Comparison of Rhizosphere Bacterial Communities of Pinus squamata, a Plant Species with Extremely Small Populations (PSESP) in Different Conservation Sites.

Pinus squamata is one of the most threatened conifer species in the world. It is endemic to northeastern Yunnan Province, China, and has been prioritized as a Plant Species with Extremely Small Populations (PSESP). The integrated study of soil properties and rhizosphere bacteria can assist conservation to understand the required conditions for the protection and survival of rare and endangered species. However, differences between the rhizospheric bacterial communities found in the soil surrounding P. squamata at different conservation sites remain unclear. In this study, Samples were collected from wild, ex situ, and reintroduced sites. Bacterial communities in different conservation sites of P. squamata rhizosphere soils were compared using Illumina sequencing. The soil physicochemical properties were determined, the relationships between the bacterial communities and soil physicochemical factors were analyzed, and the potential bacterial ecological functions were predicted. The reintroduced site Qiaojia (RQ) had the highest richness and diversity of bacterial community. Actinobacteria, Proteobacteria, and Acidobacteriota were the dominant phyla, and Bradyrhizobium, Mycobacterium, Acidothermus were the most abundant genera. Samples were scattered (R = 0.93, p = 0.001), indicating significant difference between the different conservation sites. The abundance of Mycobacterium differed between sites (0.01 < p ≤ 0.05), and the relative abundances of Bradyrhizobium and Acidothermus differed significantly among different sites (0.001 < p ≤ 0.01). Soil total potassium (TK) and available nitrogen (AN) were the main factors driving bacterial community at the phylum level (0.01 < p ≤ 0.05). This study generated the first insights into the diversity, compositions, and potential functions of bacterial communities associated with the rhizosphere soils of P. squamata in different conservation sites and provides a foundation to assess the effect of conservation based on bacterial diversity and plant growth-promoting rhizobacteria (PGPR) to guide future research into the conservation of P. squamata.

Genomic analysis of field pennycress (Thlaspi arvense) provides insights into mechanisms of adaptation to high elevation.

BACKGROUND: Understanding how organisms evolve and adapt to extreme habitats is of crucial importance in evolutionary ecology. Altitude gradients are an important determinant of the distribution pattern and range of organisms due to distinct climate conditions at different altitudes. High-altitude regions often provide extreme environments including low temperature and oxygen concentration, poor soil, and strong levels of ultraviolet radiation, leading to very few plant species being able to populate elevation ranges greater than 4000 m. Field pennycress (Thlaspi arvense) is a valuable oilseed crop and emerging model plant distributed across an elevation range of nearly 4500 m. Here, we generate an improved genome assembly to understand how this species adapts to such different environments. RESULTS: We sequenced and assembled de novo the chromosome-level pennycress genome of 527.3 Mb encoding 31,596 genes. Phylogenomic analyses based on 2495 single-copy genes revealed that pennycress is closely related to Eutrema salsugineum (estimated divergence 14.32-18.58 Mya), and both species form a sister clade to Schrenkiella parvula and genus Brassica. Field pennycress contains the highest percentage (70.19%) of transposable elements in all reported genomes of Brassicaceae, with the retrotransposon proliferation in the Middle Pleistocene being likely responsible for the expansion of genome size. Moreover, our analysis of 40 field pennycress samples in two high- and two low-elevation populations detected 1,256,971 high-quality single nucleotide polymorphisms. Using three complementary selection tests, we detected 130 candidate naturally selected genes in the Qinghai-Tibet Plateau (QTP) populations, some of which are involved in DNA repair and the ubiquitin system and potential candidates involved in high-altitude adaptation. Notably, we detected a single base mutation causing loss-of-function of the FLOWERING LOCUS C protein, responsible for the transition to early flowering in high-elevation populations. CONCLUSIONS: Our results provide a genome-wide perspective of how plants adapt to distinct environmental conditions across extreme elevation differences and the potential for further follow-up research with extensive data from additional populations and species.

Spatial genetic and epigenetic structure of Thlaspi arvense (field pennycress) in China.

