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.

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.

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.

Phylogeny and highland adaptation of Chinese species in Allium section Daghestanica (Amaryllidaceae) revealed by transcriptome sequencing.

Allium L. is one of the largest monocotyledonous genera with extensive distribution in the Northern Hemisphere. The fundamental phylogenies of Allium have been investigated using many morphological and molecular characters. However, the morphological characters may not agree with the molecular results in some Allium groups or sections (such as the Chinese Allium section Daghestanica), which may result in ambiguous species relationships and hinder further evolutionary and adaptive researches. Here, transcriptome sequences of the six Chinese endemics from Allium section Daghestanica were collected, with their single-copy genes (SCGs) were extracted. The interspecies relationships were analyzed using concatenation and coalescent methods. The branch-site model (BSM) was conducted to detect the positively selected genes (PSGs) in five highland species of this section. Based on 1644, 1281 and 1580 SCGs in flowers, leaves, and flowers-leaves combination respectively, a robust consistent and well-resolved phylogeny was generated from the concatenation method. Strong conflicts among individual gene trees were detected in the coalescent method, and morphological characters were incongruent with molecular relationships to some degree. Many PSGs were involved in responses of various stresses and stimuli (e.g. hypoxia, low temperature, aridity), DNA repair, metabolism, nutrient or energy intake, photosynthesis, and signal transduction. Our study revealed a clear interspecies relationship of Chinese endemics in Allium section Daghestanica and suggested that the discordance between morphological characters and molecular relationships might result from that the former are more susceptible to convergence compared with the latter. PSGs detected in our study may provide some insights into highland adaptation in Allium species.

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.

A transcriptome-based study on the phylogeny and evolution of the taxonomically controversial subfamily Apioideae (Apiaceae).

BACKGROUND AND AIMS: A long-standing controversy in the subfamily Apioideae concerns relationships among the major lineages, which has prevented a comprehensive study of their fruits and evolutionary history. Here we use single copy genes (SCGs) generated from transcriptome datasets to generate a reliable species tree and explore the evolutionary history of Apioideae. METHODS: In total, 3351 SCGs were generated from 27 transcriptome datasets and one genome, and further used for phylogenetic analysis using coalescent-based methods. Fruit morphology and anatomy were studied in combination with the species tree. Eleven SCGs were screened out for dating analysis with two fossils selected for calibration. KEY RESULTS: A well-supported species tree was generated with a topology [Chamaesieae, (Bupleureae, (Pleurospermeae, (Physospermopsis Clade, (Group C, (Group A, Group B)))))] that differed from previous trees. Daucinae and Torilidinae were not in the tribe Scandiceae and existed as sister groups to the Acronema Clade. Five branches (I-V) of the species tree showed low quartet support but strong local posterior probabilities. Dating analysis suggested that Apioideae originated around 56.64 Mya (95 % highest posterior density interval, 45.18-73.53 Mya). CONCLUSIONS: This study resolves a controversial phylogenetic relationship in Apioideae based on 3351 SCGs and coalescent-based species tree estimation methods. Gene trees that contributed to the species tree may undergoing rapid evolutionary divergence and incomplete lineage sorting. Fruits of Apioideae might have evolved in two directions, anemochorous and hydrochorous, with epizoochorous as a derived mode. Molecular and morphological evidence suggests that Daucinae and Torilidinae should be restored to the tribe level. Our results provide new insights into the morphological evolution of this subfamily, which may contribute to a better understanding of species diversification in Apioideae. Molecular dating analysis suggests that uplift of the Qinghai-Tibetan Plateau (QTP) and climate changes probably drove rapid speciation and diversification of Apioideae in the QTP region.

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.

Molecular phylogenetics and historical biogeography of the tribe Lilieae (Liliaceae): bi-directional dispersal between biodiversity hotspots in Eurasia.

Background and Aims: The role played by the Qinghai-Tibet Plateau (QTP) in the organismal diversification and biogeography of plants in the Northern Hemisphere has attracted much attention from evolutionary biologists. Here we use tribe Lilieae (Liliaceae), including primarily temperate and alpine lineages with disjunct distributions in the North Temperate Zone, as a case study to shed light upon these processes. Methods: Using 191 taxa (five outgroup taxa) comprising more than 60 % of extant Lilieae species across the entire geographical range, we analyse phylogenetic relationships based on three plastid markers (matK, rbcL, rpl16) and nuclear ITS. Divergence time estimation and ancestral range reconstruction were further inferred. Key Results: The results support a monophyletic Lilieae divided into four clades. Lilium is nested within Fritillaria, which is paraphyletic and partitioned into two clades, New World and Old World, in the chloroplast DNA (cpDNA) analysis. Incongruences between the ITS and cpDNA trees may be explained by divergent ITS paralogues and hybridization. Lilieae originated around 40-49 (28-67) Mya and probably diversified in the QTP region with four major clades that were established during the Oligocene and the Early Miocene. Uplift of the QTP and climatic changes probably drove early diversification of Lilieae in the QTP region. A rapid radiation occurred during the Late Miocene and the Pleistocene, coinciding temporally with recent orogenic process in the QTP region and climatic oscillations. Several lineages dispersed out of the QTP. Conclusions: Lineage persistence and explosive radiation were important processes for establishing high species diversity of Lilieae in the QTP region. Both long-distance dispersal and migration across Beringia probably contributed to the modern distribution range of Lilieae. Our study shows that biotic interchanges between the QTP region and Irano-Turanian region and the Mediterranean Basin were bi-directional, suggesting the latter was a secondary centre of diversity.

