Resolving complicated relationships of the Panax bipinnatifidus complex in southwestern China by RAD-seq data.

The P. binpinnatifidus complex included most of the Panax species distributed in Sino-Himalaya regions except for P. pseudoginseng, P. stipuleanatus and P. notoginseng. However, the delimitation and identification of these taxa within the species complex are very difficult due to the existence of morphological intermediates, and their evolutionary relationships remain unresolved despite several studies have been carried out based on traditional DNA markers. The taxonomic uncertainty hinders the identification, conservation and exploration of these wild populations of Panax. To study this species complex, we employed ddRAD-seq data of these taxa from 18 different localities of southwestern China, using two RAD analysis pipelines, STACKS and pyRAD. Based on the results of phylogenetic analysis, the species complex was divided into four clades with high supports, which largely agreed with morphologically described species. Two clades, corresponding to P. vietnamensis and P. zingiberensis, respectively, were sister groups, indicating that these two species had a closer genetic relationship; the third clade was consisted of samples with bamboo-like rhizomes named as P. wangianus clade, and the fourth one with moniliform rhizomes was named as P. bipinnatifidus clade. The population genetic structure analysis and D-statistics test showed the localized admixture among these species, which indicated that introgression had occurred among the related lineages continuously distributed in southeastern Yunnan and adjacent regions.

Climatic Refugia and Geographical Isolation Contribute to the Speciation and Genetic Divergence in Himalayan-Hengduan Tree Peonies (Paeonia delavayi and Paeonia ludlowii).

Himalaya and Hengduan Mountains (HHM) is a biodiversity hotspot, and very rich in endemic species. Previous phylogeographical studies proposed different hypotheses (vicariance and climate-driven speciation) in explaining diversification and the observed pattern of extant biodiversity, but it is likely that taxa are forming in this area in species-specific ways. Here, we reexplored the phylogenetic relationship and tested the corresponding hypotheses within Paeonia subsect. Delavayanae composed of one widespread species (Paeonia delavayi) and the other geographically confined species (Paeonia ludlowii). We gathered genetic variation data at three chloroplast DNA fragments and one nuclear gene from 335 individuals of 34 populations sampled from HHM. We performed a combination of population genetic summary statistics, isolation-with-migration divergence models, isolation by environment, and demographic history analyses. We found evidence for the current taxonomic treatment that P. ludlowii and P. delavayi are two different species with significant genetic differentiation. The significant isolation by environment was revealed within all sampled populations but genetic distances only explained by geographical distances within P. delavayi populations. The results of population divergence models and demographic history analyses indicated a progenitor-derivative relationship and the Late Quaternary divergence without gene flow between them. The coalescence of all sampled cpDNA haplotypes could date to the Late Miocene, and P. delavayi populations probably underwent a severe bottleneck in population size during the last glacial period. Genetic variation in Paeonia subsect. Delavayanae is associated with geographical and environmental distances. These findings point to the importance of geological and climatic changes as causes of the speciation event and lineage diversification within Paeonia subsect. Delavayanae.

Maintenance of species boundaries in three sympatric Ligularia (Senecioneae, Asteraceae) species.

The key process in speciation concerns the formation and maintenance of reproductive isolating barriers between diverging lineages. Although species boundaries are frequently investigated between two species across many taxa, reproductive isolating barriers among multiple species (>2) that would represent the most common phenomenon in nature, remain to be clarified. Here, we use double digest restriction-site associated DNA (ddRAD) sequencing to examine patterns of hybridization at a sympatric site where three Ligularia species grow together and verify whether those patterns contribute to the maintenance of boundaries among species. The results based on the RAD SNP datasets indicated hybridization Ligularia cyathiceps × L. duciformis and L. duciformis × L. yunnanensis were both restricted to F1 s plus a few first-generation backcrosses and no gene introgression were identified, giving rise to strong reproductive isolation among hybridizing species. Moreover, hybrid swarm simulation, using HYBRIDLAB, indicated the RAD SNP datasets had sufficient discriminatory power for accurate hybrid detection. We conclude that parental species show strong reproductive isolation and they still maintain species boundaries, which may be the key mechanism to maintain species diversity of Ligularia in the eastern Qinghai-Tibetan Plateau and adjacent areas. Moreover, this study highlights the effectiveness of RAD sequencing in hybridization studies.

