Biogeography and assembly processes of abundant and rare soil microbial taxa in the southern part of the Qilian Mountain National Park, China.

Soil microorganisms play vital roles in regulating multiple ecosystem functions. Recent studies have revealed that the rare microbial taxa (with extremely low relative abundances, which are still largely ignored) are also crucial in maintaining the health and biodiversity of the soil and may respond differently to environmental pressure. However, little is known about the soil community structures of abundant and rare taxa and their assembly processes in different soil layers on the Qinghai-Tibet Plateau (QTP). The present study investigated the community structure and assembly processes of soil abundant and rare microbial taxa on the northeastern edge of the QTP. Soil microbial abundance was defined by abundant taxa, whereas rare taxa contributed to soil microbial diversity. The results of null model show that the stochastic process ruled the assembly processes of all sub-communities. Dispersal limitation contributed more to the assembly of abundant microbial taxa in the different soil layers. In contrast, drift played a more critical role in the assembly processes of the rare microbial taxa. In addition, in contrast to previous studies, the abundant taxa played more important roles in co-occurrence networks, most likely because of the heterogeneity of the soil, the sparsity of amplicon sequencing, the sampling strategy, and the limited samples in the present study. The results of this study improve our understanding of soil microbiome assemblies on the QTP and highlight the role of abundant taxa in sustaining the stability of microbial co-occurrence networks in different soil layers.

Comparison of different internal fixation models in ankle arthrodesis using 3D finite-element analysis.

BACKGROUND: The purpose of this study is to use three-dimensional finite-element analysis to better understand the biomechanical features of various internal fixators for ankle arthrodesis. METHODS: We used finite-element analysis to compare four different types of internal fixations in ankle arthrodesis: Group A had three crossed screws (Ø6.5 mm); Group B had two crossed screws (Ø6.5 mm) and an anterior plate (Ø2.7 mm); Group C only had an anterior anatomical plate (Ø3.5 mm); Group D had one anterior anatomical plate (Ø3.5 mm) and one posterior-lateral screw (Ø6.5 mm). We adopted Ansys 21.0 software to analyze and compare the four types in terms of the displacement of the arthrodesis surface and the stress peak and stress distribution of these models under intorsion, extorsion, dorsiflexion torque, and neutral vertical load. RESULTS: ① Displacement of the arthrodesis surface: In Group A, the maximum displacement was larger than Group D under neutral vertical load and dorsiflexion torque but less than it under intorsion and extorsion torque. In Group B, the maximum displacement against dorsiflexion, neutral vertical load, intorsion, and extorsion was less than that in the other three fixation models. In Group C, the maximum displacement against the above four loading patterns were significantly higher than that in another three fixation models. ② Stress peak and stress distribution: based on the stress distribution of the four models, the peak von Mises stress was concentrated in the central sections of the compression screws, plate joints, and bending parts of the plates. CONCLUSIONS: The fixation model consisting of two crossed screws and an anterior outperformed the other three fixation models in terms of biomedical advantages; thus, this model can be deemed a safe and reliable internal fixation approach for ankle arthrodesis.

Investigation of phylogenetic relationships within Saxifraga diversifolia complex (Saxifragaceae) based on restriction-site associated DNA sequence markers.

