Plastid phylogenomics contributes to the taxonomic revision of taxa within the genus Sanicula L. and acceptance of two new members of the genus.

Introduction: The genus Sanicula L. is a taxonomically complicated taxa within Apiaceae, as its high variability in morphology. Although taxonomists have performed several taxonomic revisions for this genus, the interspecific relationships and species boundaries have not been satisfactorily resolved, especially for those endemic to China. This study mainly focused on S. giraldii var. ovicalycina, S. tienmuensis var. pauciflora, and S. orthacantha var. stolonifera and also described two new members of the genus. Methods: We newly sequenced sixteen plastomes from nine Sanicula species. Combined with eleven plastomes previously reported by us and one plastome downloaded, we performed a comprehensively plastid phylogenomics analysis of 21 Sanicula taxa. Results and Discussion: The comparative results showed that 21 Sanicula plastomes in their structure and features were highly conserved and further justified that two new species were indeed members of Sanicula. Nevertheless, eleven mutation hotspot regions were still identified. Phylogenetic analyses based on plastome data and the ITS sequences strongly supported that these three varieties were clearly distant from three type varieties. The results implied that these three varieties should be considered as three independent species, which were further justified by their multiple morphological characters. Therefore, revising these three varieties into three independent species was reasonable and convincing. Moreover, we also identified and described two new Sanicula species (S. hanyuanensis and S. langaoensis) from Sichuan and Shanxi, China, respectively. Based on their distinct morphological characteristics and molecular phylogenetic analysis, two new species were included in Sanicula. In summary, our study impelled the revisions of Sanicula members and improved the taxonomic system of the genus.

Phylogeny and diversification of genus Sanicula L. (Apiaceae): novel insights from plastid phylogenomic analyses.

BACKGROUND: The genus Sanicula L. is a unique perennial herb that holds important medicinal values. Although the previous studies on Sanicula provided us with a good research basis, its taxonomic system and interspecific relationships have not been satisfactorily resolved, especially for those endemic to China. Moreover, the evolutionary history of this genus also remains inadequately understood. The plastid genomes possessing highly conserved structure and limited evolutionary rate have proved to be an effective tool for studying plant phylogeny and evolution. RESULTS: In the current study, we newly sequenced and assembled fifteen Sanicula complete plastomes. Combined with two previously reported plastomes, we performed comprehensively plastid phylogenomics analyses to gain novel insights into the evolutionary history of this genus. The comparative results indicated that the seventeen plastomes exhibited a high degree of conservation and similarity in terms of their structure, size, GC content, gene order, IR borders, codon bias patterns and SSRs profiles. Such as all of them displayed a typical quadripartite structure, including a large single copy region (LSC: 85,074-86,197 bp), a small single copy region (SSC: 17,047-17,132 bp) separated by a pair of inverted repeat regions (IRs: 26,176-26,334 bp). And the seventeen plastomes had similar IR boundaries and the adjacent genes were identical. The rps19 gene was located at the junction of the LSC/IRa, the IRa/SSC junction region was located between the trnN gene and ndhF gene, the ycf1 gene appeared in the SSC/IRb junction and the IRb/LSC boundary was located between rpl12 gene and trnH gene. Twelve specific mutation hotspots (atpF, cemA, accD, rpl22, rbcL, matK, ycf1, trnH-psbA, ycf4-cemA, rbcL-accD, trnE-trnT and trnG-trnR) were identified that can serve as potential DNA barcodes for species identification within the genus Sanicula. Furthermore, the plastomes data and Internal Transcribed Spacer (ITS) sequences were performed to reconstruct the phylogeny of Sanicula. Although the tree topologies of them were incongruent, both provided strong evidence supporting the monophyly of Saniculoideae and Apioideae. In addition, the sister groups between Saniculoideae and Apioideae were strongly suggested. The Sanicula species involved in this study were clustered into a clade, and the Eryngium species were also clustered together. However, it was clearly observed that the sections of Sanicula involved in the current study were not respectively recovered as monophyletic group. Molecular dating analysis explored that the origin of this genus was occurred during the late Eocene period, approximately 37.84 Ma (95% HPD: 20.33-52.21 Ma) years ago and the diversification of the genus was occurred in early Miocene 18.38 Ma (95% HPD: 10.68-25.28 Ma). CONCLUSION: The plastome-based tree and ITS-based tree generated incongruences, which may be attributed to the event of hybridization/introgression, incomplete lineage sorting (ILS) and chloroplast capture. Our study highlighted the power of plastome data to significantly improve the phylogenetic supports and resolutions, and to efficiently explore the evolutionary history of this genus. Molecular dating analysis explored that the diversification of the genus occurred in the early Miocene, which was largely influenced by the prevalence of the East Asian monsoon and the uplift of the Hengduan Mountains (HDM). In summary, our study provides novel insights into the plastome evolution, phylogenetic relationships, taxonomic framework and evolution of genus Sanicula.

