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DNA barcoding of fishes from Zhoushan coastal waters using mitochondrial COI and 12S rRNA genes

Journal of Oceanology and Limnology 2023年第5期41卷 1997-2009页

作者： Yehui WANG Na SONG Shude LIU Zhi CHEN Anle XU Tianxiang GAO Fisheries College Ocean University of ChinaQingdao 266003China Shandong Fisheries Development and Resources Conservation Center Yantai 264003China College of Fisheries and Life Science Hainan Tropical Ocean UniversitySanya 572022China School of Fishery Zhejiang Ocean UniversityZhoushan 316000China

Accurate species identification is a key component of biodiversity research.DNA barcoding is an effective molecular method used for fish species identification.We aimed to study the DNA barcoding of fish in Zhoushan coastal waters,explore the differences and applicability of two gene fragments(12S rRNA and COI)of DNA barcoding in fish species identification,and established a comprehensive fish barcoding reference database.Two hundred and eighty-seven captured fish samples from Zhoushan coastal waters were identified using morphological characteristics and DNA barcoding.A total of 26412S rRNA sequences(belonging to eight orders,31 families,55 genera,and 66 species)and 188 COI sequences(belonging to seven orders,30 families,48 genera,and 58 species)were obtained.The lengths of the 12S rRNA sequences ranged from 165 to 178 bp,and the guanine-cytosine(GC)content was 45.37%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.10%and 26.66%,respectively.The length of the COI sequence ranged 574–655 bp,and the content of GC was 45.97%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.16%and 27.45%,respectively.The minimum interspecific genetic distances of 12S rRNA and COI(1.23%and 1.86%)were both greater than their maximum intraspecific genetic distances(2.42%and 8.66%).Three molecular analyses(NJ tree,ABGD,and GMYC)were performed to accurately identify and delineate species.Clustering errors occurred when the 12S rRNA sequences were delimited using the NJ tree method,and the delimitation results of ABGD and GMYC are consistent with the final species identification results.Our results demonstrate that DNA barcoding based on 12S rRNA and COI can be used as an effective tool for fish species identification,and 12S rRNA has good application prospects in the environmental dna(edna)metabarcoding of marine fish.

关键词： DNA barcoding cytochrome c oxidase subunit I(COI) 12S rRNA fish identification species delimitation Zhoushan coastal waters

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Discovery of Cladonema multiramosum sp.nov.(Cnidaria:Hydrozoa:Cladonematidae)using DNA barcoding and life cycle analyses

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Acta Oceanologica Sinica 2022年第3期41卷 44-52页

作者： Konglin Zhou Yuping Gu Lu Wang Jianming Chen Institute of Oceanography College of Geography and OceanographyMinjiang UniversityFuzhou 350108China

In contrast to typical planktonic hydromedusae,Cladonema medusae are mostly benthic,with specialised adhesive branches to adhere to the substrate.In this study,a Cladonema species discovered in a laboratory aquarium in Fuzhou,China,was confirmed as a new species,based on morphological and molecular analyses.The species was named Cladonema multiramosum sp.nov.Its medusa is distinct from that of congeners possessing substantially more adhesive branches(8-24,rarely 5-7),and tiny branches on the upper radial canals.The validity of C.multiramosumum sp.***.was also supported by molecular phylogenetic analyses based on the mitochondrial 16S rdna sequence.However,C.multiramosum sp.***.medusa also displayed considerable phenotypic plasticity with respect to its radial canals,tentacles,stinging branches per tentacle,oral tentacles,manubrium,and gonads,hindering species identification based solely on morphology.Although some morphological characteristics of hydroids(filiform tentacles and medusa buds)and nematocysts could also be used as diagnostic characters in the genus Cladonema,this information is unavailable for some Cladonema species.Thus,the taxonomy within the genus Cladonema was re-evaluated based mainly on the morphological characteristics of the medusa.Further revision of the genus Cladonema should be made when supplementary information on the life cycle and DNA barcoding are updated.

关键词： Cladonema morphology life cycle DNA barcoding 16S rdna

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Identification and DNA barcoding of a New Sillago Species in Beihai and Zhanjiang,China,with a Key to Related Species

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Journal of Ocean University of China 2022年第5期21卷 1334-1342页

作者： YU Zhengsen GUO Ting XIAO Jiaguang SONG Na GAO Tianxiang Fishery College Ocean University of ChinaQingdao 266003China Laboratory of Marine Biology and Ecology Third Institute of OceanographyMinistry of Natural ResourcesXiamen 361005China Fishery College Zhejiang Ocean UniversityZhoushan 316022China

