【作者】 张辉；

【导师】 高天翔； 张岩；

【作者基本信息】 中国海洋大学 ， 渔业资源， 2013， 博士

【摘要】 许氏平鲉和褐菖鲉为西北太平洋两种重要的卵胎生海洋经济鱼类。本研究综合运用形态学、耳石形态学、线粒体DNA控制区序列、AFLP标记和微卫星标记等多种方法，系统开展了许氏平鲉和褐菖鲉的遗传多样性和群体遗传结构及分子系统地理格局研究。主要研究结果如下：一、许氏平鲉分子系统地理学研究（1）应用单因子方差分析、判别分析和主成分分析等统计分析方法对采自烟台、连云港、日照、丹东、青岛、大连、威海、胶南和日本青森9个许氏平鲉群体244个个体的15个可量性状和11个可数性状进行比较研究。单因子方差分析结果显示日本青森群体与中国群体间存在较大差异，8个中国群体之间形态差异不显著；判别分析结果显示青森群体判别正确率为96.0%，9个群体综合判别正确率为80.7%；主成分分析结果显示前4个主成分累积贡献率为72.3%，前2个主成分主要反映鱼体头部和躯干部形态差异。（2）对中国近海8个许氏平鲉群体共228个个体的9个耳石形状指标（圆度、形态因子、环率、矩形趋近率、椭圆率、半径比、Feret比、幅形比、面密度）进行单因子方差分析、判别分析、主成分分析等统计分析，并与傅里叶分析结果进行比较。单因子方差分析结果显示，丹东群体和日照群体的9个指标存在显著差异，荣成群体和胶南、青岛、乳山三个群体分别存在9个指标的显著差异，胶南群体和乳山群体存在9个指标差异不显著（P>0.05）；第一主成分和第二主成分解释了约72%的形态差异；9个指标在判别分析中的综合判别正确率为64.9%。傅里叶分析结果显示综合判别正确率为43.8%，较第一种方法降低21.1%，9个主成分指标解释了89.2%的形态差异。多元统计分析及傅里叶分析结果均表明中国近海许氏平鲉群体之间形态差异不显著。（3）对许氏平鲉线粒体控制区全序列结构进行了分析。我们检测到了两个TAS和cTAS及保守的CSB-F、CSB-E、CSB-D、CSB1、CSB2和CSB3区。结果表明，许氏平鲉线粒体控制区序列结构与大部分海洋鱼类类似，不同的是含有两个TAS和cTAS区。所有个体的CSB1、CSB2和CSB3序列分别一致，这与大多数硬骨鱼类结果并不一致。（4）许氏平鲉是能够利用漂流海藻群生活的代表性鱼类之一，随着海藻群漂流的仔稚鱼使许氏平鲉各群体之间能够进行基因交流。我们对中日沿海的13个许氏平鲉群体进行了控制区序列分析，发现各群体的遗传多样性中等偏高。NJ系统发育树未显示明显的谱系结构，AMOVA分析、FST和确切P检验结果表明许氏平鲉各群体之间的遗传差异较小，LAMARC结果也提示各群体之间存在较强的基因流。核苷酸不配对分布和中性检验表明许氏平鲉经历了更新世晚期的群体扩张事件。（5）对许氏平鲉9个群体共180个个体的AFLP分析结果显示，许氏平鲉遗传多样性较高；AMOVA分析和群体间FST值的结果显示许氏平鲉在其分布范围内不存在显著的遗传结构，9个群体间的遗传分化水平均较低；基于Nei遗传距离构建的UPGMA树中没有检测到与采样地点相对应的分支，不存在明显的谱系结构；STRUCTURE分析结果显示所有许氏平鲉群体共享一个基因库。（6）利用8对微卫星引物对中日沿海9个许氏平鲉群体的220个个体进行了遗传多样性和群体遗传结构研究。结果表明：许氏平鲉群体的观测杂合度和多态位点信息含量值较高，呈现较高的遗传多样性。AMOVA分析、FST和确切P检验结果与AFLP结果相同，也显示许氏平鲉各群体之间的遗传差异较小；基于Nei遗传距离构建的UPGMA树中没有检测到与采样地点相对应的分支，不存在明显的谱系结构；STRUCTURE结果显示所有许氏平鲉群体共享一个基因库。二、褐菖鲉分子系统地理学研究（1）应用统计分析方法对采自中国沿海（惠州和海口）和日本沿海（横须贺和伯方岛）褐菖鲉群体的14个可量性状和11个可数性状特征进行比较研究。单因子方差分析结果显示伯方岛群体和横须贺群体间的9个指标差异不显著，与惠州群体的12个指标存在显著性差异；判别分析结果显示惠州群体判别正确率为100%，4个群体综合判别正确率为90.35%；主成分分析结果显示前4个主成分累积贡献率为53.98%，第一主成分主要反映鱼体躯干部特征，第二主成分主要反映鱼体头部和尾部特征；聚类分析结果显示日本群体聚为一支，中国群体聚为另一支。结果表明褐菖鲉中、日群体间形态差异显著。（2）利用统计分析方法对中、日4个褐菖鲉群体耳石的9个形状指标和77个椭圆傅里叶参数进行分析。判别分析结果显示中、日褐菖鲉群体判别正确率在75%~100%之间，综合判别正确率为91.1%；聚类分析结果显示中国惠州和海口的褐菖鲉群体聚为一支，日本横须贺和伯方岛群体聚为另一支；主成分分析结果显示较少的耳石形态变量即可用于褐菖鲉群体分化研究。结果表明中、日褐菖鲉群体在耳石形态上差异显著。（3）对中日沿海的16个褐菖鲉群体进行了控制区序列分析，结果发现各群体的遗传多样性中等偏高。NJ系统发育关系未呈现明显的谱系结构；AMOVA分析、FST和确切P检验结果显示许氏平鲉各群体之间的遗传差异较小，LAMARC结果提示各群体间存在较强的基因流。核苷酸不配对分布和中性检验表明褐菖鲉经历了更新世晚期的群体扩张事件。（4）对褐菖鲉8个群体共144个个体进行的AFLP分析结果显示，许氏平鲉具有较高的遗传多样性；AMOVA分析和群体间FST值的结果显示许氏平鲉分布范围内不存在显著的遗传结构，8个群体之间的遗传分化较小；基于Nei遗传距离构建的UPGMA树中没有检测到与采样地点相对应的分支，不存在明显的谱系结构；STRUCTURE分析结果显示所有许氏平鲉群体共享一个基因库。（5）运用高通量测序技术，筛选得到48对褐菖鲉微卫星引物，其中9对具有较高的种内多态。对威海群体的24个褐菖鲉个体进行了微卫星分析，结果表明：9对微卫星引物的等位基因数目为4-12，观测杂合度在0.190-0.625之间；其中6对引物显著偏离哈迪温伯格平衡，经检测是由于无效等位基因引起的。因此，上述9对引物适合进一步开展褐菖鲉的群体遗传学研究。三、基于DNA条形码的平鲉属鱼类鉴定有效性研究DNA条形码在物种鉴定中发挥了越来越重要的作用。本研究利用DNA条形码对中国沿海8种平鲉属鱼类进行鉴定。结果表明：8种平鲉属鱼类种内遗传距离小于种间差异水平，符合10×法则；基于NJ法、MP法、ML法以及贝叶斯方法构建的系统发育树显示各鱼种以较高的支持率聚为一支。除条平鲉（S.trivittatus）外，基于COI基因序列构建的系统树与形态学研究结果相一致。更多还原

【Abstract】 Sebastes schlegelii and Sebastiscus marmoratus are commercially importantmarine ovoviviparous fish in the Northwestern Pacific. In the present study,morphological, otolith morphological, mitochondrial DNA sequence, AFLP markersand microsatellite DNA markers were used to estimate their gene diversity, populationstructure and phylogeographic pattern. The main results are as follows:（A）Molecular phylogeography of Sebastes schlegelii（1）In order to study the morphological variations among nine populations of S.schlegelii (244individuals) collected from Yantai, Lianyungang, Rizhao, Dandong,Qingdao, Dalian, Weihai, Jiaonan and Aomori, eleven meristic characters and fifteenmeasureable characters were measured and the data were standardized by usinglog-transformed method and then analyzed by using one-way ANOVA, discriminantanalysis method and principal component analysis method. One-way ANOVA resultsshowed that there were no significant differences between eight Chinese populations,but significant differences were