Phylogeny and adaptive evolution of subgenus Rhizirideum (Amaryllidaceae, Allium) based on plastid genomes.

The subgenus Rhizirideum in the genus Allium consists of 38 species worldwide and forms five sections (A. sect. Rhizomatosa, A. sect. Tenuissima, A. sect. Rhizirideum, A. sect. Eduardia, and A. sect. Caespitosoprason), A. sect. Caespitosoprason being merged into A. sect. Rhizomatosa recently. Previous studies on this subgenus mainly focused on separate sections. To investigate the inter-section and inter-subgenera phylogenetic relationships and adaptive evolution of A. subg. Rhizirideum, we selected thirteen representative species, which cover five sections of this subgenus and can represent four typical phenotypes of it. We conducted the comparative plastome analysis with our thirteen plastomes. And phylogenetic inferences with CDSs and complete sequences of plastomes of our thirteen species and another fifty-four related species were also performed. As a result, the A. subg. Rhizirideum plastomes were relatively conservative in structure, IR/SC borders, codon usage, and repeat sequence. In phylogenetic results, the inter-subgenera relationships among A. subg. Rhizirideum and other genus Allium subgenera were generally similar to the previous reports. In contrast, the inter-section relationships within our subgenus A. subg. Rhizirideum were newly resolved in this study. A. sect. Rhizomatosa and A. sect. Tenuissima were sister branches, which were then clustered with A. sect. Rhizirideum and A. sect. Eduardia successively. However, Allium Polyrhizum Turcz. ex Regel, type species of A. sect. Caespitosoprason, was resolved as the basal taxon of A. subg. Rhizirideum. Allium siphonanthum J. M. Xu was also found in clade A. subg. Cyathophora instead of clade A. subg. Rhizirideum. The selective pressure analysis was also conducted, and most protein-coding genes were under purifying selection. At the same time, just one gene, ycf2, was found under positive selection, and another three genes (rbcL, ycf1a, ycf1b) presented relaxed selection, which were all involved in the photosynthesis. The low temperature, dry climate, and high altitude of the extreme habitats where A. subg. Rhizirideum species grow might impose intense natural selection forces on their plastome genes for photosynthesis. In summary, our research provides new insights into the phylogeny and adaptive evolution of A. subg. Rhizirideum. Moreover, we suggest that the positions of the A. subg. Rhizirideum species A. polyrhizum and A. siphonanthum should be reconsidered.

Plastid phylogenomic insights into relationships, divergence, and evolution of Apiales.

MAIN CONCLUSION: Members of Apiales are monophyletic and radiated in the Late Cretaceous. Fruit morphologies are critical for Apiales evolution and negative selection and mutation pressure play important roles in environmental adaptation. Apiales include many foods, spices, medicinal, and ornamental plants, but the phylogenetic relationships, origin and divergence, and adaptive evolution remain poorly understood. Here, we reconstructed Apiales phylogeny based on 72 plastid genes from 280 species plastid genomes representing six of seven families of this order. Highly supported phylogenetic relationships were detected, which revealed that each family of Apiales is monophyletic and confirmed that Pennanticeae is a member of Apiales. Genera Centella and Dickinsia are members of Apiaceae, and the genus Hydrocotyle previously classified into Apiaceae is confirmed to belong to Araliaceae. Besides, coalescent phylogenetic analysis and gene trees cluster revealed ten genes that can be used for distinguishing species among families of Apiales. Molecular dating suggested that the Apiales originated during the mid-Cretaceous (109.51 Ma), with the families' radiation occurring in the Late Cretaceous. Apiaceae species exhibit higher differentiation compared to other families. Ancestral trait reconstruction suggested that fruit morphological evolution may be related to shifts in plant types (herbaceous or woody), which in turn is related to the distribution areas and species numbers. Codon bias and positive selection analyses suggest that negative selection and mutation pressure may play important roles in environmental adaptation of Apiales members. Our results improve the phylogenetic framework of Apiales and provide insights into the origin, divergence, and adaptive evolution of this order and its members.

