Phylogeny and Taxonomic Revision of the Genus Melanosciadium (Apiaceae), Based on Plastid Genomes and Morphological Evidence.

Melanosciadium is considered a monotypic genus and is also endemic to the southwest of China. No detailed phylogenetic studies or plastid genomes have been identified in Melanosciadium. In this study, the plastid genome sequence and nrDNA sequence were used for the phylogenetic analysis of Melanosciadium and its related groups. Angelica tsinlingensis was previously considered a synonym of Hansenia forbesii. Similarly, Ligusticum angelicifolium was previously thought to be the genus Angelica or Ligusticopsis. Through field observations and morphological evidence, we believe that the two species are more similar to M. pimpinelloideum in leaves, umbel rays, and fruits. Meanwhile, we found a new species from Anhui Province (eastern China) that is similar to M. pimpinelloideum and have named it M. Jinzhaiensis. We sequenced and assembled the complete plastid genomes of these species and another three Angelica species. The genome comparison results show that M. pimpinelloideum, A. tsinlingensis, Ligusticum angelicifolium, and M. jinzhaiensis have similarities to each other in the plastid genome size, gene number, and length of the LSC and IR regions; the plastid genomes of these species are distinct from those of the Angelica species. In addition, we reconstruct the phylogenetic relationships using both plastid genome sequences and nrDNA sequences. The phylogenetic analysis revealed that A. tsinlingensis, M. pimpinelloideum, L. angelicifolium, and M. jinzhaiensis are closely related to each other and form a monophyletic group with strong support within the Selineae clade. Consequently, A. tsinlingensis and L. angelicifolium should be classified as members of the genus Melanosciadium, and suitable taxonomical treatments have been proposed. Meanwhile, a comprehensive description of the new species, M. jinzhaiensis, is presented, encompassing its habitat environment and detailed morphological traits.

Backbone phylogeny and adaptive evolution of Pleurospermum s. l.: New insights from phylogenomic analyses of complete plastome data.

Pleurospermum is a taxonomically challenging taxon of Apiaceae, as its circumscription and composition remain controversial for morphological similarities with several related genera, leading to a dispute between Pleurospermum in the broad sense and strict sense. While evidence from previous molecular studies recognized plural branching lineages within the Pleurospermum s. l., it did not support the latest delimitation of Pleurospermum s. str. by only two closely related northern species. So far, no proper delimitation for Pleurospermum has come up, and many of the plural taxa in Pleurospermum s. l. remain unresolved, which may be due to poor phylogenetic resolution yielded barely from ITS sequences. Herein, we newly assembled 40 complete plastomes from 36 species of Pleurospermum s. l. and related genera, 34 of which were first reported and generated a well-resolved backbone phylogeny in a framework of the subfamily Apioideae. From the phylogeny with greatly improved resolution, a total of six well-supported monophyletic lineages within Pleurospermum s. l. were recognized falling in different major clades of Apioideae. Combining morphological characteristics with phylogenetic inference, we suggested to re-delimit the Pleurospermum s. str. by introducing nine species mainly from the Himalayan regions and proposed its boundary features; the remaining species were suggested to be excluded from Pleurospermum to incorporate into their more related taxa being revealed. On this basis, the plastome comparison revealed not only the high conservatism but also the mild differences among lineages in plastome structure and gene evolution. Overall, our study provided a backbone phylogeny essential for further studies of the taxonomically difficult taxa within Pleurospermum s. l.

Phylogenetic position of Ligusticopsis (Apiaceae, Apioideae): evidence from molecular data and carpological characters.

Ligusticopsis (Apiaceae, Apioideae) is now considered to have an East-Asia and Sino-Himalaya distribution. The genus was not recognized as a natural and separate genus and was treated as a synonym of Ligusticum both in Flora Reipublicae Popularis Sinicae and Flora of China since first established, though Pimenov et al. have made many taxonomic revisions to Ligusticopsis, phylogenetic relationships between Ligusticopsis and Ligusticum have been in dispute. Thirty-four plastomes and 35 nrITS from Apioideae were analysed by RAxML and MrBayes to reconstruct the phylogenetic relationships, along with carpology of 10 species and comparative analyses of 17 plastomes to investigate the evidence supporting the independence of Ligusticopsis. As a result, nine species suggested to be Ligusticopsis formed a highly supported monophyletic branch (Subclade A) inside Selineae both in maximum likelihood and Bayesian inference; the results of the comparative analyses further supported the monophyly of Subclade A, mainly in the location of genes at the IRa/LSC boundary, the sequence diversity exhibited by various genes (e.g. trnH-GUG-psbA and ycf2) and same codon biases in terminator TAA (relative synonymous codon usage = 1.75). Species in Subclade A also had shared characters in mericarps, combined with other characters of the plant, 'base clothed in fibrous remnant sheaths, pinnate bracts, pinnate bracteoles longer than rays of umbellule, mericarps strongly compressed dorsally, median and lateral ribs filiform or keeled, marginal ribs winged, and numerous vittae in commissure and each furrow' should be the most important and diagnostic characters of Ligusticopsis. Our phylogenetic trees and other analyses supported the previous taxonomic treatments of Pimenov et al. that Ligusticopsis should be a natural and separate genus rather than a synonym of Ligusticum.

