DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species

There is currently international interest in the application of DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of five candidate plant DNA barcoding regions [rbcL, matK, trnH-psbA, trnL-F and internal transcribed spacer (ITS)] in Eurasian yews. This group of species is taxonomically difficult because of a lack of clear-cut morphologically differences between species and hence represents a good test case for DNA barcoding. Forty-seven accessions were analysed, representing all taxa treated in current floristic works and covering most of the distribution range of Taxus in Eurasia. As single loci, trnL-F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (= barcode taxa). Species discrimination using matK, trnH-psbA and rbcL individually was lower, with matK resolving 8 of 10, trnH-psbA 7 of 11 and rbcL 5 of 11 successfully sequenced lineages. The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Combining loci generally increased the robustness (measured by clade support) of the barcoding discrimination. Based on overall performance, trnL-F and ITS, separately or combined, are proposed as barcode for Eurasian Taxus. DNA barcoding discriminated recognized taxa of Eurasian Taxus, namely T. baccata, T. cuspidata, T. fuana and T. sumatrana, and identified seven lineages among the T. wallichiana group, some with distinct geographical distributions and morphologies, and potentially representing new species. Using the proposed DNA barcode, a technical system can be established to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade.     

DNA barcoding of Chinese snakes reveals hidden diversity and conservation needs

DNA barcoding has greatly facilitated studies of taxonomy, biodiversity, biological conservation, and ecology. Here, we establish a reliable DNA barcoding library for Chinese snakes, unveiling hidden diversity with implications for taxonomy, and provide a standardized tool for conservation management. Our comprehensive study includes 1638 cytochrome c oxidase subunit I (COI) sequences from Chinese snakes that correspond to 17 families, 65 genera, 228 named species (80.6% of named species) and 36 candidate species. A barcode gap analysis reveals gaps, where all nearest neighbour distances exceed maximum intraspecific distances, in 217 named species and all candidate species. Three species-delimitation methods (ABGD, sGMYC, and sPTP) recover 320 operational taxonomic units (OTUs), of which 192 OTUs correspond to named and candidate species. Twenty-eight other named species share OTUs, such as Azemiops feae and A. kharini, Gloydius halys, G. shedaoensis, and G. intermedius, and Bungarus multicinctus and B. candidus, representing inconsistencies most probably caused by imperfect taxonomy, recent and rapid speciation, weak taxonomic signal, introgressive hybridization, and/or inadequate phylogenetic signal. In contrast, 43 species and candidate species assign to two or more OTUs due to having large intraspecific distances. If most OTUs detected in this study reflect valid species, including the 36 candidate species, then 30% more species would exist than are currently recognized. Several OTU divergences associate with known biogeographic barriers, such as the Taiwan Strait. In addition to facilitating future studies, this reliable and relatively comprehensive reference database will play an important role in the future monitoring, conservation, and management of Chinese snakes.     

DNA barcoding of stygofauna uncovers cryptic amphipod diversity in a calcrete aquifer in Western Australia's arid zone

The arid Yilgarn region of Western Australia contains numerous subterranean calcrete aquifers with unique assemblages of obligate groundwater invertebrates (stygofauna). We aimed to establish a DNA barcoding framework for the macro-invertebrates present in a single calcrete, as a basis for future assessment of biodiversity of the Yilgarn calcretes and for investigating food webs. Intense sampling of a bore field grid in the Sturt Meadows calcrete was undertaken to obtain representatives of the entire macro-invertebrate ecosystem. A 623-bp fragment of the mitochondrial cytochrome c oxidase 1 (COI) gene was used to provide DNA barcodes for stygobiont macro-invertebrates plus terrestrial organisms that are found in the calcrete. Phylogenetic analyses revealed the existence of 12 divergent monophyletic groups of haplotypes. Subterranean amphipods (Chiltoniidae) showed three groups of COI haplotypes with sequence divergences between them of >11%. Allozyme analyses found a large number of fixed allelic differences between these three amphipod groups, indicating that there are three morphologically cryptic species within the Sturt Meadows calcrete. Unlike the sister triplet of dytiscid beetles present, the amphipods are not sister clades and are more closely related to other Yilgarn and non-Yilgarn amphipods than to each other. Our results show that the aquifer contains at least 12 macro-invertebrate species and DNA barcoding provides a useful means for discriminating species in this system.     

