Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants.

BACKGROUND: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. RESULTS: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. CONCLUSIONS: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

Assembly and comparative analysis of the complete mitochondrial and chloroplast genome of Cyperus stoloniferus (Cyperaceae), a coastal plant possessing saline-alkali tolerance.

BACKGROUND: Cyperus stoloniferus is an important species in coastal ecosystems and possesses economic and ecological value. To elucidate the structural characteristics, variation, and evolution of the organelle genome of C. stoloniferus, we sequenced, assembled, and compared its mitochondrial and chloroplast genomes. RESULTS: We assembled the mitochondrial and chloroplast genomes of C. stoloniferus. The total length of the mitochondrial genome (mtDNA) was 927,413 bp, with a GC content of 40.59%. It consists of two circular DNAs, including 37 protein-coding genes (PCGs), 22 tRNAs, and five rRNAs. The length of the chloroplast genome (cpDNA) was 186,204 bp, containing 93 PCGs, 40 tRNAs, and 8 rRNAs. The mtDNA and cpDNA contained 81 and 129 tandem repeats, respectively, and 346 and 1,170 dispersed repeats, respectively, both of which have 270 simple sequence repeats. The third high-frequency codon (RSCU > 1) in the organellar genome tended to end at A or U, whereas the low-frequency codon (RSCU < 1) tended to end at G or C. The RNA editing sites of the PCGs were relatively few, with only 9 and 23 sites in the mtDNA and cpDNA, respectively. A total of 28 mitochondrial plastid DNAs (MTPTs) in the mtDNA were derived from cpDNA, including three complete trnT-GGU, trnH-GUG, and trnS-GCU. Phylogeny and collinearity indicated that the relationship between C. stoloniferus and C. rotundus are closest. The mitochondrial rns gene exhibited the greatest nucleotide variability, whereas the chloroplast gene with the greatest nucleotide variability was infA. Most PCGs in the organellar genome are negatively selected and highly evolutionarily conserved. Only six mitochondrial genes and two chloroplast genes exhibited Ka/Ks > 1; in particular, atp9, atp6, and rps7 may have undergone potential positive selection. CONCLUSION: We assembled and validated the mtDNA of C. stoloniferus, which contains a 15,034 bp reverse complementary sequence. The organelle genome sequence of C. stoloniferus provides valuable genomic resources for species identification, evolution, and comparative genomic research in Cyperaceae.

Molecular phylogenetic reconstruction improves the taxonomic understanding of Indian Dipcadi (Asparagaceae) and reveals a new species from the bank of Hiranyakeshi River, Maharashtra, India.

Dipcadi (Scilloideae: Asparagaceae) is a genus of bulbous monocots with approximately 40 species, of which 13 occur in India. Species delimitation within the genus has been troublesome hindering a comprehensive phylogenetic analysis. The most recent phylogeny of the subfamily Ornithogaloideae included six species of Dipcadi only from Africa. Here, we reconstructed the phylogeny of Ornithogaloideae including 23 accessions comprising 13 recognized taxa (11 species and two varieties) of Indian Dipcadi. The phylogenetic analyses were based on nucleotide sequences of three plastid regions (rbcL, matK and trnL-F spacer) and one nuclear region (ITS). Pseudogaltonia clavata exhibited sister relationship to Dipcadi. Our combined nuclear + plastid dataset analyses revealed a monophyletic Dipcadi with five clades, Clade I-V. Clade I, II and III included mainly Indian species whereas Clade V included mostly African species. Clade IV comprised D. serotinum. Clade I included nine taxa including our newly described species, D. mukaianum. The new species was phylogenetically placed with D. erythraeum, D. saxorum and D. ursulae. Morphologically, the species resembled D. montanum and D. ursulae but differed in characters such as tepal cohesion, number of ovules per locule and foul-smelling flowers. Clade II and III included 11 and six taxa, respectively. D. erythraeum which has a native range from Egypt to western India was found in Clades I and V. The widespread Dipcadi species, viz. D. erythraeum and D. serotinum showed polyphyly however, the monophyly of Dipcadi is established. Our studies suggest that additional molecular markers (plastid as well as nuclear) should be tested for their taxonomy utility. Further work on the historical biogeography of Dipcadi on the subfamily Ornithogaloideae with more genetic data will yield insights how aridification of the landscape would have shaped the evolution of the geographical clades.

