Species Delimitation And Historical Biogeography Of The Genus Helice (Brachyura: Varunidae) In Marginal Seas Of The Northwestern Pacific

A unique topographic feature of the northwestern Pacific is the series of linked marginal seas. The lowering and rising of sea levels during Pliocene and Pleistocene glacial-interglacial cycles greatly affected the areas and configurations of these marginal seas, especially the intertidal zone. Intertidal mudflats are dominant habitats along the continental coast of the marginal seas. However, studies on animal phylogeography in the habitats are limited. Genus Helice is the endemic animals distributed in the mudflats of high intertidal zone in marginal seas of the northwestern Pacific. The genus Helice includes four species:H. formosensis Rathbun,1931; H. latimera Parisi,1918; H. tientsinensis Rathbun,1931; and H. tridens de Haan,1835. A recent molecular analysis identified H. formosensis and H. tientsinensis as junior synonyms of H. latimera.This study sampled 24 popultions of 4 species, and combined the morphological . and molecular data to further delineate species boundaries in Helice and to add to knowledge of the historical biogeography of the genus. Then, the COI (Cytochrome oxidase subunitⅠ) gene was chosen as molecular marker to reveal the phylogeographical pattern and demographic history of H. tientsinensis. This study will also provide scientific knowledge for the conservation of genetic diversity in marginal seas of the northwestern Pacific.The taxonomy and historical biogeography study based on combined morphological and molecular data was conducted with 157 individuals from 24 populations of 4 species. And the phylogeographical analyses were conducted based on 357 individuals from 18 populations of H. tientsinensis. Major results are listed as follows:1. Species delimitation and historical biogeography in the genus Helice1) PhylogenyThe Bayesian tree and haplotype network based on the combined dataset of 12S rRNA,16S rRNA and COI genes revealed that H. tridens and H. formosensis-latimera-tientsinensis complex were sister group with deep divergences. The haplotype network also revealed clear genetic separations among H. formosensis, H. latimera, and H. tientsinensis, with incomplete lineage sorting. 2) Genetic divergence and gene flowThe analysis of molecular variance for the combined mitochondrial dataset showed significant genetic divergence among H. formosensis, H. latimera, and H. tientsinensis, and pairwiseΦST values further indicated significant genetic differences among these species. Net average genetic distances for 16S and COI between H. tridens and the H. formosensis-latimera-tientsinensis complex were 1.25±0.48% and 4.47±0.83%, respectively. The gene flow among H. formosensis, H. latimera and H. tientsinensis was negligible.3) Divergence timeDivergence time between H. tridens and the H. formosensis-latimera-tientsinensis complex was estimated to be 1.42±0.50 Ma for the 16S gene, and 1.92±0.42 Ma for the COI gene based on the net average genetic distances.The divergence time among H. formosensis, H. latimera, and H. tientsinensis was estimated under the IM model, for 16S to be～22-730 ka (H. formosensis vs H. latimera,～120 ka; H. formosensis vs H. tientsinensis,～730 ka; H. latimera vs H. tientsinensis,～22 ka); the divergence time for COI was～40-62 ka (H. formosensis vs H. latimera,～51 ka; H. formosensis vs H. tientsinensis,～62 ka; H. latimera vs H. tientsinensis,～40 ka).4) Variation in morphologyThe denticles on the suborbital ridge were significantly different among species and sexes. The denticles on the latero-subhepatic ridge were significantly different among species and sexes except two sexes of H. formosensis, female of H. latimera and H. tridens, and two sexes of H. tridens. The scatter plot and the dendrogram based on combined data for the two characters better illustrate the morphological separation among these species. However, H. tridens was divided to two groups which related to the sex in the dendrogram.2. Phylogeography of H. tientsinensis1) Phylogenetic relationships among haplotypesThe data set of 1235 bp mitochondrial fragments of 357 specimens from 18 H. tientsinensis populations yielded 141 haplotypes. Among them,115 haplotypes were private. Three clades were revealed based on the minimum spanning haplotype network. CladeⅠonly contained individuals from East China Sea and southern Yellow Sea. CladeⅡandⅢcontained individuals from all regions. There were divergences among clades, and the divergence time among them ranged from 70 to 90 ka before present (BP).2) Genetic diversityOverall haplotype diversity was high (h= 0.92). Haplotype diversities of Bohai Sea populations except Dongying (DY) were relative low, ranging from 0.54 to 0.79. Haplotype diversities of other populations were high, ranging from 0.81 to 0.96. Overall nucleotide diversity was low (π= 0.0047). Nucleotide diversity of the Panjin (PJ) population was the lowest (0.0012) and nucleotide diversity of Rushan (RS) population was the highest (0.0048).3) Spatial genetic structure and gene flowPairwiseΦST analyses divided the H. tientsinensis populations to two groups: one contained East China Sea and southern Yellow Sea populations, the other contained northern Yellow Sea and Bohai Sea populations. There were no significant genetic divergences among populations within a group and genetic divergence to a certain extent among populations in different groups. The principal coordination analysis and spatial analysis of molecular variance divided the H. tientsinensis populations to two different groups:one contained Lianyunguang (LY) and more southern populations, the other contained Rizhao (RZ) and more northern populations. The gene flow among most neighboring populations was high.4) Demographical historyThe neutrality tests for the overall dataset of H. tientsinensis showed significant negative values for both Tajima's D and Fu's Fs, which revealed possible population expansion event in history. The expansion time of CladeⅠwas earlier than CladeⅡandⅢand the expansion times of Yellow Sea populations were earlier than other region, except few populations based on Bayesian Skyline Plot and mismatch analysis. In conclusion, the study on the genus Helice revealed:Ⅰ) H. formosensis, H. latimera, H. tientsinensis and H. tridens were valid species with incomplete lineage sorting;Ⅱ) Kuroshio Current and Tsushima Current were the reason of divergence among H. tridens and other Helice crabs and the low sea level in Pleistocene made Taiwan Strait ageographic barrier, resulting the divergence among H. formosensis, H. latimera and H. tientsinensis.The study phylogeographical pattern of H. tientsinensis revealed:Ⅰ) high haplotype diversity and low nucleotide diversity;Ⅱ) strong spatial genetic structure and divergence among southern and northern populations;Ⅲ) secondary contact of clades;Ⅳ) rapid population expansion at the Late Last Glacial Period.