Thlaspi arvense (field pennycress) is widespread in temperate regions of the northern hemisphere. We estimated the genetic and epigenetic structure of eight T. arvense populations (131 individuals) in China using amplified fragment length polymorphism and methylation-sensitive amplified polymorphism molecular-marker techniques. We detected low diversity at both genetic (mean = 0.03; total = 0.07) and epigenetic (mean = 0.04; total = 0.07) levels, while significant genetic (FST = 0.42, P < 0.001) and epigenetic (FST = 0.32, P < 0.001) divergence was found across the distribution range. Using Mantel testing, we found spatial genetic and epigenetic differentiation, consistent with isolation-by-distance models. We also identified a strong correlation between genetic and epigenetic differentiation (r = 0.7438, P < 0.001), suggesting genetic control of the epigenetic variation. Our results indicate that mating system, natural selection and gene flow events jointly structure spatial patterns of genetic and epigenetic variation. Moreover, epigenetic variation may serve as a basis of natural selection and ecological evolution to enable species to adapt to heterogeneous habitats. Our study provides novel clues for the adaptation of T. arvense.

Increased epigenetic diversity and transient epigenetic memory in response to salinity stress in Thlaspi arvense.

Epigenetic diversity could play an important role in adaptive evolution of organisms, especially for plant species occurring in new and stressful environments. Thlaspi arvense (field pennycress), a valuable oilseed crop, is widespread in temperate regions of the northern hemisphere. In this study, we investigated the effect of salinity stress on the epigenetic variation of DNA methylation and epigenetic stress memory in pennycress using methylation-sensitive amplification polymorphism (MSAP) markers. We examined how the status of DNA methylation changes across individuals in response to salinity stress and whether such an effect of maternal stress could be transferred to offspring for one or two generations in nonstressed environments. Our results based on 306 epiloci indicated no consistent change of DNA methylation status in specific epiloci across individuals within the same conditions. In contrast, we found that the epigenetic diversity at population level increased significantly in response to the stimulation of salinity stress; and this "stimulation effect" could be transferred partially in the form of stress memory to at least two generations of offspring in nonstressed environments. In addition, we observed a parallel change in functionally important traits, that is, phenotypic variation was significantly higher in plants grown under salinity stress compared with those of control groups. Taken together, our results provide novel clues for the increased spontaneous epimutation rate in response to stress in plants, of potential adaptive significance.

Medicinal dietary plants of the Yi in Mile, Yunnan, China.

BACKGROUND: The Yi is the largest ethnic group in Yunnan Province (China), with a population of five million. The Yi people tend to live in mountainous areas, and their culture includes a unique dietary system for treating and protecting people against illnesses. Medicinal plants occupy an essential place in the Yi diet because they play a key role in health and the prevention and treatment of diseases. However, few studies have addressed these medicinal dietary plants and their importance in the Yi's daily lives. The aim of this study was to (1) investigate the medicinal dietary plants used by the Yi in Mile City, (2) document the traditional knowledge held about these plants, (3) identify species with important cultural significance to the Yi in Mile City, and (4) analyze the special preparation methods and consumption habits of these plants. METHODS: Field investigations were performed in six villages in Mile City, Honghe Hani and Yi Autonomous Prefecture, Yunnan, from July 2017 to May 2018. Information was collected using direct observation, semi-structured interviews, key informant interviews, individual discussions, and focus group discussions. The use value (UV) and frequency of utilization index (FUI) of these plants were analyzed. Plant samples and voucher specimens were collected for taxonomic identification. RESULTS: This study documented 124 species belonging to 62 families and 102 genera. These plants included angiosperms (117 spp.), gymnosperms (3), pteridophytes (2), lichen (1), and fungus (1). The 20 species with the highest UV were noted as being particularly important to the Yi people's daily life in Mile City. The primary medicinal preparation method for plants recorded in the study was decoction. The most commonly used plant parts were fruits and roots. The most frequently used edible parts were fruits, and the most frequently used medicinal parts were roots. The medicinal parts were used to treat diseases such as rheumatism, edemas, kidney deficiency, spleen deficiency, gastritis, parasites, and so on. CONCLUSION: A wide variety of medicinal dietary plants are used by the Yi people in Mile City. Those plants, which have both rich nutritional and medicinal value, occupy an essential part of the Yi dietary and medicine culture. Ethnobotanical surveys of medicinal dietary plants provide a theoretical reference for the conservation and sustainable use of the plant resources and could contribute to the protection of the Yi food culture and traditional medicine in Mile City. In addition, this information provides a sound basis for developing and using Yi ethnic medicine and health products.

Complete plastome of an endemic fern species from China: Neocheiropteris palmatopedata (Polypodiaceae).

Neocheiropteris palmatopedata (Baker) Christ is an endangered fern species endemic to southwest China. In this study, we sequenced the complete plastid genome of N. palmatopedata. The gene order and structure of the N. palmatopedata plastome are similar to those published plastomes in Polypodiales. The complete plastome is 153,344 bp in length, and the GC content is 42.1%. The plastome comprises 113 unique genes (83 protein-coding genes, 29 tRNA genes and four rRNA genes).