甘肃省鱼类资源现状及DNA条形码在鱼类物种鉴定中的应用

为了摸清甘肃省土著鱼类资源与分布现状, 探索DNA条形码在鱼类辅助物种鉴定中的适用性, 2012年6-9月对甘肃境内黄河水系、嘉陵江水系和河西内陆河水系进行了较全面的鱼类调查。共采集鱼类标本3,087尾, 隶属于5目10科38属64种, 以鲤科种类最多, 为30种, 占总种数的46.88%。物种多样性分析表明, 在黄河水系的夏河和庄浪河多样性指数是所有调查点中最低的, 分别为1.38和1.09。嘉陵江水系各河段的多样性指数较高(H = 2.15-3.27), 其次为河西内陆河水系(H = 2.01-2.83)。在河西内陆河水系中, 疏勒河的均匀度指数最高, 为1.10, 黑河最低(0.68)。庄浪河的优势度指数最高, 为0.34, 而嘉陵江干流两当段的优势度指数在所有调查点中最低, 为0.04。利用DNA条形码分析了49种662尾标本的COI基因部分序列, 大部分种类在neighbor-joining系统树中形成各自的单系, 种内平均遗传距离0.88%, 种间平均遗传距离为9.99%, 在种内和种间COI序列遗传距离之间形成明显的条形码间隙, 斯氏高原鳅(Triplophysa stoliczkae)与达里湖高原鳅(T. dalaica), 甘肃高原鳅(T. robusta)与似鲇高原鳅(T. siluroides), 嘉陵裸裂尻鱼(Schizopygopsis kialingensis)与黄河裸裂尻鱼(S. pylzovi)之间的遗传距离低于2%, 甘肃高原鳅与似鲇高原鳅不能通过COI基因片段区分开, 其他两对物种可以采用核苷酸诊断法来进一步区分。斯氏高原鳅和拉氏鱼岁(Phoxinus lagowskii)种内遗传分歧较大, 揭示种内可能存在隐存种。结果表明, 对某些近缘种和不同地理种群差异较大的物种, 要将分子、形态和地理分布特点结合起来才能准确鉴定。     

Mitochondrial DNA barcoding detects some species that are real, and some that are not

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.     

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae)

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four-marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH-psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology-based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

Nuclear DNA recapitulates the cryptic mitochondrial lineages of Lumbricus rubellus and suggests the existence of cryptic species in an ecotoxological soil sentinel

Mitochondrial DNA analysis has revealed two distinct phylogenetic lineages within the ecotoxological sentinel earthworm model Lumbricus rubellus Hoffmeister, 1843. The existence of these lineages could complicate ecotoxicological studies that use the species as a sentinel for soil contamination testing, as they may respond differently to contamination; however, as mitochondrial haplotypes are not always expected to segregate in the same way as chromosomal DNA in natural populations, we further investigated this issue by using nuclear DNA markers (microsatellites) to measure genetic diversity, differentiation, and gene flow in sympatric populations of the two L. rubellus lineages at two sites in South Wales. Our results show that sympatric populations of the two lineages are more genetically differentiated than geographically distant populations of the same lineage, and Bayesian clustering analysis revealed no evidence of gene flow between the lineages at either site. Additionally, DNA sequencing of these microsatellite loci uncovered substantial differentiation between lineages at homologous flanking regions. Overall our findings indicate a high degree of nuclear genetic differentiation between the two lineages of L. rubellus, implying reproductive isolation at the two study sites and therefore the potential existence of cryptic species. The existence of two cryptic taxa has major implications for the application of L. rubellus as an ecotoxicological sentinel. It may therefore be necessary to consider the lineages as separate taxa during future ecotoxicological studies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 780–795.     

