Molecular evidence provides new insights into the evolutionary origin of an ancient traditional Chinese medicine, the domesticated "Baizhi".

Introduction: "Baizhi" is a famous herbal medicine in China, and it includes four landraces named as 'Hangbaizhi', 'Chuanbaizhi', 'Qibaizhi', and 'Yubaizhi'. Long-term artificial selection had caused serious degradation of these germplasms. Determining the wild progenitor of the landraces would be benefit for their breed improvements. Previous studies have suggested Angelica dahurica var. dahurica, A. dahurica var. formosana, or A. porphyrocaulis as potential candidates, but the conclusion remains uncertain, and their phylogenetic relationships are still in controversy. Methods: In this study, the genetic variation and phylogenetic analyses of these species and four landraces were conducted on the basis of both the nrITS and plastome datasets. Results: Genetic variation analysis showed that all 8 population of four landraces shared only one ITS haplotype, meanwhile extremely low variation occurred within 6 population at plastid genome level. Both datasets supported the four landraces might be originated from a single wild germplasm. Phylogenetic analyses with both datasets revealed largely consistent topology using Bayesian inference and Maximum likelihood methods. Samples of the four landraces and all wild A. dahurica var. dahurica formed a highly supported monophyletic clade, and then sister to the monophyly clade comprised by samples of A. porphyrocaulis, while four landraces were clustered into one clade, which further clustered with a mixed branches of A. porphyrocaulis and A. dahurica var. dahurica to form sister branches for plastid genomes. Furthermore, the monophyletic A. dahurica var. formosana was far distant from the A. dahurica var. dahurica-"Baizhi" clade in Angelica phylogeny. Such inferences was also supported by the evolutionary patterns of nrITS haplotype network and K2P genetic distances. The outcomes indicated A. dahurica var. dahurica is most likely the original plant of "Baizhi". Discussion: Considering of phylogenetic inference and evolutionary history, the species-level status of A. dahurica var. formosana should be accepted, and the taxonomic level and phylgenetic position of A. porphyrocaulis should be further confirmed. This study preliminarily determined the wild progenitor of "Baizhi" and clarified the phylogenetic relationships among A. dahurica var. dahurica, A. dahurica var. formosana and A. porphyrocaulis, which will provide scientific guidance for wild resources protections and improvement of "Baizhi".

The anti-urolithiasis activity and safety of strangury-relieving herbs: A comparative study based on fruit fly kidney stone model.

ETHNOPHARMACOLOGICAL RELEVANCE: Urolithiasis is one of the oldest and most widespread urological diseases suffered globally. In the long history of Traditional Chinese Medicine, there're numerous herbs documented with strangury-relieving properties playing crucial roles in treating various urological disorders, including dysuria, hematuria, and renal colic, etc., which may be caused by urolithiasis. Exploring these herbs may reveal safer, more effective, and cost-efficient drugs and therapies for urolithiasis. AIM OF THE STUDY: This study aims to assess the anti-urolithiasis efficacy and safety of 46 Chinese traditional and folk herbal drugs using the fruit fly (Drosophila melanogaster) kidney stone model, in order to identify the most valuable ethnomedicinal materials. MATERIALS AND METHODS: Water extract and 50% ethanol extract of each herb were prepared respectively. 0.2% (w/w) sodium oxalate was chosen as appropriate lithogenic agent through fruit fly life span study. Male fruit-flies within three days of emergence were aged for an additional three days, then were randomly divided into experimental groups, model group and control groups (n = 20). The flies in blank control group, model group and positive control group were fed with standard food, standard food containing 0.2% sodium oxalate, standard food containing 0.2% sodium oxalate and 3% (w/w) Garcinia cambogia extract, respectively. Meanwhile, flies in the experimental groups were raised on standard food containing 0.2% sodium oxalate and 3% (w/w) herbal extract. The anti-urolithiasis capability of the extracts was evaluated using stone area ratio (the stone area divided by the area of the Malpighian tubule) and stone-clearing rate. Additionally, the 7-day mortality rate was employed as an indicator of safety. RESULTS: Out of the 46 herbs, 24 exhibited significant anti-urolithiasis effects in their water extracts. Among them, Herba Nephrolepidis, Herba Humuli, Herba Desmodii Styracifolii, Cortex Plumeriae Rubrae, and Herba Mimosae Pudicae showed us a low 7-day mortality rate of fruit-flies as well. However, only a limited number of herbal extracts (8 out of 46) showed obvious anti-urolithiasis activity in their 50% ethanol extracts. CONCLUSION: Highly potential anti-urolithiasis candidates were discovered from strangury-relieving herbs recorded in classical Traditional Chinese Medicine works, highlighting the significant value of traditional and folk ethnopharmacological knowledge.

