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.

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.

Genetic diversity and population divergence of Leonurus japonicus and its distribution dynamic changes from the last interglacial to the present in China.

BACKGROUND: Leonurus japonicus, a significant medicinal plant known for its therapeutic effects on gynecological and cardiovascular diseases, has genetic diversity that forms the basis for germplasm preservation and utilization in medicine. Despite its economic value, limited research has focused on its genetic diversity and divergence. RESULTS: The avg. nucleotide diversity of 59 accessions from China were 0.00029 and hotspot regions in petN-psbM and rpl32-trnL(UAG) spacers, which can be used for genotype discrimination. These accessions divided into four clades with significant divergence. The four subclades, which split at approximately 7.36 Ma, were likely influenced by the Hengduan Mountains uplift and global temperature drop. The initial divergence gave rise to Clade D, with a crown age estimated at 4.27 Ma, followed by Clade C, with a crown age estimated at 3.39 Ma. The four clades were not showed a clear spatial distribution. Suitable climatic conditions for the species were identified, including warmest quarter precipitation 433.20 mm ~ 1,524.07 mm, driest month precipitation > 12.06 mm, and coldest month min temp > -4.34 °C. The high suitability distribution showed contraction in LIG to LGM, followed by expansion from LGM to present. The Hengduan Mountains acted as a glacial refuge for the species during climate changes. CONCLUSIONS: Our findings reflected a clear phylogenetic relationships and divergence within species L. japonicus and the identified hotspot regions could facilitate the genotype discrimination. The divergence time estimation and suitable area simulation revealed evolution dynamics of this species and may propose conservation suggestions and exploitation approaches in the future.

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.

Complete Chloroplast Genome of Hypericum perforatum and Dynamic Evolution in Hypericum (Hypericaceae).

Hypericum perforatum (St. John's Wort) is a medicinal plant from the Hypericaceae family. Here, we sequenced the whole chloroplast genome of H. perforatum and compared the genome variation among five Hypericum species to discover dynamic changes and elucidate the mechanisms that lead to genome rearrangements in the Hypericum chloroplast genomes. The H. perforatum chloroplast genome is 139,725 bp, exhibiting a circular quadripartite structure with two copies of inverted repeats (IRs) separating a large single-copy region and a small single-copy region. The H. perforatum chloroplast genome encodes 106 unique genes, including 73 protein-coding genes, 29 tRNAs, and 4 rRNAs. Hypericum chloroplast genomes exhibit genome rearrangement and significant variations among species. The genome size variation among the five Hypericum species was remarkably associated with the expansion or contraction of IR regions and gene losses. Three genes-trnK-UUU, infA, and rps16-were lost, and three genes-rps7, rpl23, and rpl32-were pseudogenized in Hypericum. All the Hypericum chloroplast genomes lost the two introns in clpP, the intron in rps12, and the second intron in ycf3. Hypericum chloroplast genomes contain many long repeat sequences, suggesting a role in facilitating rearrangements. Most genes, according to molecular evolution assessments, are under purifying selection.

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.

[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.

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.

Phylogenomics and biogeography of Catalpa (Bignoniaceae) reveal incomplete lineage sorting and three dispersal events.

Catalpa Scop. (Bignoniaceae) is a small genus (8 spp.) of trees that is disjunctly distributed among eastern Asia, eastern United States, and the West Indies. Catalpa bears beautiful inflorescences and have been cultivated as important ornamental trees for landscaping, gardening, and timber. However, the phylogenetic relationships and biogeographic history of the genus have remained unresolved. In this study, we used a large genomic dataset that includes data from the chloroplast (plastomes), and nuclear genomes (ITS and 5,759 single-copy nuclear genes) to reconstruct phylogenetic relationship within Catalpa, test interspecific gene flow events within the genus, and infer its biogeographic history. Our phylogenetic results indicate that Catalpa is monophyletic containing two main clades, section Catalpa and section Macrocatalpa. Section Catalpa is further divided into three subclades. While most relationships are congruent between the chloroplast and nuclear datasets, the position of C. ovata differs, likely due to incomplete lineage sorting. Interspecific gene flow events include C. bungei s.s. with vectors of inheritance from C. duclouxii and C. fargesii, supporting a combination of these three species and recognizing a broadly circumscribed C. bungei s.l. Our biogeographic study suggests three main dispersal events, two of which occurred during the Oligocene. The first dispersal event occurred from southwestern North America and Mexico into the Greater Antilles giving rise to the ancestor of the section of Macrocatalpa. The second dispersal event also occurred from southwestern North America and Mexico, but led to central and northern North America, subsequently reaching China through the Bering land bridge, and also reaching Europe through the North Atlantic land bridge. The third dispersal event took place in the Miocene from China to North America and gave rise to a clade composed of C. bignonioides and C. speciosa. This study uses a phylogenomic approach and biogeographical methods to infer the evolutionary history of Catalpa, highlighting issues associated with gene tree discordance, and suggesting that incomplete lineage sorting likely played an important role in the evolutionary history of Catalpa.