Functional characterization of the eugenol synthase gene (RcEGS1) in rose.

The floral volatile compound eugenol is an important constituent in many aromatic plants, being a floral attractant for pollinators as well as having antimicrobial activity. Rose flowers emit eugenol and its derivatives. We recently reported a eugenol synthase gene (RcEGS1) (JQ522949) that was present in petals of R. chinensis cv. Old Blush. RcEGS1 has its highest expression levels in the petals compared to other tissues; it has higher transcript levels at the developmental blooming stage and lower levels at budding and senescence stages. Here, we overexpressed the RcEGS1 protein in Escherichia coli, and showed by Western-blot analysis that its expression was mainly detected in stamens and petals at the flower opening stage. RcEGS1 was principally localized in the upper and lower epidermal layers, which are the major sites of scent emission in roses. Furthermore, we demonstrated that down-regulation of RcEGS1 expression in flowers by virus-induced gene silencing led to a reduction of the relative content of eugenol. We suggested that RcEGS1 was responsible for eugenol biosynthesis in roses.

Integrated taxonomy of the Asplenium normale complex (Aspleniaceae) in China and adjacent areas.

The Asplenium normale D. Don complex comprises several taxa that are either diploid or tetraploid. The tetraploids are assumed to have originated from diploid ancestors by relatively recent autopolyploidization or allopolyploidization. Some of the diploids are readily recognized morphologically but most of the taxa have until now been placed into a single species. However, phylogenetic studies have challenged this treatment and emphasized the notion that the taxonomic treatment of this complex needs to be revised. An integrative taxonomic approach was employed to delimit species in the complex using cytological, morphological, and DNA sequence data. Initially, we employed a diploid first approach to establish a robust taxonomic framework. Special efforts were made to collect and identify the diploid progenitors of each polyploid lineage identified in the plastid DNA based phylogenetic hypothesis. A total of six distinct diploid species were identified. The distinctive nature of the six diploids is strongly supported by sequence differences in plastid DNA and nuclear loci, as well as by the results of morphometric analysis. Diagnostic morphological characters were identified to distinguish the six diploid species, resulting in their revised taxonomy, which includes two novel species, namely, Asplenium normaloides and A. guangdongense. Further studies to strengthen the taxonomic classification of all of the tetraploid taxa are warranted.

Survival and Growth of Epiphytic Ferns Depend on Resource Sharing.

Locally available resources can be shared within clonal plant systems through physiological integration, thus enhancing their survival and growth. Most epiphytes exhibit clonal growth habit, but few studies have tested effects of physiological integration (resource sharing) on survival and growth of epiphytes and whether such effects vary with species. We conducted two experiments, one on individuals (single ramets) and another on groups (several ramets within a plot), with severed and intact rhizome treatments (without and with physiological integration) on two dominant epiphytic ferns (Polypodiodes subamoena and Lepisorus scolopendrium) in a subtropical montane moist forest in Southwest China. Rhizome severing (preventing integration) significantly reduced ramet survival in the individual experiment and number of surviving ramets in the group experiment, and it also decreased biomass of both species in both experiments. However, the magnitude of such integration effects did not vary significantly between the two species. We conclude that resource sharing may be a general strategy for clonal epiphytes to adapt to forest canopies where resources are limited and heterogeneously distributed in space and time.

Higher clonal integration in the facultative epiphytic fern Selliguea griffithiana growing in the forest canopy compared with the forest understorey.

BACKGROUND AND AIMS: The advantage of clonal integration (resource sharing between connected ramets of clonal plants) varies and a higher degree of integration is expected in more stressful and/or more heterogeneous habitats. Clonal facultative epiphytes occur in both forest canopies (epiphytic habitats) and forest understories (terrestrial habitats). Because environmental conditions, especially water and nutrients, are more stressful and heterogeneous in the canopy than in the understorey, this study hypothesizes that clonal integration is more important for facultative epiphytes in epiphytic habitats than in terrestrial habitats. METHODS: In a field experiment, an examination was made of the effects of rhizome connection (connected vs. disconnected, i.e. with vs. without clonal integration) on survival and growth of single ramets, both young and old, of the facultative epiphytic rhizomatous fern Selliguea griffithiana (Polypodiaceae) in both epiphytic and terrestrial habitats. In another field experiment, the effects of rhizome connection on performance of ramets were tested in small (10 × 10 cm(2)) and large (20 × 20 cm(2)) plots in both epiphytic and terrestrial habitats. KEY RESULTS: Rhizome disconnection significantly decreased survival and growth of S. griffithiana in both experiments. The effects of rhizome disconnection on survival of single ramets and on ramet number and growth in plots were greater in epiphytic habitats than in terrestrial habitats. CONCLUSIONS: Clonal integration contributes greatly to performance of facultative epiphytic ferns, and the effects were more important in forest canopies than in forest understories. The results therefore support the hypothesis that natural selection favours genotypes with a higher degree of integration in more stressful and heterogeneous environments.

