马氏珠母贝肠道及其养殖水体可培养细菌群落结构

【目的】了解马氏珠母贝(Pinctada fucata martensii)肠道及其养殖水体可培养细菌的群落组成。【方法】采用2216E平板涂布法研究海区养殖马氏珠母贝肠道与养殖水体的可培养菌群种类及丰度。【结果与结论】马氏珠母贝肠道及其养殖水体的可培养细菌归属于2门(变形菌门和厚壁菌门)3纲7目10科23属56种。属水平上,肠道中以弧菌属(74.7%)和假交替单胞菌属(18.7%)为主;养殖水体中α-变形菌纲的FJ943236_g属(40.7%)丰度最大,弧菌属(16.7%)相对肠道丰度较低。样品共有菌属为弧菌属、假交替单胞菌属、发光杆菌属和芽孢杆菌属;肠道特有菌属为希瓦氏菌属和盐单胞菌属;养殖水体特有菌属主要为FJ943236_g、鲁杰氏菌属和Nautella。在种水平上,7个种为二者共有;马氏珠母贝肠道和养殖水体特异性菌种分别为18个和31个。虽然门水平上马氏珠母贝肠道中可培养细菌群落与其养殖水体中的细菌群落大致相似,但在属、种水平上二者差异明显。     

Molecular identification and population differentiation of Aurelia spp. ephyrae in sea cucumber aquaculture ponds of northern China

Aurelia spp. ephyrae have been reported to form blooms in sea cucumber aquaculture ponds in the Bohai and Yellow Seas. To identify the species, we carried out a genetic analysis of Aurelia spp. ephyrae and medusae based on mitochondrial 16 S rRNA gene. Samples offour Aurelia sp. ephyrae populations were collected in sea cucumber aquaculture ponds and samples offour Aurelia sp. medusae populations were collected in coastal waters. Using a BLASTn search, we found that both the ephyrae collected in the aquaculture ponds and medusae collected in coastal waters belong to Aurelia coerulea. Seventeen haplotypes were recovered from the 16 S rRNA gene. The overall haplotype diversity and nucleotide diversity of the 166 A. coerulea individuals were 0.686% and 0.329%, respectively, indicating high haplotype diversity and low nucleotide diversity. Moreover, the haplotype diversity of ephyrae populations were generally lower than that of medusae populations with close sampling points. The genetic differentiation between ephyrae populations collected in the sea cucumber aquaculture ponds and A. coerulea medusae collected in coastal waters was not significant, suggesting the ephyrae populations in the sea cucumber culture ponds were part of the same genetic group as the medusae populations in the coastal waters. Phylogeographic analysis of the 16 S rRNA region revealed that there was no significant correlation between the haplotypes and the geographic distribution of populations. Pairwise fixation index values showed significant genetic differentiation and limited gene flow between A. coerulea population of Weifang and other locations.     

钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析

利用线粒体16SrRNA基因和线粒体细胞色素氧化酶亚基Ⅰ（cvtocllrome oxidase Ⅰ,COI）基因片段初步研究钦州湾牡蛎（Ostxea）的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16SrRNA基因部分长度为413bp,COI基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A＋T比例：16SrRNA基因598％;COI基因605％。对位排序比较表明,16SrRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COI片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎（Crassostrea hongkongensis）16SrRNA和COI序列与白肉牡蛎的遗传距离均为0000,有明巨牡蛎（Crassostrea ariakensis）16SrRNA和COI序列和红肉牡蛎（redmeatostxea）的遗传距离分别为0000和0011,白肉牡蛎16SrRNA和COI序列和红肉牡蛎之间的遗传距离分别为0035和0146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16SrRNA和COI基因在种间存在明显的多态性,证实了16SrRNA和COI基因序列适用于牡蛎的系统学分析。     

