Studies On Genetic Diversity And Taxonomic Status Of The Crab Genus Helice

The population genetics and molecular phylogenetics of the genus Helice (H. tientsinensis, H. latimera, H. japonica and H. wuana), as well as other six species of Grapsoidea, were analyzed by multiparameter morphometrics and mtDNA sequencing techniques to understand the status of genetic diversity and molecular phylogenetic relationship of these species. The taxonomic status of Helice was confirmed by 16S rRNA sequence analysis. The main results were as follows:Based on 33 proportional parameters of morphometric characters, three multivariation analysis methods were conducted to investigate the morphological variations among six populations of H. tientsinensis and four ones of H. latimera. The results of cluster analysis showed that H. latimera population in Quanzhou was clustered into one group, while the other nine populations were clustered into another group. Populations of H. tientsinensis and those of H. latimera were not clustered into one group respectively. Normalized Euclidean Distances of morphology of ten populations indicated close relationship each other. Discriminant analysis indicated there were no significant differences (total discriminant accuracy was only 63.4%) among ten populations. The identification accuracy of discriminant functions of Quanzhou population and Tanggu population was 88.9% and 75.0% respectively, while that of the other populations was all low. Populations could not easily be separated only by morphological characters on account of slight difference in figure. Principle component analysis revealed that ten populations were almost overlapped each other in scatter diagram of PC1 and PC2, indicating small morphological variations among ten populations. Coefficients of difference (CD) of proportional parameters of morphometric characters among populations were always smaller than 1.28, the critical value of under-subspecies - a standard of different populations by Mayr et al, which indicated that the morphological variations among populations still belonged to geographical population differences. Sympatric individuals of H. tientsinensis and H. latimera from Ningbo possessed osculant type of infraorbital crest. The case implied that H. tientsinensis and H. latimera copulated and beared offsprings, therefore, they might be one species.The phylogenetic relationship of Helice was discussed through 12S rRNA , 16S rRNA and COI sequences analyses, compared with the sequences of other Helice species in GenBank. A lot of individuals of H. tientsinensis and H. latimera inhabiting different zones shared the same haplotype of 12S rRNA, 16S rRNA and COI genes respectively, which indicated they should be the same species. One single branch(supported by 99%～100% bootstrap values) in the molecular phylogenetic trees, constructed by all individuals or haplotypes of ten populations with Neighbor-Joining and Maximum-Parsimony methods, also indicated that they were the same species.The net genetic distances of 12S rRNA, 16S rRNA and COI genes between H. japonica and H. wuana were 4.07%, 2.14% and 10.91% respectively, which were remarkably lower than those among other Helice, whose least values were 9.46%,5.12% and 17.00%. A single branch(supported by 88%～100% bootstrap values)in the molecular phylogenetic trees with high reliability were constructed by all individuals or haplotypes of H. japonica and H. wuana,prior to other species. The results demonstrated closer relationship between the two species, in accordance with those from morphological classification. That is to say, the taxon of Helice is divided into two subgenera, Helicana (H. japonica and H. wuana) and Helice (the others). As a result of high difference in figure and large net genetic distances between Helicana and Helice, it is advisable that they are updated from subgenus to genus.It is worth emphasizing that there is the least net genetic distance (1.42%) of 16S rRNA between H. tridens and H. tientsinensis / H. latimera, even close to the value (1.15%) between haplotypes in H. tientsinensis and H. latimera, so they have closer genetic relationship and later divergence time. The net genetic distance (1.42%) between H. tridens and H. tientsinensis / H. latimera is lower to the critical value of species (1.5%), so they might be the same species.AMOVA showed that molecular variation between the group of populations of H. tientsinensis and the other group of populations of H. latimera was -3.05%(P =0.53), molecular variation among populations with in groups was 34.15%(P <0.01), and molecular variation with in populations was 68.90%(P <0.01). The value ofΦCT=-0.031(P >0.05)revealed that genetics divergence between the two groups was not significant. The value ofΦSC=0.331(P <0.01)indicated that molecular variation among populations with in groups was significant. Molecular variation between H. tientsinensis and H. latimera was not significant, even smaller than that among populations with in groups. The results were similar to thoses obtained from morphology and molecular phylogenetics. All data strongly supported that H. tientsinensis and H. latimera was the same species. The difference of males'infraorbital crest between H. tientsinensis and H. latimera ought to be two different phenotypes.Two distinct lineages were found in the ten populations of H. tientsinensis and H. latimera, which might be isolated and diverged in different marginal seas of the Northwestern Pacific during middle Pleistocene when sea level fell on account of climate changes. There was significant difference in geographical distribution frequency between two lineages. Molecular variation analyses and the conventional population statistic FST revealed significant genetic structure in the ten populations of H. tientsinensis and H. latimera. These results indicated that gene flow between H. tientsinensis and H. latimera is far more restricted spatially than that predicted by the potential dispersal of the larva. The results of mismatch distribution analyses and neutrality tests indicated population expansion in two lineages. Two concurrent lineages in East China sea might be led by population reestablishment after the last Glacial Maximum.Molecular data of 16S rRNA gene showed that genetic distances between Helice and two species of Hemigrapsus are significantly smaller than those between Helice and other Grapsoidea species. Topological structure of the molecular phylogenetic tree, constructed by 16S rRNA gene with Neighbor-Joining method, revealed that all species of Helice from Sesarmidae and two species of Hemigrapsus from Varunidae were clustered into one branch, two species of Sesarma from Sesarmidae were clustered into another one, Grapsus albolineatus from Grapsidae and Plagusia tuberculata from Plagusiidae were formed one absolute branch respectively. The molecular analysis showed that the genus Helice indeed must be considered to be Varunidae, because it formed a monophyletic group with the genus Hemigrapsus yet not the Sesarmidae. Based on the above results, the taxonomic status of Helice was confirmed, and the genus Helice should be transfered from Sesarmidae to Varunidae.