A Study On Molecular Phylogeny Of Engraulidae And Population Genetic Diversity Of C. Mystus And C. Ectenes

Mitochondrial DNA was utilized to analyze the molecular phylogeny of Engraulidae and evaluated the genetic divergence and genetic structure of C.mystus and C.ectenes. In addition, polymorphic microsatellite markers were isolated from C.ectenes and used to analyze the genetic diversity of C.ectenes.1. Molecular phylogeny of Engraulidae inferred from mitochondrial DNATo evaluate the phylogenetic relationship of 11 species of Engraulidae in China, the mitochondrial 16SrRNA and Cytb genes were amplified using PCR techniques. About 600bp length 16SrRNA gene fragments and 1300bp Cytb gene fragments were obtained and sequenced. After aligned, a fragment of 472bp-501bp (16SrRNA) and 1138(Cytb) nucleotide sequence which could be used for sequence analysis was obtained. The genetic information indexes, including Kimura-2 parameter genetic distance, basal composition and phylogenetic trees were generated using MEGA3.1 software. Of the 16SrRNA aligned base pairs, 25 insertions/deletions were detected and 125 sites were variable. The mutation sites were mainly transition and transversion. Transitions were found to be more common than transversions. The genetic distances between species ranged from 0.22% (T. vitrirostris and T. mystax, C.ectenes and C.mystus) to 18.54% (S.commersonii and T. setirostris). Of the Cytb aligned base pairs, no insertions/deletions were detected and 452 sites were variable mainly due to transition and transversion. The fragment encodes 379 acids,55 of which were variable. The genetic distances between species ranged from 0.6% (T. vitrirostris and T. mystax) to 23.8% (S.commersonii and T. setirostris). Molecular phylogenetic tree was constructed by NJ methods based on the 572 bp (16SrRNA), 1138bp (Cytb) and 572bp(16SrRNA)+1138bp(Cytb) sequences, with Sardinella lemuru as the designated outgroup. The tree base on 16SrRNA gene indicated a closer relationship between T. dussumieri and T. hamiltonii. The genus Thryssa showed close relationship with Coilia, and then clustered with Setipinna. The former cluster gathered with T. kammalensis. Finally, they clustered with the Stolephorus and Engraulis.The tree base on Cytb gene indicated T. vitrirostris and T. mystax were sister species. They clustered with T. hamiltonii and T. setirostris, then cluster with T. dussumieri. The genus Thryssa gathered with Setipinna, coilia, T. kammalensis {Thryssa), Stolephorus and Engraulis in turn. The NJ tree base on 16SrRNA+Cytb sequence showed high bootstrap support. The relationship between genus was consisted with the tree based on Cytb sequence. In all, T. kammalensis was separated from Thryssa. We suggested that T. kammalensis should be considered as a new genus.2. Mitochondrial DNA diversity of C. mystus in three populationsSequence variation and genetic diversity in three populations of C. mystus, i.e., Yangtze River population, Minjiang River population, and Pearl River population, were investigated by using mtDNA 16SrRNA and Cytb gene sequence in this study. A total of 65 fish, i.e.,21 individuals from Yangtze River estuary,22 from Minjiang River estuary, and 22 from Pearl River estuary respectively, were collected. The results showed that:1)16SrRNA: In the total 65 sequences,19 distinct haplotypes were detected. We obtained 470 base pair consensus sequences of 16SrRNA.Thereinto,38 were variable sites, and 16 were parsimony sites. The content of A+T (56.3%) is bigger than G+C (43.7%). The indexes of nucleotide diversity in these three populations were Yangtze 0.108%, Minjiang 0.843%, and Pearl River 0.097%. Minjiang is the largest among these three populations. Genetic distances within the Yangtze River population were between 0.2% and 0.4%, within the Minjiang River population were between 0.2% and 4.1%, within the Pearl River population were between 0.2% and 0.6%, and the genetic distances between the Yangtze River population and the Minjiang River population was 1.9%, between the Yangtze River population and the Pearl River population was 1.5%, but that between the Pearl River population and the Minjiang River population was only 0.5%. AMOVA analysis disclosed that variation between populations accounting for 74.61% of total variation, suggesting between populations variations are the main source of total variation. This result suggests that these three populations may have isolated genetic structure respectively.2) Cytb:We obtained 607 bp consensus sequences of Cyt b. Thereinto,102 were variable sites, accounting for 16.8% of total sequence, and 68 were parsimony sites. They were all transitional and transversional variations, and no indels were found. The content of A+T(57.6%) is bigger than G+C(42.4%). The index of nucleotide diversity is largest in the Minjiang population among these three populations. Selection tests disclose that natural selection operates at molecular level within these three populations. In the total 65 sequences,34 distinct haplotypes were detected. Genetic distance within the populations was between 0.3% and 1.2%, and