Studies On The Molecular Genetic Characteristics Of Several Coilia Species

To date, the Coilia resources have decreased sharply due to the impacts of overfishing, environment destruction and pollution. And the situation is the most striking on the estuarine tapertail anchovy, C. nasus, which has been sold at a whopping price recently. For Coilia fish, it still has been debated on the molecular genetic differences and species classification, e.g., the validities of sub-species status of land-locked estuarine tapertail anchovy (C nasus taihuensis), the classification for the estuarine tapertail anchovy distributed in China and Japan (Coilia nasus is suggested as the accepted scientific name for the anchovy from both China and Japan), respectively. Genetic diversity is an important level of biological diversity and the guarantee of potential evolution. Therefore, the study of the genetic relationship among the Coilia species will facilitate the protection and management of these commecial fish. In the present study, we investigated the genetic relationship of C. nasus (include C. nasus taihuensis and the estuarine tapertail anchovy collected in China and Japan), C. mystus and C. grayii by means of molecular biological and bioinformatics approaches on a comparative study of the internal transcribed spacer-1(ITS1) sequence and Cytochrome b (Cyt b) gene sequence in Coilia mystus; a re-confirmation of the taxonomic relationship among C. nasus from China and Japan, as well as the C. nasus taihuensis using the two sequences (Cyt b gene and Mitochondrial DNA (mtDNA) control region (D-loop) sequences); and an analysis on the genetic diversity of eggs and juveniles of Coilia fishes to assesse the potential of species identification using the sequences of Cyt b gene and D-loop sequences. The main results were summarized as follows:1) Through comparison with Cyt b gene sequence, we presented the first investigation to reveal ITS1sequence characteristics and its potential utilization for genetic diversity and phylogenetic relationship measurements for this species and other Coilia fish. The results indicated that the GC content of ITS1sequence (71.19%) was higher than Cyt b gene sequence (42.96%). Also, the haplotype and nucleotide diversity of ITS1sequence (0.900/0.00997±0.00413) were higher than those of Cyt b gene sequence (0.400/0.000369±0.000469). Both maximum composite likelihood (MCL) and kimura-2-parameter (K2P) genetic distances of ITS1were at interspecies level between C. mystus and C. nasus, which were coincident with those of Cyt b gene sequences. The maximum-likelihood (ML) and unweights pair method with arithmetic means (UPGMA) trees constructed with ITS1sequences and Cyt b gene sequences suggested that C. mystus and C. nasus formed the separate monophyletic groups, respectively. Hence, it was concluded that ITS1sequence could be used as a good genetic marker for genetic diversity and phylogenetic relationship measurements for C. mystus and other Coilia species (e.g. C. nasus).2) We used Cyt b gene and D-loop sequences to re-confirm the taxonomic relationship of estuarine tapertail anchovies (C. nasus) from China and Japan, and, C. nasus taihuensis from the Taihu Lake of China. The results suggested that the nucleotide diversity of C. nasus from the river mouth of Rokkaku-gawa in Inner Ariake bay of Japan was lower than those of C. nasus from the waters of China in some previous studies, based on Cyt b gene (0.003257±0.00042) and D-loop sequences (0.006135±0.00050). All results of the high level of population differentiation index (Fst) values (Cyt b0.36111/D-loop0.54983) and low gene exchange value (Nm)(Cyt b0.44/D-loop0.16), as well as the Tajima’D and Fu’s Fs neutrality tests implied that a late Pleistocene population expansion might be occurred between C. nasus from Japan and C. nasus from China (0.80-1.26million years ago), and climatic oscillations during the Pleistocene Ice Ages might contributed to the great difference between them. The average K2P genetic distances between C. nasus from Japan and China (Cyt b0.0059293±0.0015225/D-loop0.0119264+0.0025281) and the distances between C. nasus taihuensis and C. nasus of China (Cyt b:0.00360±0.00090; D-loop:0.00423±0.00085) were at the intraspecies level. The ML trees of Cyt b gene and D-loop sequences (C mystus as outgroup) indicated that C. nasus from Japan, and C. nasus taihuensis and C. nasus from China failed to form monophyletic class and could not be separated clearly. Moreover, the corresponding analyses of molecular variance (AMOVA) and median-joining network figures could reveal that C. nasus from Japan, C. nasus taihuensis and C. nasus from China could be reconfirmed as a same species.3) It was believed that the nucleotide diversity of C. grayii (0.00274±0.00092) was lower than that of C. nasus and the diversity of C. grayii might be destroyed, based on the analysis of ca.1022bp Cyt b gene sequences. In addition, both sequences of Cyt b gene and D-loop were studied for the genetic relationship among C. grayii, C. nasus and C. mystus. The results of average K2P genetic distance and neighbour-joining (NJ) molecular dendrogram indicated that C. mystus should be the most primitive among the three valid species of Coilia, while C. grayii and C. mystus seemed to be more derived and were believed to be the sister groups [the bootstrap values were100(Cyt b) and97(D-loop), respectively]. Therefore, the proposed evaluation model will probably be that the C. nasus and C. grayii are the species diverged from C. mystus for adaptation to cold and warm living environment, respectively.4) Based on about1022bp and1322bp sequences, nucleotide sequences of Cyt b and D-loop segments of mtDNA were studied in order to develop some species identification approaches of molecular biology and bioinformatics for eggs and larvae of Coilia fishes. The genetic diversity of eggs and juveniles of Coilia fish seemed to be low (Cyt b:0.00235～0.00319; D-loop:0.00297～0.01070). The average K2P genetic distance and NJ molecular dendrogram of the Cyt b gene and D-loop sequence showed the anchovies of C. nasus and C. nasus taihuensis mixed into a cluster, and those of C mystus and C. grayii formed another two clusters. According to the above reference information, nucleotide sequences of Cyt b and D-loop of Coilia eggs and juveniles were analyzed. NJ molecular dendrogram showed that all the eggs and juveniles of C. nasus, adults of C. nasus and juveniles C. nasus taihuensis clustered together, while the eggs of C. mystus and adults of C. mystus formed a separate cluster. Thus, the eggs and juveniles of C. nasus and C. mystus could be identified by means of Cyt b and D-loop sequence analysis. However, this approach could not identify the eggs and juveniles of C. nasus and C. nasus taihuensis.