Gut microbiota regulation of T lymphocyte subsets during systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by disturbance of pro-inflammatory and anti-inflammatory lymphocytes. Growing evidence shown that gut microbiota participated in the occurrence and development of SLE by affecting the differentiation and function of intestinal immune cells. The purpose of this study was to investigate the changes of gut microbiota in SLE and judge its associations with peripheral T lymphocytes. METHODS: A total of 19 SLE patients and 16 HCs were enrolled in this study. Flow cytometry was used to detect the number of peripheral T lymphocyte subsets, and 16 s rRNA was used to detect the relative abundance of gut microbiota. Analyzed the correlation between gut microbiota with SLEDAI, ESR, ds-DNA and complement. SPSS26.0 software was used to analyze the experimental data. Mann-Whitney U test was applied to compare T lymphocyte subsets. Spearman analysis was used for calculating correlation. RESULTS: Compared with HCs, the proportions of Tregs (P = 0.001), Tfh cells (P = 0.018) and Naïve CD4 + T cells (P = 0.004) significantly decreased in SLE patients, and proportions of Th17 cells (P = 0.020) and γδT cells (P = 0.018) increased in SLE. The diversity of SLE patients were significantly decreased. Addition, there were 11 species of flora were discovered to be distinctly different in SLE group (P < 0.05). In the correlation analysis of SLE, Tregs were positively correlated with Ruminococcus2 (P = 0.042), Th17 cells were positively correlated with Megamonas (P = 0.009), γδT cells were positively correlated with Megamonas (P = 0.003) and Streptococcus (P = 0.004), Tfh cells were positively correlated with Bacteroides (P = 0.040), and Th1 cells were negatively correlated with Bifidobacterium (P = 0.005). As for clinical indicators, the level of Tregs was negatively correlated with ESR (P = 0.031), but not with C3 and C4, and the remaining cells were not significantly correlated with ESR, C3 and C4. CONCLUSION: Gut microbiota and T lymphocyte subsets of SLE changed and related to each other, which may break the immune balance and affect the occurrence and development of SLE. Therefore, it is necessary to pay attention to the changes of gut microbiota and provide new ideas for the treatment of SLE.

Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent disease that has become the third leading cause of death worldwide. Cycloastragenol (CAG), which is the genuine sapogenin of the main active triterpene saponins in Astragali radix, is a bioavailable pre-clinical candidate for chronic obstructive pulmonary disease (COPD), and it was investigated in our previous study. In order to progress medical research, it was first efficiently produced on a 2.5-kg scale via Smith degradation from astragaloside IV (AS-IV). Simultaneously, since the impurity profiling of a drug is critical for performing CMC documentation in pre-clinical development, a study on impurities was carried out. As these structures do not contain chromophores and possess weak UV absorption characteristics, HPLC-CAD and UPLC-LTQ-Orbitrap-MS were employed to carry out the quality control of the impurities. Then, column chromatography (CC), preparative thin-layer chromatography (PTLC), and crystallization led to the identification of 15 impurities from CAG API. Among these impurities, compounds 1, 4, 9, 10, 14, and 15 were elucidated via spectroscopic analysis, and 2-3, 5-8, and 11-13 were putatively identified. Interestingly, the new compounds 9 and 14 were rare 10, 19-secocycloartane triterpenoids that displayed certain anti-inflammatory activities against LPS-induced lymphocyte cells and CSE-induced MLE-12 cells. Additionally, a plausible structural transformation pathway of the degradation compounds from CAG or AS IV was proposed. The information obtained will provide a material basis to carry out the quality control and clinical safety assurance of API and related prescriptions. Reasonable guidance will also be provided regarding the compounds with weak UV absorption characteristics.

Paenibacillus turpanensis sp. nov., isolated from a salt lake of Turpan city in Xinjiang province, north-west China.

Strain YIM B00363T, a Gram-positive, aerobic, non-motile, rod-shaped, spore-forming bacterium, was isolated from saline soil samples collected from a salt lake in Xinjiang province, north-west China, and was characterized using a polyphasic approach. The optimum growth temperature was 37 °C and the optimum pH was 7.5-8.0. The major menaquinone was MK-7; anteiso-C15:0 (53.52%), iso-C15:0 (15.04%) and C16:0 (12.76%) were the predominant cellular fatty acids. The diagnostic diamino acid of the cell wall peptidoglycan was meso-diaminopimelic acid. The phospholipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, unidentified glycolipids and unknown lipids. The DNA G + C content of the type strain was 50.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain YIM B00363T belonged to a cluster comprising species of the genus Paenibacillus. The nearest relatives were P. residui MC-246T and P. senegalensis JC66T, with 93.2% and 92.8% gene sequence similarities, respectively. On the basis of its phenotypic characteristics and phylogenetic distinctivenes, strain YIM B00363T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus turpanensis sp. nov. is proposed. The type strain is YIM B00363T (= CGMCC 1.17507T = KCTC 43184T).

