•Five new eremophilane-/seco-eremophilane-type sesquiterpenoids were isolated.•Furanoeremophilane composition in Ligularia lamarum was diverse.•Major components of a pair of two sympatrically collected samples were different.•One of five samples was a hybrid, but its chemical composition was of L. lamarum.Chemical compositions and internal transcribed spacer (ITS) sequences of five samples of Ligularia lamarum collected in Sichuan Province, China, were analyzed. Fourteen compounds, including four new eremophilanes and one new seco-eremophilane, were isolated and their structures were elucidated by spectroscopic methods. Intra-specific diversity in the chemical composition was found to be higher than previously known. The result of DNA analysis suggested that one of the samples was introgressed, although its chemical composition was typical of L. lamarum.Download high-res image (175KB)Download full-size image

The evolution of photosynthesis is an important feature of mixotrophic plants. Previous inferences proposed that mixotrophic taxa tend to retain most genes relating to photosynthetic functions but vary in plastid gene content. However, no sequence data are available to test this hypothesis in Ericaceae. To investigate changes in plastid genomes that may result from a transition from autotrophy to mixotrophy, the plastomes of two mixotrophic plants, Pyrola decorata and Chimaphila japonica, were sequenced at Illumina's Genome Analyzer and compared to the published plastome of the autotrophic plant Rhododendron simsii, which also belongs to Ericaceae. The greatest discrepancy between mixotrophic and autotrophic plants was that ndh genes for both P. decorata and C. japonica plastomes have nearly all become pseudogenes. P. decorata and C. japonica also retained all genes directly involved in photosynthesis under strong selection. The calculated rate of nonsynonymous nucleotide substitutions and synonymous substitutions of protein-coding genes (dN/dS) showed that substitution rates in shade plants were apparently higher than those in sunlight plants. The two mixotrophic plastomes were generally very similar to that of non-parasitic plants, although ndh genes were largely pseudogenized. Photosynthesis genes under strong selection were retained in the two mixotrophs, however, with greatly increased substitution rates. Further research is needed to gain a clearer understanding of the evolution of autotrophy and mixotrophy in Ericaceae.

Natural hybridization has been regarded as a crucial pathway of speciation and provides the raw materials for the evolution of biodiversity. The interspecific natural hybridization of the genus Ligularia Cass. is universal and has been considered to be an important factor driving the high diversity of Ligularia species in the Hengduan Mountains, China. Although the natural hybridization between L. vellerea and L. subspicata was reported previously, the direction of hybridization was uncertain due to the limitation of sampling. Thus, in this study, we sampled more individuals and increased two fragments of chloroplast DNA on the basis of the previous study to further verify the natural hybridization between L. vellerea and L. subspicata and confirm the direction of hybridization. Based on DNA sequences (atpB-rbcL, trnL-rpl32, trnQ-5'rps16, and nuclear ribosomal internal transcribed spacer region) data, we concluded that putative hybrids were primary products of hybridization between L. vellerea and L. subspicata and the hybridization was bidirectional. Moreover, sympatric L. tongolensis was not apparently involved in the hybridization. Surprisingly, some pure L. subspicata individuals showed the disaccordance between morphology and DNA data, which might indicate that introgression occurs between L. vellerea and L. subspicata.

The dry valleys in southwest China harbor a highly varied concentration of endemic plant species. To date, the conservation of the unique ecosystem has been paid attention, whereas conservation for the regional endemic plant species has not been much focused. The dry valleys own approximately 1707 plant species among which, 37.42 % are endemic. The plant species in that dry ecosystem are currently challenged by the climate change and anthropogenic activity. To give insight into the current (and past) status of threatened species and the population conservation management strategies, we investigated the genetic diversities of endemic and endangered plant species in the dry valleys in the last decade, using nuclear or plastid molecular markers. Our data revealed that the plant species in this region have low levels of genetic diversity within populations and strong genetic structure. We thus concluded that strong geography patterns are mainly shaped by complex topography as well as their local habitats. We pointed that it is of importance to incorporate neutral and non-neutral genetic diversity when assessing the status of the endangered species. We suggest more research findings are needed to well understand and ensure regional effective conservation on the plant species occurred in the dry valleys.