found between Aomori population and eight Chinesepopulations. Discriminant analysis results showed that the correct classification ratiowas96.0%in Aomori population, and the cumulative contribution ratio was80.7%. Inthe result of principal component analysis, four principal components explained72.3%of the total variance; the first two principal components reflected the morphologicaldifference of fish’s head and trunk. The results of multivariate statistical analysismethods showed that there were significant differences between Aomori populationand eight Chinese populations. （2）Multivariate statistical analysis that includes one way analysis of variance,discriminant analysis and principle analysis was conducted for9otolith shape indicesof228individuals of Chinese S. schlegelii. Fourier analysis was used to be comparedwith shape indices method. One way analysis of variance showed significantdifferences were found between Dandong population and Rizhao population in9otolith shape indices; Rongcheng population showed significant differences in9otolith shape indices from Jiaonan, Qingdao, and Rushan population, respectively.While insignificant differences were found between Rushan and Jiaonan in their9otolith shape indices (P>0.05); the first two principle components explained71.953%of the total variance; the discriminant analysis of the9shape indices showed that thecorrect classification was64.9%among the8populations. Fourier analysis showedthat the correct classification was43.8%, which was21.1%lower than that of shapeindices analysis; the first9principle components explained89.2%of the totalvariance. Three processes of multivariate statistical analysis and Fourier analysisshowed that there was insignificant difference between eight populations of Chinese S.schlegelii.（3）The present study deals with the structure of mitochondrial DNA controlregion of the black rockfish, S. schlegelii. Two TASs and two cTASs were detectedin the species as well as CSB-F, CSB-E, CSB-D, CSB1, CSB2and CSB3. The resultsindicated that the structures of these blocks are similar with most marine fishes, but itis special that there are two TASs and two cTASs in the control region of S. schlegelii.One conserved region was found from450bp to the end of the control region, which isalso a special feature of S. schlegelii. All sequences of CSB1, CSB2and CSB3blocks were the consensus among different individuals, which is quite different frommost vertebrates. In addition, the complete mtDNA control region sequences and thefirst449bp of the control region are used to analyze the phylogenetic relationships ofS. schlegelii. The phylogenetic trees show a lack of genetic structure amongindividuals. This study also indicated that the genetic diversity is similar between thewild and cultured individuals, which are important to the fisheries management. （4）S. schlegelii is one of the representative fish that aggregate around driftingseaweed during its early developmental period. Transport of drifting seaweed byocean currents probably gives larval and juvenile S. schlegelii fish an opportunity toexpand their distribution area and to exchange genetic material between widelyseparated populations. To examine the population genetic structure of S. schlegelii, a452-bp fragment of the hypervariable portion of the mtDNA control region wassequenced and used to interpret life history characteristics and larval dispersalstrategy as well as its historical demography. Two hundred and twenty one individualsfrom13sites along the coasts of China, Japan and Korea were analyzed. Both thelevels of haplotype diversity and nucleotide diversity were not very high. Theneighbor-joining tree and the network showed that no significant genealogicalstructures corresponding to sampling locations existed. AMOVA, pair-wise FSTandexact-P test revealed no significant genetic differentiation among populations. Themigration rate among populations was strong based on the result of LAMARC. Bothmismatch distribution analysis and neutrality tests showed S. schlegelii haveexperienced a recent population expansion in the late Pleistocene. It is suggestedlarval dispersal with drifting seaweed, the current environmental factors and latePleistocene glaciations play an important role in shaping the contemporaryphylo-geographic pattern of S. schlegelii.