Species delimitation and interspecific relationships of the endangered herb genus Notopterygium inferred from multilocus variations.

Species identification and discrimination is the basis of biodiversity research. In general, it is considered that numerous nucleotide variations (e.g., whole chloroplast genomes) can identify species with higher resolution than a few loci, e.g., partial chloroplast or nuclear gene fragments. In this study, we tested this hypothesis by sampling population genetics samples of the endangered herb genus Notopterygium. We sequenced the complete plastomes, five nuclear gene regions, three chloroplast DNA fragments, and a nuclear internal transcribed spacer (nrITS) region for 18 populations sampled throughout most of the geographic ranges of all six Notopterygium species. Species identification analysis showed that four DNA barcodes (matK, rbcL, trnS-trnG, and nrITS) and/or combinations of these markers achieved Notopterygium species discrimination at higher resolution than the general plastomes and nuclear gene sequences. In particular, nrITS had the highest discriminatory power among all of the individual markers. Molecular data sets and morphological evidence indicated that all six Notopterygium species could be reclassified unambiguously to four putative species clades. N. oviforme and N. franchetii had the closest relationship. Molecular dating showed that the origin and divergence of Notopterygium species was significantly associated with geological and climatic fluctuations during the middle of the Pliocene. In conclusion, our results suggest that a few nucleotide variations can achieve species discrimination with higher resolution than numerous plastomes and general nuclear gene fragments when discerning related Notopterygium species.

Population genetics, phylogenomics and hybrid speciation of Juglans in China determined from whole chloroplast genomes, transcriptomes, and genotyping-by-sequencing (GBS).

Genomic data are a powerful tool for elucidating the processes involved in the evolution and divergence of species. The speciation and phylogenetic relationships among Chinese Juglans remain unclear. Here, we used results from phylogenomic and population genetic analyses, transcriptomics, Genotyping-By-Sequencing (GBS), and whole chloroplast genomes (Cp genome) data to infer processes of lineage formation among the five native Chinese species of the walnut genus (Juglans, Juglandaceae), a widespread, economically important group. We found that the processes of isolation generated diversity during glaciations, but that the recent range expansion of J. regia, probably from multiple refugia, led to hybrid formation both within and between sections of the genus. In southern China, human dispersal of J. regia brought it into contact with J. sigillata, which we determined to be an ecotype of J. regia that is now maintained as a landrace. In northern China, walnut hybridized with a distinct lineage of J. mandshurica to form J. hopeiensis, a controversial taxon (considered threatened) that our data indicate is a horticultural variety. Comparisons among whole chloroplast genomes and nuclear transcriptome analyses provided conflicting evidence for the timing of the divergence of Chinese Juglans taxa. J. cathayensis and J. mandshurica are poorly differentiated based our genomic data. Reconstruction of Juglans evolutionary history indicate that episodes of climatic variation over the past 4.5 to 33.80 million years, associated with glacial advances and retreats and population isolation, have shaped Chinese walnut demography and evolution, even in the presence of gene flow and introgression.

Comparative Transcriptome Analysis Reveals Adaptive Evolution of Notopterygium incisum and Notopterygium franchetii, Two High-Alpine Herbal Species Endemic to China.

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.

De novo assembly and characterization of the leaf, bud, and fruit transcriptome from the vulnerable tree Juglans mandshurica for the development of 20 new microsatellite markers using Illumina sequencing.

