Kimura 2-parameter Distances Research Articles

Identification of Neoceratitis asiatica (Becker) (Diptera: Tephritidae) based on morphological characteristics and DNA barcode.

Neoceratitis asiatica (Becker), which especially infests wolfberry (Lycium barbarum L.), could cause serious economic losses every year in China, especially to organic wolfberry production. In some important wolfberry plantings, it is difficult and time-consuming to rear the larvae or pupae to adults for morphological identification. Molecular identification based on DNA barcode is a solution to the problem. In this study, 15 samples were collected from Ningxia, China. Among them, five adults were identified according to their morphological characteristics. The utility of mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) gene sequence as DNA barcode in distinguishing N. asiatica was evaluated by analysing Kimura 2-parameter distances and phylogenetic trees. There were significant differences between intra-specific and inter-specific genetic distances according to the barcoding gap analysis. The uncertain larval and pupal samples were within the same cluster as N. asiatica adults and formed sister cluster to N. cyanescens. A combination of morphological and molecular methods enabled accurate identification of N. asiatica. This is the first study using DNA barcode to identify N. asiatica and the obtained DNA sequences will be added to the DNA barcode database.

Distinguishing Orchid Species by DNA Barcoding: Increasing the Resolution of Population Studies in Plant Biology.

Increasing the resolution of population studies in plant biology is one of the leading frontiers for omics sciences. One of the most pervasive challenges in molecular phylogenetics is the incongruence between phylogenies obtained using different data sets such as individual genes [like ribulose bisphosphate carboxylase large chain (rbcL) and maturase K (matK)] and intergenic spacers (IGS) [like nuclear ribosomal internal transcribed spacer 1 (nrITS 1) and 2 (nrITS 2), and chloroplast IGS between transfer RNA for leucine and phenylalanine (cp trnL-trnF IGS)]. To solve this challenge, we have screened the four well-established candidate gene sequences (i.e., rbcL, matK, trnL-trnF IGS, and 18S-ITS1-5.8S-ITS2-28S nrDNA) of 65 Indian orchid species. We also have included 31 different species of Dendrobium to identify the suitable locus for resolving the phylogeny-related problem below the taxonomic rank of genus. The Consortium for the Barcode of Life has recommended the locus rbcL and matK for barcoding of all land plants, including orchids. However, in this study, matK and rbcL (species resolving capacity 52% and 48%, respectively) were found to work above the taxonomic limit of genus, and thus cannot be considered a suitable tool to resolve closely related species of Dendrobium, whereas, we found that the locus 18S-ITS1-5.8S-ITS2-28S nrDNA is the best choice with the highest species resolving ability (95.23%) and the highest mean Kimura 2-parameter distance (254 for intergeneric and 144 for intrageneric) for phylogeny construction, and thus have been taken as the most promising single-locus barcode for orchids.

Craniometrics are not outdated: Interspecific morphological divergence in cryptic arvicoline rodents from Iran

We studied interspecific variability in external, cranial, and dental traits in seven species belonging to two closely related arvicoline (Arvicolinae) subgenera, the social voles (Sumeriomys) and the grey voles (Microtus). These voles were for long regarded as morphologically cryptic and the species complexity was fully appreciated only after chromosomal and molecular markers were utilized. Specimens we examined displayed clear external differences between the subgenera. Social voles had five plantar pads, relatively shorter tail (22–27% of head and body length), lighter dorsal pelage, whitish grey belly, and monochromatic or indistinctly bicolored tail. Grey voles showed six plantar pads, relatively longer tail (33–44%), dark brown back, grey belly, and sharply bicolored tail. Our results retrieved considerable heterogeneity in cranial and dental morphology. Major cranial differences between the subgenera associated with the interorbital region, the braincase and the tympanic bullae. Genetic (Kimura 2-parameter) distances, which presumably provided a priori correct estimate of the true phylogenetic divergences, explained 42% of morphometric distances between species. Below the level of a subgenus the phylogenetic signal was conserved in grey voles but it dissolved in social voles. The molecular and morphological rates of evolution were obviously decoupled in the latter possibly by selective pressures for a particular phenotype. Climatic variables however explained only 11% of interspecific heterogeneity in cranial shape. The most distinct in terms of morphology and the climatic properties of its habitat was Microtus irani which also occupies the very edge of Microtus distribution. High species richness of social and grey voles combined with heterogeneity of environmental conditions makes the Middle East an ideal region for studying diversity of developmental trajectories in voles at the levels of species and populations.