Comparative Analysis of the Chloroplast Genomes of the Chinese Endemic Genus Urophysa and Their Contribution to Chloroplast Phylogeny and Adaptive Evolution.

Urophysa is a Chinese endemic genus comprising two species, Urophysa rockii and Urophysa henryi. In this study, we sequenced the complete chloroplast (cp) genomes of these two species and of their relative Semiquilegia adoxoides. Illumina sequencing technology was used to compare sequences, elucidate the intra- and interspecies variations, and infer the phylogeny relationship with other Ranunculaceae family species. A typical quadripartite structure was detected, with a genome size from 158,473 to 158,512 bp, consisting of a pair of inverted repeats separated by a small single-copy region and a large single-copy region. We analyzed the nucleotide diversity and repeated sequences components and conducted a positive selection analysis by the codon-based substitution on single-copy coding sequence (CDS). Seven regions were found to possess relatively high nucleotide diversity, and numerous variable repeats and simple sequence repeats (SSR) markers were detected. Six single-copy genes (atpA, rpl20, psaA, atpB, ndhI, and rbcL) resulted to have high posterior probabilities of codon sites in the positive selection analysis, which means that the six genes may be under a great selection pressure. The visualization results of the six genes showed that the amino acid properties across each column of all species are variable in different genera. All these regions with high nucleotide diversity, abundant repeats, and under positive selection will provide potential plastid markers for further taxonomic, phylogenetic, and population genetics studies in Urophysa and its relatives. Phylogenetic analyses based on the 79 single-copy genes, the whole complete genome sequences, and all CDS sequences showed same topologies with high support, and U. rockii was closely clustered with U. henryi within the Urophysa genus, with S. adoxoides as their closest relative. Therefore, the complete cp genomes in Urophysa species provide interesting insights and valuable information that can be used to identify related species and reconstruct their phylogeny.

Revisiting the evolutionary events in Allium subgenus Cyathophora (Amaryllidaceae): Insights into the effect of the Hengduan Mountains Region (HMR) uplift and Quaternary climatic fluctuations to the environmental changes in the Qinghai-Tibet Plateau.

The respective roles that the Hengduan Mountains Region (HMR) uplift around 4-3 Ma and Quaternary climatic oscillations played in causing the environmental changes in the Qinghai-Tibet Plateau (QTP) remain unknown. Here, we reconstruct the evolutionary history of two varieties of Allium cyathophorum and A. spicatum of subgenus Cyathophora, restricted to the HMR and the western QTP, respectively. Forty-five populations were surveyed for chloroplast and nuclear sequence variation to evaluate phylogenetic relationships, dates of divergence and ancestral area/inflorescence reconstructions. In addition, analyses were conducted on discernable micromorphologies, cytotypes and seed size variation. Our results indicated that two varieties of A. cyathophorum are separate species, i.e. A. farreri and A. cyathophorum, and the initial split of Cyathophora was triggered by the HMR uplift around 4-3 Ma. Subsequently, A. spicatum originated through the strengthened aridification in the western QTP induced vicariance of the ancestral populations in the HMR during the early Pleistocene. A self-sustaining allotetraploid species from A. farreri and A. cyathophorum was established during an interglacial period of penultimate glaciation of the QTP. Seed size variation also supports these by the colonization-competition tradeoff among small and large seeds. Our findings appear to suggest that the HMR uplift could have strengthened the development of the Asian monsoon regimes in this region and aridification in the western QTP, while the Quaternary climatic oscillations spurred the allopatric species' range shifts and created new open microhabitat for the alloploid species.

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.

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.

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.

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.

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.

Chloroplast genomic comparison of two sister species Allium macranthum and A. fasciculatum provides valuable insights into adaptive evolution.

BACKGROUND: Allium macranthum and Allium fasciculatum are two sister species and their natural populations are separated by high mountains and deep valleys with exact opposite habitat. The chloroplast genome in angiosperms has showed useful for investigating plant evolution and systematic studies. OBJECTIVE: Comparative analysis of these genomes revealed potential markers and phylogenetic analysis, and discuss the influence of positive selected sites on adaptive evolution. METHODS: Here, we sequenced the complete chloroplast genomes of these two species and analyzed the repeat sequences components, nucleotide diversity, selection pressure and the phylogeny relationships with related species. RESULTS: A typical quadripartite structure was detected with a genome size changed from 152,148 to 152,931 bp. We identified 67 and 79 simple sequence repeats in A. macranthum and A. fasciculatum, in which the mono-nucleotide repeats A/T possess the highest percentage. Three mutational hotspots (rpl32, rps16 and matK) at the SSC and LSC regions were observed, which showed remarkably higher Pi value (> 0.03). Additionally, eight genes (rpoA, atpF, cemA, rps4, ccsA, rpoC2, rpl14 and clpP) exhibited elevated pairwise Ka/Ks ratios in alpine species. Phylogenetic analyses based on the CDS sequences and the whole complete genomes showed same topologies with high support, and A. macranthum was closely clustered with A. fasciculatum within the fourteen Amaryllidaceae species. CONCLUSION: Their coding proteins of these genes often functioned in chloroplast protein synthesis, gene transcription, energy transformation and regulation and photosynthesis. These results provide valuable insights into the alpine species adaptation and evolution.

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.