Comparative Analysis of the Complete Chloroplast Genomes in Allium Section Bromatorrhiza Species (Amaryllidaceae): Phylogenetic Relationship and Adaptive Evolution.

With the development of molecular sequencing approaches, many taxonomic and phylogenetic problems of the genus Allium L. have been solved; however, the phylogenetic relationships of some subgenera or sections, such as section Bromatorrhiza, remain unresolved, which has greatly impeded our full understanding of the species relationships among the major clades of Allium. In this study, the complete chloroplast (cp) genomes of nine species in the Allium sect. Bromatorrhiza were determined using the Illumina paired-end sequencing, the NOVOPlasty de novo assembly strategy, and the PGA annotation method. The results showed that the cp genome exhibited high conservation and revealed a typical circular tetrad structure. Among the sect. Bromatorrhiza species, the gene content, SSRs, codon usage, and RNA editing site were similar. The genome structure and IR regions' fluctuation were investigated while genes, CDSs, and non-coding regions were extracted for phylogeny reconstruction. Evolutionary rates (Ka/Ks values) were calculated, and positive selection analysis was further performed using the branch-site model. Five hypervariable regions were identified as candidate molecular markers for species authentication. A clear relationship among the sect. Bromatorrhiza species were detected based on concatenated genes and CDSs, respectively, which suggested that sect. Bromatorrhiza is monophyly. In addition, there were three genes with higher Ka/Ks values (rps2, ycf1, and ycf2), and four genes (rpoC2, atpF, atpI, and rpl14) were further revealed to own positive selected sites. These results provide new insights into the plastome component, phylogeny, and evolution of Allium species.

Molecular evolution and phylogenetic relationships of Ligusticum (Apiaceae) inferred from the whole plastome sequences.

BACKGROUND: The genus Ligusticum belongs to Apiaceae, and its taxonomy has long been a major difficulty. A robust phylogenetic tree is the basis of accurate taxonomic classification of Ligusticum. We herein used 26 (including 14 newly sequenced) plastome-scale data to generate reliable phylogenetic trees to explore the phylogenetic relationships of Chinese Ligusticum. RESULTS: We found that these plastid genomes exhibited diverse plastome characteristics across all four currently identified clades in China, while the plastid protein-coding genes were conserved. The phylogenetic analyses by the concatenation and coalescent methods obtained a more robust molecular phylogeny than prior studies and showed the non-monophyly of Chinese Ligusticum. In the concatenation-based phylogeny analyses, the two datasets yielded slightly different topologies that may be primarily due to the discrepancy in the number of variable sites. CONCLUSIONS: Our plastid phylogenomics analyses emphasized that the current circumscription of the Chinese Ligusticum should be reduced, and the taxonomy of Ligusticum urgently needs revision. Wider taxon sampling including the related species of Ligusticum will be necessary to explore the phylogenetic relationships of this genus. Overall, our study provided new insights into the taxonomic classification of Ligusticum and would serve as a framework for future studies on taxonomy and delimitation of Ligusticum from the perspective of the plastid genome.

Alliumheterophyllum (Amaryllidaceae), a new species from Henan, China.

Alliumheterophyllum D.F.Xie & X.J.He, sp. nov. (Amaryllidaceae), is a new species from Henan, China and is described based on morphological and molecular evidence. It is morphologically most similar to A.dumebuchum in the rhomboid scape in cross-section. However, distinctive differences were detected in perianth color, leaf shape and cross-section, flowers' density as well as flowering season. Similarly, the karyotype of A.heterophyllum is 2n = 2x = 16 and in A.dumebuchum is 2n = 4x = 32. Phylogenetic analysis based on nuclear ribosomal Internal Transcribed Spacers (ITS) and three cpDNA regions strongly supports that A.heterophyllum is a member of Allium section Rhizirideum and sister to the other species of this section (e.g. A.senescens, A.spirale, and A.prostratum). Currently, only one population and approximately 120 individuals were discovered; the development of scenic spots in this region may affect its growth and threaten this population. Therefore, this new species is preliminarily considered as Near Threatened (NT) according to criteria of the IUCN Red List.