Mitogen-Activated Protein Kinases Associated Sites of Tobacco Repression of Shoot Growth Regulates Its Localization in Plant Cells.

Plant defense and growth rely on multiple transcriptional factors (TFs). Repression of shoot growth (RSG) is a TF belonging to a bZIP family in tobacco, known to be involved in plant gibberellin feedback regulation by inducing the expression of key genes. The tobacco calcium-dependent protein kinase CDPK1 was reported to interact with RSG and manipulate its intracellular localization by phosphorylating Ser-114 of RSG previously. Here, we identified tobacco mitogen-activated protein kinase 3 (NtMPK3) as an RSG-interacting protein kinase. Moreover, the mutation of the predicted MAPK-associated phosphorylation site of RSG (Thr-30, Ser-74, and Thr-135) significantly altered the intracellular localization of the NtMPK3-RSG interaction complex. Nuclear transport of RSG and its amino acid mutants (T30A and S74A) were observed after being treated with plant defense elicitor peptide flg22 within 5 min, and the two mutated RSG swiftly re-localized in tobacco cytoplasm within 30 min. In addition, triple-point mutation of RSG (T30A/S74A/T135A) mimics constant unphosphorylated status, and is predominantly localized in tobacco cytoplasm. RSG (T30A/S74A/T135A) showed no re-localization effect under the treatments of flg22, B. cereus AR156, or GA3, and over-expression of this mutant in tobacco resulted in lower expression levels of downstream gene GA20ox1. Our results suggest that MAPK-associated phosphorylation sites of RSG regulate its localization in tobacco, and that constant unphosphorylation of RSG in Thr-30, Ser-74, and Thr-135 keeps RSG predominantly localized in cytoplasm.

[Complete chloroplast genome of Ligustrum lucidum and highly variable marker identification for Ligustrum].

Ligustri Lucidi Fructus, the sun-dried mature fruit of Ligustrum lucidum, is cool, plain, sweet, and bitter, which can be used as both food and medicine, with the effects of improving vision, blacking hair, and tonifying liver and kidney. It takes effect slowly. However, little is known about the genetic information of the medicinal plant and it is still a challenge to distinguish Ligustrum species. In this study, the complete chloroplast genome of L. lucidum was obtained by genome skimming and then compared with that of five other Ligustrum species, which had been reported. This study aims to evaluate the interspecific variation of chloroplast genome within the genus and develop molecular markers for species identification of the genus. The result showed that the chloroplast genome of L. lucidum was 162 162 bp with a circular quadripartite structure of two single-copy regions separated by a pair of inverted repeats. The Ligustrum chloroplast genomes were conserved with small interspecific difference. Comparative analysis of six Ligustrum chloroplast genomes revealed three variable regions(rbcL-accD, ycf1a, and ycf1b), and ycf1a and ycf1b can be used as the species-specific DNA barcode for Ligustrum. Phylogeny analysis provided the best resolution of Ligustrum and supported that L. lucidum was sister to L. gracile. This study clarified the genetic diversity of L. lucidum from provenance, which can serve as a reference for further analysis of pharmacological differences and breeding of excellent varieties with stable drug effects.

