Molecular Phylogeography Of Two Marine Ovoviviparous Fishes In Northwestern Pacific

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.