Characterization of microbial communities in sediments of the South Yellow Sea

Illumina sequencing and quantitative PCR(qPCR) based on the 16 S ribosomal RNA(rRNA) gene were conducted to characterize the vertical distribution of bacterial and archaeal communities in the sediments of two sites from the South Yellow Sea. Both bacterial and archaeal communities showed a clear stratified distribution with sediment depth. The microbial communities in the upper layers were distinct from those in the deeper layers; the relative abundances of sequences of Thaumarchaeota, Gammaproteobacteria, and Actinobacteria were higher in the upper than in the deeper sediments, whereas the sequences of Bathyarchaeia, Lokiarchaeota, Euryarchaeota, Chloroflexi, and Deltaproteobacteria were relatively more abundant in the deeper sediments. Sediment depth and total organic carbon(TOC) can significantly influence both the bacterial and archaeal communities. Furthermore, bacterial and archaeal groups potentially involved in nitrogen, sulfur, and methane metabolism were detected in both sites. In our study, both ammonia-oxidizing bacteria( Nitrospira) and ammonia-oxidizing archaea( Candidatus Nitrosopumilus) were responsible for ammonia oxidization. Additionally, sulfur-reducing bacteria SEEP-SRB1 forming consortia with anaerobic methane-oxidizing archaea ANME-2 a-2 b were capable of anaerobic methane oxidation(AOM) in the 3400-02 sediment samples.     

Studies on Bioflocculant Production by Pseudoalteromonas sp.NUM8, a Marine Bacteria Isolated from the Circulating Seawater

A bioflocculant producing potential bacteria was isolated from the circulating seawater of bio-filter using streak plate methods. The bacteria was identified through biochemical characteristics, partial 16 S ribosomal ribonucleic acids(rRNA), nucleotide sequencing, and BLAST analysis of the gene sequence that showed the bacteria have 99% similarity to Pseudoalteromonas sp.and deposited in GenBank as Pseudoalteromonas sp. NUM8 with accession number JX435820. Influences of time course assay,carbon sources, nitrogen sources, inoculum size, as well as initial pH on the bacteria producing extracellular bioflocculant activity were investigated. The results showed that the strain optimal production period of microbial bioflocculant was at 72 h(flocculating activity of 94.5%), then dropped slowly. The bacteria optimally produced the bioflocculant when 1.0% sucrose and 0.5% sodium nitrate were used as sole sources of carbon and nitrogen with flocculating activities of 92.8% and 93.8% respectively. Also, the bacteria produced the bioflocculant optimally when initial pH of the medium was 5.0(flocculating activity 93.2%), and when Ca~(2+)was used as cation(flocculating activity 93.4%). The culture condition of inoculum size of 3%(v/v) was optimal flocculant production(flocculating activity 94.4%). Composition analyses indicated the bioflocculant to be principally a glycoprotein made up of about 34.3% protein and 63.4% total carbohydrate.     

钦州湾牡蛎线粒体16S rRNA和COⅠ基因片段的序列变异分析

利用线粒体16S rRNA基因和线粒体细胞色素氧化酶亚基Ⅰ(cytochrome oxidaseⅠ,COⅠ)基因片段初步研究钦州湾牡蛎(Ostrea)的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16S rRNA基因部分长度为413bp,COⅠ基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A+T比例:16S rRNA基因59.8%;COⅠ基因60.5%。对位排序比较表明,16S rRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COⅠ片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎(Crassostrea hongkongensis)16S rRNA和COⅠ序列与白肉牡蛎的遗传距离均为0.000,有明巨牡蛎(Crassostrea ariakensis)16S rRNA和COⅠ序列和红肉牡蛎(red meat ostrea)的遗传距离分别为0.000和0.011,白肉牡蛎16S rRNA和COⅠ序列和红肉牡蛎之间的遗传距离分别为0.035和0.146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16S rRNA和COⅠ基因在种间存在明显的多态性,证实了16S rRNA和COⅠ基因序列适用于牡蛎的系统学分析。     

Spatiotemporal distributions and environmental drivers of diversity and community structure of nosZ-type denitrifiers and anammox bacteria in sediments of the Bohai Sea and North Yellow Sea,China

Denitrification and anammox processes are major nitrogen removal processes in coastal ecosystems.However,the spatiotemporal dynamics and driving factors of the diversity and community structure of involved functional bacteria have not been well illustrated in coastal environments,especially in human-dominated ecosystems.In this study,we investigated the distributions of denitrifiers and anammox bacteria in the eutrophic Bohai Sea and the northern Yellow Sea of China in May and November of2012 by constructing clone libraries employing nosZ and 16 S rRNA gene biomarkers.The diversity of nosZ-denitrifier was much higher at the coastal sites compared with the central sites,but not significant among basins or seasons.Alphaproteobacteria were predominant and prevalent in the sediments,whereas Betaproteobacteria primarily occurred at the site near the Huanghe(Yellow)River estuary.Anammox bacteria Candidatus Scalindua was predominant in the sediments,and besides,Candidatus Brocadia and Candidatus Kuenenia were also detected at the site near the Huanghe River estuary that received strong riverine and anthropogenic impacts.Salinity was the most important in structuring communities of nosZdenitrifier and anammox bacteria.Additionally,anthropogenic perturbations(e.g.nitrogen overloading and consequent high primary productivity,and heavy metal discharges)contributed significantly to shaping community structures of denitrifier and anammox bacteria,suggesting that anthropogenic activities would influence and even change the ecological function of coastal ecosystems.     