Subsect. Hirculoideae Engl. & Irmsch., belonging to Saxifraga sect. Ciliatae Haw., has high species richness. It can be divided into S. diversifolia, S. pseudohirculus, and S. sinomontana complexes based on morphological characteristics. The species with prominent leaf veins on the posterior leaf edge were placed in the S. diversifolia complex, which is mainly distributed on the eastern and southern margins of the Qinghai-Tibetan Plateau. In this study, 53 samples, representing 15 of the 33 described species in the S. diversifolia complex, were sequenced using the Restriction-site Associated DNA Sequence (RAD-seq) technique. A total of 111,938 high-quality SNP loci were screened to investigate the phylogenetic relationships within the S. diversifolia complex. The result of the neighbor-joining (NJ) tree shows that the S. diversifolia complex is a paraphyletic group. Despite of some inconsistencies as revealed by genetic structural analysis, clustering results of representative species reconstructed by both NJ and principal component analysis analyses support previous biogeographic and morphological evidences. In addition, long-distance gene flow events for 11 taxa were detected in the S. diversifolia complex, respectively from S. implicans 1 to S. implicans 2, S. diversifolia and S. maxionggouensis, and from S. maxionggouensis to S. nigroglandulifera. These findings may improve our comprehension of the phylogeny, classification, and evolution of the S. diversifolia complex.

Vegetation structural shift tells environmental changes on the Tibetan Plateau over 40 years.

Structural information of grassland changes on the Tibetan Plateau is essential for understanding alterations in critical ecosystem functioning and their underlying drivers that may reflect environmental changes. However, such information at the regional scale is still lacking due to methodological limitations. Beyond remote sensing indicators only recognizing vegetation productivity, we utilized multivariate data fusion and deep learning to characterize formation-based plant community structure in alpine grasslands at the regional scale of the Tibetan Plateau for the first time and compared it with the earlier version of Vegetation Map of China for historical changes. Over the past 40 years, we revealed that (1) the proportion of alpine meadows in alpine grasslands increased from 50% to 69%, well-reflecting the warming and wetting trend; (2) dominances of Kobresia pygmaea and Stipa purpurea formations in alpine meadows and steppes were strengthened to 76% and 92%, respectively; (3) the climate factor mainly drove the distribution of Stipa purpurea formation, but not the recent distribution of Kobresia pygmaea formation that was likely shaped by human activities. Therefore, the underlying mechanisms of grassland changes over the past 40 years were considered to be formation dependent. Overall, the first exploration for structural information of plant community changes in this study not only provides a new perspective to understand drivers of grassland changes and their spatial heterogeneity at the regional scale of the Tibetan Plateau, but also innovates large-scale vegetation study paradigm.

Bacterial communities associated with mushrooms in the Qinghai-Tibet Plateau are shaped by soil parameters / Las comunidades bacterianas asociadas con los hongos en la meseta de Qinghai-Tíbet están formadas por parámetros del suelo

Fungi capable of producing fruit bodies are essential food and medicine resources. Despite recent advances in the study of microbial communities in mycorrhizospheres, little is known about the bacterial communities contained in fruit bodies. Using high-throughput sequencing, we investigated the bacterial communities in four species of mushrooms located on the alpine meadow and saline-alkali soil of the Qinghai-Tibet Plateau (QTP). Proteobacteria (51.7% on average) and Actinobacteria (28.2% on average) were the dominant phyla in all of the sampled fairy ring fruit bodies, and Acidobacteria (27.5% on average) and Proteobacteria (25.7% on average) dominated their adjacent soils. For the Agria. Bitorquis, Actinobacteria was the dominant phylum in its fruit body (67.5% on average) and adjacent soils (65.9% on average). The alpha diversity (i.e., Chao1, Shannon, Richness, and Simpson indexes) of the bacterial communities in the fruit bodies were significantly lower than those in the soil samples. All of the fungi shared more than half of their bacterial phyla and 16.2% of their total operational taxonomic units (OTUs) with their adjacent soil. Moreover, NH4+ and pH were the key factors associated with bacterial communities in the fruit bodies and soils, respectively. These results indicate that the fungi tend to create a unique niche that selects for specific members of the bacterial community. Using culture-dependent methods, we also isolated 27 bacterial species belonging to three phyla and five classes from fruit bodies and soils. The strains isolated will be useful for future research on interactions between mushroom-forming fungi and their bacterial endosymbionts.(AU)

Chloroplast phylogenomics and the taxonomy of Saxifraga section Ciliatae (Saxifragaceae).