Further chemical investigation of two Ligularia virgaurea samples collected in China resulted in the isolation of 21 new compounds, two of which were bakkane-type sesquiterpenoids bearing an anhydride-type ring C, which was a previously unknown partial structure. These samples belonged to the V-type (the major component was virgaurenone) among the five chemotypes found in this species.

•Seventeen furanoeremophilanes were isolated from L. anoleuca and L. fischeri.•The presence of three chemotypes was detected by LCMS analysis.•Differences in chemical composition of L. anoleuca and L. fischeri are small.•L. anoleuca and L. fischeri are indistinguishable in the neutral DNA sequences.The sesquiterpenoid composition in the root and the DNA sequences of evolutionarily neutral regions were studied in Ligularia anoleuca and Ligularia fischeri (Asteraceae) collected in the Sichuan Province of China. LC–MS analysis showed that L. anoleuca populations from different localities had different chemical compositions. However, the isolated compounds were similar to each other, indicating that the differences in chemical composition were not large. The DNA analysis suggested that the two species were indistinguishable. Seventeen furanoeremophilanes and an eremophilane acetal were isolated.Graphical abstractThe two title species were indistinguishable both in the root chemicals and in neutral DNA sequences examined.

Munronia delavayi Franch. (Meliaceae) is a vulnerable perennial species that is narrowly distributed and endemic to the dry-hot valley of the middle/lower Jinsha River. However, 12 hydropower stations are currently scheduled to be built in the middle and lower Jinsha River drainage and their distribution overlaps with the range of M. delavayi. The construction of these hydropower stations will cause flooding of the adjacent habitats that support M. delavayi and will change the local ecological environment, which may result in a new and dangerous situation for the survival of this species. Our aim was to evaluate the population structure and propose the most suitable conservation strategy for M. delavayi to support its adaptive potential. In this study, we collected 70 individuals from seven populations that covered most of the geographic range along the Jinsha River and investigated the population diversity, genetic structure and demographic history of this species by analyzing the variations in one mitochondrial marker (18sf-5sr), two non-coding chloroplast DNA regions (trnT-trnL, trnS-trnG), and one nuclear DNA marker (Gs687f-994r). As a result, three mitotypes, nine chlorotypes and six haplotypes with high genetic diversity (HT = 0.580, 0.774 and 0.740) were detected based on mtDNA, cpDNA and nDNA and a strong genetic structure (FST = 0.855 and 0.545) was detected based on cpDNA and nDNA. Our study also indicated that habitat fragmentation and limited gene flow may result in the genetic differentiation of this species and that the population distributed in Qiaojia, Yongshan and Panzhihua should be proactively protected based on the conservation genetic analysis.

•Sixteen compounds were isolated from six samples of Cermanthodium lineare.•Three samples produced furanoeremophilanes, and the other three contained eremophilan-8-ones.•The DNA sequences supported the existence of two chemotypes in C. lineare.•Eremophilane sesquiterpenoids from Cremanthodium species were previously unknown.Chemical constituents and evolutionally neutral DNA sequences of six samples of Cremanthodium lineare Maxim., collected in the Sichuan Province of China, were studied. Three samples produced furanoeremophilanes and the other three, eremophilan-8-ones. The chemotypes were found to be correlated with DNA sequence types, suggesting that the chemical diversity observed has a genetic origin. Production of furanoeremophilanes by a Cremanthodium species suggests an evolutionary relationship between Cremanthodium and Ligularia species, and possibly to related genera.Three of six samples of Cremanthodium lineare produced furanoeremophilanes, and the other three contained eremophilan-8-ones; two chemotypes were thus found. These are correlated with DNA sequence types.