Manchurian walnut (Juglans mandshurica Maxim.) is a vulnerable, temperate deciduous tree valued for its wood and nut, but transcriptomic and genomic data for the species are very limited. Next generation sequencing (NGS) has made it possible to develop molecular markers for this species rapidly and efficiently. Our goal is to use transcriptome information from RNA-Seq to understand development in J. mandshurica and develop polymorphic simple sequence repeats (SSRs, microsatellites) to understand the species' population genetics. In this study, more than 47.7 million clean reads were generated using Illumina sequencing technology. De novo assembly yielded 99,869 unigenes with an average length of 747 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.32 %) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) identified 15,903 (16.9 %) unigenes. Further, we identified and characterized 63 new transcriptome-derived microsatellite markers. By testing the markers on 4 to 14 individuals from four populations, we found that 20 were polymorphic and easily amplified. The number of alleles per locus ranged from 2 to 8. The observed and expected heterozygosity per locus ranged from 0.209 to 0.813 and 0.335 to 0.842, respectively. These twenty microsatellite markers will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species. Moreover, the information uncovered in this research will also serve as a useful genetic resource for understanding the transcriptome and development of J. mandshurica and other Juglans species.

Transcriptome Sequencing and Development of Genic SSR Markers of an Endangered Chinese Endemic Genus Dipteronia Oliver (Aceraceae).

Dipteronia Oliver (Aceraceae) is an endangered Chinese endemic genus consisting of two living species, Dipteronia sinensis and Dipteronia dyeriana. However, studies on the population genetics and evolutionary analyses of Dipteronia have been hindered by limited genomic resources and genetic markers. Here, the generation, de novo assembly and annotation of transcriptome datasets, and a large set of microsatellite or simple sequence repeat (SSR) markers derived from Dipteronia have been described. After Illumina pair-end sequencing, approximately 93.2 million reads were generated and assembled to yield a total of 99,358 unigenes. A majority of these unigenes (53%, 52,789) had at least one blast hit against the public protein databases. Further, 12,377 SSR loci were detected and 4179 primer pairs were designed for experimental validation. Of these 4179 primer pairs, 435 primer pairs were randomly selected to test polymorphism. Our results show that products from 132 primer pairs were polymorphic, in which 97 polymorphic SSR markers were further selected to analyze the genetic diversity of 10 natural populations of Dipteronia. The identification of SSR markers during our research will provide the much valuable data for population genetic analyses and evolutionary studies in Dipteronia.

Negadavirga shengliensis gen. nov., sp. nov., a novel member of the family Cyclobacteriaceae isolated from oil-contaminated saline soil.

Four novel Gram-stain-negative, rod-shaped, and non-motile bacterial strains, SLG210-21(T), SLG210-4, SLG210-5 and SLG210-14, were isolated from oil-contaminated saline soil in Shengli Oilfield, China. Growth were observed at 25-42 °C (optimum 37 °C), in the presence of 0-10 % (w/v) NaCl (optimum 0-1 %) and at pH 4.0-10.0 (optimum pH 7.6-8.6). All the strains were positive for catalase and α, ß-galactosidase activities and nitrogen reduction, and negative for oxidase activity, glucose fermentation and hydrolysis of agar, starch, gelatin, Tween 40, 60 and 80. The DNA G+C contents of the four strains were 41.3-43.0 mol% and the predominant respiratory quinones were all menaquinone-7. The major fatty acids were iso-C15:0, anteiso-C15:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:1 ω5c and summed feature 9 (iso-C17:1 ω9c and/or 10-methyl C16:0), while the polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, glycolipid, two unidentified phospholipids and two unidentified amino lipids. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the four strains clustered together to form a stable branch in the family Cyclobacteriaceae, and were most closely related to the genera Cyclobacterium and Echinicola with the 16S rRNA gene sequence similarities being 88.6-90.3 and 89.6-91.4 %, respectively. DNA-DNA hybridization between SLG210-21(T) and the other three strains showed the relatedness of 93.8 ± 4.5, 96.2 ± 4.2 and 82.3 ± 4.8 %, respectively. Based on the polyphasic analysis, a novel species in a new genus, Negadavirga Shengliensis gen. nov., sp. nov., is proposed with SLG210-21(T) (=LMG 27737(T) = CGMCC1.12768(T)) [corrected] as the type strain.

Characterization of Global Transcriptome Using Illumina Paired-End Sequencing and Development of EST-SSR Markers in Two Species of Gynostemma (Cucurbitaceae).

Gynostemma pentaphyllum is an important medicinal herb of the Cucurbitaceae family, but limited genomic data have hindered genetic studies. In this study, transcriptomes of two closely-related Gynostemma species, Gynostemma cardiospermum and G. pentaphyllum, were sequenced using Illumina paired-end sequencing technology. A total of 71,607 nonredundant unigenes were assembled. Of these unigenes, 60.45% (43,288) were annotated based on sequence similarity search with known proteins. A total of 11,059 unigenes were identified in the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database. A total of 3891 simple sequence repeats (SSRs) were detected in 3526 nonredundant unigenes, 2596 primer pairs were designed and 360 of them were randomly selected for validation. Of these, 268 primer pairs yielded clear products among six G. pentaphyllum samples. Thirty polymorphic SSR markers were used to test polymorphism and transferability in Gynostemma. Finally, 15 SSR makers that amplified in all 12 Gynostemma species were used to assess genetic diversity. Our results generated a comprehensive sequence resource for Gynostemma research.