Comprising ca. 200 species, Saxifraga sect. Ciliatae is the most species-rich section of Saxifraga s.str., whose center of diversity is in the Tibeto-Himalayan region. The infra-sectional classification of sect. Ciliatae is still in debate due to the high level of species richness, as well as remarkable variations of habitat, morphology, physiology and life cycles. Subdivisions of this section proposed in various taxonomic systems have not been adequately tested in previous phylogenetic studies, partly due to low taxonomic sampling density, but also to the use of few DNA markers. In order to achieve a more robust infra-sectional classification of sect. Ciliatae, complete chloroplast genomes of 94 taxa from this section were analyzed, of which 93 were newly sequenced, assembled and annotated. The length of the 94 plastomes of sect. Ciliatae taxa range from 143,479 to 159,938 bp, encoding 75 to 79 unique protein-coding genes (PCGs). Analyses of the 94 plastomes revealed high conservation in structural organization, gene arrangement, and gene content. Gene loss and changes of IR boundaries were detected but in extremely low frequency. The molecular phylogenetic tree from concatenated PCGs and complete chloroplast genome sequences exhibited high resolution and support values and confirms that sect. Ciliatae is monophyletic. Three well-supported clades were revealed within the section that agree relatively well with the subsectional taxonomy of Gornall (1987), but some minor modifications should be made. Firstly, the monotypic subsection Cinerascentes should be abandoned and its constituent species, S. cinerascens, assigned to subsect. Gemmiparae. Secondly, subsections Rosulares and Serpyllifoliae should be merged and become subsect. Rosulares. Section Ciliatae thus comprises: subsect. Hirculoideae Engl. & Irmsch.; subsect. Rosulares Gornall; subsect. Gemmiparae Engl. & Irmsch.; subsect. Flagellares (C. B. Clarke) Engl. & Irmsch. and subsect. Hemisphaericae (Engl. & Irmsch.) Gornall.

Bacterial communities associated with mushrooms in the Qinghai-Tibet Plateau are shaped by soil parameters.

Fungi capable of producing fruit bodies are essential food and medicine resources. Despite recent advances in the study of microbial communities in mycorrhizospheres, little is known about the bacterial communities contained in fruit bodies. Using high-throughput sequencing, we investigated the bacterial communities in four species of mushrooms located on the alpine meadow and saline-alkali soil of the Qinghai-Tibet Plateau (QTP). Proteobacteria (51.7% on average) and Actinobacteria (28.2% on average) were the dominant phyla in all of the sampled fairy ring fruit bodies, and Acidobacteria (27.5% on average) and Proteobacteria (25.7% on average) dominated their adjacent soils. For the Agria. Bitorquis, Actinobacteria was the dominant phylum in its fruit body (67.5% on average) and adjacent soils (65.9% on average). The alpha diversity (i.e., Chao1, Shannon, Richness, and Simpson indexes) of the bacterial communities in the fruit bodies were significantly lower than those in the soil samples. All of the fungi shared more than half of their bacterial phyla and 16.2% of their total operational taxonomic units (OTUs) with their adjacent soil. Moreover, NH4+ and pH were the key factors associated with bacterial communities in the fruit bodies and soils, respectively. These results indicate that the fungi tend to create a unique niche that selects for specific members of the bacterial community. Using culture-dependent methods, we also isolated 27 bacterial species belonging to three phyla and five classes from fruit bodies and soils. The strains isolated will be useful for future research on interactions between mushroom-forming fungi and their bacterial endosymbionts.

Complete Chloroplast Genome Sequence of Triosteum sinuatum, Insights into Comparative Chloroplast Genomics, Divergence Time Estimation and Phylogenetic Relationships among Dipsacales.