Comparative Plastome Analysis of Three Amaryllidaceae Subfamilies: Insights into Variation of Genome Characteristics, Phylogeny, and Adaptive Evolution.

In the latest APG IV classification system, Amaryllidaceae is placed under the order of Asparagus and includes three subfamilies: Agapanthoideae, Allioideae, and Amaryllidoideae, which include many economically important crops. With the development of molecular phylogeny, research on the phylogenetic relationship of Amaryllidaceae has become more convenient. However, the current comparative analysis of Amaryllidaceae at the whole chloroplast genome level is still lacking. In this study, we sequenced 18 Allioideae plastomes and combined them with publicly available data (a total of 41 plastomes), including 21 Allioideae species, 1 Agapanthoideae species, 14 Amaryllidoideae species, and 5 Asparagaceae species. Comparative analyses were performed including basic characteristics of genome structure, codon usage, repeat elements, IR boundary, and genome divergence. Phylogenetic relationships were detected using single-copy genes (SCGs) and ribosomal internal transcribed spacer sequences (ITS), and the branch-site model was also employed to conduct the positive selection analysis. The results indicated that all Amaryllidaceae species showed a highly conserved typical tetrad structure. The GC content and five codon usage indexes in Allioideae species were lower than those in the other two subfamilies. Comparison analysis of Bayesian and ML phylogeny based on SCGs strongly supports the monophyly of three subfamilies and the sisterhood among them. Besides, positively selected genes (PSGs) were detected in each of the three subfamilies. Almost all genes with significant posterior probabilities for codon sites were associated with self-replication and photosynthesis. Our study investigated the three subfamilies of Amaryllidaceae at the whole chloroplast genome level and suggested the key role of selective pressure in the adaptation and evolution of Amaryllidaceae.

Plastid Phylogenomic Analyses Reveal the Taxonomic Position of Peucedanum franchetii.

Peucedanum franchetii is a famous folk medicinal plant in China. However, the taxonomy of the P. franchetii has not been sufficiently resolved. Due to similar morphological features between P. franchetii and Ligusticopsis members, the World Flora Online (WFO) Plant List suggested that this species transformed into the genus Ligusticopsis and merged with Ligusticopsis likiangensis. However, both species are obviously diverse in leaf shape, bracts, and bracteoles. To check the taxonomic position of P. franchetii, we newly sequenced and assembled the plastome of P. franchetii and compared it with nine other plastomes of the genus Ligusticopsis. Ten plastomes were highly conserved and similar in gene order, codon bias, RNA editing sites, IR borders, and SSRs. Nevertheless, 10 mutation hotspot regions (infA, rps8, matK, ndhF, rps15, psbA-trnH, rps2-rpoC2, psbA-trnK, ycf2-trnL, and ccsA-ndhD) were still detected. In addition, both phylogenetic analyses based on plastome data and ITS sequences robustly supported that P. franchetii was not clustered with members of Peucedanum but nested in Ligusticopsis. P. franchetii was sister to L. likiangensis in the ITS topology but clustered with L. capillacea in the plastome tree. These findings implied that P. franchetii should be transferred to genus Ligusticopsis and not merged with L. likiangensis, but as an independent species, which was further verified by morphological evidences. Therefore, transferring P. franchetii under the genus Ligusticopsis as an independent species was reasonable, and a new combination was presented.

Phylogeny and Comparative Analysis for the Plastid Genomes of Five Tulipa (Liliaceae).