Two new species of the genus Atractoscion, and resurrection of the species Atractoscion atelodus (Günther 1867) (Perciformes: Sciaenidae)

A taxonomic re-evaluation of the cosmopolitan fish species Atractoscion aequidens (Cuvier 1830) revealed that it comprises several distinct species occurring in different oceans. Here we describe two new species, Atractoscion macrolepis sp. nov. and Atractoscion microlepis sp. nov., based on specimens collected from Angola and Oman, respectively, and we resurrect the species Atractoscion atelodus (Günther 1867), from Australia. Morphological comparison of A. aequidens from South Africa (type locality) with species from geographically distant localities showed that they differed in the mode number of lateral line pored scales (76 for A. aequidens; 73 for A. macrolepis sp. nov.; 78 for A. microlepis sp. nov.) and the number of dorsal-fin soft rays (26–29 for A. aequidens; 31–33 or 34 for A. atelodus; 24–28 for A. macrolepis sp. nov.; 25–26 for A. microlepis sp. nov.). Comparison of 506 bp sequences in mitochondrial DNA cytochrome c oxidase subunit I showed that the Kimura 2-parameter distance for A. aequidens differs substantially from that for the other species (5.1–5.3% different from A. atelodus; 3.7–4.4 from A. macrolepis sp. nov.; 4.8–5.0 from A. microlepis sp. nov.). The study highlights that DNA barcoding can contribute to confirming the taxonomic status of geographic variations.

New species of Ameletus Eaton, 1885 from the Russian Far East with notes on Ameletus camtschaticus Ulmer 1927 (Ephemeroptera: Ameletidae).

The male imagoes, larvae, and eggs of Ameletus allengaensis sp. nov. and Ameletus sirotskii sp. nov. from the Russian Far East are described. Based on the structure of the male genitalia, the imago and larvae of A. allengaensis sp. nov. and A. sirotskii sp. nov. are similar to those of A. camtschaticus, but the discovery of these new species and separation from A. camtschaticus were confirmed by studies of the morphology of the larvae and male imago, as well as molecular analysis. Identity of various developmental stages of the new species were confirmed by analysis of the mitochondrial gene cytochrome oxidase 1 (COI) DNA barcode, which was also used to reconstruct the phylogenetic relationships within the genus Ameletus. The intraspecific sequence divergence based on the Kimura-2-parameter (K2P) distance ranged from 0.0-2.5%, whereas the interspecific sequence divergence based on the K2P distance ranged from 6.2-7.9% within A. sirotskii sp. nov., A. allengaensis sp. nov. and A. camtschaticus. Male imagoes of A. allengaensis sp. nov., A. sirotskii sp. nov., and A. camtschaticus can be distinguished by the size and location of small denticles on the ventral plate of the penis. The larvae of A. allengaensis sp. nov. differ from those of A. sirotskii sp. nov. by the size of gills I and II. In A. allengaensis sp. nov., gill I is almost twice as small as gill II; in A. sirotskii sp. nov., gill I is only slightly smaller than gill II. Both new species differ from A. camtschaticus by gill II, which does not have an anal rib on the anal margin.

DNA barcoding and phylogenetic analysis of five ascidians (Phlebobranchia) distributed in Gulf of Mannar, India

DNA barcoding has played a significant role in biodiversity assessment as well as its conservation. This technique involves sequencing of mitochondrial marker gene including a short COI gene, known as barcode gene. It has proved its efficiency in identifying several species and resolving the limitations incurred during identification through conventional taxonomy. This study involves the use of DNA barcoding of ascidian species belonging to order Phlebobranchia. A total of 14 individuals, covering two families, three genera and five species, were barcoded. COI gene sequences of all the five species were deposited for the first time in NCBI as well as BOLD. The NJ tree revealed identical phylogenetic relationship among the individuals collected from three different stations. Mean Kimura 2-parameter (K2P) distances within-species, genus, family and order were 0.08%, 6.69%, 9.49% and 18.58%, respectively. This result concludes that COI gene sequencing is the efficient tool in identifying ascidians of the order Phlebobranchia. We report for the first time the COI gene sequences of four species of ascidians studied.