Triosteum himalayanum, Triosteum pinnatifidum (Triosteum L., Caprifoliaceae, Dipsacales) are widely distributed in China while Triosteum sinuatum mainly occurrs in northeast China. Few reports have been determined on the genus Triosteum. In the present research, we sequenced 2 chloroplast genomes of Triosteum and analyzed 18 chloroplast genomes, trying to explore the sequence variations and phylogeny of genus Triosteum in the order Dipsacales. The chloroplast genomes of the genus Triosteum ranged from 154,579 bp to 157,178 bp, consisting of 132 genes (86 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes). Comparative analyses and phylogenetic analysis supported the division of Dipsacales into two clades, Adoxaceae and six other families. Among the six families, a clade of Valerianaceae+Dipsacaceae was recovered as a sister to a clade of Morinaceae+Linnaeaceae. A closer relationship of T. himalayanum and T. pinnatifidum among three species was revealed. Our research supported that Loniceraferdinandi and Triosteum was closely related. Zabelia had a closer relationship with Linnaea borealis and Dipelta than Morinaceae. The divergence between T. sinuatum and two other species in Triosteum was dated to 13.4 mya.

Plastome structure, phylogenomics and evolution of plastid genes in Swertia (Gentianaceae) in the Qing-Tibetan Plateau.

BACKGROUND: The genus Swertia is of great medicinal importance and one of the most taxonomically challenging taxa within Gentianaceae, largely due to the morphological similarities of species within this genus and with its closely related genera. Previous molecular studies confirmed its polyphyly but suffered from low phylogenetic resolutions because only limited sequence loci were used. Thus, we conducted the structural, gene evolutionary, and phylogenetic analyses of 11 newly obtained plastomes of Swertia. Our result greatly improved the phylogenetic resolutions in Swertia, shed new light on the plastome evolution and phylogenetic relationships of this genus. RESULTS: The 11 Swertia plastomes together with the published seven species proved highly similar in overall size, structure, gene order, and content, but revealed some structural variations caused by the expansion and contraction of the IRb region into the LSC region, due to the heterogeneous length of the ψycf1. The gene rps16 was found to be in a state flux with pseudogenes or completely lost. Similar situation was also documented in other genera of Gentianaceae. This might imply loss of the gene in the common ancestor of Gentianaceae. The distribution plot of ENC vs. GC3 showed all these plastomes arranging very close in the Wright line with an expected ENC value (49-52%), suggesting the codon usage of Swertia was mainly constrained by a GC mutation bias. Most of the genes remained under the purifying selection, however, the cemA was identified under positive selection, possibly reflecting an adaptive response to low CO2 atmospheric conditions during the Late Miocene. Our phylogenomic analyses, based on 74 protein-coding genes (CDS), supported the polyphyly of Swertia with its close allies in the subtribe Swertiinae, presumably due to recent rapid radiation. The topology inferred from our phylogenetic analyses partly supported the current taxonomic treatment. Finally, several highly variable loci were identified, which can be used in future phylogenetic studies and accurate identification of medicinal genuineness of Swertia. CONCLUSIONS: Our study confirmed the polyphyly of Swertia and demonstrated the power of plastome phylogenomics in improvement of phylogenetic resolution, thus contributing to a better understanding of the evolutionary history of Swertia.

Dispersal into the Qinghai-Tibet plateau: evidence from the genetic structure and demography of the alpine plant Triosteum pinnatifidum.

Triosteum pinnatifidum Maxim., an alpine plant, is traditionally used for several medicinal purposes. Here, both chloroplast DNA sequences and nuclear low copy sequence markers were used to investigate the genetic diversity and population structure of T. pinnatifidum. Materials were collected from thirteen localities in the northeast Qinghai-Tibet Plateau (QTP) and adjacent highlands and advanced analytical toolkits were used to access their origin and range shifts. The results revealed a higher level of population differentiation based on chloroplast DNA (cpDNA) concatenated sequences compared with the nuclear DNA sequences (F ST = 0.654 for cpDNA, F ST = 0.398 for AT103), indicating that pollen flow was still extensive in T. pinnatifidum. A decline in haplotype variation was observed from the plateau edge and adjoining highlands toward the platform of the QTP. The hypothesis "dispersal into the QTP," proposing that T. pinnatifidum experienced migration from the plateau edge and adjacent highlands to the platform, was supported. These results were in line with the hypothesis that multiple refugia exist on the plateau edge and adjacent highlands rather than on the plateau platform. Our unimodal mismatch distribution, star-like network supported a recent expansion in T. pinnatifidum.

