Genetic Differentiation And Phylogeographic Of Four Bipinnata Cycas Species

There are four Cycas species, Cycas debaoensis, C. multipinnata, C. bifida and C. micholitzii, with their leaves bipinnate. Cycas debaoensis is endemic to China; C. multipinnata and C. bifida are endemic to the Red River drainage region in the southwest China and northern Vietnam; C. micholitzii is distributed in Vietnam, Lao and Cambodia.Three intergenic spacers of chloroplast DNA (atpB-rbcL, psbA-trnH and psbB-psbH), five low copy nuclear genes (PHYP, AC5, PPRC, AAT and SAMS) and microsatellite markers were employed to assess the genetic diversity, genetic differentiation, genetic structure and population dynamics of the four species, and genealogic relationships among them. The main results were as follows:1Genetic diversityIn C. multipinnata, the1979bp combined cpDNA dataset had14parsimony-informative polymorphic sites and eight haplotypes (muHl-H8). The cpDNA haplotypes diversity (Hd) of C. multipinnata is0.7718, the nuclear diversity (Ï€) is0.00149. In C. bifida, the1990bp combined cpDNA dataset had22parsimony-informative polymorphic sites and seven haplotypes (biHl-H7). The cpDNA haplotypes diversity (Hd) of C. bifida is0.7184, and the nuclear diversity (Ï€) is0.00191. In C. micholitzii, the1960bp combined cpDNA dataset had24parsimony-informative polymorphic sites and14haplotypes (miHl-H14). The cpDNA haplotypes diversity (Hd) of C. micholitzii is0.9078, and the nuclear diversity (Ï€) is0.00107.The levels of genetic diversity based on the five low copy nuclear genes were different. AAT and SAMS had the higher diversity and recombination rates between the polymorphic sites. Less polymorphic sites were detected in PPRC gene. All the three above nuclear genes seemed to have experienced incomplete lineage sorting. PHYP and. AC5had appropriate polymorphic sites and complete lineage sorting.There were a total of402alleles of the16microsatellite loci identified in the535individuals from28populations of the four species. In details, C. micholitzii had279alleles with44private alleles; C. bifida had198alleles with21private alleles; C. multipinnata had188alleles with24private alleles; C. debaoensis had164alleles with14private alleles. It is C. micholitzii that had the most alleles and private alleles, while C. debaoensis had the fewest total and private alleles. In conclusion, the C. micholitzii had the most genetic diversity and C. debaoensis had the lowest genetic diversity.2Genetic differentiationThe three cpDNA dataset showed that all the three species, C. multipinnata, C. bifida and C. micholitzii, had significant genetic differentiation. In C. multipinnata, the AMOVA analysis indicated that90.72%genetic variation occured among populations and the FST was0.90717. In C. bifida, there was83.28%genetic variation among populations and the FST was0.83281. In C. micholitzii, there was90.72%genetic variation among populations and the FST was0.88051.The levels of genetic differentiation showed by the five low copy nuclear genes differed among the four Cycas species. It was rather significant in C. multipinnata, and the AMOVA analysis indicated that the FST was ranging from0.28426to0.75502. The genetic differentiation of C. micholitzii was moderate, and the FST was0.15861-0.31136. In C. bifida, the SAMS was abnormal and the FST was0.45432; the other four low copy nuclear genes showed low genetic differentiation, with FST ranging from0.00538to0.12108. The genetic differentiation of C. debaoensis was low, and the FST was0.10678-0.17753.The level of genetic differentiation based on microsatellite data was consistent to that of the low copy nuclear genes. Cycas multipinnata, with the FST was0.92304, shared the most significant genetic differentiation. Cycas micholitzii had moderate genetic differentiation(FST=0.21290). Cycas bifida had relatively low genetic differentiation (FST=0.11558).And C. debaoensis had the lowest genetic differentiation (FST=0.11438).There are two isolated distributions in C. multipinnata. One distribution consists of three populations in Yunnan, China, and the other consists of two populations in the northern Vietnam. The long distance isolation induced little gene flow between the two distributions, so that the genetic differentiation based on cpDNA, low copy nuclear genes and microsatellite data was rather significant. There were11C. debaoensis populations concentrated in a small area, and the more gene flow among them induced the lower genetic differentiation among populations.3Population dynamicsThe Bayesian skyline plot of cpDNA showed that the effective population size of C. multipinnata, C. bifida and C. micholitzii experienced a reduction approximately50,000-100,100years ago.The microsatellite-based Garza-Williamson indexes of all the populations were smaller than the critical value0.68, which mean all the population had experienced population contraction. So in situ and ex situ conservation should immediately be launched.4Interspecies relationshipsThe four species, C. debaoensis, C. multipinnata, C. bifida and C. micholitzii had species-specific haplotypes based on the two combined cpDNA (atpB-rbcL and psbA-trnH). As there were not enough informative polymorphic sites, the dendrogram of the cpDNA haplotypes showed that C. multipinnata, C. bifida and C. micholitzii was paraphyletic.Haplotype networks of PHYP and AC5were composed of approximately four clusters with most haplotypes were species-specific. By contrast, networks for the other loci (PPRC, AAT and SAMS) showed no obvious divergence between the four species for they might retain some ancestral haplotypes and incomplete lineage sorting.The STRUCTURE and PCoA analysis based on the microsatellite data illustrated that the four species could be divided into two parts. One part only included C. debaoensis, and the other contained C. multipinnata, C. bifida and C. micholitzii. In the second part, C. multipinnata in Vietnam was genetically close to C. micholitzii, although they were far from each other in geography at present. This pattern might be attributed to the collision from India to Eurasia, which caused massive shifting of surrounding continental block. Except for the vast expanses of land uplifting to form the Himalaya Mountains, the Indochina block, consisting of Myanmar, upper Thailand, Cambodia, Laos and most of Vietnam, shifted eastward some700km relative to the South China block along the Red River fault line. Before the chain of tectonic events above, C. multipinnata in Vietnam and C. micholitzii were distributed on the side of the "Red River Gulf", respectively. There might be the ever-existed gene flow between them by pollen.5ConclusionCycas debaoensis is endemic to a restricted area, so it has the lowest genetic diversity and genetic differentiation among the four bipinnate Cycas species. Cycas micholitzii has the highest genetic diversity due to the warmer climate in the low latitude and the large distribution range. Little gene flow made rather significant genetic differentiation between two isolated distributions of C. multipinnata.Cycas debaoensis is isolated from the other three species, as it is distributed northernmost. The nuclear genetic distance between C. multipinnata in Vietnam and C. micholitzii is smaller than that between C. multipinnata in Vietnam and C. multipinnata in China, perhaps resulting from the tectonic events of Indochina block.As all the populations of the four species have experienced bottleneck, we should protected them via both in situ and ex situ conservation.