DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.     

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'.     

Revisiting species delimitation within the genus Oxystele using DNA barcoding approach

The genus Oxystele, a member of the highly diverse marine gastropod superfamily Trochoidea, is endemic to southern Africa. Members of the genus include some of the most abundant molluscs on southern African shores and are important components of littoral biodiversity in rocky intertidal habitats. Species delimitation within the genus is still controversial, especially regarding the complex O. impervia / O. variegata. Here, we assessed species boundaries within the genus using DNA barcoding and phylogenetic tree reconstruction. We analysed 56 specimens using the mitochondrial gene COI. Our analysis delimits five molecular operational taxonomic units (MOTUs), and distinguishes O. impervia from O. variegata. However, we reveal important discrepancies between MOTUs and morphology-based species identification and discuss alternative hypotheses that can account for this. Finally, we indicate the need for future study that includes additional genes, and the combination of both morphology and genetic techniques (e.g. AFLP or microsatellites) to get deeper insight into species delimitation within the genus.     

DNA barcoding and species delimitation of butterflies (Lepidoptera) from Nigeria

Molecular Biology Reports - Accurate identification of species is a prerequisite for successful biodiversity management and further genetic studies. Species identification techniques often require...     

Assessment of the DNA barcoding for identification of Trigonostigma somphongsi,a critically endangered species in Thailand

Trigonostigma somphongsi, a critically endangered species, is a rare and endemic fish in Thailand. This species had disappeared from its natural habitat for 20 years until 2006. The DNA barcodes or the fragments of cytochrome c oxidase I (COI) of T. somphongsi were investigated for species identification. The remaining two native species in the genus Trigonostigma, T. heteromorpha and T. espei were also identified using Boraras urophthalmoides as an outgroup species. The 707-bp fragments were successfully amplified and sequenced in all fifteen fish samples. In the genus Trigonostigma, the genetic distance within and between species ranged from 0.000 to 0.005 and 0.016 to 0.039, respectively. The lowest genetic distance (0.016) was between T. heteromorpha and T. espei, while the highest genetic distance (0.039) was between T. somphongsi and T. espei, followed by T. somphongsi and T. heteromorpha (0.035). The phylogenetic analysis showed that the relationship between the three Trigonostigma species (T. somphongsi was clearly separated from T. heteromorpha and T. espei) agreed with the morphological characteristics. These results suggest that DNA barcoding is an effective approach to identify Trigonostigma species for use in the conservation and management of fisheries.     

断眼天牛DNA条码试剂盒的研制

【目的】为了加强对检疫口岸截获木材中的断眼天牛种类进行准确、快速的鉴定,克服单纯依赖于形态学方法为主的局限性,我们研发了断眼天牛属DNA条码试剂盒检测技术。【方法】本研究基于线粒体COⅠ基因(线粒体细胞色素氧化酶亚基Ⅰ),采用加拿大条码中心开发的昆虫及植物DNA提取方法,对基因序列分析,获得碱基位点规律。【结果】断眼天牛属中的9个常截获种类取得了标记性碱基位点。【结论】检疫中常截获的断眼天牛属中的9个种通过标记性碱基位点得到区分;研发了断眼天牛属DNA条码试剂盒检测技术,为进出境口岸的检疫工作提供一种新的鉴定手段。     

DNA barcoding Indian marine fishes

DNA barcoding has been adopted as a global bio-identification system for animals in recent years. A major national programme on DNA barcoding of fish and marine life was initiated in India by the authors during 2006 and 115 species of marine fish covering Carangids, Clupeids, Scombrids, Groupers, Sciaenids, Silverbellies, Mullids, Polynemids and Silurids representing 79 Genera and 37 Families from the Indian Ocean have been barcoded for the first time using cytochrome c oxidase I gene (COI) of the mtDNA. The species were represented by multiple specimens and a total of 397 sequences were generated. After amplification and sequencing of 707 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two parameter (K2P) distances within species, genera, families, orders were 0.30%, 6.60%, 9.91%, 16.00%, respectively. In addition to barcode-based species identification system, phylogenetic relationships among the species have also been attempted. The neighbour-joining tree revealed distinct clusters in concurrence with the taxonomic status of the species.     

Single-locus DNA barcoding and species delimitation of the sandfly subgenus Evandromyia (Aldamyia)

Sandfly specimens from the subgenus Evandromyia (Aldamyia) Galati, 2003 (Diptera: Psychodidae: Phlebotominae) were collected between 2012 and 2019 from nine localities in seven Brazilian states, morphologically-identified, and then DNA barcoded by sequencing the mitochondrial cytochrome c oxidase subunit I (coi) gene. Forty-four new barcode sequences generated from 10 morphospecies were combined with 49 previously published sequences from the same subgenus and analysed using sequence-similarity methods (best-match criteria) to assess their ability at specimen identification, while four different species delimitation methods (ABGD, GMYC, PTP and TCS) were used to infer molecular operational taxonomic units (MOTUs). Overall, seven of the 11 morphospecies analysed were congruent with both the well-supported clades identified by phylogenetic analysis and the MOTUs inferred by species delimitation, while the remaining four morphospecies – E. carmelinoi, E. evandroi, E. lenti and E. piperiformis – were merged into a single well-supported clade/MOTU. Although E. carmelinoi, E. evandroi and E. lenti were indistinguishable using coi DNA barcodes, E. piperiformis did form a distinct phylogenetic cluster and could be correctly identified using best-match criteria. Despite their apparent morphological differences, we propose on the basis of the molecular similarity of their DNA barcodes that these latter four morphospecies should be considered members of a recently-diverged species complex.     