Effects of Mountain Uplift and Climatic Oscillations on Phylogeography and Species Divergence of Chamaesium (Apiaceae).

Exploring the effects of orographic events and climatic shifts on the geographic distribution of organisms in the Himalayas-Hengduan Mountains (HHM) region and Qinghai-Tibetan Plateau (QTP) is crucial to understand the impact of environmental changes on organism evolution. To gain further insight into these processes, we reconstructed the evolutionary history of nine Chamaesium species distributed across the HHM and QTP regions. In total, 525 individuals from 56 populations of the nine species were analyzed based on three maternally inherited chloroplast fragments (rpl16, trnT-trnL, and trnQ-rps16) and one nuclear DNA region (internal transcribed spacer, ITS). Fifty-two chloroplast DNA (cpDNA) and 47 ITS haplotypes were identified in nine species. All of the cpDNA and ITS haplotypes were species-specific. Phylogenetic analysis suggested that all nine species form a monophyletic clade with high support. Dating analysis and ancestral area reconstruction revealed that the ancestral group of Chamaesium originated in the southern Himalayan region at the beginning of the Paleogene (60.85 Ma). The nine species of Chamaesium then separated well during the last 25 million years started in Miocene. Our maxent modeling indicated the broad-scale distributions of all nine species remained fairly stable from LIG to the present and predicted that it will remain stable into the future. The initial split of Chamaesium was triggered by climate changes following the collision of the Indian plate with the Eurasia plate during the Eocene. Subsequently, divergences within Chamaesium may have been induced by the intense uplift of the QTP, the onset of the monsoon system, and Central Asian aridification. Long evolutionary history, sexual reproduction, and habitat fragmentation could contribute to the high level of genetic diversity of Chamaesium. The higher genetic differentiation among Chamaesium populations may be related to the drastic changes of the external environment in this region and limited seed/pollen dispersal capacity.

Out of the Qinghai-Tibetan Plateau and rapid radiation across Eurasia for Allium section Daghestanica (Amaryllidaceae).

The disjunctive distribution (Europe-Caucasus-Asia) and species diversification across Eurasia for the genus Allium sect. Daghestanica has fascinating attractions for researchers aiming to understanding the development and history of modern Eurasia flora. However, no any studies have been carried out to address the evolutionary history of this section. Based on the nrITS and cpDNA fragments (trnL-trnF and rpl32-trnL), the evolutionary history of the third evolutionary line (EL3) of the genus Allium was reconstructed and we further elucidated the evolutionary line of sect. Daghestanica under this background. Our molecular phylogeny recovered two highly supported clades in sect. Daghestanica: the Clade I includes Caucasian-European species and Asian A. maowenense, A. xinlongense and A. carolinianum collected in Qinghai; the Clade II comprises Asian yellowish tepal species, A. chrysanthum, A. chrysocephalum, A. herderianum, A. rude and A. xichuanense. The divergence time estimation and biogeography inference indicated that Asian ancestor located in the Qinghai-Tibetan Plateau (QTP) and the adjacent region could have migrated to Caucasus and Europe distributions around the Late Miocene and resulted in further divergence and speciation; Asian ancestor underwent the rapid radiation in the QTP and the adjacent region most likely due to the heterogeneous ecology of the QTP resulted from the orogeneses around 4-3 million years ago (Mya). Our study provides a picture to understand the origin and species diversification across Eurasia for sect. Daghestanica.

Backbone phylogeny and evolution of Apioideae (Apiaceae): New insights from phylogenomic analyses of plastome data.