Phylogenetic positions of Thai members of Gymnema, Gymnemopsis and Sarcolobus (Apocynaceae, Asclepiadoideae, Marsdenieae), and two new Sarcolobus species uncovered by morpho-molecular evidence.

The present study assesses the phylogenetic position of certain Thai members of Gymnema, Gymnemopsis, and Sarcolobus in relation to other known Marsdenieae species. Fifteen accessions newly sequenced from Thailand were added to the dataset of the homologous sequences of 125 accessions of Marsdenieae downloaded from GenBank. In our molecular phylogeny, almost all the delimited major clades and their relationships are largely congruent with those revealed in previous studies. The monophyly of Gymnema (including the former Jasminanthes species) and that of Sarcolobus, as presently circumscribed, are confirmed. The new accessions of these two genera from Thailand are well grouped with the members of their respective genera. Our analyses provide the first molecular evidence for recognition of Gymnemopsis, a small Asian genus that has never been included in the previous phylogenetic studies, as a distinct genus. All elements of Gymnemopsis are retrieved as a well-supported monophyletic group that is strongly supported as sister to Lygisma, another small Asian genus that most closely resembles it in growth habit, color of latex, indumentum on plant parts, corona structure and follicle traits. Combined molecular phylogenetic, morphological and ecological data also support recognition of two new Sarcolobus species from Thailand, Sarcolobus busbanianus sp. nov. and S. flavus sp. nov. Similarities and differences between these new species and their close relative, S. carinatus, are discussed. In addition, this study also reveals the first record for Thailand of Gymnema lacei. Keys to the species of Gymnemopsis (for all members of the genus), Gymnema and Sarcolobus (for Thai members of these genera) are provided.

Organellar Genomes of Sargassum hemiphyllum var. chinense Provide Insight into the Characteristics of Phaeophyceae.

Sargassum hemiphyllum var. chinense, a prevalent seaweed along the Chinese coast, has economic and ecological significance. However, systematic positions within Sargassum and among the three orders of Phaeophyceae, Fucales, Ectocarpales, and Laminariales are in debate. Here, we reported the organellar genomes of S. hemiphyllum var. chinense (34,686-bp mitogenome with 65 genes and 124,323 bp plastome with 173 genes) and the investigation of comparative genomics and systematics of 37 mitogenomes and 22 plastomes of Fucales (including S. hemiphyllum var. chinense), Ectocarpales, and Laminariales in Phaeophyceae. Whole genome collinearity analysis showed gene number, type, and arrangement were consistent in organellar genomes of Sargassum with 360 SNP loci identified as S. hemiphyllum var. chinense and two genes (rps7 and cox2) identified as intrageneric classifications of Sargassum. Comparative genomics of the three orders of Phaeophyceae exhibited the same content and different types (petL was only found in plastomes of the order Fucales and Ectocarpales) and arrangements (most plastomes were rearranged, but trnA and trnD in the mitogenome represented different orders) in genes. We quantified the frequency of RNA-editing (canonical C-to-U) in both organellar genomes; the proportion of edited sites corresponded to 0.02% of the plastome and 0.23% of the mitogenome (in reference to the total genome) of S. hemiphyllum var. chinense. The repetition ratio of Fucales was relatively low, with scattered and tandem repeats (nine tandem repeats of 14-24 bp) dominating, while most protein-coding genes underwent negative selection (Ka/Ks < 1). Collectively, these findings provide valuable insights to guide future species identification and evolutionary status of three important Phaeophyceae order species.

Phylogeography and ecological niche modeling implicate multiple microrefugia of Swertia tetraptera during quaternary glaciations.

