DNA Barcode Screening and Preliminary Study on Phylogeny of Sea Snake Based on Genes COI and cytb

[Objectives]The most common gene fragment used in animal DNA barcode technology is COI,but it is not necessarily suitable for all species.This study was conducted to screen genes suitable for the DNA barcode of sea snakes.[Methods]All COI and cytb gene sequences on GenBank were searched and downloaded.After the comparison with Mega software,clustering trees of MrBayes system were established.[Results]Interspecies distances were greater than intraspecies distances for the two genes.The topological structures of their molecular hierarchical clustering trees were clear,and the support rates were high.[Conclusions]Therefore,it is concluded that not the DNA barcode of each species must be gene COI.Cytb is more suitable in terms of the mitochondrial gene of sea snakes.

Genetic Structure of the Oriental River Prawn (Macrobrachium nipponense) from the Yangtze and Lancang Rivers, Inferred from COI Gene Sequence

This study analyzed nueleotide sequences from the mitochondrial eytochrome oxidase submit （COI） gene region （450 bp） to investigate the genetic structure of the oriental river prawn （ Macrobrachium nipponense ） among nine populations from the Yangtze and Lancang Rivers. A total of 79 individuals were collected for this work. Eighty-nine nucleotides were found to be variable, resulting in 46 haplotypes. Among the nine populations, the population from Kunming shows the greatest level of variability （h = 1.000, π = 0.028）, whereas the population from Cbongqing exhibits the lowest level of variability （h = 0.700,π = 0.008）. Analysis of molecular variance suggested that of the total genetic diversity, 9.66% was attributable to inter-population diversity and the remainder （90.34%） to differences within populations. A molecular phylogenetic tree constructed using the Neighbor-joining （N J） method showed that the 46 haplotypes were assigned to two clades associated with geographic regions. These results provide basic information for the conservation and sustainable exploitation of this species.

Phylogenetic Relationships of the Genus Meretrix (Mollusca: Veneridae) Based on Mitochondrial COI Gene Sequences

Fifteen sequences from the mitochondrial cytochrome c oxidase subunit I gene （COI） were determined for 4 species of the genus Meretrix, with the homologous sequences ofM. petechialis obtained from the GenBank data library The alignment length of the sequences was 574bp after excluding ambiguous sites, including 93 parsimony informative sites. In the fragments, the percentages of A, T, C and G were 21.15%, 44.71%, 14.05% and 20.09% respectively. There were 12 haplotypes identified： 4 M. meretrix, 2 M. IamarckiL 3 M. lusoria, 1 M. lyrata and 2 M. petechialis. Furthermore, it was revealed that M.. meretrix, M. petechialis and M. lusoria shared some haplotypes. Phylogeny trees were reconstructed by Maximum-parsimony （MP） and Bayesian method using Qylina sinensis as the outgroup. Our results indicated that M. lusoria, M. petechialis and M. meretrix are closely related species. This is in accordance with the viewpoint that M. petechialis and M. lusoria should be treated as a junior synonym of M. meretrix.

Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

Mitochondrial COI Gene Sequence Variation and Taxonomic Status of Three Macrobrachium Species

Freshwater prawns （Decapoda： Caridea： Macrobrachium） play an important role in domestic fishery resources. Culturing M. rosenbergii and M. nipponense brings great economic benefits, as the two species were widely farmed in China. M. qilianensis, a native species with natural distribution limited in Gansu province, was classified into genus Macrobrachium based on external morphological characters. In order to understand the molecular genetic differences among the three species of Macrobrachium, i.e., M. rosenbergii, M. nipponense, and M. qilianensis, we analyzed sequences of mitochondrial cytochrome oxidase subunit I （COI） of them. It would provide theoretical basis of exploiting and utilizing Macrobrachium resources rationally. A total of 30 individuals （10 individuals of each species） were collected from Gansu and Zhejiang province. Samples of M. qilianensis were wild, however, that of M. rosenbergii and M. nipponense were cultured. Their mitochondrial COI gene segment sequences were obtained by using the method of PCR amplification and sequencing. After alignment, 649 bp consensus sequences of COI were obtained. One hundred and sixty-nine variable sites were detected in all 30 individuals, accounting for 26.04% of total sequence. A total of seven haplotypes were also detected. Nucleotide diversity was 0.411% within M. rosenbergii, 0.092% within M. nipponense, and 0.031% within M. qilianensis. Genetic diversity of wild M. qilianensis was much lower than that of cultured M. rosenbergii and M. nipponense. Genetic distances between different haplotypes of the three prawns ranged from 19.87% to 23.84%. It suggested that the three species were valid species, because genetic distances among them were quite great. To further determine the taxonomic status of the three prawns in family Palaemonoidae, we downloaded the corresponding COI sequences of Palaemonoidae prawns from Genbank and analyzed the phylogenetic relationships of them. Phylogenetic tree （NJ） showed that M. nipponense, M. rosenbergii and other Macrobrachium species constituted one monophyletic group. However, M. qilianensis, Exopalaemon carinicauda, and Palaemon debilis formed the other clade. Thus, results of COI sequences did not support that M. qilianensis belonged to genus Macrobrachium. The taxonomic status ofM. qilianensis should be reevaluated with more comprehensive evidences.