Traditional phylogenies inferred from chloroplast DNA fragments have not obtained a well-resolved evolutionary history for the backbone of Apioideae, the largest subfamily of Apiaceae. In this study, we applied the genome skimming approach of next-generation sequencing to address whether the lack of resolution at the tip of the Apioideae phylogenetic tree is due to limited information loci or the footprint of ancient radiation. A total of 90 complete chloroplast genomes (including 23 newly sequenced genomes and covering 20 major clades of Apioideae) were analyzed (RAxML and MrBayes) to provide a phylogenomic reconstruction of Apioideae. Dating analysis was also implemented using BEAST to estimate the origin and divergence time of the major clades. As a result, the early divergences of Apioideae have been clarified but the relationship among its distally branching clades (Group A) was only partially resolved, with short internal branches pointing to an ancient radiation scenario. Four major clades, Tordyliinae I, Pimpinelleae I, Apieae and Coriandreae, were hypothesized to have originated from chloroplast capture events induced by early hybridization according to the incongruence between chloroplast-based and nrDNA-based phylogenetic trees. Furthermore, the variable and nested distribution of junction positions of LSC (Large single copy region) and IRB (inverted repeat region B) in Group A may reflect incomplete lineage sorting within this group, which possibly contributed to the unclear phylogenetic relationships among these clades inferred from plastome data. Molecular clock analysis revealed the chloroplast capture events mainly occurred during the middle to late Miocene, providing a geological and climate context for the evolution of Apioideae.

New insights into the phylogeny and taxonomy of Chinese Physospermopsis (Apiaceae).

Physospermopsis (Apiaceae) comprises about 10 species, but its taxonomy and phylogeny are disputed. The genus is mostly distributed in the Himalayas and Hengduan Mountains at high elevation. Earlier molecular studies involving six species of Physospermopsis indicated that this genus is not monophyletic and is nested in the East Asia Clade. Therefore, the aims of this study were to re-assess the phylogenetic position of, and interspecific relationships within, Physospermopsis based on two chloroplast loci (rpl16, rps16) and one nuclear region, the internal transcribed spacers of ribosomal DNA (ITS). Eight species involving 13 populations of Physospermopsis were collected. These were sequenced and analyzed with the sequences of 31 other Apiaceae species obtained from the NCBI to determine phylogenetic relationships using Bayesian inference (BI) and Maximum likelihood (ML). Our study found that Physospermopsis is monophyletic, nested in Pleurospermeae of Apiaceae, sister to Pleurospermum. And we propose that the Physospermopsis clade should be replaced by the East Asia Clade. However, the interspecific relationships within Physospermopsis were not well resolved and the positioning of species was unclear. Diagnostic characteristics to distinguish Physospermopsis species in the field and laboratory are provided for future Physospermopsis phylogenetic studies.

New insights into the phylogeny of Sinocarum (Apiaceae, Apioideae) based on morphological and molecular data.

Sinocarum is a Sino-Himalayan endemic genus of Apiaceae and distributed in high-elevations from Nepal to SW China. In this study, morphological characteristics were combined with nuclear internal transcribed spacer (ITS) and two chloroplast DNA (cpDNA) intron sequences (rpl16 and rps16) to determine the phylogenetic placement of Sinocarum and the infrageneric relationships between five Sinocarum species. The results confirmed that Sinocarum was a polyphyletic group separated into two clades, Acronema and East Asia clades. S. coloratum, the generic type of Sinocarum, S. cruciatum, S. vaginatum and S. filicinum are in the Acronema clade. Among them, the first three species are clustered into a subclade and are closely related to the genus Acronema. While S. filicinum has a close affinity with Meeboldia. S. schizopetalum did not ally with its congeners we collected and is allied closely with members of the distantly related East Asia clade. In addition, the fruit of the Acronema clade Sinocarum species is usually oblong-ovoid or ovoid, and the pollen is super-rectangular, while the Sinocarum species in the East Asia clade have broad-ovoid fruit and sub-rhomboidal pollen. This study has furnished cumulative evidence to reduce phylogenetic uncertainty and provide a more comprehensive description of the plant morphology, fruit morphology and anatomy, and pollen morphology of these five Chinese Sinocarum species.

Complete Plastid Genome Sequencing of Eight Species from Hansenia, Haplosphaera and Sinodielsia (Apiaceae): Comparative Analyses and Phylogenetic Implications.