DNA barcoding reveals cryptic diversity in the peanut worm Sipunculus nudus

Peanut worm (Sipunculus nudus) is a cosmopolitan species mainly distributed in tropical and subtropical coastal waters. Analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences among S. nudus from GenBank revealed high genetic variation (p‐distance, 0.115–0.235; k2p, 0.128–0.297) and paraphyletic relationships. These indicated misidentification and/or cryptic diversity may be present in the genus Sipunculus. To understand the genetic diversity and to manage the recourse of S. nudus, we collected specimens from coastal waters of southern China and Taiwan. In the phylogenetic topology, specimens can be separated into four distinct clades; three of these clades (clade A, B and C) were only represented from this region (southern China and Taiwan), but the clade D grouped with individuals from Central America (Atlantic coast). Furthermore, individuals of clades A and D were collected at the same location, which does not support the hypothesis that this genetic break reflects contemporary geographical isolation. The four distinct clades observed among coastal waters of southern China and Taiwan indicated underestimated diversity. It is noteworthy that the cryptic diversity is vulnerable under high pressure of human activity.     

The genus Apsiphortica from China with DNA barcoding information (Diptera: Drosophilidae)

Two new species of the genus Apsiphortica are described from China: A. orthophallos n. sp. and A. sinuatipenis n. sp. Species delimitations are improved by integrating morphological and DNA barcoding information. The intra- and interspecific pairwise p-distances (proportional distance) are summarized for five Apsiphortica species from China. Furthermore, nucleotide sites with fixed status in the alignment of the COI sequences (639 nucleotide sites in length) are used as “pure” molecular diagnostic characters to delineate the five species. A key to all the Chinese species of the genus Apsiphortica is provided.     

DNA barcode discovers two cryptic species and two geographical radiations in the invasive drosophilid Zaprionus indianus

Comparing introduced to ancestral populations within a phylogeographical context is crucial in any study aiming to understand the ecological genetics of an invasive species. Zaprionus indianus is a cosmopolitan drosophilid that has recently succeeded to expand its geographical range upon three continents (Africa, Asia and the Americas). We studied the distribution of mitochondrial DNA (mtDNA) haplotypes for two genes (CO‐I and CO‐II) among 23 geographical populations. mtDNA revealed the presence of two well‐supported phylogenetic lineages (phylads), with bootstrap value of 100%. Phylad I included three African populations, reinforcing the African‐origin hypothesis of the species. Within phylad II, a distinct phylogeographical pattern was discovered: Atlantic populations (from the Americas and Madeira) were closer to the ancestral African populations than to Eastern ones (from Madagascar, Middle East and India). This means that during its passage from endemism to cosmopolitanism, Z. indianus exhibited two independent radiations, the older (the Eastern) to the East, and the younger (the Atlantic) to the West. Discriminant function analysis using 13 morphometrical characters was also able to discriminate between the two molecular phylads (93.34 ± 1.67%), although detailed morphological analysis of male genitalia using scanning electron microscopy showed no significant differences. Finally, crossing experiments revealed the presence of reproductive barrier between populations from the two phylads, and further between populations within phylad I. Hence, a bona species status was assigned to two new, cryptic species: Zaprionus africanus and Zaprionus gabonicus, and both were encompassed along with Z. indianus and Zaprionus megalorchis into the indianus complex. The ecology of these two species reveals that they are forest dwellers, which explains their restricted endemic distribution, in contrast to their relative cosmopolitan Z. indianus, known to be a human‐commensal. Our results reconfirm the great utility of mtDNA at both inter‐ and intraspecific analyses within the frame of an integrated taxonomical project.     

Utility of DNA barcoding in native Oreochromis species

The importance of Oreochromis in worldwide aquaculture and regional fisheries motivates the study of their genetic diversity in their native range. In this article, all mitochondrial cytochrome c oxidase subunit I gene (COI) sequences of Oreochromis species are retrieved from Barcode of Life Data system to quantify the available DNA barcoding information from wild individuals collected within the native ranges of the respective species. It is found that 70% of the known species in the genus still lack a COI barcode, and only 15% of the available sequences are from within the respective native ranges. Many of the available sequences have been produced from specimens acquired from aquaculture and introduced, naturalized populations, making the assessment of variation within the original native range challenging. Analyses of the wild-collected fraction of available sequences indicated the presence of cryptic lineages within Nile tilapia Oreochromis niloticus and O. schwebischi, the occurrence of potential introgressive hybridization between O. niloticus and blue tilapia O. aureus, and potential ancestral polymorphism between Karonga tilapia O. karongae and black tilapia O. placidus. This article also reports a case of misidentification of O. mweruensis as longfin tilapia O. macrochir. These results stress the importance of improving the knowledge of genetic variation within the native ranges of Oreochromis species for better-informed conservation of these natural resources.     