Rapid Detection of Fox-derived Components using Mitochondrial COI Gene Sequence

Fox is an important economic fur animal with a very promising prospect. The fox mitochondrial COl gene-specific primers and probes were used to opti- mize and develop a real time fluorescence PCR system for rapid detection of fox-derived components. The results showed that the real time fluorescence PCR gave a very high sensitivity, with the detection limit as low as 12 fg DNA, and it did not present any cross reaction with other common species like pig, cow, sheep, dog, soybean and maize. It could be concluded that this method can be used as an effective method for detecting the fox-derived components in food and feedstuff, and can also be applied for distinguishing the fox fur from adulterants.

Genetic Analysis of Cultured and Wild Populations of Mytilus coruscus Based on Mitochondrial DNA

DNA sequences from the mitochondrial gene cytochrome oxidase subunit I （mtDNA CO I） were used to estimate the genetic variability in two wild populations and two cultured populations of the hard shelled mussel, Mytilus coruscus. Thirty haplotypes were identified in the four populations. The cultured populations exhibited a lower number of haplotypes and genetic diversity than those of the wild populations, suggesting that a small number of effective founding breeders contributed to the genetic variation of the cultured populations. No significant differentiation was observed between the cultured population and local wild population, implying that persistent gene flow occurred in these populations. This genetic survey is intended as a baseline for future genetic monitoring of M. coruseus aquaculture stocks.

Phylogeny of the subgenus Singhardina Mahmood (Hemiptera: Cicadellidae: Typhlocybinae: Typhlocybini) from China based on nuclear and mitochondrial gene sequences

Fragments of nuclear ribosomal 28S rDNA D2 divergent domain, mitochondrial 16S rDNA, and COI partial genes of 15 species in the subgenus Singhardina Mahmood from China were amplified and sequenced. Molecular phylogenetic trees were constructed using maximum parsimony, maximum likelihood and Bayesian inference methods. Results from these methods revealed similar topologies with recognizable relationships among subclades. The phylogenetic relationship of four species groups of subgenus Singhardina Mahmood from China is discussed for the first time. The results show that Singhardina Mahmood forms a single lineage representing a monophyletic group. The Eurhadina （Singhardina） rubra species group newly proposed in this study is likely the most basal species group within Singhardina Mahmood. The E. （Singhardina） robusta species group is the sister group of the E. （Singhardina） mamata species group. Molecular evidence supports including the E. （Singhardina） vittata species group in the E. （Singhardina） punjabensis species group.

Identification of Five Sea Cucumber Species Through PCR-RFLP Analysis

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene(COI) was used to identifing five sea cucumber species(Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31 I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

A new species in the genus Synanthedon(Lepidoptera:Sesiidae)from China

A new species,Synanthedon elaeagnus sp.nov.feeding on Elaeagnus mollis,is described from Shanxi,China.This new species is similar to Synanthedon ulmicola Yang&Wang,1989 in general morphology,but it differs from this latter species by features of the forewing,female and male genitalia.Neighbor-joining and maximum likelihood analysis both recover Synanthedon elaeagnus sp.nov.as a well-supported monophyletic lineage with a high support value based on COI gene sequences.The key features of this new species are illustrated.

DNA barcode assessment of Ceramiales(Rhodophyta) in the intertidal zone of the northwestern Yellow Sea

A total of 142 specimens of Ceramiales （Rhodophyta） were collected each month from October 2011 to November 2012 in the intertidal zone of the northwestern Yellow Sea. These specimens covered 21 species, 14 genera, and four families. Cluster analyses show that the specimens had a high diversity for the three DNA markers, namely, partial large subunit rRNA gene （LSU）, universal plastid amplicon （UPA）, and partial mitochondrial cytochrome c oxidase subunit I gene （COl）. No intraspecific divergence was found in our collection for these markers, except for a 1-3 bp divergence in the COI of Ceramium kondoi, Syrnphyocladia latiuscula, and Neosiphoniajaponica. Because short DNA markers were used, the phylogenetic relationships of higher taxonomic levels were hard to evaluate with poor branch support. More than half species of our collection failed to find their matched sequences owing to shortage information of DNA barcodes for macroalgae in GenBank or BOLD （Barcode of Life Data） Systems. Three specimens were presumed as Heterosiphonia crispella by cluster analyses on DNA barcodes assisted by morphological identification, which was the first record in the investigated area, implying that it might he a cryptic or invasive species in the coastal area of northwestern Yellow Sea. In the neighbor-joining trees of all three DNA markers, Heterosiphonia japonica converged with Dasya spp. and was distant from the other Heterosiphonia spp., implying that H.japonica had affinities to the genus Dasya. The LSU and UPA markers amplified and sequenced easier than the COI marker across the Ceramiales species, but the COI had a higher ability to discriminate between species.