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.     

DNA条形码技术在珍稀濒危物种保护中的应用

<正>经过12年的发展,DNA条形码技术已经从最初的被怀疑批评转变为如今的被认可接受,科学家也从对该技术的观望转变为参与。DNA条形码技术(包括基因区域的确定、序列获取、数据库建设、鉴定算法等)虽然仍在不断完善中,但已经可用于解决实际问题。现在,DNA条形码技术到了一个新的转折期,即重点从技术探索转向实际应用。DNA条形码技术可被用于需要区分生物或确定物种名称的所有领域,在生态学、进化生物学和     

基于线粒体COI和16S片段序列的北部湾北部水螅水母DNA条形码分析

水螅水母类是浮游动物群落的重要组成部分,在近岸海洋生态系统物质循环和能量流动中扮演着重要角色。水螅水母类形态结构简单,但其物种的准确鉴定一直是分类工作中的难点。DNA条形码极大地促进了水螅水母物种的快速、准确鉴定。本研究扩增了北部湾北部28种水螅水母的线粒体COI和16S序列,分别为92条和116条;比较了2个基因片段的种内、种间K2P(Kimura 2-parameter)遗传距离;构建了基于这2个基因片段的系统发育邻接树(neighbor-joining phylogenetic tree);并结合矢量分析构建了Klee-diagram图。结果显示:COI序列的种内遗传距离为0.008±0.005(0–0.033),种间遗传距离为0.298±0.128(0.092–0.597);16S序列的种内遗传距离为0.006±0.010(0–0.047),种间遗传距离为0.394±0.195(0.068–0.898)。2个基因序列在所调查种类中,种内遗传差异均小于种间遗传差异,存在明显的条形码间隔(barcoding gap)。基于2个基因片段的NJ树均显示,单种所有个体都位于同一独立分枝。研究结果表明,以COI和16S作为DNA条形码均能对北部湾北部常见水螅水母类进行物种鉴定。     

DNA barcoding of fishes in Irtysh River China

DNA barcoding was a molecular diagnostic method that provided rapid and accurate species identification. The 650 bp-length cytochrome c oxidase subunit I (COI) gene of 33 species in Irtysh River China was sequenced and analyzed in this study. The average intra-species, -genus, -family, and -order of Kimura two parameter (K2P) distances were 0.003, 0.060, 0.163 and 0.240, respectively. The genetic distance between genus Barbatula and Cobitis was the largest whereas that between genus Hypophthalmichthys and Aristichthys was the smallest. The neighbour-joining tree constructed by all 44 haplotypes was divided into two major clusters: Cypriniformes fishes and other fishes. A cryptic species of Barbatula barbatula was detected according to 2% genetic threshold.     

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 fresh seafood in Australian markets reveals misleading labelling and sale of endangered species

Flake and shark samples were purchased from outlets in several coastal Australian regions and genetically barcoded using the cytochrome oxidase subunit 1 (CO1) gene to investigate labelling reliability and species-specific sources of ambiguously labelled fillets. Of the 41 shark fillet samples obtained, 23 yielded high-quality CO1 sequences, out of which 57% (n = 13) were labelled ambiguously (misleading) and 35% (n = 8) incorrectly. In contrast, barramundi fillets, which are widely available and sought after in Australian markets, were shown to be accurately labelled. Species identified from shark samples, including the shortfin mako (n = 3) and the scalloped hammerhead (n = 1), are assessed by the IUCN as endangered and critically endangered, respectively, with several others classified as vulnerable and near threatened.     

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.     

DNA barcoding is a useful tool for the identification of marine fishes from Japan

In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification.     

Recent discoveries of armyworms in Japan and their species identification using DNA barcoding

Long columns of migrating larval sciarid armyworms were discovered in central and northern Japan, specifically Kanagawa, Gunma, Miyagi and Akita prefectures, as well as Hokkaido. This is the first examination of armyworms in East Asia. In Europe, armyworms have been identified as Sciara militaris, belonging to the family Sciaridae (sciarid flies or black fungus gnats), by rearing them to adulthood. In Japan, we were unable to obtain live samples for rearing; therefore, DNA barcodes were obtained from the samples of armyworms collected in the Gunma and Miyagi prefectures. The DNA barcodes were compared with those obtained from the following samples: pupae of S. militaris from UK, adults of Sciara kitakamiensis, Sciara humeralis, Sciara hemerobioides, Sciara thoracica, Sciara helvola and Sciara melanostyla from Japan, and adults of one undescribed Sciara species from Malaysia. Neighbour-joining, maximum parsimony, and maximum likelihood analyses revealed that the armyworms discovered in Japan are S. kitakamiensis. Although adults of this species have been recorded in several locations in Japan, this is the first report of migrating larval armyworms. DNA barcodes were effectively used to link different life stages of this species. The average intraspecific and interspecific pairwise genetic distances of the genus Sciara were 0.3% and 12.6%, respectively. The present study illustrates that DNA barcodes are an effective means of identifying sciarid flies in Japan.     

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.     

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.