Ecological and Genetic Divergences with Gene Flow of Two Sister Species (Leucomeris decora and Nouelia insignis) Driving by Climatic Transition in Southwest China.

Understanding of the processes of divergence and speciation is a major task for biodiversity researches and may offer clearer insight into mechanisms generating biological diversity. Here, we employ an integrative approach to explore genetic and ecological differentiation of Leucomeris decora and Nouelia insignis distributed allopatrically along the two sides of the biogeographic boundary 'Tanaka Line' in Southwest China. We addressed these questions using ten low-copy nuclear genes and nine plastid DNA regions sequenced among individuals sampled from 28 populations across their geographic ranges in China. Phylogenetic, coalescent-based population genetic analyses, approximate Bayesian computation (ABC) framework and ecological niche models (ENMs) were conducted. We identified a closer phylogenetic relationship in maternal lineage of L. decora with N. insignis than that between L. decora and congeneric Leucomeris spectabilis. A deep divergence between the two species was observed and occurred at the boundary between later Pliocene and early Pleistocene. However, the evidence of significant chloroplast DNA gene flow was also detected between the marginal populations of L. decora and N. insignis. Niche models and statistical analyses showed significant ecological differentiation, and two nuclear loci among the ten nuclear genes may be under divergent selection. These integrative results imply that the role of climatic shift from Pliocene to Pleistocene may be the prominent factor for the divergence of L. decora and N. insignis, and population expansion after divergence may have given rise to chloroplast DNA introgression. The divergence was maintained by differential selection despite in the face of gene flow.

Application of RAD Sequencing for Evaluating the Genetic Diversity of Domesticated Panax notoginseng (Araliaceae).

Panax notoginseng, a traditional Chinese medicinal plant, has been cultivated and domesticated for approximately 400 years, mainly in Yunnan and Guangxi, two provinces in southwest China. This species was named according to cultivated rather than wild individuals, and no wild populations had been found until now. The genetic resources available on farms are important for both breeding practices and resource conservation. In the present study, the recently developed technology RADseq, which is based on next-generation sequencing, was used to analyze the genetic variation and differentiation of P. notoginseng. The nucleotide diversity and heterozygosity results indicated that P. notoginseng had low genetic diversity at both the species and population levels. Almost no genetic differentiation has been detected, and all populations were genetically similar due to strong gene flow and insufficient splitting time. Although the genetic diversity of P. notoginseng was low at both species and population levels, several traditional plantations had relatively high genetic diversity, as revealed by the He and π values and by the private allele numbers. These valuable genetic resources should be protected as soon as possible to facilitate future breeding projects. The possible geographical origin of Sanqi domestication was discussed based on the results of the genetic diversity analysis.

The impacts of polyploidy, geographic and ecological isolations on the diversification of Panax (Araliaceae).

BACKGROUND: Panax L. is a medicinally important genus within family Araliaceae, where almost all species are of cultural significance for traditional Chinese medicine. Previous studies suggested two independent origins of the East Asia and North America disjunct distribution of this genus and multiple rounds of whole genome duplications (WGDs) might have occurred during the evolutionary process. RESULTS: We employed multiple chloroplast and nuclear markers to investigate the evolution and diversification of Panax. Our phylogenetic analyses confirmed previous observations of the independent origins of disjunct distribution and both ancient and recent WGDs have occurred within Panax. The estimations of divergence time implied that the ancient WGD might have occurred before the establishment of Panax. Thereafter, at least two independent recent WGD events have occurred within Panax, one of which has led to the formation of three geographically isolated tetraploid species P. ginseng, P. japonicus and P. quinquefolius. Population genetic analyses showed that the diploid species P. notoginseng harbored significantly lower nucleotide diversity than those of the two tetraploid species P. ginseng and P. quinquefolius and the three species showed distinct nucleotide variation patterns at exon regions. CONCLUSION: Our findings based on the phylogenetic and population genetic analyses, coupled with the species distribution patterns of Panax, suggested that the two rounds of WGD along with the geographic and ecological isolations might have together contributed to the evolution and diversification of this genus.