A new Sillago species,Sillago parasihama sp.n.,is identified based on 127 specimens collected from the southern coast of China.We compared the morphological characters between Sillago parasihama and all other 11 Sillago species with two posterior extensions on the swim bladder.The new species is like S.sihama in the countable characters and color pattern,but is different from the latter by the distinct swim bladders.The swim bladder of S.parasihama is without lateral process.The posterior sub-extensions of anterolateral extensions are unique with some dendritic or sometimes stunted blind tubule,which are unilateral and outward,ex-tending along the abdominal,and are about one-third to half of the body of swim bladder in length.But the swim bladder of S.si-hama with 8-10 lateral processes,the posterior sub-extensions of anterolateral extensions are kinky,long and complicated,extend-ing along the abdominal wall below the peritoneum to the base of posterior extensions.S.parasihama can be distinguished from other species in this group by color pattern,meristic,and morphometric characters.Moreover,the results of genetic analysis using sequences of the mitochondrial cytochrome c oxidase subunit I(COI)fragment show significant interspecific-level genetic distances(0.159-0.231)between S.parasihama and 8 congeners in the group,which also support the validity of new species.We also provide a distribution map and a key of the related species.

关键词： new species Sillago parasihama sp.n. morphological characteristics DNA barcoding taxonomy

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DNA barcoding and molecular phylogeny of Dumasia(Fabaceae:Phaseoleae)reveals a cryptic lineage

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Plant Diversity 2020年第5期42卷 376-385页

作者： Kai-Wen Jiang Rong Zhang Zhong-Fu Zhang Bo Pan Bin Tian Key Laboratory of Biodiversity Conservation in Southwest China National Forestry and Grassland AdministrationSouthwest Forestry UniversityKunming650224China Germplasm Bank of Wild Species Kunming Institute of BotanyChinese Academy of SciencesKunming650201YunnanChina University of Chinese Academy of Sciences Beijing100049China Department of Wetland Southwest Forestry UniversityKunming650224China Center for Integrative Conservation Xishuangbanna Tropical Botanical GardenChinese Academy of SciencesMengla666303China Center of Conservation Biology Core Botanical GardensChinese Academy of SciencesMengla666303China Department of Botany and Biodiversity Research University of ViennaRennweg 14A-1030ViennaAustria

Dumasia taxonomy and classification have long been problematic.Species within this genus have few morphological differences and plants without flowers or fruits are difficult to accurately identify.In this study,we evaluated the ability of six DNA barcoding sequences,one nuclear(ITS)and five chloroplast regions(trnH-psbA,matK,rbcL,trnL-trnF,psbB-psbF),to efficiently identify Dumasia species.Most single markers or their combinations identify obvious barcoding gaps between intraspecific and interspecific genetic variation.Most combined analyses including ITS showed good species resolution and identification efficiency.We therefore suggest that ITS alone or a combination of ITS with any cpdna marker are most suitable for DNA barcoding of Dumasia.The phylogenetic analyses clearly indicated that Dumasia yunnanensis is not monophyletic and is separated as two independent branches,which may result from cryptic differentiation.Our results demonstrate that molecular data can deepen the comprehension of taxonomy of Dumasia and provide an efficient approach for identification of the species.

关键词： Cryptic species DNA barcoding Dumasia Internal transcribed spacer(ITS) Plastid genome

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Identification of three kinds of Plumeria flowers by DNA barcoding and HPLC specific chromatogram

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Journal of Pharmaceutical Analysis 2018年第3期8卷 176-180页

作者： Leilei Zhao Xiaoxue Yu Jie Shen Xinjun Xu School of Pharmaceutical Sciences Sun Yat-sen University Guangzhou 510006 China Guangdong Technology Research Center for Advanced Chinese Medicine Guangzhou China

DNA barcoding and HPLC specific chromatogram were used to identify three kinds of Plumeria flowers respectively. dnas extracted from the three Plumeria species were amplified by PCR with universal primers, and the psbA-trnH region was selected. All the amplified products were sequenced and the results were analyzed by MEGA 5.0. Chemometric methods including principal components analysis and hierarchical clustering analysis were conducted on the SAS 9.0 software to demonstrate the variability among samples. In conclusion, the psbA-trnH of all samples were successfully amplified from total dna and sequenced. These three varieties of Plumeria can be differentiated by the psbA-trnH region and clustered into three groups respectively through building neighbor joining tree, which conformed to their germplasm origins. However, it was hard to distinguish them by HPLC specific chromatograms combined with chemometrics analysis. These indicated that DNA barcoding was a promising and reliable tool for the identification of three kinds of Plumeria flowers compared to HPLC specific chromatogram generally used. It could be treated as a powerful complementary method for traditional authentication, especially for those varieties which are difficult to be identified by conventional chromatography.