A Review on the Ethnomedicinal Usage, Phytochemistry, and Pharmacological Properties of Gentianeae (Gentianaceae) in Tibetan Medicine.

Gentianaceae is a large plant family and is distributed worldwide. As the largest tribe in Gentianaceae, Gentianeae contains 939-968 species, and the Qinghai-Tibet Plateau and adjacent areas are the main centers of diversity for Gentianeae. Species from the Gentianeae are widely used in traditional Tibetan medicine. In this review, a systematic and constructive overview of the information on botany, ethnomedicinal usage, phytochemistry, and pharmacological properties of Gentianeae in Tibetan medicine is provided. The results of this study are based on a literature search, including electronic databases, books, websites, papers, and conference proceedings. Botanical studies showed that Gentianeae includes the subtribe Gentianeae and Swertiinae, and several new genera and taxa have been identified. Approximately 83 species from Gentianeae were used in Tibetan medicine, among which Gentiana and Swertia constituted the largest number of species with 42 and 24 species, respectively. The species from Gentianeae are mainly used as Bangjian (སྤང་རྒྱན།), Jieji (ཀྱི་ལྕེ།), Dida (ཏིག་ཏ།), and Ganggaqiong (གང་གྰཆུང་།) in Tibetan medicine with different clinical applications. More than 240 formulas were found containing Gentianeae species with different attending functions. Phytochemical studies showed that the main active components of Gentianeae species are iridoids, xanthones, flavonoids, and triterpenoids. The bioactivities of plants from Gentianeae include hepatic protection, upper respiratory tract protection, joint and bone protection, glucose regulation, antibacterial, antioxidant, anticancer, and antiviral effects. This review will provide a reference for future research on natural resource protection, plant-based drug development, and further clinical investigation.

Lineage-specific plastid degradation in subtribe Gentianinae (Gentianaceae).

The structure and sequence of plastid genomes is highly conserved across most land plants, except for a minority of lineages that show gene loss and genome degradation. Understanding the early stages of plastome degradation may provide crucial insights into the repeatability and predictability of genomic evolutionary trends. We investigated these trends in subtribe Gentianinae of the Gentianaceae, which encompasses ca. 450 species distributed around the world, particularly in alpine and subalpine environments. We sequenced, assembled, and annotated the plastomes of 41 species, representing all six genera in subtribe Gentianinae and all main sections of the species-rich genus Gentiana L. We reconstructed the phylogeny, estimated divergence times, investigated the phylogenetic distribution of putative gene losses, and related these to substitution rate shifts and species' habitats. We obtained a strongly supported topology consistent with earlier studies, with all six genera in Gentianinae recovered as monophyletic and all main sections of Gentiana having full support. While closely related species have very similar plastomes in terms of size and structure, independent gene losses, particularly of the ndh complex, have occurred in multiple clades across the phylogeny. Gene loss was usually associated with a shift in the boundaries of the small single-copy and inverted repeat regions. Substitution rates were variable between clades, with evidence for both elevated and decelerated rate shifts. Independent lineage-specific loss of ndh genes occurred at a wide range of times, from Eocene to Pliocene. Our study illustrates that diverse degradation patterns shape the evolution of the plastid in this species-rich plant group.

Environmental filtering affects fungal communities more than dispersal limitation in a high-elevation hyperarid basin on Qinghai-Tibet Plateau.

