Phylogeography And Population Genetics Of The East Asia Endemic Genus Kirengeshoma (Hydrangeaceae)

There are great similarities in the species and genera within the biologically rich and diverse vascular flora of China and Japan, so much so that this area is referred to by biogeographers as the Sino-Japanese Floristic Region. The mechanisms of speciation in this region have been a hot topic in the fields of systematic, ecology, evolution and plant geography for a long time. Although eastern Asia was not covered by major ice sheets during the last glacial period, climatic oscillations throughout the Quaternary and associated changes in sea level and land bridge configurations had a dramatic effect on the interaction and distribution of species, and in the abundance of organisms, in this area. To unravel some of the mysteries associated with this flora we have therefore focused our studies on the small endemic genus, Kirengeshoma (Hydrangeaceae, Cornales), which not only occurs disjunctly in the warm temperate forests of eastern China, southern Japan and South Korea, but is also the only genus in common that is endangered in all three countries. We investigated genetic diversity and population structures, determined inter-and intraspecific relationships and divergence patterns and correlated the evolution of populations with climate oscillations and changes in environments based on chloroplast DNA (cpDNA), microsatellite and SNPs data. By combining our findings with ecological field investigations, we are able to propose conservation strategies for the remaining populations of Kirengeshoma in China. The combined methods of study were useful for revealing the mechanisms of species diversification and the potential for increasing the chance of survival for small populations of endangered plants. The main results of our study are as follows:1. Interspecies divergence and population structure based on cpDNABased on the combination of six regions of chloroplast DNA (rbcL, matK, trnL-trnF, trnH-trnK, ndhF and atpB), we conducted a maximum parsimony tree for Kirengeshoma. The results supported the recognition of two species of Kirengeshoma, K. palmata in China and Japan and K. koreana in Korea, which is consistent with results based on morphology. We calculated the divergence time for each clade on the MP tree using the multiple fossil calibrations on the node. Kirengeshoma diverged in the early Pliocene and the K palmata and K. koreana lineages originated in the middle Pliocene.Among the three cpDNA intergenic spacer sequences, trnL-trnF, psbA-trnH and trnS-trnG, we detected17haplotypes based on81individuals from8populations of Kirengeshoma. At the genus-wide scale, cpDNA data revealed high estimates of haplotype diversity (hT=0.795). Nucleotide diversity (πT=0.0039). NST(0.913) was significantly higher than GST (0.697), which indicated a marked cpDNA phylogeographic structure across all populations of Kirengeshoma. Mismatch distributions indicated that the Chinese populations expanded their range around0.16mya.2. Microsatellite polymorphisms and population structureWe isolated and characterized twelve polymorphic microsatellite loci from Kirengeshoma palmata using a modified biotin-capture method. The results showed that the average of number of alleles per locus for a single population (NA) was4.6(ranging from2to8); the average of observed and expected heterozygosities (Ho and HE) and polymorphic information content (PIC) were0.497(0.091to0.909),0.624(0.091to0.879) and0.548(0.083to0.809), respectively. Only three loci (Kp48, Kp51and Kp74) displayed significant deviation from Hardy-Weinberg expectation. Significant linkage disequilibrium was not detected between any pair of loci.We detected a relative high value of expected heterozygosities (HE=0.613) across the twelve microsatellite loci. The overall value of FST was0.189. The ratio of pollen flow to seed flow was a high10to1, which indicated that seed flow between populations was limited in comparison to pollen flow. The Japanese populations appear to have undergone relatively greater genetic drift in comparison with plants in the Chinese region based on the2MOD results.3. Single nucleotide polymorphisms discovered by RAD tagWe first developed a modified RAD-tag protocol for preparing libraries in Illumina sequencing for phylogenetic and phylogeographic studies, especially for Kirengeshoma. We obtained more than201million RAD tag sequences based on26individuals, with an average of6.4million sequences for each individual. Throughout the Kirengeshoma genome, we identified106,293SNPs within the RAD tag sequences. Depth/coverage for whole data set was6.7x. The topological structure of the phylogenetic tree we constructed using SNPs data was basically congruent with the MP tree derived from the six cpDNA fragments. The SNPs data indicated that the Chinese, Japanese and Korean populations were monophyletic with100%bootstrap value. These results showed that the SNPs discovered by RAD tag were ideal for phylogenetic and phylogeographic studies in non-model species.Our studies of Kirengeshoma revealed the following. Kirengeshoma comprises two species (K. palmate and K. koreana). The divergence time of Kirengeshoma was in the early Pliocene, which can be correlated with Tsushima/Korean Strait. Kirengeshoma palmata diverged into two clades, restricted to two different regions, China and Japan, resulting from the oscillation of climate change during the Middle Pliocene. The Tianmu Shan (TM) populations of K. palmata diverged from the other three Chinese populations, LT, QL and HS, during the Middle Pleistocene episode because of moonson. Our findings not only indicate that climatic oscillations and geographic events throughout the Quaternary have played key roles in shaping the distribute pattern of Kirengeshoma, but also provide a scenario for reflecting on allopatric speciation in eastern Asia. Phylogeographic studies in the Chinese region have shown that an expansion event occurred during the Late Pleistocene. Populations in Japan were particularly sensitive to range fragmentation and to long term refugial isolation and the genetic drift effect throughout the Quaternary. Compared to studies using other molecular markers, SNPs discovered by RAD tag, is feasible, reliable and accurate. This study also yielded useful and insightful data that can provide the foundation for the conservation of the remaining wild populations of Kirengeshoma.