Chloroplast phylogenomics and divergence times of Lagerstroemia (Lythraceae).

BACKGROUND: Crape myrtles, belonging to the genus Lagerstroemia L., have beautiful paniculate inflorescences and are cultivated as important ornamental tree species for landscaping and gardening. However, the phylogenetic relationships within Lagerstroemia have remained unresolved likely caused by limited sampling and the insufficient number of informative sites used in previous studies. RESULTS: In this study, we sequenced 20 Lagerstroemia chloroplast genomes and combined with 15 existing chloroplast genomes from the genus to investigate the phylogenetic relationships and divergence times within Lagerstroemia. The phylogenetic results indicated that this genus is a monophyletic group containing four clades. Our dating analysis suggested that Lagerstroemia originated in the late Paleocene (~ 60 Ma) and started to diversify in the middle Miocene. The diversification of most species occurred during the Pleistocene. Four variable loci, trnD-trnY-trnE, rrn16-trnI, ndhF-rpl32-trnL and ycf1, were discovered in the Lagerstroemia chloroplast genomes. CONCLUSIONS: The chloroplast genome information was successfully utilized for molecular characterization of diverse crape myrtle samples. Our results are valuable for the global genetic diversity assessment, conservation and utilization of Lagerstroemia.

Chloroplast phylogenomic insights into the evolution of Distylium (Hamamelidaceae).

BACKGROUND: Most Distylium species are endangered. Distylium species mostly display homoplasy in their flowers and fruits, and are classified primarily based on leaf morphology. However, leaf size, shape, and serration vary tremendously making it difficult to use those characters to identify most species and a significant challenge to address the taxonomy of Distylium. To infer robust relationships and develop variable markers to identify Distylium species, we sequenced most of the Distylium species chloroplast genomes. RESULTS: The Distylium chloroplast genome size was 159,041-159,127 bp and encoded 80 protein-coding, 30 transfer RNAs, and 4 ribosomal RNA genes. There was a conserved gene order and a typical quadripartite structure. Phylogenomic analysis based on whole chloroplast genome sequences yielded a highly resolved phylogenetic tree and formed a monophyletic group containing four Distylium clades. A dating analysis suggested that Distylium originated in the Oligocene (34.39 Ma) and diversified within approximately 1 Ma. The evidence shows that Distylium is a rapidly radiating group. Four highly variable markers, matK-trnK, ndhC-trnV, ycf1, and trnT-trnL, and 74 polymorphic simple sequence repeats were discovered in the Distylium plastomes. CONCLUSIONS: The plastome sequences had sufficient polymorphic information to resolve phylogenetic relationships and identify Distylium species accurately.

Phylogenomic and evolutionary dynamics of inverted repeats across Angelica plastomes.

BACKGROUND: Angelica L. (family Apiaceae) is an economically important genus comprising ca. One hundred ten species. Angelica species are found on all continents of the Northern Hemisphere, and East Asia hosts the highest number of species. Morphological characters such as fruit anatomy, leaf morphology and subterranean structures of Angelica species show extreme diversity. Consequently, the taxonomic classification of Angelica species is complex and remains controversial, as the classifications proposed by previous studies based on morphological data and molecular data are highly discordant. In addition, the phylogenetic relationships of major clades in the Angelica group, particularly in the Angelica s. s. clade, remain unclear. Chloroplast (cp) genome sequences have been widely used in phylogenetic studies and for evaluating genetic diversity. RESULTS: In this study, we sequenced and assembled 28 complete cp genomes from 22 species, two varieties and two cultivars of Angelica. Combined with 36 available cp genomes in GenBank from representative clades of the subfamily Apioideae, the characteristics and evolutionary patterns of Angelica cp genomes were studied, and the phylogenetic relationships of Angelica species were resolved. The Angelica cp genomes had the typical quadripartite structure including a pair of inverted repeats (IRs: 5836-34,706 bp) separated by a large single-copy region (LSC: 76,657-103,161 bp) and a small single-copy region (SSC: 17,433-21,794 bp). Extensive expansion and contraction of the IR region were observed among cp genomes of Angelica species, and the pattern of the diversification of cp genomes showed high consistency with the phylogenetic placement of Angelica species. Species of Angelica were grouped into two major clades, with most species grouped in the Angelica group and A. omeiensis and A. sinensis grouped in the Sinodielsia with Ligusticum tenuissimum. CONCLUSIONS: Our results further demonstrate the power of plastid phylogenomics in enhancing the phylogenetic reconstructions of complex genera and provide new insights into plastome evolution across Angelica L.