Species of Tulipa (Liliaceae) are of great horticultural importance and are distributed across Europe, North Africa, and Asia. The Tien Shan Mountain is one of the primary diversity centres of Tulipa, but the molecular studies of Tulipa species from this location are lacking. In our study, we assembled four Tulipa plastid genomes from the Tien Shan Mountains, T. altaica, T. iliensis, T. patens, and T. thianschanica, combined with the plastid genome of T. sylvestris to compare against other Liliaceae plastid genomes. We focussed on the species diversity and evolution of their plastid genomes. The five Tulipa plastid genomes proved highly similar in overall size (151,691-152,088 bp), structure, gene order, and content. With comparative analysis, we chose 7 mononucleotide SSRs from the Tulipa species that could be used in further population studies. Phylogenetic analyses based on 24 plastid genomes robustly supported the monophyly of Tulipa and the sister relationship between Tulipa and Amana, Erythronium. T. iliensis, T. thianschanica, and T. altaica were clustered together, and T. patens was clustered with T. sylvestris, with our results clearly demonstrating the relationships between these five Tulipa species. Our results provide a more comprehensive understanding of the phylogenomics and comparative genomics of Tulipa.

A Combined Morphological and Molecular Evolutionary Analysis of Karst-Environment Adaptation for the Genus Urophysa (Ranunculaceae).

The karst environment is characterized by low soil water content, periodic water deficiency, and poor nutrient availability, which provides an ideal natural laboratory for studying the adaptive evolution of its inhabitants. However, how species adapt to such a special karst environment remains poorly understood. Here, transcriptome sequences of two Urophysa species (Urophysa rockii and Urophysa henryi), which are Chinese endemics with karst-specific distribution, and allied species in Semiaquilegia and Aquilegia (living in non-karst habitat) were collected. Single-copy genes (SCGs) were extracted to perform the phylogenetic analysis using concatenation and coalescent methods. Positively selected genes (PSGs) and clusters of paralogous genes (Mul_genes) were detected and subsequently used to conduct gene function annotation. We filtered 2,271 SCGs and the coalescent analysis revealed that 1,930 SCGs shared the same tree topology, which was consistent with the topology detected from the concatenated tree. Total of 335 PSGs and 243 Mul_genes were detected, and many were enriched in stress and stimulus resistance, transmembrane transport, cellular ion homeostasis, calcium ion transport, calcium signaling regulation, and water retention. Both molecular and morphological evidences indicated that Urophysa species evolved complex strategies for adapting to hostile karst environments. Our findings will contribute to a new understanding of genetic and phenotypic adaptive mechanisms of karst adaptation in plants.

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.

Comparative analysis of complete plastid genomes from Lilium lankongense Franchet and its closely related species and screening of Lilium-specific primers.

Lilium lankongense Franchet is a lily species found on the Qinghai-Tibet Plateau. It is pink with deep red spots, has a high ornamental value, and is used in hybrid breeding of horticultural lily varieties. We have insufficient knowledge of the genetic resources of L. lankongense and its phylogenetic relationships with related species. Recent molecular phylogenetic studies have shown a very close phylogenetic relationship between L. lankongense and the five species L. duchartrei, L. stewartianum, L. matangense, L. lophophorum, and L. nanum. However, molecular markers still lack sufficient signals for population-level research of the genus Lilium. We sequenced and compared the complete plastid sequences of L. lankongense and its five related species. The genomes ranged from 152,307 bp to 152,611 bp. There was a slight inconsistency detected in inverted repeat and single copy boundaries and there were 53 to 63 simple sequence repeats in the six species. Two of the 12 highly variable regions (trnC-petN and rpl32-trnL) were verified in 11 individuals and are promising for population-level studies. We used the complete sequence of 33 plastid genomes, the protein-coding region sequence, and the nuclear ITS sequence to reconstruct the phylogenetic tree of Lilium species. Our results showed that the plastid gene tree and nuclear gene tree were not completely congruent, which may be caused by hybridization, insufficient information contained in the nuclear ITS, or the small number of samples. The results of phylogenetic analysis based on plastid genomes indicated that the six Lilium species were closely related. Our study provides a preliminarily rebuilt backbone phylogeny that is significant for future molecular and morphological studies of Lilium.

Phylogeny, Age, and Evolution of Tribe Lilieae (Liliaceae) Based on Whole Plastid Genomes.

