我国近海浮游动物物种信息平台的构建及初步应用
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摘要
浮游动物是海洋生态系统物质和能量传递过程中的最重要一环,在海洋食物网中承担着枢纽的作用,将营养物质和能量从低营养级传至高营养级,其种类组成和数量变动会对整个生态系统产生影响。浮游动物的多样性和分布对海洋生态系统研究非常重要,其主要技术基础是浮游动物鉴定。传统鉴定方法是镜检计数,费时费力,对相关人员的技能要求很高,而当浮游动物处于幼体阶段的时候甚至无法以传统方法鉴定。因此,推进浮游动物鉴定规范化、快速化和提高鉴定的准确性非常重要。
本研究通过标准化的浮游动物图像与分子信息采集方法,有机结合传统形态学分类方法和现代分子生物学技术进行物种鉴定,推动了包括浮游动物形态分类信息、图像信息、生物条形码基因序列信息的我国近海浮游动物物种信息平台的建立。该平台通过建立中国近海浮游动物图像库,推动浮游动物鉴定传统方法的信息交流；通过建立DNA条形码数据库,发展前瞻性的以浮游动物DNA条形码为基础的物种多样性研究方法,推动中国近海常见浮游动物的形态和分子鉴定工作。本研究所建立的中国近海浮游动物图像库,得到7个门、26个目、68科、135个属、246个种合计1162张图片浮游动物实物图片。结合DNA条形码研究过程中遇到的问题,本研究分析了Acartia属、Pleuromamma属、原Eucalanidae科(陈清潮,1965)的一些种类的mtCOI序列多样性。来自南海同一个站位的小纺锤水蚤(Acartia negligens)、来自不同大洋的帽形真哲水蚤(Subeucalanus pileatus)和北方乳点水蚤(Pleuromamma borealis),来自中国东海和日本沿岸的(Pleuromamma xiphis)和鼻锚哲水蚤(Rhincalanus nasutus)位于NJ树的不同分支上,意味着它们在大的地理尺度上具较大的种内遗传差异。
在已有的浮游动物条形码数据库的基础上,为研究浮游动物物种组成,分别构建了胶州湾和长江口的环境样品DNA条形码文库。在胶州湾,从文库中共得到149条mtCOI序列。同种内个体间的K2P遗传距离在0到0.11之间,平均值为0.013(SE=0.003),同时,属内的种间遗传距离在0.137到0.369之间,平均值为0.265(SE=0.002)。因此种间遗传距离比种内遗传距离大了至少20倍,表明条形码序列的存在,验证了mtCOI序列用于鉴定浮游动物群落组成是可靠的。以6%作为阈值,鉴定出37个OTUs,对这些OTUs的BLAST分析结果和来自同一站位的样品进行形态学鉴定的结果进行了比对,验证了研究方法的有效性。
在长江口,研究共得到856条mtCOI片段。基因差异达到5%作为鉴定为不同种类的界限,共有70个OTUs(种类)被鉴定出来。所有种类的单系性得到了检验。将OTUs的BLAST结果与长江口种类多样性的形态学结果进行了对比。通过DNA条形码研究被鉴定出来的非终生浮游动物的浮游阶段是处于其幼体阶段。DNA条形码鉴定的结果和形态学结果是基本上一致的,在区分姊妹种方面要优于传统方法。本研究证明了借助综合性数据库,运用环境DNA条形码技术可以准确地估计浮游动物种类多样性。
Zooplankton is the most important component in material exchange and energy transfer process of marine ecosystem. It is joint of the marine food web, transporting nutrient and energy from lower trophic level to higher trophic level. The succession and quantitative changes of species can affect the whole marine ecosystem. The identification of zooplankton is the technical base of diversity and distribution of zooplankton, so it is very important to study of marine ecosystem. Traditional identifying method is examining specimen under microscope, requiring fine skill of trained identifier and time consuming. The larva may not be identified by the traditional method. Thus, promoting the identificating methods to enhance precision and reduce the consuming time of identification, is very important.