BACKGROUND: Climate fluctuations during the Pleistocene and mountain uplift are vital driving forces affecting geographic distribution. Here, we ask how an annual plant responded to the Pleistocene glacial cycles. METHODS: In this study, we analyzed the population demographic history of the annual herb Swertia tetraptera Maxim (Gentianaceae) endemic to Qinghai-Tibetan Plateau (QTP). A total of 301 individuals from 35 populations of S. tetraptera were analyzed based on two maternally inherited chloroplast fragments (trnL-trnF and trnS-trnG). Phylogeographic analysis was combined with species distribution modeling to detect the genetic variations in S. tetraptera. RESULTS: The genetic diversity of S. tetraptera was high, likely due to its wide natural range, high proportion of endemic haplotypes and evolutionary history. Fifty-four haplotypes were identified in S. tetraptera. Only a few haplotypes were widespread (Hap_4, Hap_1, Hap_3), which were dispersed throughout the present geographical range of S. tetraptera, while many haplotypes were confined to single populations. The cpDNA dataset showed that phylogeographic structuring was lacking across the distribution range of S. tetraptera. Analyses of molecular variance showed that most genetic variation was found within populations (70.51%). In addition, the relationships of the haplotypes were almost completely unresolved by phylogenetic reconstruction. Both mismatch distribution analysis and neutrality tests showed a recent expansion across the distribution range of S. tetraptera. The MaxEnt analysis showed that S. tetraptera had a narrow distribution range during the Last Glacial Maximum (LGM) and a wide distribution range during the current time, with predictions into the future showing the distribution range of S. tetraptera expanding. CONCLUSION: Our study implies that the current geographic and genetic distribution of S. tetraptera is likely to have been shaped by Quaternary periods. Multiple microrefugia of S. tetraptera existed during Quaternary glaciations. Rapid intraspecific diversification and hybridization and/or introgression may have played a vital role in shaping the current distribution patterns of S. tetraptera. The distribution range of S. tetraptera appeared to have experienced contraction during the LGM; in the future, when the global climate becomes warmer with rising carbon dioxide levels, the distribution of S. tetraptera will expand.

Organellar DNA continues to provide a rich source of information in the genomics era.

The genomics revolution continues to change how ecologists and evolutionary biologists study the evolution and maintenance of biodiversity. It is now easier than ever to generate large molecular data sets consisting of hundreds to thousands of independently evolving nuclear loci to estimate a suite of evolutionary and demographic parameters. However, any inferences will be incomplete or inaccurate if incorrect taxonomic identities and perpetuated throughout the analytical pipeline. Due to decades of research and comprehensive online databases, sequencing and analysis of mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA) and select nuclear genes can provide researchers with a cost effective and simple means to verify the species identity of samples prior to subsequent phylogeographic and population genomic analysis. The addition of these sequences to genomic studies can also shed light on other important evolutionary questions such as explanations for gene tree-species tree discordance, species limits, sex-biased dispersal patterns, adaptation, and mtDNA introgression. Although the mtDNA and cpDNA genomes often should not be used exclusively to make historical inferences given their well-known limitations, the addition of these data to modern genomic studies adds little cost and effort while simultaneously providing a wealth of useful data that can have significant implications for both basic and applied research.

Phylogenomics and genome size evolution in Amomum s. s. (Zingiberaceae): Comparison of traditional and modern sequencing methods.