Tribe Lilieae, encompassing Lilium, Notholirion, Cardiocrinum, and Fritillaria, includes economically important crops with a horticultural and medicinal value. It is considered to be a core lineage of Liliaceae, but phylogenetic relationships within it, and the timing of the origin of individual clades, remain incompletely resolved. To address these issues, we reconstructed the evolutionary history of the tribe. We sequenced 45 Liliaceae plastomes and combined them with publicly available data (for a total of 139 plastomes) to explore the systematics, origin, divergence, and evolution of Lilieae. Our taxon sampling covers all ten sections of Lilium, all Cardiocrinum species, three Notholirion species, and major phylogenetic clades of Fritillaria. Our phylogenetic analysis confirms the monophyly of major sections/subgenera of Lilium and Fritillaria with strong support. We dated the origin of Lilieae to the Eocene, with genera and species radiations inferred to have occurred in the Miocene. The reconstruction of the ancestral area implies that Lilieae may have originated from the Qinghai-Tibet Plateau (QTP): the Himalayas and Hengduan Mountains and uplifting of the QTP likely promoted divergence within the tribe. Ancestral-state reconstructions of the bulb component number (including bulblets and scales) show a strong correlation with the genus-level phylogenetic diversity in Lilieae. They also predict that the most recent common ancestor of Lilieae had bulbs with numerous bulblets. Based on these observations, we predicted that climatic oscillations associated with the QTP uplift played an important role in the evolution of the Lilieae bulb. Our findings provide a well-supported picture of evolutionary relationships and a useful framework for understanding the pathway of bulb evolution within Lilieae, contributing to a better understanding of the evolutionary history of lilies.

Plastomes of eight Ligusticum species: characterization, genome evolution, and phylogenetic relationships.

BACKGROUND: The genus Ligusticum consists of approximately 60 species distributed in the Northern Hemisphere. It is one of the most taxonomically difficult taxa within Apiaceae, largely due to the varied morphological characteristics. To investigate the plastome evolution and phylogenetic relationships of Ligusticum, we determined the complete plastome sequences of eight Ligusticum species using a de novo assembly approach. RESULTS: Through a comprehensive comparative analysis, we found that the eight plastomes were similar in terms of repeat sequence, SSR, codon usage, and RNA editing site. However, compared with the other seven species, L. delavayi exhibited striking differences in genome size, gene number, IR/SC borders, and sequence identity. Most of the genes remained under the purifying selection, whereas four genes showed relaxed selection, namely ccsA, rpoA, ycf1, and ycf2. Non-monophyly of Ligusticum species was inferred from the plastomes and internal transcribed spacer (ITS) sequences phylogenetic analyses. CONCLUSION: The plastome tree and ITS tree produced incongruent tree topologies, which may be attributed to the hybridization and incomplete lineage sorting. Our study highlighted the advantage of plastome with mass informative sites in resolving phylogenetic relationships. Moreover, combined with the previous studies, we considered that the current taxonomy system of Ligusticum needs to be improved and revised. In summary, our study provides new insights into the plastome evolution, phylogeny, and taxonomy of Ligusticum species.

Adaptation Evolution and Phylogenetic Analyses of Species in Chinese Allium Section Pallasia and Related Species Based on Complete Chloroplast Genome Sequences.

The section Pallasia is one of the components of the genus Allium subgenus Allium (Amaryllidaceae), and species relationship in this section is still not resolved very well, which hinders further evolutionary and adaptive studies. Here, the complete chloroplast genomes of five sect. Pallasia species were reported, and a comparative analysis was performed with other three related Allium species. The genome size of the eight species ranged from 151,672 bp to 153,339 bp in length, GC content changed from 36.7% to 36.8%, and 130 genes (except Allium pallasii), 37 tRNA, and 8 rRNA were identified in each genome. By analyzing the IR/LSC and IR/SSC boundary, A. pallasii exhibited differences compared with other seven species. Phylogenetic analysis achieved high supports in each branch, seven of the eight Allium species cluster into a group, and A. pallasii exhibit a close relationship with A. obliquum. Higher pairwise Ka/Ks ratios were found in A. schoenoprasoides compared to A. caeruleum and A. macrostemon while a lower value of Ka/Ks ratios was detected between A. caeruleum and A. macrostemon. This study will be a great contribution to the future phylogenetic and adaptive research in Allium.

Comparative Analysis of the Complete Chloroplast Genomes in Allium Subgenus Cyathophora (Amaryllidaceae): Phylogenetic Relationship and Adaptive Evolution.