Combining the traditional identifying methods and molecular biological technique, this study constructed the foundation of Chinese Offshore Zooplankton Species Information Platform. The platform of Chinese Zooplankton information, containing monophyly information, the images of the specimen, and the DNA barcoding sequence information, was builded by the study through standard technique for gathering images of zooplankton and their sequence. The Chinese Offshore Zooplankton Species Image Database of the platform improved communication of traditional zooplankton identifing information. The DNA barcoding Sequence Database of the platform promoted the metagenomic methods of biodiversity study based on the DNA barcoding technique. In all, the platform would improve the identification based on the morphological and DNA barcoding. The library of offshore zooplankton images of China of this platform contained1086images. These pictures belonged to246species,135genus,68families,26orders, and7phylum. Based on the analyses of DNA barcodes, mtCOI sequences of various zooplanktonic species were revised. These species belong to genus Acartia, Pleuromamma, and family Eucalanidae (as Chen Qingchao'literature in1965). Acartia negligens from the same location of the South China Sea, Subeucalanus pileatus and Pleuromamma borealis from different oceans, Pleuromamma xiphis and Rhincalanus nasutus from East China Sea and from the offshore of Japan, fell into different branchs of NJ tree, which meant they have high intraspecific genetic divergence at large geographical scale.
Based on the available zooplankton barcode database, an environmental DNA barcode library was constructed to study zooplankton composition in Jiaozhou Bay and the Changjiang estuary. In Jiaozhou Bay, A total of149mtCOI sequences were determined from the coxl gene secquences library. K2P genetic distances between individuals within the same species ranged from0to0.11, with an average of0.013(SE=0.003); meanwhile, genetic distance between species within the same genera ranged from0.137to0.369, with an average of0.265(SE=0.002). Thus the average of genetic distance between species was at least20times larger than that within species, indicating a barcode gap which ensured that mtCOI sequences can be used as reliable characters for species identification for the zooplankton assemblages. The genetic divergence of6%was adopted,37OTUs (species) was estimated. The results of BLAST of the OTUs were compared with the morphology identification work on species richness of sample from the same location of Jiaozhou Bay.The validity of the study method was assured.
In Yangzi Estuary, a total of856mitochondrial coxl gene sequences from4samples of different location were determined from the coxl gene library. The occurrence of70OTUs (species) was estimated when the genetic divergence of5%was adopted as the threshold to separate different species. The monophyly of all the species was also resolved. The results of BLAST of the OTUs were compared with the morphological study on species richness of Changjiang Estuary. The nonholozooplanktonic species identified by the DNA barcodes here were supposed to be sampled with the dispersal life history phase.The result was basically consistent with the result indentified by Morphology characters. The DNA barcoding method was better in identifying sibling species. With comprehensive database, fast estimation of zooplankton richness can be performed by the environmental DNA barcoding approach.
本研究通过标准化的浮游动物图像与分子信息采集方法,有机结合传统形态学分类方法和现代分子生物学技术进行物种鉴定,推动了包括浮游动物形态分类信息、图像信息、生物条形码基因序列信息的我国近海浮游动物物种信息平台的建立。该平台通过建立中国近海浮游动物图像库,推动浮游动物鉴定传统方法的信息交流；通过建立DNA条形码数据库,发展前瞻性的以浮游动物DNA条形码为基础的物种多样性研究方法,推动中国近海常见浮游动物的形态和分子鉴定工作。本研究所建立的中国近海浮游动物图像库,得到7个门、26个目、68科、135个属、246个种合计1162张图片浮游动物实物图片。结合DNA条形码研究过程中遇到的问题,本研究分析了Acartia属、Pleuromamma属、原Eucalanidae科(陈清潮,1965)的一些种类的mtCOI序列多样性。来自南海同一个站位的小纺锤水蚤(Acartia negligens)、来自不同大洋的帽形真哲水蚤(Subeucalanus pileatus)和北方乳点水蚤(Pleuromamma borealis),来自中国东海和日本沿岸的(Pleuromamma xiphis)和鼻锚哲水蚤(Rhincalanus nasutus)位于NJ树的不同分支上,意味着它们在大的地理尺度上具较大的种内遗传差异。
在已有的浮游动物条形码数据库的基础上,为研究浮游动物物种组成,分别构建了胶州湾和长江口的环境样品DNA条形码文库。在胶州湾,从文库中共得到149条mtCOI序列。同种内个体间的K2P遗传距离在0到0.11之间,平均值为0.013(SE=0.003),同时,属内的种间遗传距离在0.137到0.369之间,平均值为0.265(SE=0.002)。因此种间遗传距离比种内遗传距离大了至少20倍,表明条形码序列的存在,验证了mtCOI序列用于鉴定浮游动物群落组成是可靠的。以6%作为阈值,鉴定出37个OTUs,对这些OTUs的BLAST分析结果和来自同一站位的样品进行形态学鉴定的结果进行了比对,验证了研究方法的有效性。