between populations was from 0.8% to 10.8%. The largest genetic distance Was 10.8% between the Yangzte River population and the Pearl River population, and the smallest was 0.8% betwen the Minjiang River population and the Pearl River population. AMOVA analysis disclosed that variation between populations accounting for 90.25% of total variation, suggesting variation between populations is the main source of total variation. This study suggests that the genetic divergence betwen the Yangtze River population and the Minjiang River population, the Pearl River population may be arrived at subspecies level.3. Mitochondrial DNA diversity of C. ectenes in ten populations1) Cytb:C. ectenes is an important commercial fisheries species. In this study, the genetic diversity and population structure of ten Chinese populations of C. ectenes were investigated using cytochrome b (Cytb) sequences of mitochondrial DNA (mtDNA). We sequenced the complete Cytb gene of 279 individuals. One hundred and eleven polymorphic sites defined 97 distinct haplotypes. Haplotype diversity (Hd) ranged from 0.6433 to 0.9517 and nucleotide diversity (π) ranged from 0.16% to 1.23%. The Wuhan (WH) fish population had the highestπlevel. The genetic distances ranged from 0.16% to 1.26% within populations and from 0.18% to 2.66% between populations. The distances between the Fuzhou (FZ) population and other populations were greater than 2%, while the distance between the Lianyungang (LYG) and Taihu Lake (TH) populations was the smallest. The fixation index analysis indicated that there was significant genetic divergence among populations from different geographical groups. Both the neighbor-joining tree and median-joining network analysis of the haplotype data showed distinct patterns of phylogeographic structure. The hierarchical analysis of molecular variance (AMOVA) revealed that intra-group variation among populations (θSC) was highly significant. The results of this study suggest that C.ectenes populations, especially geographically isolated groups, have developed significant genetic structures. In addition, tests of neutral evolution and mismatch distribution suggest that C. ectenes may have experienced a population expansion, possibly in the past 110,000 years.2) D-loop:C.ectenes is a commercially important fishery species. In this study, intraspecific variation was examined by using mitochondrial DNA control region in 246 individuals sampled from ten localities in China. One hundred and ninety-five polymorphic sites defined 184 distinct haplotypes, revealing a moderately high haplotype diversity and relatively low nucleotide diversity in the ten localities. An excess of unique haplotypes at most sample locations were detected, which might influence genetic structure of the C.ectenes populations. Neighbor-joining tree indicated distinct patterns of phylogeographic structure among haplotypes from FZ population and those from other populations. Analyses of molecular variance (AMOVA) and Fst statistics suggested that the divergence existed among populations from ten localities, indicating that gene flow might be restricted among those regions, despite the wide dispersal. In addition, neutral tests and analysis of mismatch distribution suggested that C. ectenes might have undergone a population expansion. Our study revealed the extant population genetic structure of the C. ectenes, and was in favor of the related fishery management issues including fishery stock identification and conservation.4. Isolation of polymorphic microsatellite markers and genetic diversity evaluation of three populations of C. ectenesA repeat-echriched genomic library (the repeat units AC) was constructed by FIASCO (fast isolation by AFLP of sequences containing repeats) in C.ectenes. Ninety-seven sequences were obtained 70 pairs of primers were designed successfully, and 16 microsatellite loci were proved to be polymorphic. The applicability of these markers in a closely related species C.mystus was evaluated by the cross-species amplification. All but one (D139) microsatellite primers would be useful for amplification in C. mystus. These markers will be benefit for the studies on population genetics, conservative genetics, fishery management, and construction of genetic linkage maps of C. ectenes and C. mystus. We utilized 7 polymorphic microsatellite makers to detecte the genetic diversities of 3 C.ectenes populations, abbreviated as DD, WH and FZ. Basic data statistics showed that the alleles per locus were 4-11 alleles, the observed heterozygosity (Ho) from 0.2518 to 0.6655, and the polymorphic information contents (PIC) from 0.516 to 0.633, respectively. The Shannon index was from 1.0892 to 1.4068 and gene diversity was from 2.9887 to 4.1096, and both the Shannon index and gene diversity of FZ population were lowest, and those of DD population were highest. In addition, the data of genetic distance between populations DD and WH was the smallest and that between WH and FZ was the biggest. The UPGMA phylogenetic tree showed populations DD and WH clustered into one group, and then clustered with FZ population.