Morphological and molecular characteristics of Malayfilaria sofiani Uni, Mat Udin & Takaoka n. g., n. sp. (Nematoda: Filarioidea) from the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia) in Peninsular Malaysia

BackgroundThe filarial nematodes Wuchereria bancrofti (Cobbold, 1877), Brugia malayi (Brug, 1927) and B. timori Partono, Purnomo, Dennis, Atmosoedjono, Oemijati & Cross, 1977 cause lymphatic diseases in humans in the tropics, while B. pahangi (Buckley & Edeson, 1956) infects carnivores and causes zoonotic diseases in humans in Malaysia. Wuchereria bancrofti, W. kalimantani Palmieri, Pulnomo, Dennis & Marwoto, 1980 and six out of ten Brugia spp. have been described from Australia, Southeast Asia, Sri Lanka and India. However, the origin and evolution of the species in the Wuchereria-Brugia clade remain unclear. While investigating the diversity of filarial parasites in Malaysia, we discovered an undescribed species in the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia).MethodsWe examined 81 common treeshrews from 14 areas in nine states and the Federal Territory of Peninsular Malaysia for filarial parasites. Once any filariae that were found had been isolated, we examined their morphological characteristics and determined the partial sequences of their mitochondrial cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes. Polymerase chain reaction (PCR) products of the internal transcribed spacer 1 (ITS1) region were then cloned into the pGEM-T vector, and the recombinant plasmids were used as templates for sequencing.ResultsMalayfilaria sofiani Uni, Mat Udin & Takaoka, n. g., n. sp. is described based on the morphological characteristics of adults and microfilariae found in common treeshrews from Jeram Pasu, Kelantan, Malaysia. The Kimura 2-parameter distance between the cox1 gene sequences of the new species and W. bancrofti was 11.8%. Based on the three gene sequences, the new species forms a monophyletic clade with W. bancrofti and Brugia spp. The adult parasites were found in tissues surrounding the lymph nodes of the neck of common treeshrews.ConclusionsThe newly described species appears most closely related to Wuchereria spp. and Brugia spp., but differs from these in several morphological characteristics. Molecular analyses based on the cox1 and 12S rRNA genes and the ITS1 region indicated that this species differs from both W. bancrofti and Brugia spp. at the genus level. We thus propose a new genus, Malayfilaria, along with the new species M. sofiani.

Lacanobia oleracea nucleopolyhedrovirus (LaolNPV): A new European species of alphabaculovirus with a narrow host range.

During an insect sampling program in alfalfa crops near Montpellier, France in 2011, Lacanobia oleracea larvae were collected that died due to nucleopolyhedrovirus infection (LaolNPV). This virus was subjected to molecular and biological characterization. The virus was a multiple nucleocapsid NPV that showed similar restriction profiles to Mamestra configurata NPV-A (MacoNPV-A) but with significant differences. Polypeptide analysis demonstrated similar proteins in occlusion bodies and occlusion derived virions, to those observed in NPVs from Mamestra spp. Terminal sequencing revealed that the genome organization shared similarity with that of MacoNPV-A. The most homologous virus was MacoNPV-A 90/2 isolate (95.63% identity and 96.47% similarity), followed by MacoNPV-A 90/4 strain (95.37% and 96.26%), MacoNPV-B (89.21% and 93.53%) and M. brassicae MNPV (89.42% and 93.74%). Phylogenetic analysis performed with lef-8, lef-9, polh and a concatenated set of genes showed that LaolNPV and the Mamestra spp. NPVs clustered together with HaMNPV, but with a closer genetic distance to MacoNPV-A strains. The Kimura 2-parameter (K-2-P) distances of the complete genes were greater than 0.05 between LaolNPV and the MbMNPV/MacoNPV-B/HaMNPV complex, which indicates that LaolNPV is a distinct species. K-2-P distances were in the range 0.015–0.050 for comparisons of LaolNPV with MacoNPV-A strains, such that additional biological characteristics should be evaluated to determine species status. While MacoNPV-A was pathogenic to seven lepidopteran species tested, LaolNPV was only pathogenic to Chrysodeixis chalcites. Given these findings, Lacanobia oleracea nucleopolyhedrovirus should be considered as a new species in the Alphabaculovirus genus.

