Study In Classification Of True Limpets And Cryptic Diversity Of Circe Scripta Along Coast Of China

1. Morphological study of Patellogastropod limpets along coast of ChinaTrue limpets, which is the limpets of Order Patellogastropoda, belong to Mollusca, Gastropoda. They distribute all over the world and inhabit intertidal rocky zone mostly. In this study, true limpets were identified based on morphology of shell. These specimens were sampled from coast of China. We described shell features of every species. These features include basic parameters, shape, color, surficial textures, and we also recorded scientific names, sampling sites of each species. At last, all specimens were identified to two families (Lottiidae, Nacellidae), and four genera (Lottia、Patelloida、Nipponacmea、Cellana). Among these specimens, eleven species were identified to species level, two species were only identified to genus level.2. COI-based DNA barcoding of true limpets (Order Patellogastropoda) along coast of ChinaPatellogastropod limpets’species-identification according to morphological characters possessed uncertainty and complexity. It is necessary for more efficient and accurate identification for these common, small and shell-diverse marine molluscs. DNA barcoding is an efficient and useful tool. It may provide prominent progress for Patellogastropod limpets’discrimination and identification. In this study, a partial sequence of mitochondrial cytochrome c oxidase I (COI) gene was applied as DNA barcode to assess the viability of DNA barcoding for distinguishing Patellogastropoda. 135 COI sequences were obtained in total from 13 species belonged to Nacellidae (Cellana), Lottiidae (Lottia, Patelloida, and Nipponacmea) along coast of China. The alignment result of these sequences indicated the existence of insertions in mitochondrial COI gene of Patellogastropoda. The average Kimura 2-parameter (K2P) distances within species and genera were 0.03%-0.21%(maximum 1.01%), 18.96%-31.30%(maximum 37.80%) respectively, an obvious barcoding gap existed. All species in this study were clearly discriminated in all trees (Neighbour-joining tree, Bayesian tree, Maximum likelihood tree) with high supported clade node. The character-based barcode method successfully identified 100% of the Patellogastropod species included, and performed well in discriminating Patellogastropod genera. The results of this study affirm that DNA barcoding based on COI gene can identify species belong to Patellogastropoda rapidly and accurately.3. COI-based cryptic diversity study of Circe scripta in South China SeaDNA barcoding has demonstrated excellent effectiveness in discovering cryptic species. In this study, mitochondrial COI gene was used as barcode to examine the efficiency of the DNA barcoding for discovering cryptic diversity in Circe scripta. Eighty nine original COI sequences of Circe scripta from South China Sea were obtained and trimmed to 590 bp. Among these sequences,25 haplotypes were identified. Haplotype diversity was 0.843.87 variable sites and 46 parsimony informative sites were included among 590 sites. Nucleotide diversity is 0.02729. Haplotype 7 is probably generated from hybridization. After excluding this haplotype, both distance-based and tree-based methods reveal three clades, and Clade 3 only has one haplotype, the distances between different clades were 3.67%-8.55%, much bigger than the biggest distance intra-clade,0.85%. In addition, the numbers of CAs belonging to three clades were 17,16, and 24 respectively. Since the result of character-based method was consistent with distance-based and tree-based methods. The result of this study demonstrates that the utility of COI-based DNA barcoding for discovering cryptic diversity in Circe scripta. All analytical methods indicate that Circe scripta along coast of China possessed at least two cryptic species, meaning that it is a complex species composed of three different species.