The hybridization origin of the Chinese endemic herb genus Notopterygium (Apiaceae): Evidence from population genomics and ecological niche analysis.

Hybridization is recognized as a major force in species evolution and biodiversity formation, generally leading to the origin and differentiation of new species. Multiple hybridization events cannot easily be reconstructed, yet they offer the potential to study a number of evolutionary processes. Here, we used nuclear expressed sequence tag-simple sequence repeat and large-scale single nucleotide polymorphism variation data, combined with niche analysis, to investigate the putative independent hybridization events in Notopterygium, a group of perennial herb plants endemic to China. Population genomic analysis indicated that the four studied species are genetically well-delimited and that N. forrestii and N. oviforme have originated by hybridization. According to Approximate Bayesian Computation, the best-fit model involved the formation of N. forrestii from the crossing of N. franchetii and N. incisum, with N. forrestii further backcrossing to N. franchetii to form N. oviforme. The niche analyses indicated that niche divergence [likely triggered by the regional climate changes, particularly the intensification of East Asian winter monsoon, and tectonic movements (affecting both Qinghai-Tibetan Plateau and Qinling Mountains)] may have promoted and maintained the reproductive isolation among hybrid species. N. forrestii shows ecological specialization with respect to their parental species, whereas N. oviforme has completely shifted its niche. These results suggested that the climate and environmental factors together triggered the two-step hybridization of the East Asia herb plants. Our study also emphasizes the power of genome-wide SNPs for investigating suspected cases of hybridization, particularly unravelling old hybridization events.

Niche divergence at the intraspecific level in an endemic rare peony (Paeonia rockii): A phylogenetic, climatic and environmental survey.

Ecological factors have received increasing attention as drivers of speciation but also in the maintenance of postspeciation divergence. However, the relative significance of the responses of species to climate oscillations for driving niche divergence or conservatism in the evolution of many species that pass through diverse environments and limited geographical boundaries remains poorly understood. Paeonia rockii (one of the ancient species of Paeonia) comprising two subspecies called Paeonia rockii subsp. rockii and Paeonia rockii subsp. taibaishanica is an endemic, rare, and endangered medicinal plant in China. In this study, we integrated whole chloroplast genomes, and ecological factors to obtain insights into ecological speciation and species divergence in this endemic rare peony. RAxML analysis indicated that the topological trees recovered from three different data sets were identical, where P. rockii subsp. rockii and P. rockii subsp. taibaishanica clustered together, and molecular dating analyses suggested that the two subspecies diverged 0.83 million years ago. In addition, ecological niche modeling showed that the predicted suitable distribution areas for P. rockii subsp. rockii and P. rockii subsp. taibaishanica differed considerably, although the predicted core distribution areas were similar, where the population contracted in the last interglacial and expanded in the last glacial maximum. Under the emissions scenarios for the 2050s and 2070s, the suitable distribution areas were predicted to contract significantly, where the migration routes of the two subspecies tended to migrate toward high latitudes and elevations, thereby suggesting strong responses of the distributions of the two subspecies to climate change. These findings combined with the phylogeographic relationships provide comprehensive insights into niche variation and differentiation in this endemic rare peony, and they highlight the importance of geological and climatic changes for species divergence and changes in the population geographic patterns of rare and endangered medicinal plants in East Asia.

Complex population evolutionary history of four cold-tolerant Notopterygium herb species in the Qinghai-Tibetan Plateau and adjacent areas.