BACKGROUND AND AIMS: A targeted enrichment NGS approach was used to construct the phylogeny of Amomum Roxb. (Zingiberaceae). Phylogenies based on hundreds of nuclear genes, the whole plastome and the rDNA cistron were compared with an ITS-based phylogeny. Trends in genome size (GS) evolution were examined, chromosomes were counted and the geographical distribution of phylogenetic lineages was evaluated. METHODS: In total, 92 accessions of 54 species were analysed. ITS was obtained for 79 accessions, 37 accessions were processed with Hyb-Seq and sequences from 449 nuclear genes, the whole cpDNA, and the rDNA cistron were analysed using concatenation, coalescence and supertree approaches. The evolution of absolute GS was analysed in a phylogenetic and geographical context. The chromosome numbers of 12 accessions were counted. KEY RESULTS: Four groups were recognised in all datasets though their mutual relationships differ among datasets. While group A (A. subulatum and A. petaloideum) is basal to the remaining groups in the nuclear gene phylogeny, in the cpDNA topology it is sister to group B (A. repoeense and related species) and, in the ITS topology, it is sister to group D (the Elettariopsis lineage). The former Elettariopsis makes a monophyletic group. There is an increasing trend in GS during evolution. The largest GS values were found in group D in two tetraploid taxa, A. cinnamomeum and A. aff. biphyllum (both 2n = 96 chromosomes). The rest varied in GS (2C = 3.54-8.78 pg) with a constant chromosome number 2n = 48. There is a weak connection between phylogeny, GS and geography in Amomum. CONCLUSIONS: Amomum consists of four groups, and the former Elettariopsis is monophyletic. Species in this group have the largest GS. Two polyploids were found and GS greatly varied in the rest of Amomum.

Chimeric deletion mutation of rpoC2 underlies the leaf-patterning of Clivia miniata var. variegata.

KEY MESSAGE: The deletion mutated rpoC2 leads to yellow stripes of Clivia miniata var. variegata by down regulating the transcription of 28 chloroplast genes and disturbing chloroplast biogenesis and thylakoid membrane development. Clivia miniata var. variegata (Cmvv) is a common mutant of Clivia miniata but its genetic basis is unclear. Here, we found that a 425 bp deletion mutation of chloroplast rpoC2 underlies the yellow stripes (YSs) of Cmvv. Both RNA polymerase PEP and NEP coexist in seed-plant chloroplasts and the ß″ subunit of PEP is encoded by rpoC2. The rpoC2 mutation changed the discontinuous cleft domain required to form the PEP central cleft for DNA binding from 1103 to 59 aa. RNA-Seq revealed that 28 chloroplast genes (cpDEGs) were all down-regulated in YSs, of which, four involved in chloroplast protein translation and 21 of photosynthesis system (PS)I, PSII, cytochrome b6/f complex and ATP synthase are crucial for chloroplast biogenesis/development. The accuracy and reliability of RNA-Seq was verified by qRT-PCR. Moreover, the chlorophyll (Chl) a/b content, ratio of Chla/Chlb and photosynthetic rate (Pn) of YS decreased significantly. Meanwhile, chloroplasts of the YS mesophyll cells were smaller, irregular in shape, contain almost no thylakoid membrane, and even proplastid was found in YS. These findings indicate that the rpoC2 mutation down-regulated expression of the 28 cpDEGs, which disturb chloroplast biogenesis and its thylakoid membrane development. Thus, there are not enough PSI and II components to bind Chl, so that the corresponding areas of the leaf are yellow and show a low Pn. In this study, the molecular mechanism of three phenotypes of F1 (Cmvv ♀ × C. miniata ♂) was revealed, which lays a foundation for the breeding of variegated plants.

Geographic isolation and long-distance gene flow influence the genetic structure of the blue fan palm Brahea armata (Arecaceae).

