Key innovations and niche variation promoted rapid diversification of the widespread Juniperus (Cupressaceae).

The processes of forming lineages undergoing widespread radiations remain a knowledge gap that is fundamental to our understanding of the geographic distributions of species. Although early studies emphasized the importance of dispersal ability and historical migration events, key innovations that promote rapid diversification and/or adaptation to new habitats may also strongly influence distribution ranges. Juniperus is the second largest genus of conifers and is widely distributed throughout the Northern Hemisphere. Here, we used phylogenetic, phenotypic, and climatic data to investigate the contributions of these processes to the wide distribution and rapid diversification of Juniperus. Combining a time-scaled phylogeny and macroevolutionary theory, we show that the key innovations of berry-like seed cones and dioecy promoted the rapid diversification of Juniperus and that increased dispersal ability promoted allopatric speciation. Ecological niches had significant divergence among different clades of Juniperus. Biogeographic results supported multiple long-distance dispersal events and niche variation that contributed to the modern range of Juniperus, while both phenotypic adaptation and ecological opportunity probably drove its distribution range. Our findings suggest that the current widespread distribution is likely the result of significant divergence driven by niche variation in which ecological opportunities from key innovation and phenotypic divergence.

Chloroplast Genomes Evolution and Phylogenetic Relationships of Caragana species.

Caragana sensu lato (s.l.) includes approximately 100 species that are mainly distributed in arid and semi-arid regions. Caragana species are ecologically valuable for their roles in windbreaking and sand fixation. However, the taxonomy and phylogenetic relationships of the genus Caragana are still unclear. In this study, we sequenced and assembled the chloroplast genomes of representative species of Caragana and reconstructed robust phylogenetic relationships at the section level. The Caragana chloroplast genome has lost the inverted repeat region and wascategorized in the inverted repeat loss clade (IRLC). The chloroplast genomes of the eight species ranged from 128,458 bp to 135,401 bp and contained 110 unique genes. All the Caragana chloroplast genomes have a highly conserved structure and gene order. The number of long repeats and simple sequence repeats (SSRs) showed significant variation among the eight species, indicating heterogeneous evolution in Caragana. Selective pressure analysis of the genes revealed that most of the protein-coding genes evolved under purifying selection. The phylogenetic analyses indicated that each section forms a clade, except the section Spinosae, which was divided into two clades. This study elucidated the evolution of the chloroplast genome within the widely distributed genus Caragana. The detailed information obtained from this study can serve as a valuable resource for understanding the molecular dynamics and phylogenetic relationships within Caragana.

Historical climate change and vicariance events contributed to the intercontinental disjunct distribution pattern of ash species (Fraxinus, Oleaceae).

The Northern Hemisphere temperate forests exhibit a disjunct distributional pattern in Europe, North America, and East Asia. Here, to reveal the promoter of intercontinental disjunct distribution, Fraxinus was used as a model organism to integrate abundant fossil evidence with high-resolution phylogenies in a phytogeographic analysis. We constructed a robust phylogenetic tree using genomic data, reconstructed the geographic ancestral areas, and evaluated the effect of incorporating fossil information on the reconstructed biogeographic history. The phylogenetic relationships of Fraxinus were highly resolved and divided into seven clades. Fraxinus originated in western North America during Eocene, and six intercontinental dispersal events and five intercontinental vicariance events were occured. Results suggest that climate change and vicariance contributed to the intercontinental disjunct distribution pattern of Fraxinus. Moreover, results highlight the necessity of integrating phylogenetic relationship and fossil to improve the reliability of inferred biogeographic events and our understanding of the processes underlying disjunct distributions.

[Species identification of Ligustrum lucidum].

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.

Maternal Donor and Genetic Variation of Lagerstroemia indica Cultivars.