Isolation and characterization of polymorphic microsatellite loci in the endangered plant Dipteronia sinensis (Sapindaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in Dipteronia sinensis to investigate the population genetics of this endangered plant. • METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, 19 microsatellite loci were developed in D. sinensis and evaluated for their variability in 29 samples from a natural population. For the 15 polymorphic loci, the number of alleles ranged from nine to 33, while the observed and expected heterozygosities ranged from 0.3793 to 0.9655 and from 0.6029 to 0.9609, respectively. Their cross-taxa transferability was also investigated in Acer miaotaiense, A. palmatum, and A. pictum subsp. mono, and 10 to 15 loci proved amplifiable in these species. • CONCLUSIONS: These microsatellite markers could be employed to investigate the population genetics of D. sinensis and may potentially be applicable to other related species.

Genetic structure within and among populations of Saruma henryi, an endangered plant endemic to China.

The endangered perennial plant Saruma henryi Oliv. is endemic to China and has phylogenetic, ecological, and medicinal value. We used 10 microsatellite (SSR) loci to investigate genetic diversity and differentiation in 16 natural populations. Genetic diversity was high at the species level (H(E) = 0.9427, h = 0.9410, I = 3.0213) and low at the population level (H(E) = 0.4441, h = 0.4307, I = 0.6822). Pronounced genetic differentiation was detected among populations (G(ST) = 0.5428, F(ST) = 0.5524), in line with the limited among-population gene flow (Nm = 0.21). The significant isolation-by-distance pattern revealed by a Mantel test (r = 0.311, P = 0.001) suggested a clear geographic tendency in the distribution of genetic variability. Bayesian assignment and principal coordinates analyses supported the clustering of 16 populations into three groups. The present SSR results were also compared with previously published ISSR results. These results have significant implications for conservation of the species.

Chloroplast DNA phylogeography of Clintoniaudensis Trautv. & Mey. (Liliaceae) in East Asia.

In this paper, we report the phylogeographic history of Clintoniaudensis Trautv. & Mey. (Liliaceae) inferred from two types of chloroplast DNA markers, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequences. Eighty samples were collected from 20 natural populations, 19 located in China and one in Japan, across the entire range of the species in East Asia. High genetic diversity (h(T)(PCR-RFLP)=0.972, h(T)(sequencing)=0.874) and significant differentiation (N(ST)(PCR-RFLP)=0.893, N(ST)(sequencing)=0.988 and G(ST)(PCR-RFLP)=0.777, G(ST)(sequencing)=0.978) were detected at the species level. These findings are consistent with a scenario of clonal reproduction and locally restricted gene exchange. The level of diversity of tetraploid populations was slightly greater than that of diploid populations. Significant molecular variation was found between tetraploids and diploids. Tetraploids may be autopolyploids and may have formed repeatedly in different locations. At least three regions of origin could be recognized. Twenty-six distinct haplotypes were identified. Haplotype frequency distributions were strongly skewed, with most haplotypes (n=25) represented by only one sample each and thus restricted to a single population. Non-overlapping distributions of cpDNA haplotypes and strong genetic differentiation among populations and/or different ploid level were consistent with the findings of a nested clade analysis, which inferred long-distance colonization as the major process influencing the spatial haplotype distribution of this species. Nested clade analysis showed that the 26 haplotypes formed two 3-step, four 2-step and eleven 1-step clades, with twelve clades showing significant geographical associations. Higher N(ST) than G(ST) (P<0.05) suggested a distinct phylogeographical pattern. Based on mismatch distribution analysis and neutrality tests, we found no evidence of population expansion in the species. Our analyses indicate that the history of C.udensis involved both long-distance migration and the tectonic events of Mountains in East Asia.

Strong phylogeographic pattern of cpDNA variation reveals multiple glacial refugia for Saruma henryi Oliv. (Aristolochiaceae), an endangered herb endemic to China.