Phylogenomic analysis of Bupleurum in Western Sichuan, China, including an overlooked new species.

A comparative analysis of chloroplast (cp) genomes and 45s nuclear ribosomal DNA (nrDNA), and a phylogenomic study of six closely related species (including an overlooked new species) of genus Bupleurum from the western part of Sichuan Province in southwestern China were performed. The six species are similar morphologically and it is difficult to identify them; moreover, their genetic relationships remain unclear. It was found that the cp genomes of the six Bupleurum species were extremely similar, and they were highly homogeneous in terms of cp genome structure, genes and its arrangement. Intergenic spacer rpl32-trnL, petA-psbJ, trnK-rps16, and the coding gene ycf1 were considered highly variable. In phylogenetic trees constructed based on the complete cp genome, protein-coding sequences, nrDNA and ITS sequences, Chinese Bupleurum species all formed two major clades; among these trees, nrDNA tree had the best species resolution; the highly variable regions showed no advantage over other molecular markers. Among the six Bupleurum species, B. malconense, B. sichuanense were close relatives to B. chinense and B. yinchowense, B. chaishoui may also be a consanguinity, while B. microcephalum, B. wenchuanense, and the new species B. pseudochaishoui were closely related. At the end, the new species B. pseudochaishoui Z. Chao sp. nov. was described and illustrated, and a key to the six species was tabulated.

The novel developed microsatellite markers revealed potential hybridization among Cymbidium species and the interspecies sub-division of C. goeringii and C. ensifolium.

BACKGROUND: Orchids (Cymbidium spp.) exhibit significant variations in floral morphology, pollinator relations, and ecological habitats. Due to their exceptional economic and ornamental value, Cymbidium spp. have been commercially cultivated for centuries. SSR markers are extensively used genetic tools for biology identification and population genetics analysis. RESULT: In this study, nine polymorphic EST-SSR loci were isolated from Cymbidium goeringii using RNA-Seq technology. All nine SSR loci showed transferability in seven other congeneric species, including 51 cultivars. The novel SSR markers detected inter-species gene flow among the Cymbidium species and intra-species sub-division of C. goeringii and C. ensifolium, as revealed by neighborhood-joining and Structure clustering analyses. CONCLUSION: In this study, we developed nine microsatellites using RNA-Seq technology. These SSR markers aided in detecting potential gene flow among Cymbidium species and identified the intra-species sub-division of C. goeringii and C. ensifolium.

Molecular quantification for differentiation of fresh and dried Jinqian Baihua She.

Freshly-used crude drugs have unique functions and advantages in TCM practice of treating diseases. Jinlong Capsule is a patent traditional Chinese medicine product effective for treatment of hepatocarcinoma, and fresh Jinqian Baihua She (JBS, the body of juvenile Bungarus multicinctus) is one of its important ingredients. The emergence of counterfeit fresh JBS, often identified as dried JBS with almost identical appearance, poses a difficult problem in the quality control of the product. Herein we report a molecular quantification-based method for differentiation of fresh and dried JBS by determining the copy number of a specific DNA marker in the samples. Using species-specific primers and TaqMan probes, we established a real-time quantitative PCR system for amplification of a fragment in the 658-bp cytochrome oxidase subunit I (COI) region from JBS specimens. The amplicon copy number in the muscle tissues ranged from 1.14 × 107 to 4.83 × 107 copies/mg in fresh JBS samples, as compared with 1.13 × 105-8.91 × 106 copies/mg in dried JBS samples. Based upon Fisher discriminant analysis, we used 1.27 × 107 copies/mg as the cut-off value for differentiating fresh and dried JBS, which was validated in the single-blinded validation test of fresh and dried JBS samples. This qPCR system may provide an efficient means for accurate identification of fresh JBS to improve the quality control of the medicinal product.

