DNA barcoding of the genus Lepidion (Gadiformes: Moridae) with recognition of Lepidion eques as a junior synonym of Lepidion lepidion

DNA sequences of cytochrome c oxidase I gene (COI) from Lepidion spp. were employed to test the efficiency of species identification. A sample of 32 individuals from five Lepidion species was sequenced and combined with 26 sequences from other BOLD projects. As a result, 58 Lepidion DNA sequences of the COI gene belonging to eight of the nine recognized Lepidion species were analysed. Sequences were aligned and formed seven clades in a Bayesian phylogenetic tree, where Lepidion lepidion and Lepidion eques grouped jointly. The Kimura 2‐parameter genetic distances, among congeners were, on average, 4.28%, 16 times greater than among conspecifics (0.27%). The main diagnostic meristic data of Lepidion spp. were compiled and a detailed morphological revision of the congeneric species L. eques and L. lepidion was made. The eye diameter was significantly different between L. eques and L. lepidion (P < 0.001). The number of anal fin rays ranged from 45 to 51 in L. lepidion and from 47 to 54 in L. eques, but no significant differences were obtained in the mean values of this variable (P = 0.07). According to the morphological and genetic analyses, the results strongly suggest that the Mediterranean codling L. lepidion and the North Atlantic codling L. eques are conspecific, making L. eques a junior synonym of L. lepidion.     

Incomplete estimates of genetic diversity within species: Implications for DNA barcoding

DNA barcoding has greatly accelerated the pace of specimen identification to the species level, as well as species delineation. Whereas the application of DNA barcoding to the matching of unknown specimens to known species is straightforward, its use for species delimitation is more controversial, as species discovery hinges critically on present levels of haplotype diversity, as well as patterning of standing genetic variation that exists within and between species. Typical sample sizes for molecular biodiversity assessment using DNA barcodes range from 5 to 10 individuals per species. However, required levels that are necessary to fully gauge haplotype variation at the species level are presumed to be strongly taxon‐specific. Importantly, little attention has been paid to determining appropriate specimen sample sizes that are necessary to reveal the majority of intraspecific haplotype variation within any one species. In this paper, we present a brief outline of the current literature and methods on intraspecific sample size estimation for the assessment of COI DNA barcode haplotype sampling completeness. The importance of adequate sample sizes for studies of molecular biodiversity is stressed, with application to a variety of metazoan taxa, through reviewing foundational statistical and population genetic models, with specific application to ray‐finned fishes (Chordata: Actinopterygii). Finally, promising avenues for further research in this area are highlighted.     

DNA barcoding as a tool for coral reef conservation

DNA Barcoding (DBC) is a method for taxonomic identification of animals that is based entirely on the 5′ portion of the mitochondrial gene, cytochrome oxidase subunit I (COI-5). It can be especially useful for identification of larval forms or incomplete specimens lacking diagnostic morphological characters. DBC can also facilitate the discovery of species and in defining “molecular taxonomic units” in problematic groups. However, DBC is not a panacea for coral reef taxonomy. In two of the most ecologically important groups on coral reefs, the Anthozoa and Porifera, COI-5 sequences have diverged too little to be diagnostic for all species. Other problems for DBC include paraphyly in mitochondrial gene trees and lack of differentiation between hybrids and their maternal ancestors. DBC also depends on the availability of databases of COI-5 sequences, which are still in early stages of development. A global effort to barcode all fish species has demonstrated the importance of large-scale coordination and is yielding promising results. Whether or not COI-5 by itself is sufficient for species assignments has become a contentious question; it is generally advantageous to use sequences from multiple loci.     

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.     