蛤蜊科3种贝类16S rRNA基因片段及ITS2核苷酸序列分析

利用PCR技术分别扩增连云港及启东沿海蛤蜊科的西施舌(Coelomactra antiquata)、中国蛤蜊(Mactrachinensis)和四角蛤蜊(Mactra veneriformis)3种双壳贝的16S rRNA基因片段和ITS2核苷酸序列,测序后用DNA star软件分析了核苷酸差异。结果显示:三种贝类16S rRNA基因片段长度相同,均为306bp(去除引物),核苷酸存在多态性,共有45个变异位点,54个核苷酸发生了变异,全部为碱基置换。西施舌与中国蛤蜊此片段核苷酸的同源性为88.9%,与四角蛤蜊的同源性为88.6%,中国蛤蜊与四角蛤蜊的同源性为90.6%。三种蛤蜊ITS2序列分别为390 bp(西施舌)4、41 bp(四角蛤蜊)和466 bp(中国蛤蜊),存在长度多态性,ITS2核苷酸差异分析结果显示,西施舌与中国蛤蜊的同源性为70.9%-71.1%,西施舌与四角蛤蜊的为70.5%-71.0%,中国蛤蜊与四角蛤蜊的同源性为88.1%-88.8%。ITS2序列分析结果与16S rRNA基因片段分析结果一致,2种分子分析法均显示中国蛤蜊与四角蛤蜊的亲缘关系近。     

Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

Using shotgun sequencing data, the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao, China). There are two group I introns in the 16S rRNA gene, which is structurally similar to that of Caulerpa sertularioides (Bryopsidales, Chlorophyta). The chloroplast-encoded tufA gene sequence is 1 230 bp long, very AT-rich (61.5%), and is similar to previously published 16S rRNA sequences of bryopsidinean algae. Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae, Halimedineae, and Ostreobidineae are three distinct lineages. These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae. Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however, this clade lacked robust nodal support. Moreover, the phylogenetic tree inferred from rbcL GenBank sequences, combined with the geographical distributions of Bryopsis species, identified a strongly supportive clade for three differently distributed Asian Bryopsis species. The preliminary results suggesting that these organisms are of distinct regional endemism.     

Gel microbead cultivation with a subenrichment procedure can yield better bacterial cultivability from a seawater sample than standard plating method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%-100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%-100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.     

一株海水氨氧化细菌的系统发育分析

应用PCR技术扩增16S rRNA基因和amoA(ammonia monooxygenase subunit A)的基因片段,并测定其序列,对一株源于海水养殖水体的高效氨氧化细菌(ammonia-oxidizing bacteria,AOB)进行了系统发育分析。结果表明,经PCR扩增得到了1 098 bp的16S rRNA基因片段和491 bp的amoA基因片段,将其序列用NCBI-Blast软件在GenBank数据库中进行同源性检索后发现,该菌株的16S rRNA基因序列和amoA基因序列分别与亚硝化单胞菌Nitrosomonas sp.NS20的相对应基因片段相似性分别为98.4%和96.7%。在此基础上构建了系统发育树,表明用该菌株的16S rRNA基因片段和amoA基因片段构建的系统发育树均与亚硝化单胞菌属类聚在一起,结合该菌株形态和生理生化特性,鉴定该株氨氧化细菌属亚硝化单胞菌。     

Identification of Microplankton in South China Sea with Image-Matching Individual PCR

In this study, marine microplankton were identified by combining standard light microscopy with Sanger 18S rRNA gene sequencing. The image-matching individual PCR technique was applied to identify the image collectable unicellular microplankton to genera. Instead of pure strain culture and morphological identification, microplankton individual cells were isolated and fixed with glutaraldehyde, frozen and stored for months. Finally, they were imaged under a microscope and molecularly identified via phylogenetic analysis of their 18S ribosomal RNA gene(18S rDNA). Microplankton cells were collected at 30 locations in South China Sea, and were assigned to 21 known and 4 unidentified genera(2 uncultured fungi and 2 uncultured stramenopiles) with phylogenetic analysis in parallel to the morphological identification.     