A specific SNP-based multiplex PCR assay for the simultaneous identification of two biological ingredients for the Chinese patent medicine, Danggui Buxue pill.

Background: An increasing number of Chinese patent medicines (CPM) have been widely used in East Asian and North American countries, and the safety and efficacy of CPM have highly attracted public attention. However, it is difficult to supervise the authenticity of multiple biological ingredients within CPM based on microscopic inspection and physical and chemical detection. The raw materials may have similar characteristics of tissue structures and ergastic substances or similar chemical composition and contents when substitutes and/or adulterants are added. DNA molecular markers have been used to distinguish the biological ingredients within CPM based on conventional PCR assay. However, it was proved to be time- and labor-consuming and reagent-wasting, as multiple PCR amplification strategies were required for identifying the complex species composition within CPM. Here, we took the CPM (Danggui Buxue pill) as an example and aimed to establish a specific SNP-based multiplex PCR assay and simultaneously determine the authenticity of the two biological ingredients (Angelicae Sinensis Radix and Astragali Radix) within this CPM. Methods: We, respectively, designed the species-specific primers based on highly variable nrITS for discriminating Angelicae Sinensis Radix and Astragali Radix from their common substitutes and adulterants. The specificity of the primers was checked through conventional PCR assay and multiplex PCR assay. Furthermore, we used a handcrafted Danggui Buxue pill sample (DGBXP) to optimize annealing temperatures for the primers with multiplex PCR, and the sensitivity was also assessed. Finally, fourteen batches of commercial Danggui Buxue pills were used to verify the stability and practicability of the established multiplex PCR assay. Results: Two pairs of highly species-specific primers for amplifying Angelicae Sinensis Radix and Astragali Radix were screened, and our established multiplex PCR assay showed high specificity and sensitivity (lowest detection concentration: 4.0 × 10-3 ng/µL) at an optimal annealing temperature of 65°C. The method could simultaneously identify both biological ingredients within the Danggui Buxue pill. Conclusion: The specific SNP-based multiplex PCR provided a simple, time-, and labor-saving method for the simultaneous identification of the two biological ingredients within Danggui Buxue pills. This study was expected to provide a novel qualitative quality control strategy for CPM.

High genetic diversity and low population differentiation of a medical plant Ficus hirta Vahl., uncovered by microsatellite loci: implications for conservation and breeding.

BACKGROUND: Wuzhimaotao (Radix Fici Hirtae) originates from the dry root of Ficus hirta (Moraceae), which is widely known as a medical and edible plant distributed in South China. As the increasing demand for Wuzhimaotao, the wild F. hirta has been extremely reduced during the past years. It is urgent to protect and rationally develop the wild resources of F. hirta for its sustainable utilization. However, a lack of genetic background of F. hirta makes it difficult to plan conservation and breeding strategies for this medical plant. In the present study, a total of 414 accessions of F. hirta from 7 provinces in southern China were evaluated for the population genetics using 9 polymorphic SSR markers. RESULTS: A mean of 17.1 alleles per locus was observed. The expected heterozygosity (He) varied from 0.142 to 0.861 (mean = 0.706) in nine SSR loci. High genetic diversity (He = 0.706, ranged from 0.613 to 0.755) and low genetic differentiation among populations (G'ST = 0.147) were revealed at population level. In addition, analysis of molecular variance (AMOVA) indicated that the principal molecular variance existed within populations (96.2%) was significantly higher than that among populations (3.8%). Meanwhile, the three kinds of clustering methods analysis (STRUCTURE, PCoA and UPGMA) suggested that the sampled populations were clustered into two main genetic groups (K = 2). Mantel test showed a significant correlation between geographic and genetic distance among populations (R2 = 0.281, P < 0.001). Pollen flow, seed flow and/or geographical barriers might be the main factors that formed the current genetic patterns of F. hirta populations. CONCLUSIONS: This is a comprehensive study of genetic diversity and population structure of F. hirta in southern China. We revealed the high genetic diversity and low population differentiation in this medicinal plant and clarified the causes of its current genetic patterns. Our study will provide novel insights into the exploitation and conservation strategies for F. hirta.

