Studies On Molecular Phylogeography And Biogeography Of The Genus Hydrilla

Aquatic vascular plants are a special group that differs from terrestrial plants with distinctive growth forms and habitats. Among the various aquatic plant life forms, the evolutionary processes of freshwater submerged species are most likely distinct due to their exclusive occurrence in the discrete and patchy aquatic habitats. Many phylogeographic studies have been conducted on aquatic plants to explore their evolutionary processes, whereas cosmopolitan species of aquatic plants are seldom studied with a broader sampling scheme. In this study, we focused on the worldwide submerged genus Hydrilla, a monotypic genus in the family Hydrocharitaceae. We collected samples throughout China to investigate its detailed geographic distribution; examined the genetic diversity and phylogeographic structure based one chroloplast fragment and eight microsatellite loci; inferred biogeographic history of Hydilla by combining with cpDNA sequences of samples from other countries; compared morphological data of different genetic clades to explore the cryptic species in Hydrilla. The main results were as follows:1) Hydrilla was widespread in the region to the east of the line from Northeast China to Southwest China. Sequences of the cpDNA trnL-F fragment were obtained from 681 individuals in 123 populations. High genetic diversity (Hd= 0.608, Pi= 0.0038) and genetic differentiation (FST= 0.820) were revealed. AMOVA revealed that 17.97% of the total variation occurred within populations, and 82.03% occurred between populations. If these populations divided into nine river basins, the highest genetic diversity was present in the Yangtze River Basin and the river basins in Southeast China, whereas no genetic variation was found in the Amur-Heilong River Basin, the Liao River and Hai River Basin and the Yellow River Basin. A total of 9 haplotypes were obtained, and these haplotypes formed four genetic clusters (A, B, C and D). Each cluster had respective distribution range, indicating Hydrilla had significant phylogeographical structure (Nst> GsT,p< 0.01).2) The genetic diversity and population genetic structure were further investigated using 8 microsatellite loci and the trnL-F fragment.. We genotyped834 individuals from 57 populations covered its distribution range. A total of 66 genotypes were found, and 22 populations consisted of a single genotype, suggesting that vegetative reproduction is dominant in Hydrilla. AMOVA analyses indicated that more genetic differentiation distributed within populations (52%) than that among populations (48%). Two genetic clusters (Northern and Southern) separated by the Yangtze River were identified based on the microsatellite data, which were corresponding to A plus B clades and C plus D clades of cpDNA data. Significant correlation between genetic and geographic distances among populations was detected based on both two molecular markers, whereas no correlation was found among populations in each genetic cluster, indicating limited gene flow between these two clusters. While the discordance between the two distinct genetic clusters may be caused by the different vectors of gene flow of the two markers.3) The biogeographic history of Hydrilla was inferred by combined our data with trnL-F sequences of samples from other countries. A total of 14 haplotypes were obtained, which formed four distinct clades with robust support in phylogenetic analyses. The divergence of Hydrilla was estimated to have occurred in the late Miocene, and the diversification of various clades was dated to the Pleistocene epoch. Ancestral areas reconstruction suggested an East Asian origin of Hydrilla and its subsequent dispersal throughout the world. Combined with the results of previous studies, the presence of all four clades in China indicates that China is most likely the centre of Hydrilla genetic diversity.4) Morphological data were measured in 118 samples from China and other countries. Data were grouped based on samples’ genetic evidence, and comparisons were conducted among different genetic groups. Significant differentiation between genetic groups were revealed at leaf number, leaf length, leaf width or leaf form, indicating new species in Hydrilla.In conclusion, Hydrilla had high genetic diversity, and differed greatly among different river basins. Significant phylogeographic structure was present in China and great genetic differentiation was found between northern and southern populations. Hydrilla diverged in East Asia in the late Miocene and subsequently dispersed to other areas. China is most likely the centre of Hydrilla genetic diversity. Cryptic species likely exist in Hydrilla. This study revealed high genetic diversity and significant phylogeographic structure in widespread species in aquatic plants. As the first case in the biogeographic studies on widespread species in aquatic plants, this study also provides a good example for us to understand the evolutionary processes that occur in submerged macrophytes.