Lagerstroemia indica L. is a well-known ornamental plant with large pyramidal racemes, long flower duration, and diverse colors and cultivars. It has been cultivated for nearly 1600 years and is essential for investigating the germplasm and assessing genetic variation to support international cultivar identification and breeding programs. In this study, 20 common Lagerstroemia indica cultivars from different varietal groups and flower morphologies, as well as multiple wild relative species, were analyzed to investigate the maternal donor of Lagerstroemia indica cultivars and to discover the genetic variation and relationships among cultivars based on plastome and nuclear ribosomal DNA (nrDNA) sequences. A total of 47 single nucleotide polymorphisms (SNPs) and 24 insertion/deletions (indels) were identified in the 20 L. indica cultivars' plastome and 25 SNPs were identified in the nrDNA. Phylogenetic analysis based on the plastome sequences showed that all the cultivars formed a clade with the species of L. indica, indicating that L. indica was the maternal donor of the cultivars. Population structure and PCA analyses supported two clades of cultivars, which exhibited significant genetic differences according to the plastome dataset. The results of the nrDNA supported that all 20 cultivars were divided into three clades and most of the cultivars had at least two genetic backgrounds and higher gene flow. Our results suggest that the plastome and nrDNA sequences can be used as molecular markers for assessing the genetic variation and relationships of L. indica cultivars.

Insights into the phylogeny and chloroplast genome evolution of Eriocaulon (Eriocaulaceae).

BACKGROUND: Eriocaulon is a wetland plant genus with important ecological value, and one of the famous taxonomically challenging groups among angiosperms, mainly due to the high intraspecific diversity and low interspecific variation in the morphological characters of species within this genus. In this study, 22 samples representing 15 Eriocaulon species from China, were sequenced and combined with published samples of Eriocaulon to test the phylogenetic resolution using the complete chloroplast genome. Furthermore, comparative analyses of the chloroplast genomes were performed to investigate the chloroplast genome evolution of Eriocaulon. RESULTS: The 22 Eriocaulon chloroplast genomes and the nine published samples were proved highly similar in genome size, gene content, and order. The Eriocaulon chloroplast genomes exhibited typical quadripartite structures with lengths from 150,222 bp to 151,584 bp. Comparative analyses revealed that four mutation hotspot regions (psbK-trnS, trnE-trnT, ndhF-rpl32, and ycf1) could serve as effective molecular markers for further phylogenetic analyses and species identification of Eriocaulon species. Phylogenetic results supported Eriocaulon as a monophyletic group. The identified relationships supported the taxonomic treatment of section Heterochiton and Leucantherae, and the section Heterochiton was the first divergent group. Phylogenetic tree supported Eriocaulon was divided into five clades. The divergence times indicated that all the sections diverged in the later Miocene and most of the extant Eriocaulon species diverged in the Quaternary. The phylogeny and divergence times supported rapid radiation occurred in the evolution history of Eriocaulon. CONCLUSION: Our study mostly supported the taxonomic treatment at the section level for Eriocaulon species in China and demonstrated the power of phylogenetic resolution using whole chloroplast genome sequences. Comparative analyses of the Eriocaulon chloroplast genome developed molecular markers that can help us better identify and understand the evolutionary history of Eriocaulon species in the future.

Dynamic evolution of the plastome in the Elm family (Ulmaceae).

