Anchovy Morphological Variation, Genetic Diversity And Phylogenetic Study

Genus Coilia (Gray 1831) fishes are distributed mainly in the northwest and western Pacific, extending southward toward Canton in the north of southern China and northward to the Ariake Sound of southwestern Japan. They are classified into Engraulidae, Clupeiforms. In China, four kinds of Coilia fishes were found. They are named C. ectenes, C. mystus, C. grayii and C. brachygnathus. Some scientist treated the residual group of C. ectenes as a subspecies, C. e. taihuensis, but this opinion is still controversial. Two of them, namely, C. ectenes and C. mystus, are of higher economic value.In order to understand the morphological differences between four populations of genus Coilia (Teleostei: Clupeiforms) and identify them conveniently, truss network data were used to conduct multivariate analysis. Nineteen morphometric measurements were made for each individual. Burnaby's multivariate method was used to obtain size-adjusted shape data. The cluster analysis and discriminant analysis were used to discriminate among populations. The results indicated that 1) the four populations were clustered into three distinct groups; the first group included Changjiang C. mystus and Taihu C. ectenes, the second one included Zhujiang C. mystus, the last one included Changjiang C. ectenes, and 2) discriminant analysis with selected 4 morphological parameters showed that the identification accuracy was between 88% and 100%, and global identification accuracy was 95%. Our result showed that populations of different Coilia species living in geographic proximity to one another are more similar than conspecifics living farther apart. Separation and adaption are important to morphological difference. The taxonomy of genus Coilia should be reconsidered. This study also showed that the method to obtain size-adjusted data is important to acquire right conclusion.The genetic differentiation between C. ectenes and C. e. taihuensis have been investigated at the mtDNA level. RFLP analysis of Cytochrome b(cytb) gene amplified by PCR was used. Seven restriction enzymes were found to have at least one recognition site at this gene. Six different haplotypes weredetected between the populations studied. About one to two restriction patterns of each enzyme were revealed. The haplotype diversity of C. ectenes and C. e. taihuensis were 0. 1613 and 0. 2713, respectively. The nucleotide diversity of C. ectenes and C. e. taihuensis were 0.6173% and 1.3298%, respectively. An hierarchical analysis of molecular variance(AMOVA) revealed that very low levels of genetic partitioning, with less than 0.1% of the total variation attributed to between population differences, and lack of genetic structure. Our results supported that C. e. taihuensis is a geographical population of C. ectenes, their difference has not rise to subspecies level.Also, sequences analysis of cytochrome b (cytb) gene segments amplified by PCR was used to explore genetic diversity and genetic differentiation between C. ectenes and C. e. taihuensis. Five different haplotypes were detected between the populations studied. The haplotype diversity of C. ectenes and C. e. taihuensis were 0.9000 and 0.7000, respectively. The nucleotide diversity of C. ectenes and C. e. taihuensis were 0.3763% and 0.4301%, respectively. An hierarchical analysis of molecular variance (AMOVA) revealed that very low levels of genetic partitioning, with 0. 00% of the total variation attributed to between population differences, and lack of genetic structure. This result also supported that C. e. taihuensis is a geographical population of C. ectenes; their difference has not risen to subspecies level.Some differences maybe exist between different populations of C. mystus due to isolation and adaption. The great morphological differences between two geographical populations of C. mystus, Yangtze River population and Pearl River population, were revealed by multivariate analysis. In general, fishes demonstrate greater variance in morphometric traits both within and between populations than other vertebrate, and are more susceptible to environmentally-induced morphological variation. Thus, it is necessary to reveal the differences between different geographical populations by using stable molecular markers.Genetic diversity and evolutionary characters in two populations of C.mystus, Yangtze River population and Pearl River population, have been investigated at the mtDNA level. A total of ten fish, i. e., five fish at each site, were collected. Sequences of cytochrome b (cytb) gene segments amplified by PCR was used. 44 variable sites were detected, accounting for 11.5% of total sequence. Genetic distances within Yangtze River population were between 0.3% and 1.0%, within Pearl River population were between 0.5% and 0.8%, and genetic distances between Yangtze River population and Pearl River population were from 9.4% to 10.9%. The large genetic distances between these two populations suggest their divergence (?)aybe arrived at subspecies level. Five different haplotypes were detected within Yangtze River C. mystus and four within Pearl River C. mystus. The haplotype diversity index of Yangtze River C. mystus and Pearl River C. mystus were 1. 000 and 0. 900, respectively. The nucleotide diversity of Yangtze River population and Pearl River population were 0.677% and 0.573%, respectively. These indexes suggest that these two populations of C. mystus axe abundant in genetic diversity.Two methods, Ka/Ks test and McDonald-Kreitman test, were used to detect if natural selection operate at DNA level during the formation of divergence between these two populations. Significant results were revealed by the two methods (the ratios of K_?/K_? are all less than 1 and the G value and P value of MK test is equal to 6. 715 and 0. 00956, respectively), and it suggests that natural selection operates on this gene during its evolution. The cytb gene is an important gene in mitochondrial DNA, and severe diseases can be caused once the nonsynonymous substitution happens in this gene. Due to natural selection, the individual which has this damaged gene can not be fixed in the population and was removed from this population. Thus, it is reasonable to observe the action of natural selection.Molecular Phylogenetic relationships and evolutionary characters of four Coilia fishes are studied using the combined cytochrome b(cytb), 12SrRNA and 16SrRNA gene segment sequences dataset. The results are as follows: (1) Farris-test suggests that these three gene segment sequences can be merged to perform phylogenetic analysis; (2) Based on hLRTs criterion, modeltest software selects HKY+G model as the most appropriate model to build molecularphylogenetic trees; (3) Three bootstrapped trees, neighbour-joining(NJ) tree, maximum parsimony tree(MP) and maximum likelihood tree, were built with 1000 replicates. They all supported that Coilia fishes are constituted of two clades. Four samples of C. grayii are monophyletic group with bootstrap support value between 88% and 100%, and the last thirteen samples are the other monophyletic group, and their bootstrap values are between 83% and 98%; (4) The topology differences between NJ tree, MP tree and ML tree are checked by Templeton-test. It showed that ML tree is the best one, MP the second and NJ the worst. But their differences are not significant(P>0.05); (5) Relative-rate test which based on Li-Bousquet method suggested that local molecular clock hold within Coilia fishes. Based on the phylogenetic trees, we present a hypothesis on the origin of genus Coilia fishes.