The formation of the Baja California Peninsula (BCP) has impacted the microevolutionary dynamics of different species in ways that depend on biological traits such as dispersal capacity. Plants with relatively low levels of vagility have exhibited high genetic divergence between the BCP and Continental mainland. Brahea armata (Arecaceae) is a palm species inhabiting the northern part of the BCP and Sonora; its distribution occurs in isolated oases of vegetation. We aimed to evaluate the influence of the formation of the BCP on the genetic structure of B. armata using nuclear microsatellites and chloroplast markers (cpDNA) to compare patterns of genetic diversity and structure with previous published studies. Because gene flow through seeds is usually more limited compared to pollen flow, we expect to find stronger genetic structure at (cpDNA) than at nuclear markers. Moreover, larger genetic structure might also be explained by the smaller effective population size of cpDNA. We analyzed six microsatellite markers and two cpDNA regions. The main results indicated high levels of genetic differentiation among isolated populations located in the BCP, while low genetic differentiation was found between southern populations of the BCP and Sonora, suggesting long distance gene flow. In contrast, chloroplast markers indicated high levels of genetic structure between BCP and Sonora populations, suggesting asymmetrical gene flow between pollen (measured by nuclear microsatellites) and seed (cpDNA markers). This study provides valuable information on genetic diversity of B. armata that can be relevant for conservation and management; and develops microsatellites markers that can be transferred to other Brahea species.

Comparative plastome analysis of Musaceae and new insights into phylogenetic relationships.

BACKGROUND: Musaceae is an economically important family consisting of 70-80 species. Elucidation of the interspecific relationships of this family is essential for a more efficient conservation and utilization of genetic resources for banana improvement. However, the scarcity of herbarium specimens and quality molecular markers have limited our understanding of the phylogenetic relationships in wild species of Musaceae. Aiming at improving the phylogenetic resolution of Musaceae, we analyzed a comprehensive set of 49 plastomes for 48 species/subspecies representing all three genera of this family. RESULTS: Musaceae plastomes have a relatively well-conserved genomic size and gene content, with a full length ranging from 166,782 bp to 172,514 bp. Variations in the IR borders were found to show phylogenetic signals to a certain extent in Musa. Codon usage bias analysis showed different preferences for the same codon between species and three genera and a common preference for A/T-ending codons. Among the two genes detected under positive selection (dN/dS > 1), ycf2 was indicated under an intensive positive selection. The divergent hotspot analysis allowed the identification of four regions (ndhF-trnL, ndhF, matK-rps16, and accD) as specific DNA barcodes for Musaceae species. Bayesian and maximum likelihood phylogenetic analyses using full plastome resulted in nearly identical tree topologies with highly supported relationships between species. The monospecies genus Musella is sister to Ensete, and the genus Musa was divided into two large clades, which corresponded well to the basic number of n = x = 11 and n = x =10/9/7, respectively. Four subclades were divided within the genus Musa. A dating analysis covering the whole Zingiberales indicated that the divergence of Musaceae family originated in the Palaeocene (59.19 Ma), and the genus Musa diverged into two clades in the Eocene (50.70 Ma) and then started to diversify from the late Oligocene (29.92 Ma) to the late Miocene. Two lineages (Rhodochlamys and Australimusa) radiated recently in the Pliocene /Pleistocene periods. CONCLUSIONS: The plastome sequences performed well in resolving the phylogenetic relationships of Musaceae and generated new insights into its evolution. Plastome sequences provided valuable resources for population genetics and phylogenetics at lower taxon.

Insights into adaptive evolution of plastomes in Stipa L. (Poaceae).

BACKGROUND: The study presents results of research on the evolution of plastid genomes in Stipa L. which is a large genus of the Poaceae family, comprising species diverse in terms of geographic distribution, growing under highly variated habitat conditions. Complete plastome sequences of 43 taxa from Stipeae and Ampelodesmae tribes were analyzed for the variability of the coding regions against the background of phylogenetic relationships within the genus Stipa. The research hypothesis put forward in our research was that some of coding regions are affected by a selection pressure differentiated between individual phylogenetic lines of Stipa, potentially reducing the phylogenetic informativeness of these CDS. The study aimed to answer the question, which genes evolve in Stipa most rapidly and what kind of changes in the properties of encoded amino acids this entails. Another goal of this research was to find out whether individual genes are affected by positive selection and finally, whether selective pressure is uniform within the genus or does it vary between particular evolutionary lines within the genus. RESULTS: Results of our study proved the presence of selective pressure in 11 genes: ccsA, matK, ndhC, ndhF, ndhK, rbcL, rpoA rpoC1, rpoC2, rps8 and rps11. For the first time the effect of positive selection on the rps8, rps11, and ndhK genes was documented in grasses. The varied pace of evolution, different intensity and effects of selective pressure have been demonstrated between particular phylogenetic lines of the genus tested. CONCLUSIONS: Positive selection in plastid genome in Stipa mostly affects photosynthetic genes. The potential strongest adaptive pressure was observed in the rbcL gene, especially in the oldest evolutionary group comprising Central Asian high-mountain species: S. basiplumosa, S. klimesii, S. penicillata and S. purpurea, where adaptive pressure probably affected the amino acids directly related to the efficiency of CO2 assimilation.