Saruma henryi Oliv. (Aristolochiaceae) is an endangered herb endemic to China. In this study, chloroplast microsatellites (cpSSRs) and sequences of the atpB-rbcL intergenic spacers were employed to reveal its genetic diversity and phylogeographic patterns. We detected high within-species genetic diversity (H(T)=0.939 for cpSSR; H(T)=0.862 for atpB-rbcL) and pronounced among-population genetic differentiation (H(S)=0.182, G(ST)=0.811, R(ST)=0.9, F(ST)=0.93 for cpSSR; H(S)=0.238, G(ST)=0.724, N(ST)=0.758, F(ST)=0.79 for atpB-rbcL) with a strong phylogeographic pattern (R(ST)>G(ST), P<0.01 for cpSSR; N(ST)>G(ST), U=0.25 for atpB-rbcL). Eleven haplotypes were distinguished by cpSSRs and atpB-rbcL intergenic spacers, respectively. The molecular phylogenetic data, together with the geographic distribution of the haplotypes, suggested the existence of multiple localized glacial refugia in Mts. Qinling, eastern Mts. Bashan and the southeastern edge of Yunnan-Guizhou Plateau. Nested clade analysis (NCA) and population genetic analyses supported the limited gene flow (caused by low dispersal capacity and complex topography of its habitats) as the major factor responsible for the strong population differentiation and phylogeographic pattern. Past fragmentation and allopatric fragmentation were inferred as important processes responsible for the modern phylogeograhpic pattern. In addition, contiguous range expansions occurred in western Mts. Qinling and eastern Mts. Bashan.

Evolutionary legacy of a forest plantation tree species (Pinus armandii): Implications for widespread afforestation.

Many natural systems are subject to profound and persistent anthropogenic influence. Human-induced gene movement through afforestation and the selective transportation of genotypes might enhance the potential for intraspecific hybridization, which could lead to outbreeding depression. However, the evolutionary legacy of afforestation on the spatial genetic structure of forest tree species has barely been investigated. To do this properly, the effects of anthropogenic and natural processes must be examined simultaneously. A multidisciplinary approach, integrating phylogeography, population genetics, species distribution modeling, and niche divergence would permit evaluation of potential anthropogenic impacts, such as mass planting near-native material. Here, these approaches were applied to Pinus armandii, a Chinese endemic coniferous tree species, that has been mass planted across its native range. Population genetic analyses showed that natural populations of P. armandii comprised three lineages that diverged around the late Miocene, during a period of massive uplifts of the Hengduan Mountains, and intensification of Asian Summer Monsoon. Only limited gene flow was detected between lineages, indicating that each largely maintained is genetic integrity. Moreover, most or all planted populations were found to have been sourced within the same region, minimizing disruption of large-scale spatial genetic structure within P. armandii. This might be because each of the three lineages had a distinct climatic niche, according to ecological niche modeling and niche divergence tests. The current study provides empirical genetic and ecological evidence for the site-species matching principle in forestry and will be useful to manage restoration efforts by identifying suitable areas and climates for introducing and planting new forests. Our results also highlight the urgent need to evaluate the genetic impacts of large-scale afforestation in other native tree species.

Framework Phylogeny, Evolution and Complex Diversification of Chinese Oaks.

Oaks (Quercus L.) are ideal models to assess patterns of plant diversity. We integrated the sequence data of five chloroplast and two nuclear loci from 50 Chinese oaks to explore the phylogenetic framework, evolution and diversification patterns of the Chinese oak's lineage. The framework phylogeny strongly supports two subgenera Quercus and Cerris comprising four infrageneric sections Quercus, Cerris, Ilex and Cyclobalanopsis for the Chinese oaks. An evolutionary analysis suggests that the two subgenera probably split during the mid-Eocene, followed by intergroup divergence within the subgenus Cerris around the late Eocene. The initial diversification of sections in the subgenus Cerris was dated between the mid-Oligocene and the Oligocene-Miocene boundary, while a rapid species radiation in section Quercus started in the late Miocene. Diversification simulations indicate a potential evolutionary shift on section Quercus, while several phenotypic shifts likely occur among all sections. We found significant negative correlations between rates of the lineage diversification and phenotypic turnover, suggesting a complex interaction between the species evolution and morphological divergence in Chinese oaks. Our infrageneric phylogeny of Chinese oaks accords with the recently proposed classification of the genus Quercus. The results point to tectonic activity and climatic change during the Tertiary as possible drivers of evolution and diversification in the Chinese oak's lineage.