Historical geological and climatic events are the most important drivers of population expansions/contractions, range shifts, and interspecific divergence in plants. However, the species divergence and spatiotemporal population dynamics of alpine cold-tolerant herbal plants in the high-altitude Qinghai-Tibetan Plateau (QTP) and adjacent areas remain poorly understood. In this study, we investigated population evolutionary history of four endangered Notopterygium herb species in the QTP and adjacent regions. We sequenced 10 nuclear loci, 2 mitochondrial DNA regions, and 4 chloroplast DNA regions in a total of 72 natural populations from the 4 species, and tested the hypothesis that the population history of these alpine herbs was markedly affected by the Miocene-Pliocene QTP uplifts and Quaternary climatic oscillations. We found that the four Notopterygium species had generally low levels of nucleotide variability within populations. Molecular dating and isolation-with-migration analyses suggested that Notopterygium species diverged ~1.74-7.82 million years ago and their differentiation was significantly associated with recent uplifts of the eastern margin of the QTP. In addition, ecological niche modeling and population history analysis showed that N. incisum and N. franchetii underwent considerable demographic expansions during the last glacial period of the Pleistocene, whereas a demographic contraction and a expansion occurred for N. forrestii and N. oviforme during the antepenultimate interglacial period and penultimate glacial period, respectively. These findings highlight the importance of geological and climatic changes during the Miocene-Pliocene and Pleistocene as causes of species divergence and changes in population structure within cold-tolerant herbs in the QTP biodiversity hotspot.

Effects of Mountain Uplift and Climatic Oscillations on Phylogeography and Species Divergence in Four Endangered Notopterygium Herbs.

Mountain uplift and climatic fluctuations are important driving forces that have affected the geographic distribution and population dynamics history of organisms. However, it is unclear how geological and climatic events might have affected the phylogeographic history and species divergence in high-alpine herbal plants. In this study, we analyzed the population demographic history and species differentiation of four endangered Notopterygium herbs on the high-altitude Qinghai-Tibetan Plateau (QTP) and adjacent areas. We combined phylogeographic analysis with species distribution modeling to detect the genetic variations in four Notopterygium species (N. incisum, N. franchetii, N. oviforme, and N. forrestii). In total, 559 individuals from 74 populations of the four species were analyzed based on three maternally inherited chloroplast fragments (matK, rbcL, and trnS-trnG) and one nuclear DNA region (internal transcribed spacer, ITS). Fifty-five chloroplast DNA (cpDNA) and 48 ITS haplotypes were identified in the four species. All of the cpDNA and ITS haplotypes were species-specific, except N. franchetii and N. oviforme shared one cpDNA haplotype, H32. Phylogenetic analysis suggested that all four species formed a monophyletic clade with high bootstrap support, where N. franchetii and N. oviforme were sisters. In addition, each Notopterygium species generated an individual clade that corresponded to their respective species in the ITS tree. Population dynamics analyses and species distribution modeling showed that the two widely distributed herbs N. incisum and N. franchetii exhibited obvious demographic expansions during the Pleistocene ice ages. Molecular dating suggested that the divergence of the four Notopterygium species occurred approximately between 3.6 and 1.2 Mya, and it was significantly associated with recent extensive uplifts of the QTP. Our results support the hypothesis that mountain uplift and Quaternary climatic oscillations profoundly shaped the population genetic divergence and demographic dynamics of Notopterygium species. The findings of this and previous studies provide important insights into the effects of QTP uplifts and climatic changes on phylogeography and species differentiation in high altitude mountainous areas. Our results may also facilitate the conservation of endangered herbaceous medicinal plants in the genus Notopterygium.

Comparative Transcriptome Analysis Reveals Adaptive Evolution of Notopterygium incisum and Notopterygium franchetii, Two High-Alpine Herbal Species Endemic to China.

