DNA barcoding: error rates based on comprehensive sampling

DNA barcoding has attracted attention with promises to aid in species identification and discovery; however, few well-sampled datasets are available to test its performance. We provide the first examination of barcoding performance in a comprehensively sampled, diverse group (cypraeid marine gastropods, or cowries). We utilize previous methods for testing performance and employ a novel phylogenetic approach to calculate intraspecific variation and interspecific divergence. Error rates are estimated for (1) identifying samples against a well-characterized phylogeny, and (2) assisting in species discovery for partially known groups. We find that the lowest overall error for species identification is 4%. In contrast, barcoding performs poorly in incompletely sampled groups. Here, species delineation relies on the use of thresholds, set to differentiate between intraspecific variation and interspecific divergence. Whereas proponents envision a “barcoding gap” between the two, we find substantial overlap, leading to minimal error rates of ~17% in cowries. Moreover, error rates double if only traditionally recognized species are analyzed. Thus, DNA barcoding holds promise for identification in taxonomically well-understood and thoroughly sampled clades. However, the use of thresholds does not bode well for delineating closely related species in taxonomically understudied groups. The promise of barcoding will be realized only if based on solid taxonomic foundations.     

Applying DNA barcoding for the study of geographical variation in host-parasitoid interactions

Studies on the biogeography of host-parasitoid interactions are scarce, mainly because of technical difficulties associated with rearing and species identification. DNA barcoding is increasingly recognized as a valuable tool for taxon identification, allowing to link different life history stages of a species. We evaluate the usefulness of a protocol based on cytochrome oxidase I (COI) sequencing for the study of geographical variation of host-parasitoid interactions. Larvae of Acroclita subsequana (Lepidoptera: Tortricidae) were collected in Macaronesia and dissected to search for parasitoid larvae. Both hosts and parasitoids were sequenced and assigned to molecular operational taxonomic units (MOTUs) based on pairwise genetic distances, tree-based and similarity-based methods. Hosts were grouped into six MOTUs, usually with an allopatric distribution, while parasitoids clustered into 12 MOTUs, each of which was mostly found attacking a single host MOTU. Available COI sequence databases failed to provide identification to species level for these MOTUs. Three challenges related to the applicability of DNA barcoding in this type of studies are identified and discussed: (i) more suitable primers need to be developed for both parasitoids and hosts; (ii) the most commonly used approaches for inferring MOTUs have different limitations (e.g. arbitrary nature of defining a threshold to separate MOTUs) and need to be improved or replaced by other techniques; and (iii) for the identification of MOTUs, it is imperative to increase the range of sequenced taxa in the currently available reference databases. Finally, in spite of these difficulties, we discuss how DNA barcoding will help ecological and biogeographical studies of host-parasitoid interactions.     

DNA barcoding gap: reliable species identification over morphological and geographical scales

The philosophical basis and utility of DNA barcoding have been a subject of numerous debates. While most literature embraces it, some studies continue to question its use in dipterans, butterflies and marine gastropods. Here, we explore the utility of DNA barcoding in identifying spider species that vary in taxonomic affiliation, morphological diagnosibility and geographic distribution. Our first test searched for a ‘barcoding gap’ by comparing intra‐ and interspecific means, medians and overlap in more than 75 000 computed Kimura 2‐parameter (K2P) genetic distances in three families. Our second test compared K2P distances of congeneric species with high vs. low morphological distinctness in 20 genera of 11 families. Our third test explored the effect of enlarging geographical sampling area at a continental scale on genetic variability in DNA barcodes within 20 species of nine families. Our results generally point towards a high utility of DNA barcodes in identifying spider species. However, the size of the barcoding gap strongly depends on taxonomic groups and practices. It is becoming critical to define the barcoding gap statistically more consistently and to document its variation over taxonomic scales. Our results support models of independent patterns of morphological and molecular evolution by showing that DNA barcodes are effective in species identification regardless of their morphological diagnosibility. We also show that DNA barcodes represent an effective tool for identifying spider species over geographic scales, yet their variation contains useful biogeographic information.     