Toward finally unraveling the phylogenetic relationships of Juncaceae with respect to another cyperid family, Cyperaceae.

Juncaceae is a cosmopolitan family belonging to the cyperid clade of Poales together with Cyperaceae and Thurniaceae. These families have global economic and ethnobotanical significance and are often keystone species in wetlands around the world, with a widespread cosmopolitan distribution in temperate and arctic regions in both hemispheres. Currently, Juncaceae comprises more than 474 species in eight genera: Distichia, Juncus, Luzula, Marsippospermum, Oreojuncus, Oxychloë, Patosia and Rostkovia. The phylogeny of cyperids has not been studied before in a complex view based on most sequenced species from all three families. In this study, most sequenced regions from chloroplast (rbcL, trnL, trnL-trnF) and nuclear (ITS1-5.8S-ITS2) genomes were employed from more than a thousand species of cyperids covering all infrageneric groups from their entire distributional range. We analyzed them by maximum parsimony, maximum likelihood, and Bayesian inference to revise the phylogenetic relationships in Juncaceae and Cyperaceae. Our major results include the delimitation of the most problematic paraphyletic genus Juncus, in which six new genera are recognized and proposed to recover monophyly in this group: Juncus, Verojuncus, gen. nov., Juncinella, gen. et stat. nov., Alpinojuncus, gen. nov., Australojuncus, gen. nov., Boreojuncus, gen. nov. and Agathryon, gen. et stat. nov. For these genera, a new category, Juncus supragen. et stat. nov., was established. This new classification places most groups recognized within the formal Juncus clade into natural genera that are supported by morphological characters.

A turn in species conservation for hairpin banksias: demonstration of oversplitting leads to better management of diversity.

PREMISE: Understanding evolutionary history and classifying discrete units of organisms remain overwhelming tasks, and lags in this workload concomitantly impede an accurate documentation of biodiversity and conservation management. Rapid advances and improved accessibility of sensitive high-throughput sequencing tools are fortunately quickening the resolution of morphological complexes and   thereby improving the estimation of species diversity. The recently described and critically endangered Banksia vincentia is morphologically similar to the hairpin banksia complex (B. spinulosa s.l.), a group of eastern Australian flowering shrubs whose continuum of morphological diversity has been responsible for taxonomic controversy and possibly questionable conservation initiatives. METHODS: To assist conservation while testing the current taxonomy of this group, we used high-throughput sequencing to infer a population-scale evolutionary scenario for a sample set that is comprehensive in its representation of morphological diversity and a 2500-km distribution. RESULTS: Banksia spinulosa s.l. represents two clades, each with an internal genetic structure shaped through historical separation by biogeographic barriers. This structure conflicts with the existing taxonomy for the group. Corroboration between phylogeny and population statistics aligns with the hypothesis that B. collina, B. neoanglica, and B. vincentia should not be classified as species. CONCLUSIONS: The pattern here supports how morphological diversity can be indicative of a locally expressed suite of traits rather than relationship. Oversplitting in the hairpin banksias is atypical since genomic analyses often reveal that species diversity is underestimated. However, we show that erring on overestimation can yield negative consequences, such as the disproportionate prioritization of a geographically anomalous population.

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.

Developing an efficient DNA barcoding system to differentiate between Lilium species.