植物DNA条形码技术的发展及应用

在对DNA条形码技术的发展过程进行归纳分析的基础上,对植物DNA条形码技术的研究进展、工作流程及分析方法、影响其鉴定准确性的因素及其在植物分类学研究中的应用现状及存在的争议进行了综合分析和阐述,并展望了植物DNA条形码技术的发展趋势及应用前景。通过具体实例说明将植物DNA条形码技术与传统植物学知识相结合可作为民族植物学的研究手段之一。认为:目前常用的植物DNA条形码主要有单一片段和多片段组合2种方式,这2种方式各有优缺点;常用的DNA序列有matK、trnH-psbA、rbcL和ITS等,但均有一定的局限性;针对不同的使用目的,应选择不同的植物DNA条形码标准;影响植物DNA条形码鉴定准确性的因素包括物种的类型和数量、系统树构建方法、杂交/基因渗入、物种起源时间的差异、分子进化速率差异等;当前植物DNA条形码研究工作的重点是选择合适的DNA片段并对其进行评价。     

COI barcoding of Nebelid testate amoebae (Amoebozoa: Arcellinida): extensive cryptic diversity and redefinition of the Hyalospheniidae Schultze

We used Cytochrome Oxidase Subunit 1 (COI) to assess the phylogenetic relationships and taxonomy of Nebela sensu stricto and similar taxa (Nebela group, Arcellinida) in order to clarify the taxonomic validity of morphological characters. The COI data not only successfully separated all studied morphospecies but also revealed the existence of several potential cryptic species. The taxonomic implications of the results are: (1) Genus Nebela is paraphyletic and will need to be split into at least two monophyletic assemblages when taxon sampling is further expanded. (2) Genus Quadrulella, one of the few arcellinid genera building its shell from self-secreted siliceous elements, and the mixotrophic Hyalosphenia papilio branch within the Nebela group in agreement with the general morphology of their shell and the presence of an organic rim around the aperture (synapomorphy for Hyalospheniidae). We thus synonymise Hyalospheniidae and Nebelidae. Hyalospheniidae takes precedence and now includes Hyalosphenia, Quadrulella (previously in the Lesquereusiidae) and all Nebelidae with the exception of Argynnia and Physochila. Leptochlamys is Arcellinida incertae sedis. We describe a new genus Padaungiella Lara et Todorov and a new species Nebela meisterfeldi n. sp. Heger et Mitchell and revise the taxonomic position (and rank) of several taxa. These results show that the traditional morphology-based taxonomy underestimates the diversity within the Nebela group, and that phylogenetic relationships are best inferred from shell shape rather than from the material used to build the shell.     

There is more to maerl than meets the eye: DNA barcoding reveals a new species in Britain,Lithothamnion erinaceum sp. nov. (Hapalidiales,Rhodophyta)

Due to the high plasticity of coralline algae, identification based on morphology alone can be extremely difficult, so studies increasingly use a combination of morphology and genetics in species delimitation. A DNA barcoding study was carried out on maerl-forming coralline algae using the mitochondrial cytochrome oxidase 1 gene, CO1, and the plastid gene, psbA, on field specimens from Falmouth and Oban together with herbarium specimens from the Natural History Museum, UK, and the Smithsonian Institution, Washington, USA. Results revealed the presence in the north of Britain of a new species, Lithothamnion erinaceum Melbourne & J. Brodie, sp. nov., which was previously misidentified as Lithothamnion glaciale. The results also indicated that Lithothamnion lemoineae, which had earlier been recorded from Britain, was not present. One of the biggest concerns at present is how organisms will respond to climate change and ocean acidification, and it is imperative that investigations are put on a firm taxonomic basis. Our study has highlighted the importance of using molecular techniques to aid in the elucidation of cryptic diversity.     