Reef-associated crustacean fauna: biodiversity estimates using semi-quantitative sampling and DNA barcoding

The cryptofauna associated with coral reefs accounts for a major part of the biodiversity in these ecosystems but has been largely overlooked in biodiversity estimates because the organisms are hard to collect and identify. We combine a semi-quantitative sampling design and a DNA barcoding approach to provide metrics for the diversity of reef-associated crustacean. Twenty-two similar-sized dead heads of Pocillopora were sampled at 10 m depth from five central Pacific Ocean localities (four atolls in the Northern Line Islands and in Moorea, French Polynesia). All crustaceans were removed, and partial cytochrome oxidase subunit I was sequenced from 403 individuals, yielding 135 distinct taxa using a species-level criterion of 5% similarity. Most crustacean species were rare; 44% of the OTUs were represented by a single individual, and an additional 33% were represented by several specimens found only in one of the five localities. The Northern Line Islands and Moorea shared only 11 OTUs. Total numbers estimated by species richness statistics (Chao1 and ACE) suggest at least 90 species of crustaceans in Moorea and 150 in the Northern Line Islands for this habitat type. However, rarefaction curves for each region failed to approach an asymptote, and Chao1 and ACE estimators did not stabilize after sampling eight heads in Moorea, so even these diversity figures are underestimates. Nevertheless, even this modest sampling effort from a very limited habitat resulted in surprisingly high species numbers.     

Assessing the effect of varying sequence length on DNA barcoding of fungi

DNA barcoding shows enormous promise for the rapid identification of organisms at the species level. There has been much recent debate, however, about the need for longer barcode sequences, especially when these sequences are used to construct molecular phylogenies. Here, we have analysed a set of fungal mitochondrial sequences - of various lengths - and we have monitored the effect of reducing sequence length on the utility of the data for both species identification and phylogenetic reconstruction. Our results demonstrate that reducing sequence length has a profound effect on the accuracy of resulting phylogenetic trees, but surprisingly short sequences still yield accurate species identifications. We conclude that the standard short barcode sequences ( approximately 600 bp) are not suitable for inferring accurate phylogenetic relationships, but they are sufficient for species identification among the fungi.     

Local scale DNA barcoding of bivalves (Mollusca): a case study

Divergence in cytochrome c oxidase 1 (COI), the genetic marker proposed for DNA barcoding, was investigated in marine bivalves from the genera Ennucula , Nucula , Yoldiella and Thyasira . No overlap in levels of intra- and interspecific variation was found. The levels of divergence found suggest that barcodes from COI will be useful in distinguishing between the species investigated in this study. The insufficiency of blast searches in GenBank to assign many of the obtained sequences to correct phylum was noted and clearly demonstrates the need for better search strategies specifically targeted at identification using DNA barcodes.     

DNA barcoding of billfishes

DNA barcoding is a method promising fast and accurate identification of animal species based on the sequencing of the mitochondrial c oxidase subunit (COI) gene. In this study, we explore the prospects for DNA barcoding in one particular fish group, the billfishes (suborder Xiphioidei--swordfish, marlins, spearfishes, and sailfish). We sequenced the mitochondrial COI gene from 296 individuals from the 10 currently recognized species of billfishes, and combined these data with a further 57 sequences from previously published projects. We also sequenced the rhodopsin gene from a subset of 72 individuals to allow comparison of mitochondrial results against a nuclear marker. Five of the 10 species are readily distinguishable by COI barcodes. Of the rest, the striped marlin (Kajikia audax) and white marlin (K. albida) show highly similar sequences and are not unambiguously distinguishable by barcodes alone, likewise are the three spearfishes Tetrapturus angustirostris, T. belone, and T. pfluegeri. We discuss the taxonomic status of these species groups in light of our and other data, molecular and morphological.     

DNA barcoding of blastocystis

We have developed a simple method for subtyping the intestinal protistan parasite Blastocystis using an approach equivalent to DNA barcoding in animals. Amplification of a 600 bp region of the small subunit ribosomal RNA gene followed by single primer sequencing of the PCR product provides enough data to assign isolates to specific subtypes unambiguously. We believe that this approach will prove useful in future epidemiological studies.     

真菌DNA条形码技术研究进展

DNA条形码(DNA barcoding)技术作为一门新兴的物种鉴定方法以其灵敏、精确、方便和客观的优势,在动植物和微生物的分类鉴定中已经得到广泛应用.真菌鉴定中常用作标准条形码的是核核糖体DNA内转录间隔区(Internal transcribed spacer,ITS),如今也有一些新型条形码被发现和应用到实际操作中,如微条形码、ND6、EF3.本文对DNA条形码技术的产生和发展做出了总结,通过研究其在真菌中应用的实际案例分析了DNA条形码技术的优缺点及发展趋势,并指出DNA条形码技术将以全新的视角来弥补传统分类学的不足,最终实现生物自身的序列变异信息与现有形态分类学的结合.     

