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.

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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.