The Morphylogy, Genetic Difference And Phylogenetic Analysis Of Several Typical Nanoplanktonic Diatom Species In China Sea

The marine nanoplanktonic diatoms are important component of marine phytoplankton and marine resources,and some species can cause red tide.In the diatoms,Skeletonema,Thalassiosira and Chaetoceros are relatively common red tide causative species.The study on the classification and genetic characteristic of these importantly dominant nanoplanktonic diatom species is the base of marine ecology and red tide research with emphasis on the analysis of genetic diversity of inter-species,intra-species,and geographical genetic differentiation of typical species.This dissertation focused on Skeletonema,Thalassiosira and Chaetoceros as the typical genus of nanoplanktonic diatoms along the coast of China.The single isolates of the diatoms were isolated and cultivated.And the morphological studies were carried out using LM,SEM,and TEM.These studies include the morphological observation under the electron microscope,the description of morphological characteristics and classification.And then the SSU nrDNA and LSU nrDNA were used as DNA marker for molecular studies.The main purpose of this dissertation centered on the morphological classification and molecular identification of these typical nanoplanktonic diatoms,and analyzed the interspecies difference,intraspecies difference,geographical genetic difference,and phylogenetic relation.The main results were showed as follows:1.The morphology,genetic difference and the phylogenetic analysis on four species of SkeletonemanrDNA sequences analysis were carried out on 21 strains of Skeletonema isolated from the coast of China including Shenzhen,Hongkong,Qingdao,North Huanghai sea,Xiamen,Yangtse River estuary,Guangzhou etc.And what should be mentioned is that one of the strains is from Korea.According to the morphological characteristics, four species were identified:Skeletonema marinoi Sarno et Zingone,Skeletonema dohrnii Sarno et Kooistra,Skeletonema subsalsum(Cleve) Bethge and Skeletonema tropicum Cleve.The analyses of the nrDNA sequences and the phylogenetic tree indicated that S.subsalsum and S.tropicum were monophyletic and the S.marinoi and S.dohrnii cannot be distinguished.The latter may be attributed to the very small morphological difference between S.marinoi and S.dohrnii,which is further confirmed by the high similarity of SSU nrDNA sequences between them(above 99.62%).Nevertheless,the similarity of SSU nrDNA between other species was about 98.37%～98.93%.The similarity of LSU nrDNA between the the S.marinoi and S.dohrnii was above 99.11%,markedly higher than the similarity,about 92.47%～96.3%,between other species.2.The morphology,genetic difference and the phylogenetic analysis on eight species of ThalassiosiraThe morphological study and nrDNA sequences analysis were carried out on 20 strains of Thalassiosira which were mainly isolated from Hongkong,Hainan, Zhoushan,Taiwan Strait,Jiaozhou Bay etc.Based on the traditionally morphological characteristics,eight Thalassiosira species were identified:Thalassiosira minima Gaarder,Thalassiosira weissflogii(Grun.)Fryxell & Hasle,Thalassiosira rotula Meunier,Thalassiosira subtilis(Ostenfele) Gran,Thalassiosira diporocyclus Hasle, Thalassiosira pseudonana Hasle et Heimdal,Thalassiosira mala Takano and Thalassiosira allenii Takano.On the molecular level,the analyses of the nrDNA sequences and the phylogeny showed that T.subtilis,T.pseudonana,T.weissflogii,T. allenii,T.minima,T.mala and T.diporocyclus were monophyletic.And T.weissflogii was the earliest divergence from other species,which suggested that the genetic distance between T.weissflogii and other seven species was comparatively large.SSU and LSU sequences analysis both revealed that the difference between the T. diporocyclus and other seven species was most distinct.The most distinct difference was between the T.weissflogii and the T.diporocyclus(SSU nrDNA,6.57%;LSU nrDNA,12.82%),while,the least was between the T.rotula and T.ubtilis(SSU nrDNA,1.52%;LSU nrDNA,1.71%).Both SSU and LSU sequence implied that difference between T.ubtilis and T.seudonana was not distinct(2.00%～2.60%). According to the SSU nrDNA gene analysis,the difference of Thalassiosira species under this study was from 0.13%to 2.53%,which was coherent to the LSU nrDNA gene analysis.3.The morphology,genetic difference and phylogenetic analysis of two species of ChaetocerosThe morphological study an nrDNA sequences analysis were carried out on four strains of Chaetoceros isolated from the Hongkong,Hainan,Xiamen,Huanghai etc.. According to the traditionally morphological characteristics,two species were identified:Chaetoceros curvisetius Cleve and Chaetoceros brevis Schütt.On the molecular level,the analysis results of the nrDNA sequences and the phylogenetic tree indicated that the difference between C curvisetius and C brevis,based on the nrDNA gene analysis,was from 6.64%to 10.77%,while within C curvisetius species, the difference was from 0.18%to 0.59%.The nucleotide polymorphism existed in the SSU nrDNA gene sequences of species of Chaetoceros.The average nucleotide polymorphism of C curvisetius approximated to 0.45%.According to the LSU nrDNA gene sequences,the difference between C curvisetius and C brevis was from 17.15%～19.42%and the difference also existed in the strains from different locations.