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Landscape features are effective geographical barriers resulting inpopulation differentiation of plant species. Taihang Mountains in China possess complexly geographical topology and specific landscape characteristics. Two closely related syntopic Opisthopappus species, mainly distribute in different areas of Taihang Mountains, respectively. How the landscape of Taihang Mountains affects the gene exchange between these two species still unclear. Combined SNP data from restriction-site associated DNA sequencing (RAD-seq) and recently developed landscape genetic methods (EEMS, Samβada, LFMM), we conducted a landscape genetic analysis of these two species. It found that the diversity of O. longilobus was higher than that of O. taihangensis, the gene flow was mostly from north to south along Taihang Mountains. However, a general north–south gene exchange barrier between O. longilobus and O. taihangensis was detected. Among the landscape factors of Taihang Mountains, eight was found to be the important ones, including average precipitation in August, October, and November, solar radiation in August, soil PH, built-up land, rain-fed cultivated land, and workability. And these eight factors were closely related to the occurred barriers, indicating that climatic conditions and human activities rather than geographical environment resulted in these barriers. Twenty-nine selected SNPs were identified to be significant correlated with the eight factors, especially average precipitation in November. Thus, the average precipitation in November could be regarded as an ecological indicator for O. longilobus and O. taihangensis. The results revealed the effect of landscape features on two species and the adaption on the landscape environment of Taihang Mountains during the long-term evolution.

Opisthopappus is a major wild source of Asteraceae with good cold and drought resistance. Two species of this genus (Opisthopappus taihangensis and Opisthopappus longilobus) have been employed as model systems to address the evolutionary history of perennial herb biomes in the Taihang Mountains of China. However, further studies on the adaptive divergence processes of these two species are currently impeded by the lack of genomic resources. To elucidate the molecular mechanisms involved, a comparative analysis of these two species was conducted. Among the identified transcription factors, the bHLH members were most prevalent, which exhibited significantly different expression levels in the terpenoid metabolic pathway. O. longilobus revealed a higher expression than did O. taihangensis in terms of terpenes biosynthesis and metabolism, particularly regarding monoterpenoids and diterpenoids. Analyses of the positive selection genes (PSGs) identified from O. taihangensis and O. longilobus, 1203 genes were found that related to adaptative divergence, which were under rapid evolution and/or have signs of positive selection. Different PSG expressions occurred primarily in the mitochondrial electron transport, starch degradation, secondary metabolism, as well as nucleotide synthesis and S-metabolism pathway processes. Two PSGs were obviously differentially expressed in terpenes biosynthesis that might result in the fragrances divergence between O. longilobus and O. taihangensis, which would provide insights as to how the two species adapted to different environments, characterized by sub-humid warm temperate and temperate continental monsoon climates. The comparative analysis for these two species of Opisthopappus not only revealed how the divergence occurred from molecular perspective, but also provided novel insights into how differential adaptations occurred in Taihang Mountains.

Clintonia udensis is a perennial herb possessing two cytotypes diploid (2n=14) and tetraploid (4n=28). In the Hualongshan Mountains, the diploid of C. udensis primarily grows in 2450 m areas on the south slopes, while the tetraploid grows mainly in 1900 m areas on the north slopes. So, this intra-polyploidy is regarded as an excellent material to study the origination, evolution and adaption of plant polyploidy. Through field investigations and molecular genotyping, we initially analyzed the bud bank spatial characteristics, clonal growth, and spatial genetic structure populations between the different ploidy of C. udensis. It found that the rhizome knot styles of C. udensis had zigzag, C, V, and Y models between the two cytotypes. There was no dominated clone present in the diploid or tetraploid. The clone architectures of two ploidies were both phalanxes. However, the number of rhizome knots, the number of buds of each rhizome knot, the ratio of rhizome branches, and average tetraploid clones were higher than that of the diploid. The diversity indices of the tetraploid, such as clone diversity index, genetic distribution uniformity, and genetic diversity index, were also slightly higher than that of the diploid. Thus, clonal reproduction differentiation and significant genetic variations occurred between the diploid and tetraploid of C. udensis. These two cytotypes, through seed reproduction and clonal growth, became a facultative clonal species and maintained its survival stability and reproduction. During the evolutionary process, the tetraploid of C. udensis with higher clonal diversity and genetic diversity responded and adapted to new surroundings that different from the ancestral diploid in the Hualongshan Mountains.

Opisthopappus Shih is an endemic and endangered genus restricted to the Taihang Mountains that has important ornamental and economic value. According to the Flora Reipublicae Popularis Sinicae (FRPS, Chinese version), this genus contains two species (Opisthopappus longilobus and Opisthopappus taihangensis), whereas in the Flora of China (English version) only one species O. taihangensis is present. The interspecific phylogenetic relationship remains unclear and undefined, which might primarily be due to the lack of specific molecular markers for phylogenetic analysis. For this study, 2644 expressed sequence tag-simple sequence repeats (EST-SSRs) from 33,974 unigenes using a de novo transcript assembly of Opisthopappus were identified with a distribution frequency of 7.78% total unigenes. Thereinto, mononucleotides (1200, 45.39%) were the dominant repeat motif, followed by trinucleotides (992, 37.52%), and dinucleotides (410, 15.51%). The most dominant trinucleotide repeat motif was ACC/GGT (207, 20.87%). Based on the identified EST-SSRs, 245 among 1444 designed EST-SSR primers were selected for the development of potential molecular markers. Among these markers, 63 pairs of primers (25.71%) generated clear and reproducible bands with expected sizes. Eventually, 11 primer pairs successfully amplified all individuals from the studied populations. Through the EST-SSR markers, a high level of genetic diversity was detected between Opisthopappus populations. A significant genetic differentiation between the O. longilobus and O. taihangensis populations was found. All studied populations were divided into two clusters by UPGMA, NJ, STRUCTURE, and PCoA. These results fully supported the view of the FRPS, namely, that O. longilobus and O. taihangensis should be regarded as two distinct species. Our study demonstrated that transcriptome sequences, as a valuable tool for the quick and cost-effective development of molecular markers, was helpful toward obtaining comprehensive EST-SSR markers that could contribute to an in-depth assessment of the genetic and phylogenetic relationships between Opisthopappus species.