A Hybrid Zone And Reticulate Evolution In Ranunculus Cantonensis Polyploid Complex

Polyploidization is the most common and characteristic cell genetics process influencing plant evolution. Polyploidization and hybridization occured cooperatively will have a greater impact on plant evolution. Hybridization can form new genetic combination, while polyploidization can stabilize the new genetic combination through reducing genetic segregation and eliminating hybrid sterility, which finally result in hybrid speciation, speices swarming and hybrid zones. Therefore, polyploidization and hybridization are hot issues in plant evolutionary biology. It is proved that in the presence of gene flow, disruptive selection can cause assortative mating and generate reproduction isolation, then reproductive isolation result in sympatric speciation. However, there are many debates on gene flow dynamic during speciation and the impaction of gene flow to adaptive evolution. Natural hybrid zones provide natural laboratories for studying on gene flow dynamic, species cohesive and hybrid adaptive evolution. Closely related species in hybrid zones apply key models for studying sympatric speciation and mechanism of genetic divergence. Thus, research on genetic recombination and adaptive introgression caused by gene flow in hybrid zone can clarify the process of speciation and enrich the theory of hybrid speciation.Ranunculus cantoniensis polyploid complex mainly includes Ranunculus chinensis(2x), R. trigonus(2x), R. silerifolius var. silerifolius(2x), R. silerifolius var. dolichanthus(2x), R. cantoniensis(4x), R. diffusus(4x), R. vaginatus(5x) and R. sieboldii(6x, 8x), widespreads in tropical and subtropical Asia. There are many sympatric areas among different taxa of this complex, and this complex is still in young phdases of polyploid complex evolutionary process. Field investigation and karyotype analysis found that there may be many hybrid individuals in sympatric area, for example, four diploid species distribute sympatrically in Puer of Yunan province. Taxa of R. cantoniensis polyploid complex have a short growth cycle, about 6-7 month, and present a complete evolution process from low-ploids to high-ploids. Those characteristics mean that this complex is an ideal materal for polyploidizaiton and hybridization research.This study took R. cantoniensis polyploid complex as a model system for studying on polyploidizaiton and hybridization, using several molecular markers, FISH and transcriptomic data, to clarify the reticulate relationship of taxa in this complex; to detect hybrids in sympatric aera, and to explore hybridization pattern and consequence of the hybrid zone through analyzing gene flow and genetic structure of the hybrid zone; to obtain a large number of genes using transcriptome sequencing, and to attempt to reconstruct phylogeny of four diploids using these data; to detect ingrogression between four diploids and adaptive gene spreading among different species through gene flow. The key results of the research are summarized as follows:1. The reticulate relationship of R. cantoniensis polyploid complex. Phylogenetic analysis of 10 taxa in R. cantoniensis polyploid complex was conducted using ribosomal DNA internal transcribed spacer(ITS) and FISH. The results show that R. shuichengensis and R. repens bear the unique genotypes, have a distant relationship with other taxa in R. cantoniensis polyploid complex. The other 8 taxa are closely related, and 6 of 8 taxa bear the same ITS genotype of R. chinensis, so we considered that R. cantoniensis polyploid complex mainly includes this 8 taxa, and R. chinensis may play a pivotal genome role in this complex, by which other taxa can banding together. Moreover, the results of FISH and phylogeny based on ITS show that R. vaginatus(5x) is derived from hybridization between R. diffusus(4x) and R. sieboldii(6x). Based on the results above, we drawed a reticulate relationship figure of this complex.2. The discovery of a multispecies hybrid zone and pattern of hybridization in this hybrid zone. ITS and two chloroplast gene was used to study a multispecies sympatry in Puer of Yunnan province, and results showed that PCR products of ITS have many heterozygous sites, so we cloned these sequences and sequenced them, and conducted a phylogentic network analysis. The results showed that many individuals from the multispecies sympatry have more than two kinds of ITS genotypes and remarkable conficts between ITS data and chloroplast data. Moreover, the fertility of pollen gain of these individuals shows a remarkable reduction. All the results showed that there are lots of hybridization in this multispecies