关键词： Plumeria DNA barcoding HPLC specific chromatogram Chemometrics analysis Identification

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Morphology versus DNA barcoding： two sides of the same coin. A case study of Ceutorhynchus erysimi and C. contractus identification

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Insect Science 2016年第4期23卷 638-648页

作者： Svetlana Stepanovic Andrea Kosovac Oliver Krstic Jelena Jovic lvo Tosevski Faculty of Science Institute of Biology and Ecology University of Kragujevac 34000 Kragujevac Serbia Department of Plant Pests Institute for Plant Protection and Environment Banatska 33 11080 Zemun Serbia CABI 1 Rue des Grillons 2800 Delemont Switzerland

Genotyping of 2 well-known weevil species from the genus Ceutorhynchus （Coleoptera： Curculionidae） distributed in west Palearctic, C. erysimi and C. contractus, revealed phenotype versus genotype inconsistencies in a set of 56 specimens （25 C. erysimi and 31 C. contractus） collected from 25 locations in Serbia and Montenegro. An analysis of the mitochondrial cytochrome oxidase subunit I gene （CO1）, widely used as a barcoding region, and a nuclear gene, elongation factor-v （EF-1α）, revealed stable genetic divergence among these species. The average uncorrected pairwise distances for the CO1 and EF-1α genes were 3.8%, and 1.3%, respectively, indicating 2 genetically well-segregated species. However, the genetic data were not congruent with the phenotypic characteristics of the studied specimens. In the first place, C. erysimi genotypes were attached to specimens with phenotypic characteristics of C. contractus. Species-specific PCR-RFLP assays for the barcoding gene COIwere applied for the molecular identification of 101 additional specimens of both morphospecies （33 C. erysimi and 68 C. contractus） and were found to confirm this incongruity. The discrepancy between the genetic and morphological data raises the question of the accuracy of using a barcoding approach, as it may result in misleading conclusions about the taxonomic position of the studied organism. Additionally, the typological species concept shows considerable weakness when genetic data are not supported with phenotypic characteristics as in case of asymmetric introgression, which may cause certain problems, especially in applied studies such as biological control programs in which the biological properties of the studied organisms are the main focus.

关键词： Ceutorhynchus contractus Ceutorhynchus erysimi DNA barcoding molecular identification morphology PCR-RFLP

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dna-barcoding of Some Medicinal Plant Species in Saudi Arabia Using rbcL and matK Genes

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Phyton-International Journal of Experimental Botany 2020年第4期89卷 1059-1081页

作者： Abdulrahman Alasmari Department of Biology Faculty of ScienceUniversity of TabukTabuk71491Saudi Arabia

In the Kingdom of Saudi Arabia(KSA),thousands of plants are considered to have therapeutic value.The ambiguous use of identification mainly morphological characteristics of many plants has resulted in the adulteration and displacement of plant products which undermine their therapeutic value and weak documentation of plant resources.The aims of this study were therefore to evaluate genetic variability and explore the phylogeographic architecture for Saudi medicinal plant samples using rbcL and matK genes as barcodes for genomic identification.The matK and rbcL sequences collected for these samples were used as key markers for examining the relationship between Saudi medicinal plant species based on genetic diversity.During our study we were successful in identifying and documenting 4 different species(Foeniculum vulgare,Nitraria retusa,Dodonaea viscosa,and Rumex nervosus)located in Saudi Arabia using DNA barcoding technique.A total number of 8 sequences were obtained with a total sequence length of 6176 bp,where it ranged from 617 bp to 878 bp with an aver-age length of 772 bp.The total number of rbcL sequences length is 2801 bp,where it ranges from 617 bp to 807 bp with an average length of 700.2 bp.Out of the 4 plant samples used,only three samples were identified correctly on the species level with an identity percentage higher than 95%using rbcL gene.Additionally,4 matK sequences have been retrieved belong to 4 species.The total number of matK sequences length is 3375 bp,where it ranges from 819 bp to 878 bp with an average length of 843.8 bp.Out of the 4 plant samples used,only two samples were identified correctly on the species level with an identity percentage higher than 98% using matK gene.Both rbcL and matK have been able to identify most of our collected plant samples by genus,and some by species.Using only one dna-barcoding technique was not reliable for plant identification,where matK and rbcL must be used as a dual dna-barcoding procedure.