Recent advances in molecular phylogenetics provide us with information of Allium L. taxonomy and evolution, such as the subgenus Cyathophora, which is monophyletic and contains five species. However, previous studies detected distinct incongruence between the nrDNA and cpDNA phylogenies, and the interspecies relationships of this subgenus need to be furtherly resolved. In our study, we newly assembled the whole chloroplast genome of four species in subgenus Cyathophora and two allied Allium species. The complete cp genomes were found to possess a quadripartite structure, and the genome size ranged from 152,913 to 154,174 bp. Among these cp genomes, there were subtle differences in the gene order, gene content, and GC content. Seven hotspot regions (infA, rps16, rps15, ndhF, trnG-UCC, trnC-GCA, and trnK-UUU) with nucleotide diversity greater than 0.02 were discovered. The selection analysis showed that some genes have elevated Ka/Ks ratios. Phylogenetic analysis depended on the complete chloroplast genome (CCG), and the intergenic spacer regions (IGS) and coding DNA sequences (CDS) showed same topologies with high support, which revealed that subgenus Cyathophora was a monophyletic group, containing four species, and A. cyathophorum var. farreri was sister to A. spicatum with 100% bootstrap value. Our study revealed selective pressure may exert effect on several genes of the six Allium species, which may be useful for them to adapt to their specific living environment. We have well resolved the phylogenetic relationship of species in the subgenus Cyathophora, which will contribute to future evolutionary studies or phylogeographic analysis of Allium.

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.

Insights into phylogeny, age and evolution of Allium (Amaryllidaceae) based on the whole plastome sequences.

BACKGROUND AND AIMS: The genus Allium L., one of the largest monocotyledonous genera and one that includes many economically important crops with nutritional and medicinal value, has been the focus of classification or phylogeny studies for centuries. Recent studies suggested that the genus can be divided into 15 subgenera and 72 sections, which were further classified into three evolutionary lineages. However, the phylogenetic relationships reconstructed by one or two loci showed weaker support, especially for the third evolutionary lineage, which might not show the species relationships very clearly and could hinder further adaptive and evolutionary study. METHODS: In this study, a total of 39 complete chloroplast genomes of Allium (covering 12 Allium subgenera) were collected, and combining these with 125 species of plastomes from 19 other families of monocots, we reconstructed the phylogeny of the genus Allium, estimated the origin and divergence time of the three evolutionary lineages and investigated the adaptive evolution in this genus and related families. RESULTS: Our phylogenetic analysis confirmed the monophyly and three evolutionary lineages of Allium, while new species relationships were detected within the third evolutionary lineage. The divergence time of the three evolutionary lineages was estimated to be in the early Eocene to the middle Miocene, and numerous positive selected genes (PSGs) and PSGs with high average Ka/Ks values were found in Allium species. CONCLUSIONS: Our results detected a well-supported phylogenetic relationship of Allium. The PSGs and PSGs with high Ka/Ks values, as well as diversified morphologies, complicated chromosome characteristics and unique reproductive modes may play important roles in the adaptation and evolution of Allium species. This is the first study that conducted phylogenetic and evolutionary analyses on the genus Allium combined with the plastome and morphological and cytological data. We hope that this study can contribute to further analysis of Allium for other researchers.

Advances in Isolation and Culture of Chicken Embryonic Stem Cells In Vitro.

Chicken embryonic stem cells (cESCs) isolated from the egg at the stage X hold great promise for cell therapy, tissue engineering, pharmaceutical, and biotechnological applications. They are considered to be pluripotent cells with the capacity to self-renewal and differentiate into specialized cells. However, long-term maintenance of cESCs cannot be realized now, which impedes the establishment of cESC line and limits their applications. Therefore, the separation locations, isolation methods, and culture conditions especially the supplements and action mechanisms of cytokines, including leukemia inhibitory factor, fibroblast growth factor, transforming growth factor beta, bone morphogenic protein, and activin for cESCs in vitro, have been reviewed here. These defined strategies will contribute to identify the key mechanism on the self-renewal of cESCs, facilitate to optimize system that supports the derivation and longtime maintenance of cESCs, establish the cESC line, and develop the biobank of genetic resources in chicken.