Molecular diversity of Germany's freshwater fishes and lampreys assessed by DNA barcoding

This study represents the first comprehensive molecular assessment of freshwater fishes and lampreys from Germany. We analysed COI sequences for almost 80% of the species mentioned in the current German Red List. In total, 1056 DNA barcodes belonging to 92 species from all major drainages were used to (i) build a reliable DNA barcode reference library, (ii) test for phylogeographic patterns, (iii) check for the presence of barcode gaps between species and (iv) evaluate the performance of the barcode index number (BIN) system, available on the Barcode of Life Data Systems. For over 78% of all analysed species, DNA barcodes are a reliable means for identification, indicated by the presence of barcode gaps. An overlap between intra‐ and interspecific genetic distances was present in 19 species, six of which belong to the genus Coregonus. The Neighbour‐Joining phenogram showed 60 nonoverlapping species clusters and three singleton species, which were related to 63 separate BIN numbers. Furthermore, Barbatula barbatula, Leucaspius delineatus, Phoxinus phoxinus and Squalius cephalus exhibited remarkable levels of cryptic diversity. In contrast, 11 clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. The analysis of our barcode library together with public data resulted in 89 BINs, of which 56% showed taxonomic conflicts. Most of these conflicts were caused by the use of synonymies, inadequate taxonomy or misidentifications. Moreover, our study increased the number of potential alien species in Germany from 14 to 21 and is therefore a valuable groundwork for further faunistic investigations.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

DNA条形码及其在海洋浮游动物生态学研究中的应用

浮游动物的准确鉴定是浮游动物生态学研究的基础.传统的基于形态特征的鉴定不仅费时费力,而且部分类群特别是浮游幼体由于形态差异细微,鉴定存在困难,导致物种多样性被低估.DNA条形码(DNA barcodes)技术为浮游动物物种鉴定提供了一个有力工具,已迅速应用于海洋浮游动物生态学研究.本文介绍了DNA条形码的基本概念、优势及局限性,总结了该技术(主要是基于线粒体细胞色素C氧化酶第一亚基(mtCOI)基因序列片段的DNA条形码)在海洋浮游动物物种快速鉴定、隐种发现、营养关系研究、生物入侵种监测、群落历史演变反演、种群遗传学以及生物地理学中的成功应用.随着DNA条形码数据库信息量覆盖率的不断提高和新一代测序技术的快速发展,DNA条形码将提供除了种类鉴定外更加丰富的信息,从而帮助人们更好地理解海洋浮游动物的多样性及其在生态系统中的功能,推动海洋浮游动物生态学的发展.     

DNA barcoding of Neotropical bats: species identification and discovery within Guyana

Sequence diversity in the cytochrome c oxidase subunit 1 gene has been shown to be an effective tool for species identification and discovery in various groups of animals, but has not been extensively tested in mammals. We address this gap by examining the performance of DNA barcodes in the discrimination of 87 species of bats from Guyana. Eighty‐one of these species showed both low intraspecific variation (mean = 0.60%), and clear sequence divergence from their congeners (mean = 7.80%), while the other six showed deeply divergent intraspecific lineages suggesting that they represent species complexes. Although further work is needed to examine patterns of sequence diversity at a broader geographical scale, the present study validates the effectiveness of barcoding for the identification of regional bat assemblages, even highly diverse tropical faunas.     

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.     

DNA barcoding of the ichthyofauna of Taal Lake, Philippines

This study represents the first molecular survey of the ichthyofauna of Taal Lake and the first DNA barcoding attempt in Philippine fishes. Taal Lake, the third largest lake in the Philippines, is considered a very important fisheries resource and is home to the world's only freshwater sardine, Sardinella tawilis. However, overexploitation and introduction of exotic fishes have caused a massive decline in the diversity of native species as well as in overall productivity of the lake. In this study, 118 individuals of 23 native, endemic and introduced fishes of Taal Lake were barcoded using the partial DNA sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene. These species belong to 21 genera, 17 families and 9 orders. Divergence of sequences within and between species was determined using Kimura 2-parameter (K2P) distance model, and a neighbour-joining tree was generated with 1000 bootstrap replications using the K2P model. All COI sequences for each of the 23 species were clearly discriminated among genera. The average within species, within genus, within family and within order percent genetic divergence was 0.60%, 11.07%, 17.67% and 24.08%, respectively. Our results provide evidence that COI DNA barcodes are effective for the rapid and accurate identification of fishes and for identifying certain species that need further taxonomic investigation.     