Evolutionary directions of single nucleotide substitutions and structural mutations in the chloroplast genomes of the family Calycanthaceae.

BACKGROUND: Chloroplast genome sequence data is very useful in studying/addressing the phylogeny of plants at various taxonomic ranks. However, there are no empirical observations on the patterns, directions, and mutation rates, which are the key topics in chloroplast genome evolution. In this study, we used Calycanthaceae as a model to investigate the evolutionary patterns, directions and rates of both nucleotide substitutions and structural mutations at different taxonomic ranks. RESULTS: There were 2861 polymorphic nucleotide sites on the five chloroplast genomes, and 98% of polymorphic sites were biallelic. There was a single-nucleotide substitution bias in chloroplast genomes. A â†’ T or T â†’ A (2.84%) and G â†’ C or C â†’ G (3.65%) were found to occur significantly less frequently than the other four transversion mutation types. Synonymous mutations kept balanced pace with nonsynonymous mutations, whereas biased directions appeared between transition and transversion mutations and among transversion mutations. Of the structural mutations, indels and repeats had obvious directions, but microsatellites and inversions were non-directional. Structural mutations increased the single nucleotide mutations rates. The mutation rates per site per year were estimated to be 0.14-0.34 × 10- 9 for nucleotide substitution at different taxonomic ranks, 0.64 × 10- 11 for indels and 1.0 × 10- 11 for repeats. CONCLUSIONS: Our direct counts of chloroplast genome evolution events provide raw data for correctly modeling the evolution of sequence data for phylogenetic inferences.

Interspecific chloroplast genome sequence diversity and genomic resources in Diospyros.

BACKGROUND: Fruits of persimmon plants are traditional healthy food in China, Korea and Japan. However, due to the shortage of morphological and DNA markers, the development of persimmon industry has been heavily inhibited. RESULTS: Chloroplast genomes of Diospyros cathayensis, D. virginiana, D. rhombifolia and D. deyangensis were newly sequenced. Comparative analyses of ten chloroplast genomes including six previously published chloroplast genomes of Diospyros provided new insights into the genome sequence diversity and genomic resources of the genus. Eight hyper-variable regions, trnH-psbA, rps16-trnQ, rpoB-trnC, rps4-trnT-trnL, ndhF, ndhF-rpl32-trnL, ycf1a, and ycf1b, were discovered and can be used as chloroplast DNA markers at/above species levels. The complete chloroplast genome sequences provided the best resolution at inter-specific level in comparison with different chloroplast DNA sequence datasets. CONCLUSION: Diospyros oleifera, D. deyangensis, D. virginiana, D. glaucifolia, D. lotus and D. jinzaoshi are important wild species closely related to the cultivated persimmon D. kaki. The hyper-variable regions can be used as DNA markers for global genetic diversity detection of Diospyros. Deeper study on these taxa would be helpful for elucidating the origin of D. kaki.

Resolving the systematic positions of enigmatic taxa: Manipulating the chloroplast genome data of Saxifragales.

Accurately resolving the phylogeny of enigmatic taxa is always a challenge in phylogenetic inference. Such uncertainties could be due to systematic errors or model violations. Here, we provide an example demonstrating how these factors affect the positioning of Paeoniaceae within Saxifragales based on chloroplast genome data. We newly assembled 14 chloroplast genomes from Saxifragales, and by combining these genomes with those of 63 other angiosperms, three datasets were assembled to test different hypotheses proposed by recent studies. These datasets were subjected to maximum parsimony, maximum likelihood and Bayesian analyses with site-homogeneous/heterogeneous models, different data partitioning strategies, and the inclusion/exclusion of weak phylogenetic signals. Three datasets exhibited remarkable heterogeneity among sites and among taxa of Saxifragales. Phylogenetic analyses under homogeneous models or maximum parsimony showed a closer relationship of Paeoniaceae with herbaceous families in the order. Data partitioning strategies did not change the general tree topology. However, PhyloBayes analysis under the CAT+GTR model resulted in a relationship closer to woody families. We conclude that although genomic data significantly increase the phylogenetic resolution of enigmatic taxa with high support, the phylogenetic results inferred from such data might be analysis or signal dependent. The analytical pipeline outlined here combines phylogenomic inference methods with evaluation of lineage-specific rates of substitution, model selection, and assessment of systematic error. These methods would be applicable to resolve similar difficult questions in the tree of life.