The Qaidam Basin is the most extensive (120 000 km2) basin on the Qinghai-Tibet Plataea (QTP). Recent studies have shown that environmental selection and dispersal limitation influence the soil fungal community significantly in a large-scale distance. However, less is known about large-scale soil fungal community assemblages and its response to the elevation gradient in the high-elevation basin ecosystems. We studied fungal assemblages using Illumina sequencing of the ITS1 region from 35 sites of the Qaidam Basin. As the increase of elevation, fungal species richness and Chao1 index also increased. The Ascomycota was the most abundant phylum (more than 70% of total sequences), and six of the 10 most abundance fungal family was detected in all 35 soil samples. The key factors influencing the soil fungal community composition in the Qaidam Basin were environmental filtering (soil properties and climate factors). The Mantel test showed no significant relationship between geographic distance and community similarity (r = 0.05; p = 0.81). The absence of the distance effect might be caused by lacking dispersal limitation for the soil fungal community.

Large-scale distribution of bacterial communities in the Qaidam Basin of the Qinghai-Tibet Plateau.

Many studies have investigated patterns of soil microbial communities over large spatial scales. However, these studies mainly focused on a few sites. Here, we studied the near-surface (0-30 cm) soil microbial communities of 35 soil samples collected from most of the areas of the Qaidam Basin, which is the largest basin on the Qinghai-Tibet Plateau. A total of 32 phyla and 838 genera were detected from all the samples, in which Actinobacteria, Proteobacteria, Bacteroidetes, and Acidobacteria were the most dominant and cosmopolitan phyla. The most abundant phyla (relative abundance > 5%) detected in all 35 soil samples were also the most dominant, which could be explained by their great dispersal ability. The microbial community structures correlated strongly with variations in pH and Mg2+ and were distinct between the high Mg2+ content (>20 g/kg) samples and other samples (Acidobacteria, Actinobacteria, and Chloroflexi were significantly less abundant in the high Mg2+ content group, but the abundance of Firmicutes was significantly greater). Finally, the microbial spatial pattern was influenced by both the local environment and spatial distance, but environmental factors were the primary drivers of microbial spatial patterns in the Qaidam Basin.

Development of EST-SSR markers in Saxifraga sinomontana (Saxifragaceae) and cross-amplification in three related species.

PREMISE: Saxifraga sinomontana (Saxifragaceae) is a widespread alpine species in the Qinghai-Tibetan Plateau and its flanking mountains. We developed a set of expressed sequence tag-simple sequence repeat (EST-SSR) markers to investigate the genetic diversity and evolutionary history of the species. METHODS AND RESULTS: We initially designed 50 EST-SSR markers based on transcriptome data of S. sinomontana. Nineteen of 50 loci (38%) were successfully amplified, 13 of which were polymorphic. These were tested on 71 individuals from four populations. Three to 18 alleles per locus were detected, and the levels of observed and expected heterozygosity ranged from 0.2817 to 0.9155 and 0.2585 to 0.8495, respectively. In addition, cross-amplification was successful for all 13 loci in three congeneric species, S. tangutica, S. heleonastes, and S. congestiflora. CONCLUSIONS: These EST-SSR markers will be useful for studying the genetic diversity of S. sinomontana and disentangling the phylogenetic relationships of related species.

A population genetics perspective on the evolutionary histories of three clonal, endemic, and dominant grass species of the Qinghai-Tibet Plateau: Orinus (Poaceae).