MAIN CONCLUSION: This study compared the plastomes of Ulmaceae allowing analyses of the dynamic evolution, including genome structure, codon usage bias, repeat sequences, molecular mutation rates, and phylogenetic inferences. Ulmaceae is a small family in the order Rosales. This family consists of seven genera, including Ulmus, Zelkova, Planera, Hemiptelea, Phyllostylon, Ampelocera, and Holoptelea. Ulmaceae is an interesting lineage from plant biogeographic, systematic, evolutionary, and paleobotanic perspectives. It is also a good model to investigate the evolution of the plastomes in woody plants. In this study, we sequenced and assembled the complete plastomes of the six Ulmaceae genera to compare genomic structures and reveal the molecular evolutionary patterns. The size of the quadripartite plastomes ranged from 158,290 bp to 161,886 bp. The genomes contained 131 genes, including 87 coding genes, 36 tRNA, and 8 rRNA. The gene number, gene content, and genomic structure were highly consistent among the Ulmaceae genera. Nine variable regions including ndhA intron, ndhF-rpl32, ycf1, psbK-trnS, rps16-trnQ, trnT-trnL, trnT-psbD, trnS-trnG, and rpl32-trnL, were identified in Ulmaceae plastomes according to the nucleotide diversity values. Condon usage was biased among the genes and showed consistent trends in the seven genera. Molecular evolution analyses revealed that most of the genes and all gene groups were under widespread purifying selection. Twelve genes (ccsA, matK, psbH, psbK, rbcL, rpl22, rpl32, rpoA, rps12, rps15, rps16, and ycf2) were under positive selection. Phylogenetic analyses supported that Ulmaceae should be divided into two main clades, such as the temperate clade, including Ulmus, Zelkova, Planera, and Hemiptelea and the tropical clade, including Phyllostylon, Ampelocera and Holoptelea. This study reports the structure and evolutionary characteristics of the Elm family. These new genomic data will benefit assessments of genomic evolution and provide information to elucidate the phylogenetic relationships among Ulmaceae species.

Characterization of the Dicranostigma leptopodum chloroplast genome and comparative analysis within subfamily Papaveroideae.

BACKGROUND: Dicranostigma leptopodum (Maxim.) Fedde is a perennial herb with bright yellow flowers, well known as "Hongmao Cao" for its medicinal properties, and is an excellent early spring flower used in urban greening. However, its molecular genomic information remains largely unknown. Here, we sequenced and analyzed the chloroplast genome of D. leptopodum to discover its genome structure, organization, and phylogenomic position within the subfamily Papaveroideae. RESULTS: The chloroplast genome size of D. leptopodum was 162,942 bp, and D. leptopodum exhibited a characteristic circular quadripartite structure, with a large single-copy (LSC) region (87,565 bp), a small single-copy (SSC) region (18,759 bp) and a pair of inverted repeat (IR) regions (28,309 bp). The D. leptopodum chloroplast genome encoded 113 genes, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The dynamics of the genome structures, genes, IR contraction and expansion, long repeats, and single sequence repeats exhibited similarities, with slight differences observed among the eight Papaveroideae species. In addition, seven interspace regions and three coding genes displayed highly variable divergence, signifying their potential to serve as molecular markers for phylogenetic and species identification studies. Molecular evolution analyses indicated that most of the genes were undergoing purifying selection. Phylogenetic analyses revealed that D. leptopodum formed a clade with the tribe Chelidonieae. CONCLUSIONS: Our study provides detailed information on the D. leptopodum chloroplast genome, expanding the available genomic resources that may be used for future evolution and genetic diversity studies.

Phylogenomics and Genetic Diversity of Arnebiae Radix and Its Allies (Arnebia, Boraginaceae) in China.

Arnebiae Radix is a traditional medicine with pleiotropic properties that has been used for several 100 years. There are five species of Arnebia in China, and the two species Arnebia euchroma and Arnebia guttata are the source plants of Arnebiae Radix according to the Chinese Pharmacopoeia. Molecular markers that permit species identification and facilitate studies of the genetic diversity and divergence of the wild populations of these two source plants have not yet been developed. Here, we sequenced the chloroplast genomes of 56 samples of five Arnebia species using genome skimming methods. The Arnebia chloroplast genomes exhibited quadripartite structures with lengths from 149,539 and 152,040 bp. Three variable markers (rps16-trnQ, ndhF-rpl32, and ycf1b) were identified, and these markers exhibited more variable sites than universal chloroplast markers. The phylogenetic relationships among the five Arnebia species were completely resolved using the whole chloroplast genome sequences. Arnebia arose during the Oligocene and diversified in the middle Miocene; this coincided with two geological events during the late Oligocene and early Miocene: warming and the progressive uplift of Tianshan and the Himalayas. Our analyses revealed that A. euchroma and A. guttata have high levels of genetic diversity and comprise two and three subclades, respectively. The two clades of A. euchroma exhibited significant genetic differences and diverged at 10.18 Ma in the middle Miocene. Three clades of A. guttata diverged in the Pleistocene. The results provided new insight into evolutionary history of Arnebia species and promoted the conservation and exploitation of A. euchroma and A. guttata.