Chloroplast genome structure and phylogenetic analysis of Glycosmis parviflora (Sims) Little 1948, a folk medicinal plant featured in Lingnan Region, China.

Glycosmis parviflora is the most widely spread and the most morphologically varied species of Chinese Glycosmis, and its roots and leaves serve as folk medicines. We sequenced the complete chloroplast (cp) genome of G. parviflora. The cp genome obtained was a circular DNA molecule of 159,825 bp in length, containing one large and one small single copy region (LSC and SSC) of 87,517 and 18,352 bp separated by a pair of 26,978 bp inverted repeat regions (IRs). The overall GC content of the cp genome was 38.40%. The phylogenetic analysis revealed that Glycosmis was strongly supported as a monophyletic group belonging to Clauseneae, and G. parviflora was closely related to G. pentaphylla. The results will provide the basis for the further study of molecular markers and phylogeny of G. parviflora.

Limited genetic diversity and high differentiation in Angelica dahurica resulted from domestication: insights to breeding and conservation.

BACKGROUND: Angelica dahurica belongs to the Apiaceae family, whose dry root is a famous traditional Chinese medicine named as "Bai zhi". There are two cultivars (A. dahurica cv. 'Hangbaizhi' and A. dahurica cv. 'Qibaizhi'), which have been domesticated for thousands of years. Long term artificial selection has led to great changes in root phenotypes of the two cultivars, and also decreased their adaptability to environment. We proposed hypothesis that the cultivars may have lost some of the genetic diversity found in the wild species and may be highly differentiated from the latter during the domestication process. However, few studies have been carried out on how domestication affected the genetic variation of this species. Here, we accessed the levels of genetic variation and differentiation within and between wild A. dahurica populations and two cultivars using 12 microsatellite markers. RESULTS: The results revealed that the genetic diversity of the cultivars was much lower than that of wild A. dahurica, and A. dahurica cv. 'Qibaizhi' had lower genetic diversity compared to A. dahurica cv. 'Hangbaizhi'. AMOVA analysis showed significant genetic differentiation between the wild and cultivated A. dahurica populations, and between A. dahurica cv. 'Hangbaizhi' and A. dahurica cv. 'Qibaizhi'. Results from Bayesian, UPGMA, NJ and PcoA clustering analysis indicated that all 15 populations were assigned to two genetic clusters corresponding to the wild and cultivated populations. Bayesian clustering analysis further divided the cultivated populations into two sub-clusters corresponding to the two cultivars. CONCLUSIONS: Our study suggests that the domestication process is likely the major factor resulting in the loss of genetic diversity in cultivated A. dahurica populations and in significant genetic differentiation from the wild populations due to founder effect and/or artificially directional selections. This large-scale analysis of population genetics could provide valuable information for genetic resources conservation and breeding programs of Angelica dahurica.

Molecular quantification, a new strategy for quality control of Chinese patent medicine containing animal-derived crude drug: Qi She in Jinlong capsule as an example.