BACKGROUND: Lilium is an important ornamental bulb, possesses medicinal properties, and is also edible. Species within the Lilium genus share very similar morphology and macroscopic characteristics, thus they cannot be easily and clearly distinguished from one another. To date, no efficient species-specific markers have been developed for classifying wild lily species, which poses an issue with further characterizing its medicinal properties. RESULTS: To develop a simple and reliable identification system for Lilium, 45 representative species from 6 sections were used to develop a DNA barcoding system, which was based on DNA sequence polymorphisms. In this study, we assessed five commonly used DNA barcode candidates (ITS, rbcL, ycf1b, matK and psbA-trnH) and five novel barcode candidates obtained from highly variable chloroplast genomic regions (trnL-trnF, trnS-trnG, trnF-ndhJ, trnP-psaJ-rpI33 and psbB-psbH). We showed that a set of three novel DNA barcodes (ITS + trnP-psaJ-rpI33 + psbB-psbH) could be efficiently used as a genetic marker to distinguish between lily species, as assessed by methods including DNAsp, BI and ML tree, and Pair Wise Group (PWG). CONCLUSIONS: A rapid and reliable DNA barcoding method was developed for all 45 wild Lilium species by using ITS, trnP-psaJ-rpI33, and psbB-psbH as DNA barcoding markers. The method can be used in the classification of wild Lilium species, especially endangered species, and also provides an effective method for selective lily breeding.

Evolutionary history of Hemerocallis in Japan inferred from chloroplast and nuclear phylogenies and levels of interspecific gene flow.

The perennial herb genus Hemerocallis (Asphodelaceae) shows four flowering types: diurnal half-day, diurnal one-day, nocturnal half-day, and nocturnal one-day flowering. These flowering types are corresponding to their main pollinators, and probably act as a primary mechanism of reproductive isolation. To examine how the four flowering types diverged, we reconstructed the phylogeny of the Japanese species of Hemerocallis using 1615 loci of nuclear genome-wide SNPs and 2078 bp sequences of four cpDNA regions. We also examined interspecific gene flows among taxa by an Isolation-with-Migration model and a population structure analysis. Our study revealed an inconsistency between chloroplast and nuclear genome phylogenies, which may have resulted from chloroplast capture. Each of the following five clusters is monophyletic and clearly separated on the nuclear genome-wide phylogenetic tree: (I) two nocturnal flowering species with lemon-yellow flowers, H. citrina (half-day flowering) and H. lilioasphodelus (one-day flowering); (II) a diurnal one-day flowering species with yellow-orange flowers, H. middendorffii; (III) a variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. disticha; (IV) another variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. aurantiaca, and a diurnal one-day flowering species with yellow-orange flowers, H. major; (V) a diurnal half-day flowering species with yellow-orange flowers, H. hakuunensis. The five clusters are consistent with traditional phenotype-based taxonomy (cluster I, cluster II, and clusters III-V correspond to Hemerocallis sect. Hemerocallis, Capitatae, and Fulvae, respectively). These findings could indicate that three flowering types (nocturnal flowering, diurnal one-day flowering, and diurnal half-day flowering) diverged in early evolutionary stages of Hemerocallis and subsequently a change from diurnal half-day flowering to diurnal one-day flowering occurred in a lineage of H. major. While genetic differentiation among the five clusters was well maintained, significant gene flow was detected between most pairs of taxa, suggesting that repeated hybridization played a role in the evolution of those taxa.

Plastomes in the holoparasitic family Balanophoraceae: Extremely high AT content, severe gene content reduction, and two independent genetic code changes.