Spatial Genetic Structure and Demographic History of the Dominant Forest Oak Quercus fabri Hance in Subtropical China.

Oak trees (Quercus L.) are important models for estimating abiotic impacts on the population structure and demography of long life span tree species. In this study, we generated genetic data for 17 nuclear microsatellite loci in 29 natural populations of Quercus fabri to estimate the population genetic structure. We also integrated approximate Bayesian computation (ABC) and ecological niche analysis to infer the population differentiation processes and demographic history of this oak species. The genetic analyses indicated two genetic clusters across the 29 populations collected, where most approximately corresponded to the intraspecific differentiation among populations from western and eastern China, whereas admixed populations were mainly found in central mountains of China. The best model obtained from hierarchical ABC simulations suggested that the initial intraspecific divergence of Q. fabri potentially occurred during the late Pliocene (ca. 3.99 Ma) to form the two genetic clusters, and the admixed population group might have been generated by genetic admixture of the two differentiated groups at ca. 53.76 ka. Ecological analyses demonstrated clear differentiation among the Q. fabri population structures, and association estimations also indicated significant correlations between geography and climate with the genetic variation in this oak species. Our results suggest abiotic influences, including past climatic changes and ecological factors, might have affected the genetic differentiation and demographic history of Q. fabri in subtropical China.

Polyploid origins in Gynostemma pentaphyllum (Cucurbitaceae) inferred from multiple gene sequences.

The genus Gynostemma (Cucurbitaceae) constitutes a polyploid group of perennial creeping herbs, in whose evolution polyploidization is a key component. With the largest variety of cytotypes (2n=22, 44, 66 and 88) in Gynostemma, G. pentaphyllum is also the most widespread species in this genus. In the present study, we inferred the origins of polyploids in G. pentaphyllum using sequences of the plastid intergenic spacers (trnL-trnF, psbB-psbF and rpl20-rps12) and cloned DNA sequences from two nuclear regions (RPB2 and nrDNA ITS). Phylogenetic analyses of the separate and the combined nuclear gene datasets all supported autoploid origins of polyploids in G. pentaphyllum. Three polyploid populations were more closely related, indicating that significant genetic differentiation may have occurred between diploids and polyploids. We concluded that polyploidization might be an important evolutionary mechanism in the diversification of G. pentaphyllum. On the other hand, no chloroplast DNA (cpDNA) variation was detected in ingroups except the octoploid DL 8x, which possessed a different cpDNA haplotype from the other populations of G. pentaphyllum. This can be explained by limited sample sizes, possible extinction of its diploid progenitors and/or the occurrence of chloroplast transfer through hybridization with other Gynostemma species. However, the distribution of cytotypes in G. pentaphyllum was not as typical as many other autopolyploid complexes. Polyploidization failed to contribute significantly to the expansion of its geographic range. The geographic distribution of diploids and polyploids in G. pentaphyllum may be associated with the past ecological environments of different areas, especially during the glacial period.

[Plasma levels of nociceptin/orphanin FQ in patients with bipolar disorders and health adults].

OBJECTIVE: To investigate the plasma levels of nociceptin/orphanin FQ (OGQ) in patients with bipolar depression and bipolar mania and to analyze the relationship of plasma OFQ to bipolar disorders. METHODS: Peripheral blood samples were collected from 21 patients with bipolar II depression (BD group), 26 patients with bipolar I mania (BM group), and 31 health adults (control group). The concentrations of plasma OFQ were measured by radioimmunoassay (RIA). The psychological status was examined by Hamilton Depression Scale (HAMD), Bech-Rafaelsen Mania Rating Scale (BRMS), and Montgomery and Asberg Depression Rating Scale (MADRS). RESULTS: The plasma OFQ level of the BD group was (20+/-4) ng/L, significantly higher than that of the control group [(14+/-5) ng/L, t=5.28, P<0.01], and the OFQ level of the BM group was (11+/-3) ng/L, significantly lower than that of the control group (t=-2.47, P<0.05). There were not significant differences in the plasma OFQ between the males and females in all groups (all P>0.05). In the BD group the plasma OFQ level was significantly positively correlated with HAMD and MADRS scores (r=0.607, P<0.01; r=0.541, P<0.05). In the BM group the plasma OFQ level was significantly negatively correlated with the BRM score (r=-0.750, P<0.01). CONCLUSION: The changes of plasma OFQ level may play a role in the pathogenesis of bipolar disorder and indicate the severity of disease. Plasma OFQ may be a new biological parameter of bipolar disorder.