Culex (Culiciomyia) sasai (Diptera: Culicidae), senior synonym of Cx. spiculothorax and a new country record for Bhutan

Culex (Culiciomyia) spiculothorax was described from Thailand based on the presence of spiculation on the thorax of larvae. Adult females are characterized but are indistinguishable from those of related species, such as Cx. pallidothorax. Phylogenetic analysis of mitochondrial oxidase subunit I (COI) sequences revealed that specimens identified as Cx. spiculothorax from Thailand, Japan and Bhutan form a single clade with Cx. sasai from Japan (Kimura 2-parameter genetic distances 0–0.9%) that is clearly distinct from clades comprised of other species of subgenus Culiciomyia. Attempts to collect Cx. sasai from several locations in Japan were unsuccessful – only larvae with thoracic vesicular-like spicules identified as Cx. spiculothorax were collected. Careful examination of specimens collected near the type locality of Cx. sasai revealed the presence of spicules on the thorax. Based on these findings, Cx. spiculothorax is formally synonymized with Cx. sasai, which replaces the former as the species present in Thailand and is a new country record for Bhutan.

Genetic evidence supporting the taxonomic separation of the Arabian and Northwest African subspecies of the desert hedgehog (Paraechinus aethiopicus)

Traditional subspecies call attention to differences between geographic populations with research potential, but their value is often in need of revision. Genetic data can be useful for evaluating the taxonomic validity of historical species and subspecies designations or for identifying morphologically cryptic divergent lineages worthy of further in-depth taxonomic study. The desert hedgehog (Paraechinus aethiopicus) has a wide but fragmented distribution in arid and semi-arid habitats from the northwest to the northeast of Africa and southwestern Asia, and its taxonomy is still unclear. We used mitochondrial (cytochrome b, Cyt b, and 12S ribosomal RNA, 12S) and nuclear (breast cancer type 1 susceptibility protein, BRCA1, and apolipoprotein B, Apob) DNA sequence data to assess the degree of genetic divergence between two of its three major proposed subspecies: Arabian (P. a. dorsalis) and Northwest African (P. a. deserti); this is the first molecular evaluation of the taxonomy of P. aethiopicus. Phylogenetic analyses, comparison of interspecific and intraspecific genetic distances observed across hedgehog species, and molecular species delimitation methods (distance-based clustering and tree-based), all indicate a level of genetic differentiation between dorsalis and deserti that is compatible with their taxonomic separation. Their divergence in the studied genes were consistently comparable to, or greater than, several intrageneric and a few intergeneric distances in hedgehogs. The Cyt b net Kimura 2-parameter distance between dorsalis and deserti was 10.8±1.3%, which is about the mean between congeneric species in reviews of Cyt b distances for mammals. This study, as a test of the genetic distinctiveness of dorsalis and deserti, suggests that they represent evolutionarily significant units and flags them for future phylogeographic and taxonomic investigations.

Identification of four Indian ascidians based on COI gene sequences

DNA barcoding involving the sequencing of a short mitochondrial DNA segment, cytochrome c oxidase subunit 1 (COI) gene, is a specialized technique for the identification of species even at the early embryonic and larval stages, which is quite difficult in morphology-based taxonomy. Ascidians are sessile invertebrate chordates possessing numerous biochemical as well as pharmacological activities. In this study, a total of 36 ascidian samples belonging to the family Didemnidae were sequenced for a 650 bp region of the mitochondrial COI gene. All the species were represented by multiple specimens. The barcode sequences showed no stop-codons and indels in the alignments. The aligned sequences were submitted in Barcode submission tool, NCBI, and the accession numbers were obtained. The minimum intraspecific distance was found to be 0.00% and the maximum was 2.23%. Mean Kimura 2-parameter (K2P) distances within-species, genus, and family were 0.88, 5.98, and 20.03%, respectively. The mean interspecific distance is more than the mean intraspecific divergence, which indicates efficiency of DNA barcoding for identification of ascidians.