The transition to a heterotrophic lifestyle in angiosperms is characterized by convergent evolutionary changes. Plastid genome remodeling includes dramatic functional and physical reductions with the highest degrees observed in fully heterotrophic plants. Genes related to photosynthesis are generally absent or pseudogenized, while a few genes related to other metabolic processes that take place within the plastid are almost invariably maintained. The family Balanophoraceae consists of root holoparasites that present reduced plastid genomes with an extraordinarily elevated AT content and the single genetic code change ever documented in land plant plastomes (the stop codon TAG now codes for tryptophan). Here, we studied the plastomes of Lophophytum leandri and Ombrophytum subterraneum (Balanophoraceae) that showed the remarkable absence of the gene trnE, a highly biased nucleotide composition, and an independent genetic code change (the standard stop codon TGA codes for tryptophan). This is the second genetic code change identified in land plant plastomes. Analysis of the transcriptome of Lophophytum indicated that the entire C5 pathway typical of plants is conserved despite the lack of trnE in its plastome. A hypothetical model of plastome evolution in the Balanophoraceae is presented.

Genetic diversity of Oxytropis section Xerobia (Fabaceae) in one of the centres of speciation.

The genetic diversity and phylogenetic relationships of Oxytropis caespitosa, O. grandiflora, O. eriocarpa, O. mixotriche, O. nitens, O. peschkovae and O. triphylla, section Xerobia subgenus Oxytropis, in one of the main speciation centres of the genus Oxytropis (Baikal Siberia and adjacent territories of Northeastern Mongolia) were studied based on sequence analysis of the psbA-trnH, trnL-trnF and trnS-trnG intergenic spacers of cpDNA, as well as the ITS nrDNA. Most populations are characterized by a high level of chloroplast genetic diversity (h varied from 0.327 to 1.000 and π from 0.0001 to 0.0090) due to the ancient origin for some species and to hybridization and polyploidy for others. 67 haplotypes were identified, of which six were shared. Phylogenetic relationships among species could not be satisfactorily resolved. Only the haplotypes of O. triphylla formed a group with rather high support. Probably, O. caespitosa, O. grandiflora, O. mixotriche and O. nitens constitute a single genetic complex. As regards the ITS nrDNA polymorphism, we detected only two ribotypes (RX1, RX2). Both were found in O. caespitosa, O. eriocarpa, O. mixotriche and O. peschkovae, while RX1 was present in O. nitens and O. triphylla, RX2 in O. grandiflora. The absence of diagnostic species-specific variants for the markers studied, together with the sharing of cpDNA haplotypes and nrDNA ribotypes between species, and the resulting polytomies on the phylogenetic trees, confirm the hypothesis on the hybrid origin of some of them. Obviously, the reproductive barriers within the sect. Xerobia are weak. However, morphological differences between the species of the sect. Xerobia are clearly pronounced, even when they grow in sympatry.

Phylogeography of a gypsum endemic plant across its entire distribution range in the western Mediterranean.

PREMISE: Gypsum soils in the Mediterranean Basin house large numbers of edaphic specialists that are adapted to stressful environments. The evolutionary history and standing genetic variation of these taxa have been influenced by the geological and paleoclimatic complexity of this area and the long-standing effect of human activities. However, little is known about the origin of Mediterranean gypsophiles and the factors affecting their genetic diversity and population structure. METHODS: Using phylogenetic and phylogeographic approaches based on microsatellites and sequence data from nuclear and chloroplast regions, we evaluated the divergence time, genetic diversity, and population structure of 27 different populations of the widespread Iberian gypsophile Lepidium subulatum throughout its entire geographic range. RESULTS: Lepidium subulatum diverged from its nearest relatives ~3 million years ago, and ITS and psbA/matK trees supported the monophyly of the species. These results suggest that both geological and climatic changes in the region around the Plio-Pleistocene promoted its origin, compared to other evolutionary processes. We found high genetic diversity in both nuclear and chloroplast markers, but a greater population structure in the chloroplast data. These results suggest that while seed dispersal is limited, pollen flow may be favored by the presence of numerous habitat patches that enhance the movement of pollinators. CONCLUSIONS: Despite being an edaphic endemic, L. subulatum possesses high genetic diversity probably related to its relatively old age and high population sizes across its range. Our study highlights the value of using different markers to fully understand the phylogeographic history of plant species.