Molecular species identification with rich floristic sampling: DNA barcoding the pteridophyte flora of Japan

Background

DNA barcoding is expected to be an effective identification tool for organisms with heteromorphic generations such as pteridophytes, which possess a morphologically simple gametophyte generation. Although a reference data set including complete coverage of the target local flora/fauna is necessary for accurate identification, DNA barcode studies including such rich taxonomic sampling on a countrywide scale are lacking.

Methodology/Principal Findings

The Japanese pteridophyte flora (733 taxa including subspecies and varieties) was used to test the utility of two plastid DNA barcode regions (rbcL and trnH-psbA) with the intention of developing an identification system for native gametophytes. DNA sequences were obtained from each of 689 (94.0%) taxa for rbcL and 617 (84.2%) taxa for trnH-psbA. Mean interspecific divergence values across all taxon pairs (K2P genetic distances) did not reveal a significant difference in rate between trnH-psbA and rbcL, but mean K2P distances of each genus showed significant heterogeneity according to systematic position. The minimum fail rate of taxon discrimination in an identification test using BLAST (12.52%) was obtained when rbcL and trnH-psbA were combined, and became lower in datasets excluding infraspecific taxa or apogamous taxa, or including sexual diploids only.

Conclusions/Significance

This study demonstrates the overall effectiveness of DNA barcodes for species identification in the Japanese pteridophyte flora. Although this flora is characterized by a high occurrence of apogamous taxa that pose a serious challenge to identification using DNA barcodes, such taxa are limited to a small number of genera, and only minimally detract from the overall success rate. In the case that a query sequence is matched to a known apogamous genus, routine species identification may not be possible. Otherwise, DNA barcoding is a practical tool for identification of most Japanese pteridophytes, and is especially anticipated to be helpful for identification of non-hybridizing gametophytes.     

Fast phylogenetic DNA barcoding

We present a heuristic approach to the DNA assignment problem based on phylogenetic inferences using constrained neighbour joining and non-parametric bootstrapping. We show that this method performs as well as the more computationally intensive full Bayesian approach in an analysis of 500 insect DNA sequences obtained from GenBank. We also analyse a previously published dataset of environmental DNA sequences from soil from New Zealand and Siberia, and use these data to illustrate the fact that statistical approaches to the DNA assignment problem allow for more appropriate criteria for determining the taxonomic level at which a particular DNA sequence can be assigned.     

DNA barcoding Korean birds

DNA barcoding, an inventory of DNA sequences from a standardized genomic region, provides a bio-barcode for identifying and discovering species. Several recent studies suggest that the sequence diversity in a 648 bp region of the mitochondrial gene for cytochrome c oxi- dase I (COI) might serve as a DNA barcode for identify- ing animal species such as North American birds, in- sects and fishes. The present study tested the effective- ness of a COI barcode in discriminating Korean bird species. We determined the 5' terminus of the COI bar- code for 92 species of Korean birds and found that spe- cies identification was unambiguous; the genetic differ- ences between closely related species were, on average, 25 times higher than the differences within species. We identified only one misidentified species out of 239 specimens in a genetic resource bank, so confirming the accuracy of species identification in the banking system. We also identified two potential composite species, calling for further investigation using more samples. The finding of large COI sequence differences between species confirms the effectiveness of COI barcodes for identifying Korean bird species. To bring greater reliability to the identification of species, increased in- tra- and interspecies sampling, as well as supplementa- tion of the mitochondrial barcodes with nuclear ones, is needed.     

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.     

