple sequence alignments. Specimens comprising DNA‐based candidate species were evaluated using traditional taxonomically diagnostic phenotypic characters to identify specimens to integrative species hypotheses and link these, where possible, to currently described species. Despite the limited alpine habitat (ca. 3,250 ha), we document the most diverse alpine lichen community known to date from the southern Rocky Mountains, with up to 240 candidate species/species‐level lineages of lichen‐forming fungi. 139 species were inferred using integrative taxonomy, plus an additional 52 candidate species within 29 different putative species complexes. Over 68% of sequences could not be assigned to species‐level rank with statistical confidence, corroborating the limited utility of current sequence repositories for species‐level DNA barcoding of lichen‐forming fungi. By integrating vouchered specimens, DNA sequence data, and photographic documentation, we provide an important baseline of lichen‐forming fungal diversity for the limited alpine habitat in the Colorado Plateau. These data provide an important resource for subsequent research in the ecology and evolution of lichens alpine habitats, including DNA barcodes for most putative species/species‐level lineages occurring in the La Sal Mountains, and vouchered collections representing any potentially undescribed species that can be used for future taxonomic studies.     

DNA条形码分析方法研究进展

DNA条形码是一段短的、标准化的DNA序列,DNA条形码技术通过对DNA条形码序列分析实现物种的有效鉴定.随着生物DNA条形码序列的大量测定,DNA条形码分析方法得到迅速发展,推动了其在生物分子鉴定中的应用.2003年以来,DNA条形码技术已广泛应用于动物、植物和真菌等物种的鉴定,并有力地推动了生物分类学、生物多样性和生态学等学科的发展.本文在综述DNA条形码技术的基础上,总结了5类主要的DNA条形码分析方法,即基于遗传距离的分析、基于遗传相似度的分析、基于系统发育树的分析、基于序列特征的分析和基于统计分类法的分析,并进一步展望了DNA条形码技术的发展与应用.     

Identifying species of moths (Lepidoptera) from Baihua Mountain,Beijing, China,using DNA barcodes

DNA barcoding has become a promising means for the identification of organisms of all life‐history stages. Currently, distance‐based and tree‐based methods are most widely used to define species boundaries and uncover cryptic species. However, there is no universal threshold of genetic distance values that can be used to distinguish taxonomic groups. Alternatively, DNA barcoding can deploy a “character‐based” method, whereby species are identified through the discrete nucleotide substitutions. Our research focuses on the delimitation of moth species using DNA‐barcoding methods. We analyzed 393 Lepidopteran specimens belonging to 80 morphologically recognized species with a standard cytochrome c oxidase subunit I (COI) sequencing approach, and deployed tree‐based, distance‐based, and diagnostic character‐based methods to identify the taxa. The tree‐based method divided the 393 specimens into 79 taxa (species), and the distance‐based method divided them into 84 taxa (species). Although the diagnostic character‐based method found only 39 so‐identifiable species in the 80 species, with a reduction in sample size the accuracy rate substantially improved. For example, in the Arctiidae subset, all 12 species had diagnostics characteristics. Compared with traditional morphological method, molecular taxonomy performed well. All three methods enable the rapid delimitation of species, although they have different characteristics and different strengths. The tree‐based and distance‐based methods can be used for accurate species identification and biodiversity studies in large data sets, while the character‐based method performs well in small data sets and can also be used as the foundation of species‐specific biochips.     

Cryptic diversity,high host specificity and reproductive synchronization in army ant‐associated Vatesus beetles

Army ants and their arthropod symbionts represent one of the most species‐rich animal associations on Earth, and constitute a fascinating example of diverse host–symbiont interaction networks. However, despite decades of research, our knowledge of army ant symbionts remains fragmentary due to taxonomic ambiguity and the inability to study army ants in the laboratory. Here, we present an integrative approach that allows us to reliably determine species boundaries, assess biodiversity, match different developmental stages and sexes, and to study the life cycles of army ant symbionts. This approach is based on a combination of community sampling, DNA barcoding, morphology and physiology. As a test case, we applied this approach to the staphylinid beetle genus Vatesus and its different Eciton army ant host species at La Selva Biological Station, Costa Rica. DNA barcoding led to the discovery of cryptic biodiversity and, in combination with extensive community sampling, revealed strict host partitioning with no overlap in host range. Using DNA barcoding, we were also able to match the larval stages of all focal Vatesus species. In combination with studies of female reproductive physiology, this allowed us to reconstruct almost the complete life cycles of the different beetle species. We show that Vatesus beetles are highly adapted to the symbiosis with army ants, in that their reproduction and larval development are synchronized with the stereotypical reproductive and behavioural cycles of their host colonies. Our approach can now be used to study army ant‐symbiont communities more broadly, and to obtain novel insights into co‐evolutionary and ecological dynamics in species‐rich host–symbiont systems.     

DNA barcoding of stylommatophoran land snails: a test of existing sequences

DNA barcoding has attracted attention because it is a potentially simple and universal method for taxonomic assignment. One anticipated problem in applying the method to stylommatophoran land snails is that they frequently exhibit extreme divergence of mitochondrial DNA sequences, sometimes reaching 30% within species. We therefore trialled the utility of barcodes in identifying land snails, by analysing the stylommatophoran cytochrome oxidase subunit I sequences from GenBank. Two alignments of 381 and 228 base pairs were used to determine potential error rates among a test data set of 97 or 127 species, respectively. Identification success rates using neighbour‐joining phylogenies were 92% for the longer sequence and 82% for the shorter sequence, indicating that a high degree of mitochondrial variation may actually be an advantage when using phylogeny‐based methods for barcoding. There was, however, a large overlap between intra‐ and interspecific variation, with assignment failure (per cent of samples not placed with correct species) particularly associated with a low degree of mitochondrial variation (Kimura 2‐parameter distance < 0.05) and a small GenBank sample size (< 25 per species). Thus, while the optimum intra/interspecific threshold value was 4%, this was associated with an overall error of 32% for the longer sequences and 44% for the shorter sequences. The high error rate necessitates that barcoding of land snails is a potentially useful method to discriminate species of land snail, but only when a baseline has first been established using conventional taxonomy and sample DNA sequences. There is no evidence for a barcoding gap, ruling out species discovery based on a threshold value alone.