Quality control for Chinese patent medicine (CPM) containing animal-derived crude drug(s) is rather difficult. The methods based on chemical composition analysis, which are commonly used in CPM consisted of plant-derived crude drugs, are often not applicable for CPM containing animal-derived crude drug, because the effective constituents of most animal-derived crude drugs remain unknown. Even if there are such methods, they are usually qualitative rather than quantitative, and the specificity is generally poor. Here we proposed a molecular quantification method for CPM containing animal-derived crude drug, based upon the hypothesis that the amount of remnant DNA fragments could reflect feeding quantity of the crude drugs and thus ensure the quality of the CPM. Take Jinlong capsule [a hepatocellular carcinoma-resisting Chinese patent medicine comprising of three fresh animal drugs, i.e. Shougong (Peking gecko, Gekko swinhonis), Qi She (sharp-snouted pitviper, Deinagkistrodon acutus), and Jinqian Baihua She (many-banded krait, Bungarus multicinctus)] as an example, we established a qPCR assay for Qi She in the capsule, which verified the feasibility of the quality control method based on molecular quantification. Species-specific primers and TaqMan probe for Qi She were designed, and the qPCR assay system was then established. The assay exhibited a good specificity; there's a good linearity between Ct values and logarithm of the target amplicon copy numbers within the range of 8.8 × 101 to 8.8 × 106 copies/µL, and the limit of detection was 88 copies/µL. The method was validated through reproducibility, stability assessment. Recovery of spiked samples was between 91.59% and 101.69%. It was verified that the copy numbers reflected the original feeding amount of an animal-derived crude drug by self-made Jinlong capsules. The assay was successfully applied in Qi She-specific amplicon determination in 20 batches of Jinlong capsule. The study was expected to provide a new strategy for quality control of CPM containing animal-derived crude drug.

Chloroplast genome sequencing based on genome skimming for identification of Eriobotryae Folium.

BACKGROUND: Whole chloroplast genome (cpDNA) sequence is becoming widely used in the phylogenetic studies of plant and species identification, but in most cases the cpDNA were acquired from silica gel dried fresh leaves. So far few reports have been available to describe cpDNA acquisition from crude drugs derived from plant materials, the DNA of which usually was seriously damaged during their processing. In this study, we retrieved cpDNA from the commonly used crude drug Eriobotryae Folium (Pipaye in Chinese, which is the dried leaves of Eriobotrya japonica, PPY) using genome skimming technique. RESULTS: We successfully recovered cpDNA sequences and rDNA sequences from the crude drug PPY, and bioinformatics analysis showed a high overall consistency between the cpDNA obtained from the crude drugs and fresh samples. In the ML tree, each species formed distinct monophyletic clades based on cpDNA sequence data, while the phylogenetic relationships between Eriobotrya species were poorly resolved based on ITS and ITS2. CONCLUSION: Our results demonstrate that both cpDNA and ITS/ITS2 are effective for identifying PPY and its counterfeits derived from distantly related species (i.e. Dillenia turbinata and Magnolia grandiflora), but cpDNA is more effective for distinguishing the counterfeits derived from the close relatives of Eriobotrya japonica, suggesting the potential of genome skimming for retrieving cpDNA from crude drugs used in Traditional Chinese Medicine for their identification.

The chloroplast genomes of four Bupleurum (Apiaceae) species endemic to Southwestern China, a diversity center of the genus, as well as their evolutionary implications and phylogenetic inferences.

BACKGROUND: As one of the largest genera in Apiaceae, Bupleurum L. is well known for its high medicinal value. The genus has frequently attracted the attention of evolutionary biologist and taxonomist for its distinctive characteristics in the Apiaceae family. Although some chloroplast genomes data have been now available, the changes in the structure of chloroplast genomes and selective pressure in the genus have not been fully understood. In addition, few of the species are endemic to Southwest China, a distribution and diversity center of Chinese Bupleurum. Endemic species are key components of biodiversity and ecosystems, and investigation of the chloroplast genomes features of endemic species in Bupleurum will be helpful to develop a better understanding of evolutionary process and phylogeny of the genus. In this study, we analyzed the sequences of whole chloroplast genomes of 4 Southwest China endemic Bupleurum species in comparison with the published data of 17 Bupleurum species to determine the evolutionary characteristics of the genus and the phylogenetic relationships of Asian Bupleurum. RESULTS: The complete chloroplast genome sequences of the 4 endemic Bupleurum species are 155,025 bp to 155,323 bp in length including a SSC and a LSC region separated by a pair of IRs. Comparative analysis revealed an identical chloroplast gene content across the 21 Bupleurum species, including a total of 114 unique genes (30 tRNA genes, 4 rRNA genes and 80 protein-coding genes). Chloroplast genomes of the 21 Bupleurum species showed no rearrangements and a high sequence identity (96.4-99.2%). They also shared a similar tendency of SDRs and SSRs, but differed in number (59-83). In spite of their high conservation, they contained some mutational hotspots, which can be potentially exploited as high-resolution DNA barcodes for species discrimination. Selective pressure analysis showed that four genes were under positive selection. Phylogenetic analysis revealed that the 21 Bupleurum formed two major clades, which are likely to correspond to their geographical distribution. CONCLUSIONS: The chloroplast genome data of the four endemic Bupleurum species provide important insights into the characteristics and evolution of chloroplast genomes of this genu, and the phylogeny of Bupleurum.