Cosmopolitan or Cryptic Species? A Case Study of Capitella teleta (Annelida: Capitellidae).

Capitella teleta Blake et al., 2009 is an opportunistic capitellid originally described from Massachusetts (USA), but also reported from the Mediterranean, NW Atlantic, and North Pacific, including Japan. This putatively wide distribution had not been tested with DNA sequence data; intraspecific variation in morphological characters diagnostic for the species had not been assessed with specimens from non-type localities, and the species status of the Japanese population(s) was uncertain. We examined the morphology and mitochondrial COI (cytochrome c oxidase subunit I) gene sequences of Capitella specimens from two localities (Ainan and Gamo) in Japan. Specimens from Ainan and Gamo differed from C. teleta from Massachusetts in methyl-green staining pattern, shape of the genital spines, and shape of the capillary chaetae; we concluded that these characters vary intraspecifically. Species delimitation analyses of COI sequences suggested that worms from Ainan and Massachusetts represent C. teleta; these populations share a COI haplotype. The specimens from Gamo may represent a distinct species and comprise a sister group to C. teleta s. str.; we refer to the Gamo population as Capitella aff. teleta. The average Kimura 2-parameter (K2P) distance between C. teleta s. str. and C. aff. teleta was 3.7%. The COI data indicate that C. teleta actually occurs in both the NW Atlantic and NW Pacific. Given the short planktonic larval duration of C. teleta, this broad distribution may have resulted from anthropogenic dispersal.

Identification of Sphaeroma terebrans via morphology and the mitochondrial cytochrome c oxidase subunit I (COI) gene.

Sphaeroma terebrans, a wood-boring isopoda, is distributed worldwide in tropical and subtropical mangroves. The taxonomy of S. terebrans is usually based on morphological characteristics, with its molecular identification still poorly understood. The number of teeth on the uropodal exopod and the length of the propodus of the seventh pereopod are considered as the major morphological characteristics in S. terebrans, which can cause difficulty in regards to accurate identification. In this study, we identified S. terebrans via molecular and morphological data. Furthermore, the validity of the mitochondrial cytochrome c oxidase subunit I (COI) gene as a DNA barcode for the identification of genus Sphaeroma, including species S. terebrans, S. retrolaeve, and S. serratum, was examined. The mitochondrial COI gene sequences of all specimens were sequenced and analysed. The interspecific Kimura 2-parameter distances were higher than intraspecific distances and no intraspecific-interspecific distance overlaps were observed. In addition, genetic distance and nucleotide diversity (π) exhibited no differences within S. terebrans. Our results revealed that the mitochondrial COI gene can serve as a valid DNA barcode for the identification of S. terebrans. Furthermore, the number of teeth on the uropodal exopod and the length of the propodus of the seventh pereopod were found to be unreliable taxonomic characteristics for S. terebrans.

DNA barcoding of Clarias gariepinus, Coptodon zillii and Sarotherodon melanotheron from Southwestern Nigeria

DNA barcoding has been adopted as a gold standard rapid, precise and unifying identification system for animal species and provides a database of genetic sequences that can be used as a tool for universal species identification. In this study, we employed mitochondrial genes 16S rRNA (16S) and cytochrome oxidase subunit I (COI) for the identification of some Nigerian freshwater catfish and Tilapia species. Approximately 655 bp were amplified from the 5′ region of the mitochondrial cytochrome C oxidase subunit I (COI) gene whereas 570 bp were amplified for the 16S rRNA gene. Nucleotide divergences among sequences were estimated based on Kimura 2-parameter distances and the genetic relationships were assessed by constructing phylogenetic trees using the neighbour-joining (NJ) and maximum likelihood (ML) methods. Analyses of consensus barcode sequences for each species, and alignment of individual sequences from within a given species revealed highly consistent barcodes (99% similarity on average), which could be compared with deposited sequences in public databases. The nucleotide distance between species belonging to different genera based on COI ranged from 0.17% between Sarotherodon melanotheron and Coptodon zillii to 0.49% between Clarias gariepinus and C. zillii, indicating that S. melanotheron and C. zillii are closely related. Based on the data obtained, the utility of COI gene was confirmed in accurate identification of three fish species from Southwest Nigeria.