（5）AFLP marker was used to investigate the population genetic structure of S.schlegelii among populations from China and Japan. The results indicated S.schlegelii showed high Nei’s gene diversity. No significant genealogical branches orclusters corresponding to sampling localities were detected by UPGMA tree. Theresults of AMOVA analysis and pairwise FSTvalues showed no genetic divergenceamong different geographic populations and high gene flow was existed. The result ofSTRUCTURE showed all the populations of S. schlegelii shared one gene pool. Theresults of AFLP were in accordance with those of mtDNA.（6）Eight microsatellite DNAloci were used to investigate the genetic diversityand population structure of S. schlegelii. The results indicated polymorphism information content (PIC) and observed heterozygosity were high, indicating a highlevel in gene diversity of S. schlegelii. No significant genealogical branches orclusters corresponding to sampling localities were detected by UPGMA tree. Theresults of AMOVA analysis and pairwise FSTvalues showed no genetic divergenceamong different geographic populations and high gene flow was existed. The result ofSTRUCTURE showed all the populations of S. schlegelii shared one gene pool. Theresults of AFLP were in accordance with the results of mtDNA and AFLP.（B）Molecular phylogeography of Sebastiscus marmoratus（1）In order to study the morphological variations among seven populations ofS. marmoratus (114individuals) collected from China (Huizhou and Haikou) andJapan (Hakata Island and Yokosuka), eleven meristic characters and fourteenmorphological characteristics were measured and the data were analyzed by usingone-way ANOVA, discriminant analysis method, principal component analysismethod and cluster analysis method. One-way ANOVA results showed that there wereno significant differences between Hakata Island and Yokosuka in nine indices, butsignificant differences were found between Hakata Island and Huizhou in twelveindices. Discriminant analysis results showed that the correct classification ratio was100%in Huizhou, and the cumulative contribution ratio was90.35%. In the result ofprincipal component analysis, four principal components explained53.977%of thetotal variance, the first principal component reflected the feature of fish’s trunk, andthe second principal component reflected the feature of fish’s head and rump. Clusteranalysis results showed that there were significant differences among populations inChina and Japan, Chinese and Japanese populations were pooled in one groupseparately. The results of multivariate statistical analysis methods showed that therewere significant differences among populations in China and Japan.（2）In order to study the otolith morphological variations among fourpopulations of S. marmoratus (101individuals) collected from China (Huizhou andHaikou) and Japan (Hakata Island and Yokosuka), nine otolith shape index andseventy-seven elliptic Fourier parameters were measured. The data were analyzed by using discriminant analysis method, principal component analysis method and clusteranalysis method. The results of discriminant analysis showed that the correctclassification ratio ranged from75%to100%, and the average classification ratio was91.1%. The results of cluster analysis showed that there was significant differenceamong populations in China and Japan, and Chinese and Japanese populations werepooled in one group respectively. According to the result of principal componentanalysis, the divergence of populations of S. marmoratus could be measured by fewerotolith morphological variables. The results of multivariate statistical analysismethods showed that there was significant difference in otolith morphology amongpopulations in China and Japan.