Phylogenetic position of Bupleurum sikangense inferred from the complete chloroplast genome sequence.

Bupleurum sikangense is an endemic species to China distributed in Xizang (Tibet), which has high saikosaponin content and potential medicinal value. Morphologically, it extremely resembles B. commelynoideum. In order to get a better understanding of the relationship between B. sikangense and B. commelynoideum, and on the phylogenetic status of the two species in the genus, the complete chloroplast (cp) genomes of them were sequenced. The genome organization, repeat sequences, codon usage, RNA-editing sites, and variation of their cp genomes revealed high similarity between the species. Some highly variable regions like trnK-UUU_rps16, rps16_trnQ-UUG, ndhC_trnV-UAC, petA_psbJ, accD_psaI, and petL_psbE were identified, providing potential molecular markers for differentiating the two species. Phylogenetic analysis indicated that B. commelynoideum has a closer relationship to B. chinese than that to B. sikangense. Overall, this study will not only improve our knowledge about cp genomes of these two species, and but also provide data for further research on species identification, safe medical application, conservation genetics, etc., of Bupleurum plants.

Chloroplast genomes of two Mediterranean Bupleurum species and the phylogenetic relationship inferred from combined analysis with East Asian species.

MAIN CONCLUSION: The chloroplast genomes of Mediterranean Bupleurum species are reported for the first time. Phylogenetic analysis supports the species as a basal clade of Bupleurum with divergence time at 35.40 Ma. Bupleurum is one of the most species-rich genus with high medicinal value in Apiaceae. Although infrageneric classifications of Bupleurum have been the subject of numerous studies, it still remains controversial. Chloroplast genome information will prove essential in advancing our understanding on phylogenetic study. Here we report cp genomes of two woody Bupleurum species (Bupleurum gibraltaricum and B. fruticosum) endemic to Mediterranean. The complete cp genomes of the two species were 157,303 and 157,391 bp in size, respectively. They encoded 114 unique genes including 30 tRNA genes, 4 rRNA genes and 80 protein coding genes. Genome structure, distributions of SDRs and SSRs, gene content exhibited similarities among Bupleurum species. High variable hotspots were detected in eight intergenic spacers and four genes. Most of genes were under purifying selection with two exceptions: atpF and clpP. The phylogenetic analysis based on 80 coding genes revealed that the genus was divided into 2 distinct clades corresponding to the 2 subgenera (subg. Penninervia, subg. Bupleurum) with divergence time at the end of collision of India with Eurasia. Most species diversified mainly during the later period of uplift of Qinghai-Tibetan Plateau. The cp genomes of the two Bupleurum species can be significant complementary to insights into the cp genome characteristics of this genus. The comparative chloroplast genomes and phylogenetic analysis advances our understanding of the evolution of cp genomes and phylogeny in Bupleurum.

Low host specificity and broad geographical ranges in a community of parasitic non-pollinating fig wasps (Sycoryctinae; Chalcidoidea).