Climate changes during glacial periods have had significant effects on the current distribution of plant species. Palaeontologial data suggest that modern cycads originated in southwest China. Cycas debaoensis (Cycadaceae) is an endangered species restricted to a small area of southwest China. This species has been classified into two types: sand and karst, according to the soil matrix they grow on. To determine the locations of its glacial refugia and its genetic structure, we examined chloroplast sequence variation of the atpB-rbcL and psbA-trnH intergenic spacers. Four chloroplast DNA haplotypes were obtained from 120 individuals collected from 11 populations covering the entire extant distribution range of the species. Significant population subdivision was detected (GST = 0.684 and FST = 0.74160), suggesting low levels of gene flow between regions and populations. There was marked haplotype differentiation between populations in the sand and karst regions, with only one haplotype being present in both. The molecular phylogenetic data, together with the geographic distribution of the haplotypes, suggest that C. debaoensis experienced range contraction during glacial periods, and that the current populations are still confined to the areas of the original refugia. These results implied that isolated refugia might have maintained in both sand and karst regions during the last glacial maximum and even earlier glaciations. The low within-population diversity of C. debaoensis suggested that there were strong bottleneck events or founder effects within each separate region during the Quaternary climatic oscillations. These findings are important for the conservation of this endangered species.

Citrus hongheensis is a critically endangered species endemic to the Honghe river region in southeastern Yunnan, China. Its genetic diversity and differentiation were investigated using Inter-Simple Sequence Repeat (ISSR) markers. One hundred primers were screened, and a total of 245 loci were amplified from seven natural populations by 13 informative and reliable primers. Of these 245 ISSR loci, 233 were polymorphic and the detected variations revealed a relatively high level of intraspecific genetic diversity. At the population level, the mean percentage of polymorphic loci (PPB) was 36.50%, while the average expected heterozygosity (He) and Shannon diversity index (Ho) were 0.1327 and 0.1972, respectively. At the species level (across all populations), PPB was 95.10%, while He and Ho were 0.3520 and 0.5195, respectively. A high Gst value (0.6247) indicated that there is significant differentiation among populations, which was confirmed by AMOVA analysis (Φst = 0.6420). Pairwise genetic identity (I) values among populations ranged from 0.6341 to 0.7675, with a mean of 0.7008. We propose that the high level of genetic differentiation may be the result of habitat fragmentation and limited gene flow (Nm = 0.1502). For effective in situ conservation and population restoration of C. hongheensis it will be important to maintain historical processes, including high outbreeding rates, sufficient gene flow, and large effective population sizes.

Intra-specific diversity in Liularia vellerea growing in the northwestern to central Yunnan province of China was studied by chemical and genetic approaches. Samples collected in the Jianchuan, Lijiang, and Zhongdian areas contained 6,15-dioxygenated furanoeremophilanes as their major components (type A); whereas samples from the Luguhu area accumulated 1,6-dioxygenated furanoeremophilanes (type B); a sample from near Kunming, however, contained 6,15-dioxygenated eremophilanolides (type C). 11βH- and 11αH-6β-angeloyloxy-15-carboxyeremophil-7-en-12,8-olides (eremofarfugins D and E) were also isolated and their structures were determined. A correlation between the composition and the DNA sequence was observed in the ITSs.L. vellerea of Zhongdian, Luguhu, and Kunming areas in Yunnan Province of China could be distinguished on the basis of chemical constituents and DNA sequences.

Geological activities and climate oscillations during the Quaternary period profoundly impacted the distribution of species in Southwest China. Some plant species may be harbored in refugia, such as the dry-hot valleys of Southwest China. Cycas chenii X. Gong & W. Zhou, a critically endangered cycad species, which grows under the canopy in subtropical evergreen broad-leaved forests along the upstream drainage area of the Red River, is endemic to this refugium. In this study, 60 individuals of C. chenii collected from six populations were analyzed by sequencing two chloroplast intergenic spacers (cpDNA: psbA-trnH and trnL-trnF) and two nuclear genes (PHYP and RBP-1). Results showed high genetic diversity at the species level, but low within-population genetic diversity and high interpopulation genetic differentiation. A Bayesian phylogenetic tree based on cpDNA showed that five chloroplast haplotypes were clustered into two clades, which corresponds to the division of the western and eastern bank of the Red River. These data indicate a possible role for the Red River as a geographic barrier to gene flow in C. chenii. Based on our findings, we propose appropriate in situ and ex situ conservation strategies for C. chenii.