Hansenia Turcz., Haplosphaera Hand.-Mazz. and Sinodielsia H.Wolff are three Apiaceae genera endemic to the Hengduan Mountains and the Himalayas, which usually inhabit elevations greater than 2000 m. The phylogenetic relationships between and within the genera were uncertain, especially the placement of Hap. himalayensis and S. microloba. Therefore, we aimed to conduct comparative (simple sequence repeat (SSR) structure, codon usage bias, nucleotide diversity (Pi) and inverted repeat (IR) boundaries) and phylogenetic analyses of Hansenia, Haplosphaera and Sinodielsia (also compared with Chamaesium and Bupleurum) to reduce uncertainties in intergeneric and interspecific relationships. We newly assembled eight plastid genomes from Hansenia, Haplosphaera and Sinodielsia species, and analyzed them with two plastid genomes from GenBank of Hap. phaea,S. yunnanensis. Phylogenetic analyses used these ten genomes and another 22 plastid genome sequences of Apiaceae. We found that the newly assembled eight genomes ranged from 155,435 bp to 157,797 bp in length and all had a typical quadripartite structure. Fifty-five to 75 SSRs were found in Hansenia, Haplosphaera and Sinodielsia species, and the most abundant SSR was mononucleotide, which accounted for 58.47% of Hansenia, 60.21% of Haplosphaera and 48.01% of Sinodielsia. There was no evident divergence of codon usage frequency between the three genera, where codons ranged from 21,134 to 21,254. The Pi analysis showed that trnE(UUC)-trnT(GGU), trnH(GUG)-psbA and trnE(UUC)-trnT(GGU) spacer regions had the highest Pi values in the plastid genomes of Hansenia (0.01889), Haplosphaera (0.04333) and Sinodielsia (0.01222), respectively. The ndhG-ndhI spacer regions were found in all three genera to have higher diversity values (Pi values: 0.01028-0.2), and thus may provide potential DNA barcodes in phylogenetic analysis. IR boundary analysis showed that the length of rps19 and ycf1 genes entering IRs were usually stable in the same genus. Our phylogenetic tree demonstrated that Hap. himalayensis is sister to Han. weberbaueriana; meanwhile, Haplosphaera and Hansenia are nested together in the East Asia clade, and S. microloba is nested within individuals of S. yunnanensis in the Acronema clade. This study will enrich the complete plastid genome dataset of the Apiaceae genera and has provided a new insight into phylogeny reconstruction using complete plastid genomes of Hansenia, Haplosphaera and Sinodielsia.

Phylogeny and Comparative Analysis of Chinese Chamaesium Species Revealed by the Complete Plastid Genome.

Chamaesium H. Wolff (Apiaceae, Apioideae) is a small genus mainly distributed in the Hengduan Mountains and the Himalayas. Ten species of Chamaesium have been described and nine species are distributed in China. Recent advances in molecular phylogenetics have revolutionized our understanding of Chinese Chamaesium taxonomy and evolution. However, an accurate phylogenetic relationship in Chamaesium based on the second-generation sequencing technology remains poorly understood. Here, we newly assembled nine plastid genomes from the nine Chinese Chamaesium species and combined these genomes with eight other species from five genera to perform a phylogenic analysis by maximum likelihood (ML) using the complete plastid genome and analyzed genome structure, GC content, species pairwise Ka/Ks ratios and the simple sequence repeat (SSR) component. We found that the nine species' plastid genomes ranged from 152,703 bp (C. thalictrifolium) to 155,712 bp (C. mallaeanum), and contained 133 genes, 34 SSR types and 585 SSR loci. We also found 20,953-21,115 codons from 53 coding sequence (CDS) regions, 38.4-38.7% GC content of the total genome and low Ka/Ks (0.27-0.43) ratios of 53 aligned CDS. These results will facilitate our further understanding of the evolution of the genus Chamaesium.

Comparative Analysis of the Complete Plastid Genome of Five Bupleurum Species and New Insights into DNA Barcoding and Phylogenetic Relationship.

Bupleurum L. (Apiaceae) is a perennial and herbal genus, most species of which have high medicinal value. However, few studies have been performed using plastome data in this genus, and the phylogenetic relationships have always been controversial. In this study, the plastid genomes of Bupleurum chinense and Bupleurum commelynoideum were sequenced, and their gene content, order, and structure were counted and analyzed. The only three published Bupleurum species (B. boissieuanum, B. falcatum, and B. latissimum) and other fifteen allied species were selected to conduct a series of comparative and phylogenetic analyses. The genomes of B. chinense and B. commelynoideum were 155,869 and 155,629 bp in length, respectively, both of which had a typical quadripartite structure. The genome length, structure, guanine and cytosine (GC) content, and gene distribution were highly similar to the other three Bupleurum species. The five Bupleurum species had nearly the same codon usages, and eight regions (petN-psbM, rbcL-accD, ccsA-ndhD, trnK(UUU)-rps16, rpl32-trnL(UAG)-ccsA, petA-psbJ, ndhF-rpl32, and trnP(UGG)-psaJ-rpl33) were found to possess relatively higher nucleotide diversity, which may be the promising DNA barcodes in Bupleurum. Phylogenetic analysis revealed that all Bupleurum species clustered into a monophyletic clade with high bootstrap support and diverged after the Chamaesium clade. Overall, our study provides new insights into DNA barcoding and phylogenetic relationship between Bupleurum and its related genera, and will facilitate the population genomics, conservation genetics, and phylogenetics of Bupleurum in Apiaceae.