Plants, phytophagous insects and their parasitoids form the most diverse assemblages of macroscopic organisms on earth. Enclosed assemblages in particular represent a tractable system for studying community assembly and diversification. Communities associated with widespread plant species are especially suitable as they facilitate a comparative approach. Pantropical fig-wasp communities represent a remarkably well-replicated system, ideal for studying these historical processes. We expect high dispersal ability in non-pollinating fig wasps to result in lower geographical turnover in comparison to pollinating fig wasps. The ability of non-pollinating wasps to utilise a number of hosts (low host specificity) is a key determinant of overall geographical range, with intraspecific competition becoming a constraining factor should diet breadth overlap among species. Finally, we expect conserved community structure throughout the host range. We aim to test these expectations, derived from population genetic and community studies, using the multi-trophic insect community associated with Ficus hirta throughout its 3,500 km range across continental and insular Asia. We collect molecular evidence from one coding mitochondrial gene, one non-coding nuclear gene and multiple microsatellites across 25 geographical sites. Using these data, we establish species boundaries, determine levels of host specificity among non-pollinating fig wasps and quantify geographical variation in community composition. We find low host specificity in two genera of non-pollinating fig wasps. Functional community structure is largely conserved across the range of the host fig, despite limited correspondence between the ranges of non-pollinator and pollinator species. While nine pollinators are associated with Ficus hirta, the two non-pollinator tribes developing in its figs each contained only four species. Contrary to predictions, we find stronger isolation by distance in non-pollinators than pollinators. Long-lived non-pollinators may disperse more gradually and be less reliant on infrequent long-distance dispersal by wind currents. Segregation among non-pollinating species across their range is suggestive of competitive exclusion and we propose that this may be a result of increased levels of local adaptation and moderate, but regular, rates of dispersal. Our findings provide one more example of lack of strict codiversification in the geographical diversification of plant-associated insect communities.

Development and characterization of 16 novel microsatellite markers by Transcriptome sequencing for Angelica dahurica and test for cross-species amplification.

BACKGROUND: Angelica dahurica (Apiaceae) is an important herb in traditional Chinese medicine. Because of its important medicinal and economic values, its wild resources were over-exploited and increasingly reduced. Meanwhile, the diversity of cultivars of A. dahurica has decreased as a result of long-term artificial cultivation. However, there are no population genetics studies of natural A. dahurica reported yet, especially for using microsatellite markers (SSRs) to investigate population genetics of the species. RESULTS: Sixteen polymorphic EST-SSR loci were isolated from A. dahurica with transcriptome sequencing technology (RNA-Seq). The number of alleles varied from 2 to 15 per polymorphic locus over populations with the observed and expected heterozygosities ranging from 0.000 to 1.000 and from 0.000 to 0.829, respectively. Significant deviations from Hardy-Weinberg equilibrium were observed at 8 loci. Tests of linkage disequilibrium showed 11 informative locus pairs were significant across all populations. Cross-species amplification showed that 14 out of 16 SSR loci have the transferability in cultivar-A. dahurica cv. 'Hangbaizhi' and A. decursiva. CONCLUSIONS: The 16 newly developed loci microsatellite primers with RNA-Seq will be useful for further investigating population genetics of A. dahurica, cultivars and other members of this genus.

The complete chloroplast genome sequence of Centella asiatica (Linnaeus) Urban.

The apiaceous species Centella asiatica (Linnaeus) Urban is attractive not only to pharmaceutical researchers for its versatile medicinal uses, but also to botanists for its phylogenetic significance. We acquired its whole chloroplast genome (CP) through genome skimming. The CP genome ofCentella asiatica was 154,771 bp in length, including a large single-copy (LSC) region with 86176 bp, a small single copy (SSC) region with 18107 bp, and a pair of inverted repeats (IR) regions with 25,343 bp. The whole AT content of the CP genome was 62.3%. Phylogenomic analysis revealed that Centella asiatica formed a separate clade sister to Saniculoideae and Apioideae species in the family Apiaceae. The work provides beneficial data for following researches on the genetic variation, species identification, phylogeny, and classification of Centella.

Multiple parapatric pollinators have radiated across a continental fig tree displaying clinal genetic variation.