The extreme conditions (e.g., cold, low oxygen, and strong ultraviolet radiation) of the high mountains provide an ideal natural laboratory for studies on speciation and the adaptive evolution of organisms. Up to now, few genome/transcriptome-based studies have been carried out on how plants adapt to conditions at extremely high altitudes. Notopterygium incisum and Notopterygium franchetii (Notopterygium, Apiaceae) are two endangered high-alpine herbal plants endemic to China. To explore the molecular genetic mechanisms of adaptation to high altitudes, we performed high-throughput RNA sequencing (RNA-seq) to characterize the transcriptomes of the two species. In total, more than 130 million sequence reads, 81,446 and 63,153 unigenes with total lengths of 86,924,837 and 62,615,693 bp, were generated for the two herbal species, respectively. OrthoMCL analysis identified 6375 single-copy orthologous genes between N. incisum and N. franchetii. In total, 381 positively-selected candidate genes were identified for both plants by using estimations of the non-synonymous to synonymous substitution rate. At least 18 of these genes potentially participate in RNA splicing, DNA repair, glutathione metabolism and the plant-pathogen interaction pathway, which were further enriched in various functional gene categories possibly responsible for environment adaptation in high mountains. Meanwhile, we detected various transcription factors that regulated the material and energy metabolism in N. incisum and N. franchetii, which probably play vital roles in the tolerance to stress in surroundings. In addition, 60 primer pairs based on orthologous microsatellite-containing sequences between the both Notopterygium species were determined. Finally, 17 polymorphic microsatellite markers (SSR) were successfully characterized for the two endangered species. Based on these candidate orthologous and SSR markers, we detected that the adaptive evolution and species divergence of N. incisum and N. franchetii were significantly associated with the extremely heterogeneous environments and climatic oscillations in high-altitude areas. This work provides important insights into the molecular mechanisms of adaptation to high-altitudes in alpine herbal plants.

Comparative Analysis of the Complete Chloroplast Genome of Four Endangered Herbals of Notopterygium.

Notopterygium H. de Boissieu (Apiaceae) is an endangered perennial herb endemic to China. A good knowledge of phylogenetic evolution and population genomics is conducive to the establishment of effective management and conservation strategies of the genus Notopterygium. In this study, the complete chloroplast (cp) genomes of four Notopterygium species (N. incisum C. C. Ting ex H. T. Chang, N. oviforme R. H. Shan, N. franchetii H. de Boissieu and N. forrestii H. Wolff) were assembled and characterized using next-generation sequencing. We investigated the gene organization, order, size and repeat sequences of the cp genome and constructed the phylogenetic relationships of Notopterygium species based on the chloroplast DNA and nuclear internal transcribed spacer (ITS) sequences. Comparative analysis of plastid genome showed that the cp DNA are the standard double-stranded molecule, ranging from 157,462 bp (N. oviforme) to 159,607 bp (N. forrestii) in length. The circular DNA each contained a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRs). The cp DNA of four species contained 85 protein-coding genes, 37 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes, respectively. We determined the marked conservation of gene content and sequence evolutionary rate in the cp genome of four Notopterygium species. Three genes (psaI, psbI and rpoA) were possibly under positive selection among the four sampled species. Phylogenetic analysis showed that four Notopterygium species formed a monophyletic clade with high bootstrap support. However, the inconsistent interspecific relationships with the genus Notopterygium were identified between the cp DNA and ITS markers. The incomplete lineage sorting, convergence evolution or hybridization, gene infiltration and different sampling strategies among species may have caused the incongruence between the nuclear and cp DNA relationships. The present results suggested that Notopterygium species may have experienced a complex evolutionary history and speciation process.

Characterization of Global Transcriptome Using Illumina Paired-End Sequencing and Development of EST-SSR Markers in Two Species of Gynostemma (Cucurbitaceae).

Gynostemma pentaphyllum is an important medicinal herb of the Cucurbitaceae family, but limited genomic data have hindered genetic studies. In this study, transcriptomes of two closely-related Gynostemma species, Gynostemma cardiospermum and G. pentaphyllum, were sequenced using Illumina paired-end sequencing technology. A total of 71,607 nonredundant unigenes were assembled. Of these unigenes, 60.45% (43,288) were annotated based on sequence similarity search with known proteins. A total of 11,059 unigenes were identified in the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database. A total of 3891 simple sequence repeats (SSRs) were detected in 3526 nonredundant unigenes, 2596 primer pairs were designed and 360 of them were randomly selected for validation. Of these, 268 primer pairs yielded clear products among six G. pentaphyllum samples. Thirty polymorphic SSR markers were used to test polymorphism and transferability in Gynostemma. Finally, 15 SSR makers that amplified in all 12 Gynostemma species were used to assess genetic diversity. Our results generated a comprehensive sequence resource for Gynostemma research.