Phylogenomic approaches untangle early divergences and complex diversifications of the olive plant family.

BACKGROUND: Deep-branching phylogenetic relationships are often difficult to resolve because phylogenetic signals are obscured by the long history and complexity of evolutionary processes, such as ancient introgression/hybridization, polyploidization, and incomplete lineage sorting (ILS). Phylogenomics has been effective in providing information for resolving both deep- and shallow-scale relationships across all branches of the tree of life. The olive family (Oleaceae) is composed of 25 genera classified into five tribes with tribe Oleeae consisting of four subtribes. Previous phylogenetic analyses showed that ILS and/or hybridization led to phylogenetic incongruence in the family. It was essential to distinguish phylogenetic signal conflicts, and explore mechanisms for the uncertainties concerning relationships of the olive family, especially at the deep-branching nodes. RESULTS: We used the whole plastid genome and nuclear single nucleotide polymorphism (SNP) data to infer the phylogenetic relationships and to assess the variation and rates among the main clades of the olive family. We also used 2608 and 1865 orthologous nuclear genes to infer the deep-branching relationships among tribes of Oleaceae and subtribes of tribe Oleeae, respectively. Concatenated and coalescence trees based on the plastid genome, nuclear SNPs and multiple nuclear genes suggest events of ILS and/or ancient introgression during the diversification of Oleaceae. Additionally, there was extreme heterogeneity in the substitution rates across the tribes. Furthermore, our results supported that introgression/hybridization, rather than ILS, is the main factor for phylogenetic discordance among the five tribes of Oleaceae. The tribe Oleeae is supported to have originated via ancient hybridization and polyploidy, and its most likely parentages are the ancestral lineage of Jasmineae or its sister group, which is a "ghost lineage," and Forsythieae. However, ILS and ancient introgression are mainly responsible for the phylogenetic discordance among the four subtribes of tribe Oleeae. CONCLUSIONS: This study showcases that using multiple sequence datasets (plastid genomes, nuclear SNPs and thousands of nuclear genes) and diverse phylogenomic methods such as data partition, heterogeneous models, quantifying introgression via branch lengths (QuIBL) analysis, and species network analysis can facilitate untangling long and complex evolutionary processes of ancient introgression, paleopolyploidization, and ILS.

Phylogenomics and Biogeography of Populus Based on Comprehensive Sampling Reveal Deep-Level Relationships and Multiple Intercontinental Dispersals.

Populus not only has significant economic and ecological values, but also serves as a model tree that is widely used in the basic research of tree growth, physiology, and genetics. However, high levels of morphological variation and extensive interspecific hybridization of Populus pose an obstacle for taxonomy, and also to the understanding of phylogenetic interspecific relationships and biogeographical history. In this study, a total of 103 accessions representing almost all the wild species of Populus were collected and whole-genome re-sequenced to examine the phylogenetic relationships and biogeography history. On the basis of 12,916,788 nuclear single nucleotide polymorphisms (SNPs), we reconstructed backbone phylogenies using concatenate and coalescent methods, we highly disentangled the species relationships of Populus, and several problematic taxa were treated as species complexes. Furthermore, the phylogeny of the chloroplast genome showed extensive discordance with the trees from the nuclear genome data, and due to extensive chloroplast capture and hybridization of Populus species, plastomes could not accurately evaluate interspecies relationships. Ancient gene flow between clades and some hybridization events were also identified by ABBA-BABA analysis. The reconstruction of chronogram and ancestral distributions suggested that North America was the original region of this genus, and subsequent long dispersal and migration across land bridges were contributed to the modern range of Populus. The diversification of Populus mainly occurred in East Asia in recent 15 Ma, possibly promoted by the uplift of the Tibetan Plateau. This study provided comprehensive evidence on the phylogeny of Populus and proposed a four-subgeneric classification and a new status, subgenus Abaso. Meanwhile, ancestral distribution reconstruction with nuclear data advanced the understanding of the biogeographic history of Populus.