The ways that plant-feeding insects have diversified are central to our understanding of terrestrial ecosystems. Obligate nursery pollination mutualisms provide highly relevant model systems of how plants and their insect associates have diversified and the over 800 species of fig trees (Ficus) allow comparative studies. Fig trees can have one or more pollinating fig wasp species (Agaonidae) that breed within their figs, but factors influencing their number remain to be established. In some widely distributed fig trees, the plants form populations isolated by large swathes of sea, and the different populations are pollinated by different wasp species. Other Ficus species with continuous distributions may present genetic signatures of isolation by distance, suggesting more limited pollinator dispersal, which may also facilitate pollinator speciation. We tested the hypothesis that Ficus hirta, a species for which preliminary data showed genetic isolation by distance, would support numerous pollinator species across its range. Our results show that across its range F. hirta displays clinal genetic variation and is pollinated by nine parapatric species of Valisia. This is the highest number of pollinators reported to date for any Ficus species, and it is the first demonstration of the occurrence of parapatric pollinator species on a fig host displaying continuous genetic structure. Future comparative studies across Ficus species should be able to establish the plant traits that have driven the evolution of pollinator dispersal behaviour, pollinator speciation and host plant spatial genetic structure.

The complete chloroplast genome sequence of Ficus hirta (Moraceae).

The dry root (Radix Fici Hirtae) of Ficus hirta has been used as a traditional herbal medicine in Ling nan regions of China for a long time. As its large market demand, the wild resources of F. hirta have sharply reduced. It is necessary to conduct the study of conservation genetics. However, there is still lack of complete genome information for the research on evolutionary biology, population genetics and phylogeography of this species. Here, we sequenced the complete chloroplast (CP) genome of F. hirta using Next Generation Sequencing technology (NGS). The CP genome of F. hirta is 160,374 bp in length, which contains a large single-copy (LSC) region of 88,446 bp, a small sing-copy (SSC) region of 18,134 bp, and two inverted repeat (IRa and IRb) regions of 26,897 bp. A total of 130 genes were successfully annotated containing 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Phylogenetic analysis support genus Ficus is monophyletic and F. hirta is closely related to F. carica within this genus.

Plant geographic phenotypic variation drives diversification in its associated community of a phytophagous insect and its parasitoids.

BACKGROUND: While the communities constituted by phytophageous insects and their parasites may represent half of all terrestrial animal species, understanding their diversification remains a major challenge. A neglected idea is that geographic phenotypic variation in a host plant may lead to heterogeneous evolutionary responses of the different members of the associated communities. This could result in diversification on a host plant by ecological speciation in some species, leading to geographic variation in community composition. In this study we investigated geographic variation of inflorescence receptacle size in a plant, Ficus hirta, and how the hymenopteran community feeding in the inflorescences has responded. Our predictions were: 1) Inflorescence size variation affects wasp species differently depending on how they access oviposition sites. 2) In some affected lineages of wasps, we may observe vicariant, parapatric species adapted to different inflorescence sizes. RESULTS: We show that fig (the enclosed inflorescence of Ficus) wall thickness varies geographically. The fig-entering pollinating wasp was not affected, while the parasites ovipositing through the fig wall were. Two parapatric species of Philotrypesis, exhibiting strikingly different ovipositor lengths, were recorded. One species of Sycoscapter was also present, and it was restricted, like the shorter-ovipositor Philotrypesis, to the geographic zone where fig walls were thinner. CONCLUSIONS: Previous work on fig wasps suggested that parapatric geographic ranges among congenerics were due to adaptation to variation in abiotic factors, complemented by interspecific competition. Our results show that parapatric ranges may also result from adaptation to variation in biotic factors. Within an insect community, differences among species in their response to geographic phenotypic variation of their host plant may result in geographically heterogeneous community structure. Such heterogeneity leads to heterogeneous interaction networks among sites. Our results support the hypothesis that plant geographic phenotypic variation can be a driver of diversification in associated insect communities, and can complement other diversification processes.