We performed analyses of amplified fragment length polymorphism (AFLP) in order to characterize the evolutionary history of Orinus according to its population genetic structure, as well as to investigate putative hybrid origins of O. intermedius and to provide additional insights into relationships among species. The genus Orinus comprises three clonal grasses that are dominant species within xeric alpine grasslands of the Qinghai-Tibet Plateau (QTP). Here, we used eight selectively obtained primer pairs of EcoRI/MseI to perform amplifications in 231 individuals of Orinus representing 48 populations and all three species. We compared our resulting data to genetic models of hybridization using a Bayesian algorithm within NewHybrids software. We determined that genetic variation in Orinus was 56.65% within populations while the among-species component was 30.04% using standard population genetics statistics. Nevertheless, we detected that species of Orinus were clustered into three highly distinct genetic groups corresponding to classic species identities. Our results suggest that there is some introgression among species. Thus, we tested explicit models of hybridization using a Bayesian approach within NewHybrids software. However, O. intermedius likely derives from a common ancestor with O. kokonoricus and is probably not the result of hybrid speciation between O. kokonoricus and O. thoroldii. We suspect that recent isolation of species of Orinus in allopatry via vicariance may explain the patterns in diversity that we observed, and this is corroborated by a Mantel test that showed significant positive correlation between geographic and genetic distance (r = 0.05, p < 0.05). Recent isolation may explain why Orinus differs from many other clonal species by exhibiting the highest diversity within populations rather than among them.

Genetic Structure and Eco-Geographical Differentiation of Lancea tibetica in the Qinghai-Tibetan Plateau.

The uplift of the Qinghai-Tibetan Plateau (QTP) had a profound impact on the plant speciation rate and genetic diversity. High genetic diversity ensures that species can survive and adapt in the face of geographical and environmental changes. The Tanggula Mountains, located in the central of the QTP, have unique geographical significance. The aim of this study was to investigate the effect of the Tanggula Mountains as a geographical barrier on plant genetic diversity and structure by using Lancea tibetica. A total of 456 individuals from 31 populations were analyzed using eight pairs of microsatellite makers. The total number of alleles was 55 and the number per locus ranged from 3 to 11 with an average of 6.875. The polymorphism information content (PIC) values ranged from 0.2693 to 0.7761 with an average of 0.4378 indicating that the eight microsatellite makers were efficient for distinguishing genotypes. Furthermore, the observed heterozygosity (Ho), the expected heterozygosity (He), and the Shannon information index (I) were 0.5277, 0.4949, and 0.9394, respectively, which indicated a high level of genetic diversity. We detected high genetic differentiation among all sampling sites and restricted gene flow among populations. Bayesian-based cluster analysis (STRUCTURE), principal coordinates analysis (PCoA), and Neighbor-Joining (NJ) cluster analysis based on microsatellite markers grouped the populations into two clusters: the southern branch and the northern branch. The analysis also detected genetic barriers and restricted gene flow between the two groups separated by the Tanggula Mountains. This study indicates that the geographical isolation of the Tanggula Mountains restricted the genetic connection and the distinct niches on the two sides of the mountains increased the intraspecific divergence of the plants.

Deep Intraspecific Divergence in the Endemic Herb Lancea tibetica (Mazaceae) Distributed Over the Qinghai-Tibetan Plateau.

Qinghai-Tibetan Plateau (QTP) is an important biodiversity hub, which is very sensitive to climate change. Here in this study, we investigated genetic diversity and past population dynamics of Lancea tibetica (Mazaceae), an endemic herb to QTP and adjacent highlands. We sequenced chloroplast and nuclear ribosomal DNA fragments for 429 individuals, collected from 29 localities, covering their major distribution range at the QTP. A total of 19 chloroplast haplotypes and 13 nuclear genotypes in two well-differentiated lineages, corresponding to populations into two groups isolated by Tanggula and Bayangela Mountains. Meanwhile, significant phylogeographical structure was detected among sampling range of L. tibetica, and 61.50% of genetic variations was partitioned between groups. Gene flow across the whole region appears to be restricted by high mountains, suggesting a significant role of geography in the genetic differences between the two groups. Divergence time between the two lineages dated to 8.63 million years ago, which corresponded to the uplifting of QTP during the late Miocene and Pliocene. Ecological differences were found between both the lineages represent species-specific characteristics, sufficient to keep the lineages separated to a high degree. The simulated distribution from the last interglacial period to the current period showed that the distribution of L. tibetica experienced shrinkage and expansion. Climate changes during the Pleistocene glacial-interglacial cycles had a dramatic effect on L. tibetica distribution ranges. Multiple refugia of L. tibetica might have remained during the species history, to south of the Tanggula and north of Bayangela Mountains, both appeared as topological barrier and contributed to restricting gene flow between the two lineages. Together, geographic isolation and climatic factors have played a fundamental role in promoting diversification and evolution of L. tibetica.