DNA barcoding and genetic diversity of phyllostomid bats from the Yucatan Peninsula with comparisons to Central America

The mitochondrial cytochrome c oxidase subunit I gene is the standard DNA barcoding region used for species identification and discovery. We examined the variation of COI (454 bp) to discriminate 20 species of bats in the family Phyllostomidae that are found in the Yucatan Peninsula of southeastern Mexico and northern Guatemala and compared them genetically to other samples from Central America. The majority of these species had low intraspecific variation (mean = 0.75%), but some taxa had intraspecific variation ranging to 8.8%, suggesting the possibility of cryptic species (i.e. Desmodus rotundus and Artibeus jamaicensis). There was a recurring biogeographic pattern in eight species with a separation of northern and southern Middle American localities. The Yucatan Peninsula was a discrete area identified in four species, whereas Panama was recovered in five species of phyllostomid bats. Our study establishes a foundation for further molecular work incorporating broader taxonomic and geographic coverage to better understand the phylogeography and genetic diversity that have resulted from the ecological constraints in this region and the remarkable differentiation of bats in the Neotropics.     

DNA extraction techniques for DNA barcoding of minute gall-inhabiting wasps

DNA extraction from minute hymenopterans and their larvae is difficult and challenging because of their small size indicating a low amount of starting material. Hence, 11 DNA extraction methods were compared to determine their efficacy in isolating DNA. Success of each method was scored on a 2% agarose gel after PCR of the cox 1 mitochondrial locus. A silica-membrane-based approach was the most successful, followed by a method using a combination of incubation buffers and a method using magnetic beads. The method using buffers was the most cost- and time effective. Using this method, larvae from Eucalyptus seed capsule galls could be assigned a role (parasitoid, gall former or inquiline) in the gall-inhabiting complex.     

Identification of Odontamblyopus lacepedii via morphology and DNA barcoding

To evaluate taxonomic and genetic variation in Odontamblyopus lacepedii collected in East Asia, morphometric characters and molecular analysis were described. The body was ribbon-like. The body and head were grayish-purple with no embedded scales. Eyes were rudimentary, but distinct, covered by skin. The dorsal and anal fins were continuous with the caudal fin. The edges of the dorsal, anal, and caudal fins were dark chocolate-brown. Genetic analysis of four species revealed higher mean A + T (55.4%) than G + C (44.6%). Twenty-two haplotype sequences were identified, containing 22 haplotypes, 131 variable sites and 122 parsimony-informative sites, with haplotype and nucleotide diversity differing among species. The intraspecies genetic distances ranged from 0.2% to 0.5% (average 0.3%) and interspecies differences from 1.1% to 17.6% (average 14.6%), respectively, indicating that these four species of Amblyopinae can be accurately identified. The NJ tree constructed using COI fragments based on 22 haplotypes was composed of two main branches: the upper branch comprising O. lacepedii and O. sp. showing a close relationship, with Trypauchen vagina and Ctenotrypauchen microcephalus clustering on the second branch. Thus, the results validated the identification of O. lacepedii at the morphological and genetic level.     

Identifying the true oysters (Bivalvia: Ostreidae) with mitochondrial phylogeny and distance-based DNA barcoding

Oysters (family Ostreidae), with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. As a useful tool for molecular species identification, DNA barcoding might offer significant potential for oyster identification and taxonomy. This study used two mitochondrial fragments, cytochrome c oxidase I (COI) and the large ribosomal subunit (16S rDNA), to assess whether oyster species could be identified by phylogeny and distance-based DNA barcoding techniques. Relationships among species were estimated by the phylogenetic analyses of both genes, and then pairwise inter- and intraspecific genetic divergences were assessed. Species forming well-differentiated clades in the molecular phylogenies were identical for both genes even when the closely related species were included. Intraspecific variability of 16S rDNA overlapped with interspecific divergence. However, average intra- and interspecific genetic divergences for COI were 0-1.4% (maximum 2.2%) and 2.6-32.2% (minimum 2.2%), respectively, indicating the existence of a barcoding gap. These results confirm the efficacy of species identification in oysters via DNA barcodes and phylogenetic analysis.     