关键词： Phylogeographic architecture genomic identification DNA barcoding

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Species delimitation in the green algal genus Codium (Bryopsidales) from Korea using DNA barcoding

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Acta Oceanologica Sinica 2015年第4期34卷 114-124页

作者： LEE Hyung Woo KIM Myung Sook Department of Biology Jeju National University

Codium, one of the largest marine green algal genera, is difficult to delimit species boundary accurately based on morphological identification only. DNA barcoding is a powerful tool for discriminating species of seaweeds. The plastid elongation factor TU （tufA） is considered as maker to perform DNA barcoding of green algal species than rbcL gene due to universality and rapid evolution rate. We conducted DNA barcoding application to Codium specimens from the Jeju Island, Korea to overcome the limit of morphological identification and to confirm the species diversity. As a result of applying tufA marker, we newly generated fifty-five tufA barcodes to resolve eight species. TufA marker exhibited 6.1%–21.8% interspecific divergences, wider than the gap of rbcL exon 1, 3.5%–11.5%. Molecular analysis of rbcL exon 1 sequences of Codium revealed eight distinct species like tufA analysis separated in five phylogenetic groups. DNA barcoding of the genus Codium using tufA marker is more helpful to overcome the limit of morphological identification, and this is more potential to reveal cryptic species and to resolve the relationships among subspecies than rbcL analysis alone. The complement of tufA barcoding and rbcL analyses including morphology for the genus Codium in the northwestern Pacific will give much more reliable achievement for discovering species diversity and resolving the phylogenetic relationships.

关键词： Codium DNA barcoding rbcL Species delimitation tufA

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First record of abnormal body coloration in a rockfish Sebastes koreanus(Scorpaenoidei:Sebastidae)from coastal water of China based on morphological characteristics and DNA barcoding

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Journal of Oceanology and Limnology 2024年第2期42卷 640-646页

作者： Ang LI Huan WANG Changting AN Shufang LIU Yellow Sea Fisheries Research Institute Chinese Academy of Fishery SciencesQingdao 266071China Laboratory for Marine Fisheries Science and Food Production Processes Laoshan LaboratoryQingdao 266071China

The first record of abnormal body coloration in Sebastes koreanus Kim and Lee,1994,from the Yellow Sea of China,was documented based on morphological characteristics and DNA barcoding.The two rockfish specimens were collected from the coastal waters of Qingdao,China,and the whole body and all fins of them were red.Of the two red-colored rockfish,there were tiny deep red spots on each fin,2 red radial stripes behind and below the eyes and 1 large deep red blotch on the opercula,while the similar stripe and spot patterns are also present in the S.koreanus specimens with normal body coloration.The countable characteristics of the two specimens are in the range of the morphometry of S.koreanus.To further clarify the species identity and taxonomic status of the two specimens,dna barcode analysis was carried out.The genetic distance between the red-colored rockfish and S.koreanus was 0,and the minimum net genetic distances between the red-colored rockfish and other Sebastes species except for S.koreanus were 3.0%,which exceeds the threshold of species delimitation.The phylogenetic analysis showed that the DNA barcoding sequences of the two red-colored rockfish clustered with the S.koreanus sequences.The above results of dna barcode analysis also support that the two red-colored rockfish could be identified as the species of S.koreanus.The mechanism of color variation in S.koreanus is desirable for further research and the species could be an ideal model to study the color-driven speciation of the rockfishes.

关键词： abnormal body coloration Sebastes koreanus coastal water of China Yellow Sea morphological characteristics DNA barcoding

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16S rRNA is a better choice than COI for DNA barcoding hydrozoans in the coastal waters of China

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Acta Oceanologica Sinica 2014年第4期33卷 55-76页

作者： ZHENG Lianming HE Jinru LIN Yuanshao CAO Wenqing ZHANG Wenjing College of Ocean and Earth Sciences Xiamen University Marine Biodiversity and Global Change Research Center Xiamen University Fujian Provincial Key Laboratory for Coastal Ecology and Environmental Studies (CEES) XiamenUniversity

Identification of hydrozoan species is challenging, even for taxonomic experts, due to the scarcity of distinct morphological characters and phenotypic plasticity. DNA barcoding provides an efficient method for species identification, however, the choice between mitochondrial cytochrome c oxidase subunit I（COI） and large subunit ribosomal RNA gene（16S） as a standard barcode for hydrozoans is subject to debate. Herein, we directly compared the barcode potential of COI and 16S in hydrozoans using 339 sequences from 47 pelagic hydrozoan species. Analysis of Kimura 2-parameter genetic distances（K2P） documented the mean intraspecific/interspecific variation for COI and 16S to be 0.004/0.204 and 0.003/0.223, respectively. An obvious ＂barcoding gap＂ was detected for all species in both markers and all individuals of a species clustered together in both the COI and 16S trees. These results suggested that the species within the studied taxa can be efficiently and accurately identified by COI and 16S. Furthermore, our results confirmed that 16S was a better phylogenetic marker for hydrozoans at the genus level, and in some cases at the family level. Considering the resolution and effectiveness for barcoding and phylogenetic analyses of Hydrozoa, we strongly recommend 16S as the standard barcode for hydrozoans.

关键词： DNA barcoding hydrozoan COI 16S rRNA

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