Target separation of flavonoids from Saxifraga tangutica using two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography.

An orthogonally (80.3%) preparative two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography method has been established for the isolation and purification of flavonoids from Saxifraga tangutica. Initially, flavonoids were enriched by means of a middle-pressure chromatographic tower (containing middle chromatogram isolated gel). In the first dimension, a XION preparative column was used to separate the flavonoid fractions under the guidance of characteristic ultraviolet absorption spectra of flavonoids and nine flavonoid fractions were obtained. Then, the coeluted flavonoid fractions were selected for further purification via reversed-phase liquid chromatography with the parent ion peak of quercetin (303), kaempferol (287), or isorhamnetin (317). Several flavonoids could be separated from each hydrophilic interaction chromatography fraction; furthermore, flavonoids with poor resolution in one-dimensional liquid chromatography were isolated in two-dimensional liquid chromatography due to the orthogonality. In addition, this technique was valuable for trace flavonoids, which were concentrated in the first stage and separated in the second stage. In total, 18 flavonoids with either quercetin, kaempferol, or isorhamnetin parent nuclei were targetedly obtained, and 15 flavonoids were obtained for the first time from S. tangutica. These results established that the off-line two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography technique was efficient for the isolation of flavonoids from Saxifraga tangutica.

Rapid Intraspecific Diversification of the Alpine Species Saxifraga sinomontana (Saxifragaceae) in the Qinghai-Tibetan Plateau and Himalayas.

An increasing number of phylogeographic studies have been conducted for plant species in the Qinghai-Tibetan Plateau (QTP) and its flanking mountains. However, these studies have mainly focused on the determination of glacial refugia and routes of inter-/post-glacial expansions. Rapid intraspecific diversification of plants in this region have not been thoroughly discussed. Herein, we investigate the effects of the Quaternary climate changes on population genetic structure and diversifications of a herbaceous alpine species, Saxifraga sinomontana, which may have an evolutionary time scale <5 million years in the QTP and Himalayan regions. Using a total of 350 individuals from 29 populations, we studied the evolutionary history of S. sinomontana by analyzing cpDNA trnL-trnF, rpl16 and nrDNA ITS sequences. A total of 89 haplotypes and 158 genotypes were detected for cpDNA and ITS sequences, respectively. Only a few haplotypes/genotypes were widespread, while an extremely large number of haplotypes/genotypes were restricted to single populations, which were scattered throughout the current geographical range of S. sinomontana. This suggests the existence of microrefugia of this species during the Quaternary glaciations. In addition, the relationships of the haplotypes/genotypes were almost completely not resolved by phylogenetic reconstruction. Combining characteristics in terms of high haplotype richness, large proportion of private haplotypes, and shallow haplotype divergence, we speculate that recent intraspecific diversification has occurred in S. sinomontana. Molecular clock analysis estimated that the onset diversification within S. sinomontana to be 1.09 Ma (95% HPD = 0.80-1.45), coinciding with the extensive Quaternary glaciations on the QTP which started ca. 1.17 Ma. The Quaternary climatic oscillations may have triggered rapid intraspecific diversification in this QTP-Himalayan species. However, large niche breadth, as well as introgression/hybridization between the studied species and its closely related sympatric saxifrages, may also played a role to some extent on the current genetic structure of S. sinomontana, which need to be further studied.