The utility of DNA barcoding as a tool to assess the success of ecological restoration using Hemiptera as a biological indicator

DNA barcoding uses a short, standardized DNA fragment to sort individuals into species. This molecular technique has applications in fields including ecology, evolution, conservation, and biogeography. In ecological applications such as species monitoring and habitat restoration, its potential has not been fully realized and implemented. Invertebrates are excellent biological indicators, as changes in species diversity or community assemblage provide important insights into the condition of, or changes in, the environment. This information is particularly useful within the context of restoration ecology. In this study, DNA barcoding is used to assess the potential of Hemiptera as a biological indicator of restoration success for the Buffelsdraai Landfill Site Community Reforestation Project (Durban, South Africa). A total of 393 Hemiptera specimens were collected from sites reforested at distinct phases (plots reforested in 2010, 2012, and 2015) and two reference sites (natural forest and grassland). The Hemiptera species composition and assemblage were assessed by analyzing diversity indices, ordination, unweighted pair‐group average cluster analysis, and phylogenetic analysis. Hemiptera species composition varied significantly across the chronologically different reforested sites, with a higher species richness observed in the older reforested plots. This suggests that Hemiptera diversity can be used to track restoration success, even over the small temporal scale used in this study. This study highlights the utility of DNA barcoding as a taxonomic sorting tool both to monitor ecological restoration and to discover specific taxa within Hemiptera that may be useful biological indicators.     

Testing the potential of DNA barcoding in vertebrate radiations: the case of the littoral cichlids (Pisces,Perciformes, Cichlidae) from Lake Tanganyika

We obtained 398 cytochrome c oxidase subunit I barcodes of 96 morphospecies of Lake Tanganyika (LT) cichlids from the littoral zone. The potential of DNA barcoding in these fishes was tested using both species identification and species delineation methods. The best match (BM) and best close match (BCM) methods were used to evaluate the overall identification success. For this, three libraries were analysed in which the specimens were categorized into Operational Taxonomic Units (OTU) in three alternative ways: (A) morphologically distinct, including undescribed, species, (B) valid species and (C) complexes of morphologically similar or closely related species. For libraries A, B and C, 73, 73 and 96% (BM) and 72, 70 and 94% (BCM) of the specimens were correctly identified. Additionally, the potential of two species delineation methods was tested. The General Mixed Yule Coalescent (GMYC) analysis suggested 70 hypothetical species, while the Automatic Barcode Gap Discovery (ABGD) method revealed 115 putative species. Although the ABGD method had a tendency to oversplit, it outperformed the GMYC analysis in retrieving the species. In most cases where ABGD suggested oversplitting, this was due to intraspecific geographical variation. The failure of the GMYC method to retrieve many species could be attributed to discrepancies between mitochondrial gene trees and the evolutionary histories of LT cichlid species. Littoral LT cichlids have complex evolutionary histories that include instances of hybridization, introgression and rapid speciation. Nevertheless, although the utility of DNA barcoding in identification is restricted to the level of complexes, it has potential for species discovery in cichlid radiations.     

DNA barcodes for 1/1000 of the animal kingdom

This study reports DNA barcodes for more than 1300 Lepidoptera species from the eastern half of North America, establishing that 99.3 per cent of these species possess diagnostic barcode sequences. Intraspecific divergences averaged just 0.43 per cent among this assemblage, but most values were lower. The mean was elevated by deep barcode divergences (greater than 2%) in 5.1 per cent of the species, often involving the sympatric occurrence of two barcode clusters. A few of these cases have been analysed in detail, revealing species overlooked by the current taxonomic system. This study also provided a large-scale test of the extent of regional divergence in barcode sequences, indicating that geographical differentiation in the Lepidoptera of eastern North America is small, even when comparisons involve populations as much as 2800 km apart. The present results affirm that a highly effective system for the identification of Lepidoptera in this region can be built with few records per species because of the limited intra-specific variation. As most terrestrial and marine taxa are likely to possess a similar pattern of population structure, an effective DNA-based identification system can be developed with modest effort.     

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.     

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.     

Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.