The effect of Hengduan Mountains Region (HMR) uplift to environmental changes in the HMR and its eastern adjacent area: Tracing the evolutionary history of Allium section Sikkimensia (Amaryllidaceae).

Exploring the effects of orographic events and climatic shifts on geographic distribution of organism in the Hengduan Mountains Region (HMR) and its eastern adjacent area is crucial to the understanding of the environmental changes to organismal evolution. To gain further insight into these processes, we reconstruct evolutionary history of ten species in Allium section Sikkimensia, distributed across regions abovementioned. Using chloroplast and nuclear sequence variation of 79 populations of these ten Allium species with known morphological preferences, we elucidate the phylogenetic relationship, divergence time, ancestral area and genetic structures. Climatic variables analysis, Isolation by distance (IBD) and environment (IBE) and Species distribution modeling (SDM) were analyzed along different genetic clades. These analyses indicated that the initial split of Sikkimensia was triggered by climate changes following Qinghai-Tibet Plateau sensu lato (QTPsl) uplift during the late Miocene. Subsequently, divergences within lineage (lineage A)/among lineages (lineage C and D) in Sikkimensia may be induced by the intense uplift of the HMR around 3-4 Ma and abrupt intensifying of the Asian monsoon regimes. Furthermore, Sikkimensia populations exhibited lopsided demographic history in the Last Glacial Maximum (LGM), as was indicated by the expansion of their range in the QDM and contraction in the HMR. Our findings appear to suggest that the HMR uplift could have strengthened the orographic difference between the HMR and its eastern adjacent area and led to a colder climate in the HMR, while geological topography also played an important role for taxa to respond the climate change that had taken place in the HMR and its eastern adjacent area during the Pleistocene.

The complete chloroplast genome of Meeboldia yunnanensis (Apiaceae).

Meeboldia yunnanensis Wolff (Apiaceae) is a perennial species naturally distributed in Yunnan and Xizang. The complete chloroplast genome sequence of M. yunnanensis was generated by de novo assembly using whole-genome next-generation sequencing data. The complete chloroplast genome of M. yunnanensis was 154,865 bp in total sequence length and divided into four distinct regions: small single-copy region (17,370 bp), large single-copy region (84,641 bp), and a pair of inverted repeat regions (26,427 bp). The genome annotation displayed a total of 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that M. yunnanensis has close relationship to Pterygopleurum neurophyllum.

Distribution of hepatitis B virus genotype in 5 cities of Fujian province and the clinical implications of HBV genotype / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the prevalence of hepatitis B virus (HBV) genotype in 5 cities of Fujian province and the clinical implications of distinct genotypes in HBV-related liver diseases.</p><p><b>METHODS</b>HBV genotype was determined by the restriction fragment length polymorphism analysis in patients with chronic HBV infection in 5 cities of Fujian province. The relationship between HBV genotype and its clinical implications was studied by multinomal logistic regression and correspondence analysis.</p><p><b>RESULTS</b>Of the 431 HBV DNA positive patients detected by PCR, 275 (63.8%) belonged to HBV genotype B, 100 (23.2%) to genotype C, 51 (11.8%) to genotype D and D-mixed genotype. Genotype A, E and F were not found. Multinomal logistic regression showed that genotype B was more prevalent in Quanzhou and Sanming cities than in Fuzhou (P = 0.002, P = 0.006), and genotype B appeared significantly more common in asymptomatic carriers (ASC), chronic hepatitis B (CHB) and severe hepatitis (SH). Genotype C was most prevalent in patients with liver cirrhosis (LC) (47.0%) than in those with ASC (14.5%) and SH (14.7%) (P = 0.009, P < 0.001). The positive rate of hepatitis B e antigen was higher in patients with genotype C than in those with genotype B and genotype D (56.0% vs. 52.4%, P = 0.008, and 56.0% vs. 30.8%, P = 0.051, respectively). By correspondence analysis, genotype D and D-mixed genotype seemed to be correlated with hepatocellular carcinoma (HCC).</p><p><b>CONCLUSIONS</b>(1) The major popular genotypes of HBV were B, C and D in Fujian. (2) Data of our study suggested that the geographic distribution of genotype B and C might be different in some cities of Fujian. (3) Genotype B might have a tendency to lead to SH in younger patients with chronic hepatitis B and the development of LC might be associated with genotype C among the elder patients. (4) Genotype D appeared to associate with development of HCC, which called for further study to confirm.</p>