DNA barcoding of Clarias gariepinus, Coptodon zillii and Sarotherodon melanotheron from Southwestern Nigeria.

DNA barcoding has been adopted as a gold standard rapid, precise and unifying identification system for animal species and provides a database of genetic sequences that can be used as a tool for universal species identification. In this study, we employed mitochondrial genes 16S rRNA (16S) and cytochrome oxidase subunit I (COI) for the identification of some Nigerian freshwater catfish and Tilapia species. Approximately 655 bp were amplified from the 5' region of the mitochondrial cytochrome C oxidase subunit I (COI) gene whereas 570 bp were amplified for the 16S rRNA gene. Nucleotide divergences among sequences were estimated based on Kimura 2-parameter distances and the genetic relationships were assessed by constructing phylogenetic trees using the neighbour-joining (NJ) and maximum likelihood (ML) methods. Analyses of consensus barcode sequences for each species, and alignment of individual sequences from within a given species revealed highly consistent barcodes (99% similarity on average), which could be compared with deposited sequences in public databases. The nucleotide distance between species belonging to different genera based on COI ranged from 0.17% between Sarotherodonmelanotheron and Coptodon zillii to 0.49% between Clarias gariepinus and C. zillii, indicating that S. melanotheron and C. zillii are closely related. Based on the data obtained, the utility of COI gene was confirmed in accurate identification of three fish species from Southwest Nigeria.

Estimating diversity of crabs (Decapoda: Brachyura) in a no-take marine protected area of the SW Atlantic coast through DNA barcoding of larvae

DNA barcoding was used to identify crab larvae from the Marine Biological Reserve of Arvoredo, encompassing a coastal archipelago off the SW Atlantic coast (27°S, 48°W). Partial mitochondrial COI or 16S rRNA gene sequences were obtained for 488 larvae, leading to the identification of 20 species. The COI sequences generated 13 barcode index numbers (BINs) within Barcode of Life Data Systems (BOLD), among which 11 were concordant with single species. DNA from ∼ 6% of the larvae did not amplify using the primers tested; based on external morphological characteristics, these larvae represented four possible additional operational taxonomic units (OTUs) at the family level. Intraspecific variation for the COI and 16S rRNA genes was found to be < 2.6% and < 2.1% respectively (Kimura 2-parameter distance), whereas interspecific divergence ranged from 7.9% to 21.5% and 6.4% to 14.5%, respectively. These results imply that both genes are suitable for use in species identification of brachyuran crabs of this area. Molecular identification of this group successfully enabled the diagnosis of larvae of closely related species, including congeners in Mithrax, Achelous and Callinectes. In addition, eight out of 20 species recognized represent new records for the reserve suggesting that the brachyuran fauna in the area has been underestimated based on traditional biodiversity measures. The availability of primers suited to the targeted species, and the development of a taxonomically comprehensive DNA barcoding database are the major recommendations to improve the accuracy and feasibility of using DNA barcoding for species identification of SW Atlantic brachyuran crabs.

Identification of Tibetan medicine "Dida" of Gentianaceae using DNA barcoding

The ITS2 barcode was used toidentify Tibetan medicine "Dida", and tosecure its quality and safety in medication. A total of 13 species, 151 experimental samples for the study from the Tibetan Plateau, including Gentianaceae Swertia, Halenia, Gentianopsis, Comastoma, Lomatogonium ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V3.7.1. The Kimura 2-Parameter (K2P) distances were calculated using MEGA 6.0. The neighbor-joining (NJ) phylogenetic trees were constructed. There are 31 haplotypes among 231 bp after alignment of all ITS2 sequence haplotypes, and the average G±C content of 61.40%. The NJ tree strongly supported that every species clustered into their own clade and high identification success rate, except that Swertia bifolia and Swertia wolfangiana could not be distinguished from each other based on the sequence divergences. DNA barcoding could be used as a fast and accurate identification method to distinguish Tibetan medicine "Dida" to ensure its safe use.