DNA barcoding of Dutch birds

The mitochondrial cytochrome c oxidase subunit I (COI) can serve as a fast and accurate marker for the identification of animal species, and has been applied in a number of studies on birds. We here sequenced the COI gene for 387 individuals of 147 species of birds from the Netherlands, with 83 species being represented by > 2 sequences. The Netherlands occupies a small geographic area and 95% of all samples were collected within a 50 km radius from one another. The intraspecific divergences averaged 0.29% among this assemblage, but most values were lower; the interspecific divergences averaged 9.54%. In all, 95% of species were represented by a unique barcode, with 6 species of gulls and skua (Larus and Stercorarius) having at least one shared barcode. This is best explained by these species representing recent radiations with ongoing hybridization. In contrast, one species, the Lesser Whitethroat Sylvia curruca showed deep divergences, averaging 5.76% and up to 8.68% between individuals. These possibly represent two distinct taxa, S. curruca and S. blythi, both clearly separated in a haplotype network analysis. Our study adds to a growing body of DNA barcodes that have become available for birds, and shows that a DNA barcoding approach enables to identify known Dutch bird species with a very high resolution. In addition some species were flagged up for further detailed taxonomic investigation, illustrating that even in ornithologically well-known areas such as the Netherlands, more is to be learned about the birds that are present.     

DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification

DNA barcoding employs short, standardized gene regions (5' segment of mitochondrial cytochrome oxidase subunit I for animals) as an internal tag to enable species identification. Prior studies have indicated that it performs this task well, because interspecific variation at cytochrome oxidase subunit I is typically much greater than intraspecific variation. However, most previous studies have focused on local faunas only, and critics have suggested two reasons why barcoding should be less effective in species identification when the geographical coverage is expanded. They suggested that many recently diverged taxa will be excluded from local analyses because they are allopatric. Second, intraspecific variation may be seriously underestimated by local studies, because geographical variation in the barcode region is not considered. In this paper, we analyse how adding a geographical dimension affects barcode resolution, examining 353 butterfly species from Central Asia. Despite predictions, we found that geographically separated and recently diverged allopatric species did not show, on average, less sequence differentiation than recently diverged sympatric taxa. Although expanded geographical coverage did substantially increase intraspecific variation reducing the barcoding gap between species, this did not decrease species identification using neighbour-joining clustering. The inclusion of additional populations increased the number of paraphyletic entities, but did not impede species-level identification, because paraphyletic species were separated from their monophyletic relatives by substantial sequence divergence. Thus, this study demonstrates that DNA barcoding remains an effective identification tool even when taxa are sampled from a large geographical area.     

DNA barcoding Australia's fish species

Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (barcoded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (cox1). Most species were represented by multiple specimens, and 754 sequences were generated. The GC content of the 143 species of teleosts was higher than the 61 species of sharks and rays (47.1% versus 42.2%), largely due to a higher GC content of codon position 3 in the former (41.1% versus 29.9%). Rays had higher GC than sharks (44.7% versus 41.0%), again largely due to higher GC in the 3rd codon position in the former (36.3% versus 26.8%). Average within-species, genus, family, order and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18% and 23.27%, respectively. All species could be differentiated by their cox1 sequence, although single individuals of each of two species had haplotypes characteristic of a congener. Although DNA barcoding aims to develop species identification systems, some phylogenetic signal was apparent in the data. In the neighbour-joining tree for all 754 sequences, four major clusters were apparent: chimaerids, rays, sharks and teleosts. Species within genera invariably clustered, and generally so did genera within families. Three taxonomic groups-dogfishes of the genus Squalus, flatheads of the family Platycephalidae, and tunas of the genus Thunnus-were examined more closely. The clades revealed after bootstrapping generally corresponded well with expectations. Individuals from operational taxonomic units designated as Squalus species B through F formed individual clades, supporting morphological evidence for each of these being separate species. We conclude that cox1 sequencing, or 'barcoding', can be used to identify fish species.     

Plant DNA barcoding in China

Identification is the keystone of biology (Bell, 1986).However, to biologists and students of biology, the total numbers of species that must be identified far outnumber the names commonly used in English, Chinese, or other living languages.In addition, the identification cues vary greatly between different taxonomical groups.Even for the taxonomists with long training and experience, it is difficult to remember all specific terms for a given group, e.g., Orchidaceae or Poaceae, without help of floristic books or monographs.It takes much time and effort to train a taxonomist, at a time when fewer and fewer young students are interested in this "classical" and "out-of-style", but extremely important, discipline.Many students elect to learn the more "advanced' and "modem" biological disciples like molecular biology and biochemistry.Thus, in China and therest of the world, taxonomists are themselves becoming "endangered".The rise of the DNA barcoding is expected to mitigate, at least in part, this dilemma.