Complete chloroplast genome sequence of Salix sinopurpurea (Salicaceae).

Salix sinopurpurea is a morphologically special shrubby willow characterizing opposite leaves. Here, we reported the complete chloroplast (cp) genome sequence of Salix sinopurpurea. The cp genome is 155,546 bp in length, including a large single-copy (LSC) region of 84,412 bp, a small single-copy (SSC) region of 16,216 bp, and a pair of inverted repeated regions of 27,459 bp. The cp genome of Salix sinopurpurea encodes 130 genes, including 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic tree showed that Salix sinopurpurea is closely related to Salix psammophila and Salix suchowensis.

Soluble Expression, One-Step Purification and Characterization of Recombinant Human Growth Hormone Fused with ompA3 in Escherichia coli.

BACKGROUND: Human growth hormone (hGH) is the first recombinant protein approved for the treatment of human growth hormone deficiency. However, expression in inclusion bodies and low expression levels are enormous challenges for heterologous expression of hGH in Escherichia coli. OBJECTIVE: To increase the soluble expression of recombinant hGH with correct folding in E. coli. METHODS: We constructed a new recombinant expression plasmid containing the coding sequence of the outer membrane protein A (ompA3) which was used for the expression in Transetta (DE3) E. coli. In order to simplify the purification process and cleavage of recombinant proteins, the fusion sequence should contain hexahistidine-tag (His6) and enterokinase recognition sites (D4K). The effect of different expression conditions on recombinant hGH expression was optimized in flask cultivations. Furthermore, the periplasmic solution containing soluble hGH was purified by Ni-NTA affinity chromatography. Circular dichroism (CD), western blot and mass spectrometry analyses were used to characterize the protein. Moreover, the growth-promoting effect of the purified hGH was also evaluated by cell proliferation assay. RESULTS: High-level expression (800 µg/mL) was achieved by induction with 0.5 mM IPTG at 30°C for 10 hours. The purity of hGH was over 90%. The immunological activity, secondary structure and molecular weight of the purified hGH were consistent with native hGH. The purified hGH was found to promote the growth of MC3T3-E1 cells, and was found to show the highest activity at a concentration of 100 ng/mL. CONCLUSION: Our research provides a feasible and convenient method for the soluble expression of recombinant hGH in E. coli, and may lay a foundation for the production and application of hGH in the industry.

Strigolactones: a plant phytohormone as novel anti-inflammatory agents.

Strigolactones (SLs) are a novel class of plant hormones with enormous potential for the prevention and treatment of inflammation. To further investigate the anti-inflammatory activities of SLs, a representative SL, GR24, and the reductive products of its D-ring were synthesized and their anti-inflammatory activities were fully evaluated on both in vitro and in vivo models. Among these compounds, the two most active optical isomers (2a and 6a) demonstrated strong inhibitory activity on the release of inflammatory cytokines, including nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) by blocking the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways; they also greatly inhibited the migration of neutrophils and macrophages in fluorescent protein labeled zebrafish larvae. These results identified the promising anti-inflammatory effects of SLs, and suggested that both the absolute configuration of SL and the α,ß-unsaturated D-ring structure are essential for the observed anti-inflammatory activity.