Genetic and chemical discrimination of traditional Tibetan medicine seabuckthorn based on DNA barcode and 1H-NMR metabolic method

To differentiate three medicinal Hippopahe species of seabuckthorn, a combined genetic and chemical identification method was established in this study. ITS2 and psbA-trnH were tested for identification of 3 species of seabuckthorn. Detection of the kimura 2-parameter (K2P) distance, the neighbor-joining (NJ) tree and the barcoding gap were used to assess the identification efficiency. ¹H-NMR based metabolic method was applied to acquire the profile of metabolites. PCA was used to analysis the metabolite data. The results indicated that DNA barcode combined ¹H-NMR based metabolic method is a powerful tool for the identification of 3 medicinal Hippopahe species of seabuckthorn. The finding demonstrated that different genetic variation and chemical constituents existed among 3 medicinal Hippopahe species of seabuckthorn. The combined identification method will improve the reliability of species discrimination and could be applicable to much other ethnic medicine which has various origins in China.

Identification, Discrimination, and Discovery of Species of Marine Planktonic Ostracods Using DNA Barcodes.

The Ostracoda (Crustacea; Class Ostracoda) is a diverse, frequently abundant, and ecologically important component of the marine zooplankton assemblage. There are more than 200 described species of marine planktonic ostracods, many of which (especially conspecific species) can be identified only by microscopic examination and dissection of fragile morphological characters. Given the complexity of species identification and increasing lack of expert taxonomists, DNA barcodes (short DNA sequences for species discrimination and identification) are particularly useful and necessary. Results are reported from analysis of 210 specimens of 78 species of marine planktonic ostracods, including two novel species, and 51 species for which barcodes have not been previously published. Specimens were collected during 2006 to 2008 from the Atlantic, Indian, and Southern Oceans, Greenland Sea and Gulf of Alaska. Samples were collected from surface to 5,000 m using various collection devices. DNA sequence variation was analyzed for a 598 base-pair region of the mitochondrial cytochrome oxidase subunit I (COI) gene. Kimura-2-Parameter (K2P) genetic distances within described species (mean = 0.010 ± 0.017 SD) were significantly smaller than between species (0.260 + 0.080), excluding eight taxa hypothesized to comprise cryptic species due to morphological variation (especially different size forms) and/or collection from different geographic regions. These taxa showed similar K2P distance values within (0.014 + 0.026) and between (0.221 ± 0.068) species. All K2P distances > 0.1 resulted from comparisons between identified or cryptic species, with no overlap between intra- and interspecific genetic distances. A Neighbor Joining tree resolved nearly all described species analyzed, with multiple sequences forming monophyletic clusters with high bootstrap values (typically 99%). Based on taxonomically and geographically extensive sampling and analysis (albeit with small sample sizes), the COI barcode region was shown to be a valuable character for discrimination, recognition, identification, and discovery of species of marine planktonic ostracods.

DNA Barcode for Identifying Folium Artemisiae Argyi from Counterfeits.

Folium Artemisiae Argyi is an important herb in traditional Chinese medicine. It is commonly used in moxibustion, medicine, etc. However, identifying Artemisia argyi is difficult because this herb exhibits similar morphological characteristics to closely related species and counterfeits. To verify the applicability of DNA barcoding, ITS2 and psbA-trnH were used to identify A. argyi from 15 closely related species and counterfeits. Results indicated that total DNA was easily extracted from all the samples and that both ITS2 and psbA-trnH fragments can be easily amplified. ITS2 was a more ideal barcode than psbA-trnH and ITS2+psbA-trnH to identify A. argyi from closely related species and counterfeits on the basis of sequence character, genetic distance, and tree methods. The sequence length was 225 bp for the 56 ITS2 sequences of A. argyi, and no variable site was detected. For the ITS2 sequences, A. capillaris, A. anomala, A. annua, A. igniaria, A. maximowicziana, A. princeps, Dendranthema vestitum, and D. indicum had single nucleotide polymorphisms (SNPs). The intraspecific Kimura 2-Parameter distance was zero, which is lower than the minimum interspecific distance (0.005). A. argyi, the closely related species, and counterfeits, except for Artemisia maximowicziana and Artemisia sieversiana, were separated into pairs of divergent clusters by using the neighbor joining, maximum parsimony, and maximum likelihood tree methods. Thus, the ITS2 sequence was an ideal barcode to identify A. argyi from closely related species and counterfeits to ensure the safe use of this plant.