Effects of a Potential Autochthonous Probiotic Bacillus subtilis 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber,Apostichopus japonicus Selenka

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber(Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0(control), 10~5, 10~7, and 10~9 CFU g~(-1) for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 10~9 CFU g~(-1)(P 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber(P 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 105 and 107 CFU g~(-1) inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 10~9 CFU g~(-1) not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.     

海洋弧菌鉴定与分类相关功能基因研究进展

【目的】探究功能基因在海洋弧菌物种鉴定中的作用,为海洋弧菌多样性研究以及致病性弧菌的监测和防控提供参考。【方法】根据已有的弧菌分类学研究成果,阐述16S rRNA基因、管家基因和毒力基因等相关功能基因在海洋弧菌鉴定与分类研究中的应用进展。【结果】16S rRNA基因和功能基因可用于海洋弧菌的分类学及其快速检测鉴定的研究,对致病性弧菌的诊治具有重要的意义。【结论】功能基因和16S rRNA基因在海洋弧菌菌种鉴定中的广泛应用,提高了海洋弧菌种间分类的准确性,对海洋弧菌的多样性研究以及水产养殖和人类健康的风险评估有重要价值。探索新型功能基因在海洋弧菌分类学研究和快速鉴定中的应用有现实意义。     

Distribution of nutrients and eutrophication assessment in the Bohai Sea of China

Water samples were collected in 120 stations in the Bohai Sea of China to analyze the distribution of dissolved nutrients and assess the degree of eutrophication in August 2002. The result shows that the average concentration of DIN increased and the PO4-P concentration sharply decreased compared to the previous data of corresponding period. The high concentrations of DIN and PO4-P occurred in coastal waters, especially in the bays and some river estuaries, while the high concentrations of SiO3-Si in the surface and middle depth occurred in the central area of the Bohai Sea. The average ratio of DIN/ PO4-P was much higher than the Redfield Ratio (16:1). Apparently, PO4-P was one of the limiting nutrient for phytoplankton growing in the sea. The average concentrations of DON and DOP were higher than their inorganic forms. The results of eutrophication assessment show that 22.1% of all stations were classified as violating the concentration levels of the National Seawater Quality Standard (GB 3097-1997) for DIN and only 3.9% for PO4-P. The average eutrophication index in the overall area was 0.21±0.22 and the high values occurred in Bohai Bay, Liaodong Bay and near the Yellow River estuary. This means that the state of eutrophication was generally mesotrophic in the Bohai Sea, but relatively worse in the bays, especially some river estuaries.     

Foraminiferal Changes and Response to the Channel Diversion and Discharge Fluctuation of the Yellow River in the North Yellow Sea During the Past Century

Diversion of the Yellow River is a unique geological event in offshore China, causing changes of the sedimentary environment in eastern China Seas. The last diversion took place in AD 1855, with the estuary diverted from the Yellow Sea into the Bohai Sea. The identification of the river diversion events in the shelf sediments would not only provide the definite ages for the sediments, but also give a clue for better understanding of the sedimentation in that area. In this study, 210 Pb, grain size, geochemical element, and foraminiferal data in core H205 from the north Yellow Sea were systematically investigated. A high-resolution sedimentary record was established, which was coupled with the Yellow River diversion and runoff changes. The results show that the foraminiferal composition and foraminiferal abundance of the sediments from the north Yellow Sea had good response to the Yellow River diversion in 1855. Before the change, shallow water assemblages dominated the foraminifera, and the abundance of each foraminiferal species was very low. After the diversion event, the abundance of most foraminifera increased sharply, with a maximum increase of 16 times, and the assemblage was still dominated by shallow water species. Furthermore, the changes in foraminiferal abundance in the core sediments corresponded well with the discharge fluctuation of the Yellow River since 1855. When the Yellow River began entering the Bohai Sea, the Yellow River water, which is rich in nutrients, along with the coastal currents affected the north Yellow Sea, increased the primary productivity in the north Yellow Sea, which is the main reason for the abrupt increase and fluctuation of foraminiferal abundance in this area. At the meantime, the East Asian winter monsoon could also promote the development of nearshore foraminiferal species by enhancing the coastal currents.     