Next Generation Sequencing-Based Molecular Marker Development: A Case Study in Betula Alnoides.

Betula alnoides is a fast-growing valuable indigenous tree species with multiple uses in the tropical and warm subtropical regions in South-East Asia and southern China. It has been proved to be tetraploid in most parts of its distribution in China. In the present study, next generation sequencing (NGS) technology was applied to develop numerous SSR markers for B. alnoides, and 64,376 contig sequences of 106,452 clean reads containing 164,357 candidate SSR loci were obtained. Among the derived SSR repeats, mono-nucleotide was the main type (77.05%), followed by di- (10.18%), tetra- (6.12%), tri- (3.56%), penta- (2.14%) and hexa-nucleotide (0.95%). The short nucleotide sequence repeats accounted for 90.79%. Among the 291 repeat motifs, AG/CT (46.33%) and AT/AT (44.15%) were the most common di-nucleotide repeats, while AAT/ATT (48.98%) was the most common tri-nucleotide repeats. A total of 2549 primer sets were designed from the identified putative SSR regions of which 900 were randomly selected for evaluation of amplification successfulness and detection of polymorphism if amplified successfully. Three hundred and ten polymorphic markers were obtained through testing with 24 individuals from B. alnoides natural forest in Jingxi County, Guangxi, China. The number of alleles (NA) of each marker ranged from 2 to 19 with a mean of 5.14. The observed (HO) and expected (HE) heterozygosities varied from 0.04 to 1.00 and 0.04 to 0.92 with their means being 0.64 and 0.57, respectively. Shannon-Wiener diversity index (I) ranged from 0.10 to 2.68 with a mean of 1.12. Cross-species transferability was further examined for 96 pairs of SSR primers randomly selected, and it was found that 48.96⁻84.38% of the primer pairs could successfully amplify each of six related Betula species. The obtained SSR markers can be used to study population genetics and molecular marker assisted breeding, particularly genome-wide association study of these species in the future.

A chloroplast genomic strategy for designing taxon specific DNA mini-barcodes: a case study on ginsengs.

BACKGROUND: Universal conventional DNA barcodes will become more and more popular in biological material identifications. However, in many cases such as processed medicines or canned food, the universal conventional barcodes are unnecessary and/or inapplicable due to DNA degradation. DNA mini-barcode is a solution for such specific purposes. Here we exemplify how to develop the best mini-barcodes for specific taxa using the ginseng genus (Panax) as an example. RESULTS: The chloroplast genome of P. notoginseng was sequenced. The genome was compared with that of P. ginseng. Regions of the highest variability were sought out. The shortest lengths which had the same discrimination powers of conventional lengths were considered the best mini-barcodes. The results showed that the chloroplast genome of P. notoginseng is 156,387 bp. There are only 464 (0.30%) substitutions between the two genomes. The intron of rps16 and two regions of the coding gene ycf1, ycf1a and ycf1b, evolved the quickest and served as candidate regions. The mini-barcodes of Panax turned out to be 60 bp for ycf1a at a discrimination power of 91.67%, 100 bp for ycf1b at 100%, and 280 bp for rps16 at 83.33%. CONCLUSIONS: The strategy by searching the whole chloroplast genomes, identifying the most variable regions, shortening the focal regions for mini-barcodes are believed to be efficient in developing taxon-specific DNA mini-barcodes. The best DNA mini-barcodes are guaranteed to be found following this strategy.

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.