在长江口,研究共得到856条mtCOI片段。基因差异达到5%作为鉴定为不同种类的界限,共有70个OTUs(种类)被鉴定出来。所有种类的单系性得到了检验。将OTUs的BLAST结果与长江口种类多样性的形态学结果进行了对比。通过DNA条形码研究被鉴定出来的非终生浮游动物的浮游阶段是处于其幼体阶段。DNA条形码鉴定的结果和形态学结果是基本上一致的,在区分姊妹种方面要优于传统方法。本研究证明了借助综合性数据库,运用环境DNA条形码技术可以准确地估计浮游动物种类多样性。
Zooplankton is the most important component in material exchange and energy transfer process of marine ecosystem. It is joint of the marine food web, transporting nutrient and energy from lower trophic level to higher trophic level. The succession and quantitative changes of species can affect the whole marine ecosystem. The identification of zooplankton is the technical base of diversity and distribution of zooplankton, so it is very important to study of marine ecosystem. Traditional identifying method is examining specimen under microscope, requiring fine skill of trained identifier and time consuming. The larva may not be identified by the traditional method. Thus, promoting the identificating methods to enhance precision and reduce the consuming time of identification, is very important.
Combining the traditional identifying methods and molecular biological technique, this study constructed the foundation of Chinese Offshore Zooplankton Species Information Platform. The platform of Chinese Zooplankton information, containing monophyly information, the images of the specimen, and the DNA barcoding sequence information, was builded by the study through standard technique for gathering images of zooplankton and their sequence. The Chinese Offshore Zooplankton Species Image Database of the platform improved communication of traditional zooplankton identifing information. The DNA barcoding Sequence Database of the platform promoted the metagenomic methods of biodiversity study based on the DNA barcoding technique. In all, the platform would improve the identification based on the morphological and DNA barcoding. The library of offshore zooplankton images of China of this platform contained1086images. These pictures belonged to246species,135genus,68families,26orders, and7phylum. Based on the analyses of DNA barcodes, mtCOI sequences of various zooplanktonic species were revised. These species belong to genus Acartia, Pleuromamma, and family Eucalanidae (as Chen Qingchao'literature in1965). Acartia negligens from the same location of the South China Sea, Subeucalanus pileatus and Pleuromamma borealis from different oceans, Pleuromamma xiphis and Rhincalanus nasutus from East China Sea and from the offshore of Japan, fell into different branchs of NJ tree, which meant they have high intraspecific genetic divergence at large geographical scale.
Based on the available zooplankton barcode database, an environmental DNA barcode library was constructed to study zooplankton composition in Jiaozhou Bay and the Changjiang estuary. In Jiaozhou Bay, A total of149mtCOI sequences were determined from the coxl gene secquences library. K2P genetic distances between individuals within the same species ranged from0to0.11, with an average of0.013(SE=0.003); meanwhile, genetic distance between species within the same genera ranged from0.137to0.369, with an average of0.265(SE=0.002). Thus the average of genetic distance between species was at least20times larger than that within species, indicating a barcode gap which ensured that mtCOI sequences can be used as reliable characters for species identification for the zooplankton assemblages. The genetic divergence of6%was adopted,37OTUs (species) was estimated. The results of BLAST of the OTUs were compared with the morphology identification work on species richness of sample from the same location of Jiaozhou Bay.The validity of the study method was assured.
In Yangzi Estuary, a total of856mitochondrial coxl gene sequences from4samples of different location were determined from the coxl gene library. The occurrence of70OTUs (species) was estimated when the genetic divergence of5%was adopted as the threshold to separate different species. The monophyly of all the species was also resolved. The results of BLAST of the OTUs were compared with the morphological study on species richness of Changjiang Estuary. The nonholozooplanktonic species identified by the DNA barcodes here were supposed to be sampled with the dispersal life history phase.The result was basically consistent with the result indentified by Morphology characters. The DNA barcoding method was better in identifying sibling species. With comprehensive database, fast estimation of zooplankton richness can be performed by the environmental DNA barcoding approach.
引文
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