3种星虫线粒体16S rRNA、COI和Cyt b基因片段的序列比较

对裸体方格星虫(Sipunculus nudus)、可口革囊星虫(Phascolosoma esculenta)和澳洲管体星虫(Siphonosoma australe)的线粒体16S rRNA、COI和细胞色素b(Cytb)基因片段序列进行比较,并对其系统发生进行了初步探讨。采用PCR方法得到总长度分别为531～544bp(16S)、652～675bp(COI)和406～453bp(Cytb)的线粒体片段。片段碱基A+T比例较高(16S rRNA基因58.3%,COI基因56.9%,Cytb基因59.5%)。16S rRNA片段存在169个碱基变异位点(其中包括167个简约信息位点)和44个碱基插入/缺失,种内个体间变异较小;COI片段有512个碱基(333个简约信息位点)存在变异,79个碱基插入/缺失;Cytb片段存在347个碱基(318个简约信息位点)变异位点,16个碱基插入/缺失。数据分析结果支持3种星虫和环节动物的分类地位较近,与软体动物较远的分类观点。此外,裸体方格星虫与澳洲管体星虫之间亲缘关系较近(D=0.3159、0.3156、0.2361)。认为3种星虫线粒体16S rRNA、COI和Cytb基因在种间存在明显的多态性,证实了三种基因序列均普遍适用于星虫种及以上阶元的系统学分析。     

Phylogenetic analysis of bacteria in sea ice brine sampled from the Canada Basin, Arctic Ocean

Bacterial diversity in sea ice brine samples which collected from four stations located at the Canada Basin, Arctic Ocean was analyzed by PCR-DGGE. Twenty-three 16S rDNA sequences of bacteria obtained from DGGE bands were cloned and sequenced. Phylogenetic analysis clustered these sequences within γ-proteobacteria, Cytophaga-Flexlbacter-Bacteroides （CFB） group, Firmicutes and Actinobacteria. The phylotype of Pseudoalteromonas in the γ-proteobacteria was predominant and members of the CFB group and γ-proteobacteria were highly abundant in studied sea ice brine samples.     

粤西镇海湾近江牡蛎线粒体16S rRNA基因序列变异分析

采用聚合酶链式反应 (PCR)技术对粤西镇海湾水域的近江牡蛎Crassostrearivularis(Gould)群体 2 7个个体的线粒体DNA16SrRNA基因序列片段进行扩增 ,获得大约 5 0 0bp的扩增产物。PCR产物经纯化后进行序列测定 ,经ClustalX同源排序 ,除去引物及部分端部序列 ,得到4 14bp的核苷酸片段。 2 7个个体共检测到 2个变异位点 ,均为颠换位点 ,没发现碱基位点插入、缺失及转换位点 ,共 3种单倍型 ,每个单倍型只有一个碱基的差异。运用DNASP软件计算得该群体的核苷酸多样性和平均核苷酸差异数分别为 0 .0 0 0 36和 0 .14 815。此结果提示镇海湾近江牡蛎群体遗传多样性已很低 ,很有必要从其他分布区引进近江牡蛎亲贝来扩大该种群的遗传多样性     

3种柔鱼线粒体基因与核核糖体基因片段序列比较分析

为准确检测柔鱼（Ommαstrephesbartram川、茎柔鱼（ Dosidicus gigas）与阿根廷滑柔鱼（ IIIex argentinus ） 的种间遗传差异,对线粒体16SrRNA、细胞色素b（Cytb）与编码核糖体大亚基的基因（28SrDNA）片段序列进 行测定。经比对获得同源片段序列的长度分别为444、430、464坤,其中16SrRNA与28SrDNA基因片段上分别存在3处和47处碱基插入/缺失。核昔酸组成分析表明;3种柔鱼在3个基因片段上的核音酸组成差异不显著, 在线粒体2个基因片段上的A＋T含量（16SrRNA;69.90%、72.01%、74.66%; Cytb; 63.61%、68.91%、71.65% ） 均明显高于C＋C含量（16SrRNA;30.10%、27.99%、25.34%; Cytb; 36.39%、31.09%、28.35% ）,而在28SrDNA 基因片段上的A＋T含量（37.16%、36.74%、38.29% ）明显低于C＋C含量（62.84%、63.26%、61.71 %）0 3种柔鱼 在28SrDNA基因片段上检测到的核昔酸替代率最低,为6.68%,而蛋白质编码基因Cytb核昔酸替代率最高,为 20.93%,核营酸替代均发生在密码子第3位点上,而且未引起氨基酸替代。基于邻接法、最大简约法与最大似然 法重建的系统树显示,柔鱼与茎柔鱼的亲缘关系较近。根据C严b基因片段序列分析,柔鱼与茎柔鱼和阿根廷滑柔鱼的分歧时间分别为653-790万a和765 - 925万a,种间分化事件发生在中新世至上新世间。