Deeply divergent lineages of the widespread New Zealand amphipod Paracalliope fluviatilis revealed using allozyme and mitochondrial DNA analyses

1. We evaluated the population genetic structure of the common New Zealand amphipod Paracalliope fluviatilis using eight allozyme loci, and the mitochondrial cytochrome oxidase c subunit I (COI) gene locus. Morphological analyses were also conducted to evaluate any phenotypic differences. Individuals belonging to P. fluviatilis were collected from a total of 14 freshwater fluvial habitats on the North and South Islands, New Zealand. 2. We found evidence for strong genetic differentiation among locations (Wright's F_ST > 0.25), and fixed differences (non‐shared alleles) at two of the eight allozyme loci indicating the possibility of previously unknown species. Analysis of a 545‐bp fragment of the COI locus was mostly congruent with the allozyme data and revealed the same deeply divergent lineages (sequence divergences up to 26%). 3. Clear genetic breaks were identified between North Island and South Island populations. North Island populations separated by <100 km also showed genetic differences between east and west draining watersheds (sequence divergence >12%). Accordingly, present‐day dispersal among hydrologically isolated habitats appears minimal for this taxon. 4. Although population differences were clearly shown by allozyme and mtDNA analyses, individuals were morphologically indistinguishable. This suggests that, as in North American and European taxa (e.g. Hyalella and Gammarus), morphological conservatism may be prevalent among New Zealand's freshwater amphipods. We conclude that molecular techniques, particularly the COI gene locus, may be powerful tools for resolving species that show no distinctive morphological differences.     

DNA barcoding highlights a cryptic species of grenadier Macrourus in the Southern Ocean

Although three species of the genus Macrourus are recognized in the Southern Ocean, DNA sequencing of the mitochondrial COI gene revealed four well-supported clades. These barcode data suggest the presence of an undescribed species, a conclusion supported by meristic and morphometric examination of specimens.     

DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts

Despite ongoing efforts to protect species and ecosystems in Cuba, habitat degradation, overuse and introduction of alien species have posed serious challenges to native freshwater fish species. In spite of the accumulated knowledge on the systematics of this freshwater ichthyofauna, recent results suggested that we are far from having a complete picture of the Cuban freshwater fish diversity. It is estimated that 40% of freshwater Cuban fish are endemic; however, this number may be even higher. Partial sequences (652 bp) of the mitochondrial gene COI (cytochrome c oxidase subunit I) were used to barcode 126 individuals, representing 27 taxonomically recognized species in 17 genera and 10 families. Analysis was based on Kimura 2-parameter genetic distances, and for four genera a character-based analysis (population aggregation analysis) was also used. The mean conspecific, congeneric and confamiliar genetic distances were 0.6%, 9.1% and 20.2% respectively. Molecular species identification was in concordance with current taxonomical classification in 96.4% of cases, and based on the neighbour-joining trees, in all but one instance, members of a given genera clustered within the same clade. Within the genus Gambusia, genetic divergence analysis suggests that there may be at least four cryptic species. In contrast, low genetic divergence and a lack of diagnostic sites suggest that Rivulus insulaepinorum may be conspecific with Rivulus cylindraceus. Distance and character-based analysis were completely concordant, suggesting that they complement species identification. Overall, the results evidenced the usefulness of the DNA barcodes for cataloguing Cuban freshwater fish species and for identifying those groups that deserve further taxonomic attention.     

DNA barcoding of Gaultheria L.in China (Ericaceae: Vaccinioideae)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer (ITS),were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based analyses.This study included 186 individuals from 89 populations representing 30 species.For all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species resolvability.Polymerase chain reaction amplification and sequencing were successful for all four loci.Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination ofmatK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for Chinese Gaultheria,by which only 11 out of 30 species can be separated.Loci rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for Chinese Gaultheria.DNA barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

DNA barcoding of Gaultheria L. in China (Ericaceae: Vaccinioideae)

Abstract Four DNA barcoding loci, chloroplast loci rbcL, matK, trnH‐psbA, and nuclear locus internal transcribed spacer (ITS), were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P‐distance, Wilcoxon signed rank test, and tree‐based analyses. This study included 186 individuals from 89 populations representing 30 species. For all individuals, single locus markers showed high levels of sequencing universality but were ineffective for species resolvability. Polymerase chain reaction amplification and sequencing were successful for all four loci. Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH‐psbA. A combination of matK and ITS was the most efficient DNA barcode among all studied regions, however, they do not represent an appropriate candidate barcode for Chinese Gaultheria, by which only 11 out of 30 species can be separated. Loci rbcL, matK, and trnH‐psbA, which were recently proposed as universal plant barcodes, have a very poor capacity for species separation for Chinese Gaultheria. DNA barcodes may be reliable tools to identify the evolutionary units of this group, so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.