The Taxonomic Status of Japanese Threadfin Bream Nemipterus japonicus(Bloch, 1791)(Perciformes: Nemipteridae) with a Redescription of this Species from the South China Sea Based on Morphology and DNA Barcodes

Because of its importance as a food source, Nemipterus japonicus （Bloch, 1791） （Nemipteridae） or Japanese threadfin bream is the best studied of these taxa, and numerous investigations have examined its fisheries, its biology and biochemistry. De- spite such intensive work, the taxonomic status of N. japonicus has never been seriously questioned and it is regarded as a common species, widely distributed throughout the Indo-Westem Pacific Ocean. In fact, Bloch＇s description of the type specimen of N.ja- ponicus has ambiguous collection data and lacks a designation for the type locality, though it is probably Java. In this paper, DNA barcode results based on COl gene support the existence of two geographically separated lineages of the Japanese threadfin bream, both being an Indian Ocean and western Pacific lineage, with 2.7% sequence divergence, and the results indicate a possible existing of some cryptic species. The two lineages also possess a diagnostic difference in their belly color, with specimens in the South China Sea having a silver belly, while those from the Indian Ocean isolate specimen have a yellow coloration. Based upon new collections from the South China Sea, this species from the western Pacific is morphologically redescribed and its details of DNA barcode diver- sity are shown for the future investigations.

One new species and two new records in the subgenus Stegana(sensu stricto)(Diptera:Drosophilidae)from China,with DNA barcoding information

One new and two known species of the subgenus Stegana(s.str.)from China are described or redescribed:S.(S.)helvipecta sp.nov.,S.(S.)furta(Linnaeus,1767)and S.(S.)taba Okada,1971.A total of 41 DNA sequences of partial mitochondrial cytochrome c oxidase subunit I(COI)gene of 18 species of this subgenus are newly obtained in this study.These sequences along with other available barcoding sequences for this subgenus are incorporated in a molecular analysis using the neighbor-joining(NJ)method in order to assess the efficacy of DNA barcoding for delimiting these species.Habitus and a key to all Chinese Stegana(s.str.)species are provided.

Genetic differentiation of Bemisia tabaci （Gennadius） （Hemiptera： Aleyrodidae） biotype Q based on mitochondrial DNA markers

In the present study, genetic differentiation of Bemisia tabaci （Gennadius） biotype Q was analyzed based on mitochondrial cytochrome oxidase I （mt CO1） gene sequence. The results showed that B. tabaci biotype Q could be separated into two subclades, which were labeled as subclades Q1 and Q2. Subclade Q1 was probably indigenous to the regions around the Mediterranean area and subclade Q2 to Israel or Cyprus. It was because B. tabaci was composed of several genetically distinct groups with a strong geographical association between more closely related biotypes. Not all of the B. tabaci biotype Q in the non-Mediterranean countries come from the same regions. Until now, all B. tabaci biotype Q in China were grouped into subclade Q 1. The B. tabaci biotype Q introduced into the US included both subclades Q 1 and Q2. The genetic structure analysis showed higher genetic variation of subclade Q 1 than that of subclade Q2.

Phylogeny of the green lacewing Chrysoperla nipponensis species-complex （Neuroptera： Chrysopidae）in China, based on mitochondrial sequences and AFLP data

Severalstudieshaveindicatedthatthegreenlacewing, Chrysoperlanipponensis （Neuroptera： Chrysopidae） may include more than one valid species. We investigated the phylogenetic status of Chrysoperla nipponensis s.l. in China and Japan using mitochondrial sequences and AFLP data. The molecular phylogenetic analyses based on mitochondrial genes showed that the C. nipponensis species-complex comprises four clades, each having high support values. In addition, the phylogenetic tree based on AFLP data indicates that the species-complex comprises three groups. These results confirm that C. nipponensis s.1. comprises at least three genetically distinct clades and suggests that two of these clades may be closely related to populations of C. nipponensis in Japan. However, these clades cannot be recognized as species until analysis of their courtshin songs has been cornnleted