sympatry. Meanwhile, the results showed that the reproduction isolation between R. trigonus and R. silerifolius var. dolichanthus is relatively low, and which lead to a high ratio of hybrids and dissymmetry gene flow between the two taxa in this hybrid zone. Four species in this hybrid zone can hybridize with each other, and formed seven different hybrid genotyes, including F1, backcross progenys and a three-species hybrid. We confirmed that this region is a four-species hybrid zone, and an extraordinarily abundant hybrid swarm was formed, which increased the genotype diversity of this region.3. Development of microsatellite marker. The research conducted six microsatellite sequences enrichment using the magnetic beads enrichment procedure. Thirteen pairs of primers with high stability were screened by polyacrylamide gel electrophoresis. Universality of these primers was tested using four diploid populations respectively, and the results show that these primers have a good universality in the four diploid taxa. Moreover, this research also finds that many of these primers significantly deviate from the hardy-weinberg equilibrium, which may be related to selfing.4. Genetic structure of the hybrid zone. Genetic structure analyses of populations collected in hybrid zone and outside were conducted by microsatellite marker developed above, and results show that hybridizations were extensive in the entire sympatry. Hybrid individuals were detected in the populations collected from the hybrid zone and reference populations outside the hybrid zone. Thus, we speculated that gene flow is very common in the distribution aera of this complex, which will result in adaptive gene scattering over the species barrier in the hybrid zone, leading to collective evolution. This result also proved that gene flow in the hybrid zone is not symmetric, R. trigonus always as female parents, and even leading to an extreme phenomenon of introgression-chloroplast capture.5. Comparative transcriptomes of diploids in the hybrid zone. Four individuals collected from the hybrid zone were used to transcriptome sequencing. After assembling and gene annotation, we found Ranunculus transcriptomes are most similar with Vitis vinifera genome. 1687 orthologs shared by four diploids were infered by a new orthology inference method published recently. Then we attempted to construct phylogentic tree using these orthologs. For testing whether or not the introgression between species in the hybrid zone exists, this research calculated the Patterson’s D-statistic values between R. trigonus, R. chinensis and R. silerifolius var. dolichanthus. Moreover the results further proved that there is a remarkable introgression between R. trigonus- R. chinensis and R. trigonus – R. silerifolius var. dolichanthus. In order to detect positive selection genes between different taxa, Ka/Ks ratio of each ortholog was calculated, and positive selection genes were annotated by software Blast2 go. The results showed that the functional enrichment of positive selection genes is mainly related to banding, and enrichment of biological process is mainly related to metabolism and cell metabolism. Further analysis showed that some of positive selection genes are related to stress tolerance and reproduction isolation. Thus, these genes may be directly related to adaptive evolution. Pairwise sequence alignment of these positive selective genes showed that many positive selection genes of R. trigonus- R. chinensis and R. trigonus – R. silerifolius var. dolichanthus are completely same, which meaned that introgression between different taxa results in adaptive gene spread.In conclusion, this study got an early picture of reticulate relationship of R. cantoniensis polyploid complex; developed 13 pairs of microsatellite primers suitable for R. cantoniensis polyploid complex; discovered a multispecies hybrid zone, and proved dissymmetric gene flow and a great diversity of genotypes in the hybrid zone; obtained a large number of genes using transcriptomics sequencing, and laid a good foundation for late-stage study; tried to infer ortholog clusters using a new orthology inference method, and proved phylogenetic tree constructed based on transscriptome have a higher resolution; determined introgression between different species pairs in the hybrid zone, and considered that adaptive genes can spread to other species over the species barrier. All the results indicated that hybridization between low-ploid taxa and adaptive gene ingrogression are very extensive in polyploid complex, and which lead to collective evolution in polyploid complex. This study further enriched the theory of hybrid speciation.