Phylogeny of Chinese Polystichum (Dryopteridaceae) based on chloroplast DNA sequence data (trnL-F and rps4-trnS).

Polystichum is one of the largest and most taxonomically complex fern genera in China. The evolutionary relationships of Chinese Polystichum and related genera, and the relationship between our Polystichum phylogeny and ecogeographic distribution, were tested by the use of DNA sequence data. Fifty-one species of Polystichum and 21 species in allied genera were sequenced for the plastid intergenic spacers rps4-trnS and trnL-F. Maximum parsimony and Bayesian phylogenetic analyses of both individual and combined data sets showed that Chinese Polystichum as commonly recognized was paraphyletic: one clade (the CCPC clade) included Cyrtomidictyum lepidocaulon, two Cyrtogonellum species, three Cyrtomium species, and a small number of Polystichum species usually occurring on limestone. A second clade, Polystichum sensu stricto, included the remainder of the Polystichum species; these often occur on non-limestone substrates. The remaining Cyrtomium species formed the third clade. Three subclades resolved within Polystichum sensu stricto (s.s.) clade do not correspond with recent sectional classifications, and we outline the issues relevant to a new classification for the genus.

First report of chromosome numbers of the Carlemanniaceae (Lamiales).

The Carlemanniaceae comprises two small genera that are restricted to East Asia: the Carlemannia and Silvianthus. These genera were previously placed in the Rubiaceae or Caprifoliaceae, but are now considered a distinct family that is probably related to the Oleaceae in the Lamiales. The family is still poorly understood with respect to its morphological characteristics. Here, we present the first report of the chromosome numbers of the family using species from both genera, i.e., Carlemannia tetragona, Silvianthus bracteatus ssp. bracteatus, and S. bracteatus ssp. clerodendroides. The species were compared with the chromosome numbers of Oleaceae and associated families using a Bayesian tree that was generated from rbcL and ndhF sequence data from Genbank. C. tetragona had 2n = 30 (x = 15), whereas the two subspecies of Silvianthus had 2n = 38 (x = 19). Comparisons of chromosome numbers support the distinctness of the Carlemanniaceae, not only from the Oleaceae (x = 11, 13, 23), but also from the Tetrachondraceae (x = 10, 11), a family that is possibly related to the Carlemanniaceae and/or Oleaceae in the Lamiales. The notable difference in chromosome number between Carlemannia and Silvianthus, as well as the differences in other characteristics (pollen, seed, and fruit morphology), suggests that the family split early in its evolution.

Phylogenetics of Chinese Dryopteris (Dryopteridaceae) based on the chloroplast rps4-trnS sequence data.

Dryopteris is one of the largest and most taxonomically complex fern genera in the Dryopteridaceae, with 127 species occurring throughout temperate, sub-temperate, subtropical, and tropical China. Investigations of the evolutionary relationships of a subset of these Chinese Dryopteris species, using DNA sequence-based methods, specifically tested the monophyly of the genus and the validity of the previous subgeneric classifications. Sixty species of Dryopteris, four closely related non-Dryopteris and three species of Arachniodes, were used as outgroup taxa. The rps4-trnS region of the chloroplast genome was sequenced in these species for the first time. Both maximum parsimony (MP) and neighbor-joining (NJ) analyses identified six polyphyletic clades that contained Dryopteris species. These results were supported by a Bayesian analysis of the same data set. The phylogenetic patterns strongly suggest the polyphyletic status of Dryopteris; the monophyletic groupings of the species do not correspond with either Fraser-Jenkins [In: Bull Brit Mus (Nat Hist) Bot 14(3):183-218, 1986} or Wu (In: Flora Reipublica Popularis Sinicae Tomus 5 (1) pp 1-241, 2000] subgeneric classification of Dryopteris, except in a few specific cases. This work represents the first molecular systematic analyses of Chinese Dryopteris, and we propose the next steps necessary to recognize new subgenera of the genus.