（3）To examine the population genetic structure of S. marmoratus, a458-bpfragment of the hypervariable portion of the mtDNA control region was sequencedand used to interpret life history characteristics and larval dispersal strategy as well asits historical demography. Two hundred and forty four individuals from16sites alongthe coasts of China and Japan were analyzed. Both the levels of haplotype diversityand nucleotide diversity were high. The neighbor-joining tree and the network showedthat no significant genealogical clades corresponding to sampling locations existed.AMOVA, pair-wise FSTand exact-P test revealed no significant genetic differentiationamong populations. Both mismatch distribution analysis and neutrality tests showed S.marmoratus have experienced a recent population expansion in the late Pleistocene.（4）AFLP was used to investigate the population genetic structure of S.marmoratus among populations from China and Japan. The results indicated S.schlegelii showed high Nei’s gene diversity. No significant genealogical branches orclusters corresponding to sampling localities were detected by UPGMA tree. Theresults of AMOVA analysis and pairwise FSTvalues showed no genetic divergenceamong different geographic populations and high gene flow was existed. The result ofSTRUCTURE showed all the populations of S. marmoratus shared one gene pool.The results of AFLP were in accordance with the results of mtDNA. （5）S. marmoratus is a typical sedentary fish and usually lives in the bottom ofthe coastal zone from Japan to the East China Sea. Next-generation sequencingplatform was used to obtain microsatellite loci sequences of S. marmoratus, andforty-eight perfect microsatellite repeats were suitable for primer design. Ninepolymorphic microsatellite markers were exploited and characterized for S.marmoratus. The allele number of all polymorphic microsatellite markers rangedfrom4to12. Expected and observed heterozygosity varied from0.302to0.909and0.190to0.625, respectively. Six of the nine markers deviated obviously from Hardy–Weinberg equilibrium (P<0.05). In further studies, the genetic variation andpopulation structure of this species would be investigated using these microsatelliteloci.（C）DNAbarcodes of eight species in genus SebastesDNA barcode is effective for biological taxonomy and is able to identifyingspecies from any life-history stage. In the present study, eight species which belong tofour different subgenera of genus Sebastes found in China sea waters were identifiedby cytochrome c oxidase I (COI) barcode. The results indicated that the intra-speciesvariation in DNA barcode was less than inter-species variation. When thephylogenetic trees were reconstructed by neighbor-joining (NJ), maximum parsimony(MP), maximum likelihood (ML) and Bayesian methods, all the species clustered intheir groups distinguishable by high bootstrap values, which proved that COI barcodeis a powerful means to differentiate between species of Sebastes and supports theiridentification. When the molecular tree was compared to the morphological tree, onlyS. trivittatus in subgenus Sebastocles settled in the different positions. It is suggestedthat S. trivittatus is one of the shallowest occurring species in the Northwest Pacificdue to its life characters.更多还原