[Development of a Genus Identification Method for Poisonous Plants Using Real-Time PCR].

We have developed a rapid genus identification method for poisonous plants. The real-time PCR using the TaqMan® probe method was employed for detection, with the amplified targets being the "trnL (UAA)-intron" or "trnL-trnF intergenic spacer" regions of chloroplast DNA. The targeted plants were selected six genera (Aconitum, Colchicum, Veratrum, Brugmansia, Scopolia and Narcissus), which have been implicated in many instances of food poisoning in Japan. A tissue lysis solution was used for DNA extraction, which can be completed within approximate 30 min. A master mix corresponding to the tissue lysis solution was used for real-time PCR reagents. As a result, we were able to complete the entire process from DNA extraction to genus identification in 4 to 5 hr. The detection sensitivity was estimated at approximately 1 pg of DNA for all six plant genera. Remarkably, an amplification plot was discerned even with the crude cell lysates of all samples. It was also possible to obtain amplification curves for three plant samples that had been subjected to simulated cooking (boiling). This study suggests that the developed method can rapidly identify six genera of poisonous plants.

Unlocking the Complete Chloroplast Genome of a Native Tree Species from the Amazon Basin, Capirona (Calycophyllum Spruceanum, Rubiaceae), and Its Comparative Analysis with Other Ixoroideae Species.

Capirona (Calycophyllum spruceanum Benth.) belongs to subfamily Ixoroideae, one of the major lineages in the Rubiaceae family, and is an important timber tree. It originated in the Amazon Basin and has widespread distribution in Bolivia, Peru, Colombia, and Brazil. In this study, we obtained the first complete chloroplast (cp) genome of capirona from the department of Madre de Dios located in the Peruvian Amazon. High-quality genomic DNA was used to construct libraries. Pair-end clean reads were obtained by PE 150 library and the Illumina HiSeq 2500 platform. The complete cp genome of C. spruceanum has a 154,480 bp in length with typical quadripartite structure, containing a large single copy (LSC) region (84,813 bp) and a small single-copy (SSC) region (18,101 bp), separated by two inverted repeat (IR) regions (25,783 bp). The annotation of C. spruceanum cp genome predicted 87 protein-coding genes (CDS), 8 ribosomal RNA (rRNA) genes, 37 transfer RNA (tRNA) genes, and one pseudogene. A total of 41 simple sequence repeats (SSR) of this cp genome were divided into mononucleotides (29), dinucleotides (5), trinucleotides (3), and tetranucleotides (4). Most of these repeats were distributed in the noncoding regions. Whole chloroplast genome comparison with the other six Ixoroideae species revealed that the small single copy and large single copy regions showed more divergence than inverted regions. Finally, phylogenetic analyses resolved that C. spruceanum is a sister species to Emmenopterys henryi and confirms its position within the subfamily Ixoroideae. This study reports for the first time the genome organization, gene content, and structural features of the chloroplast genome of C. spruceanum, providing valuable information for genetic and evolutionary studies in the genus Calycophyllum and beyond.

Evolution of floral characters and biogeography of Heloniadeae (Melanthiaceae): an example of breeding system shifts with inflorescence change.

Heloniadeae (Melanthiaceae) presents an East Asia-North America disjunct distribution. Different molecular and morphological data nevertheless support the tribe as a monophyletic group. However, their phylogenetic relationships and biogeographic history, together with the character evolution, are not clear. Therefore, we constructed a Bayesian phylogenetic tree for Heloniadeae using cpDNA and inferred the historical biogeography and floral character evolution. The results revealed that Heloniadeae was distributed in high-latitudes of East Asia and North America, originating since 22.2 mya. The East Asia clade migrated into southwest China, and subsequently colonized the Korean Peninsula, Taiwan, the Ryukyus, and spread northward to Japan and southern Sakhalin. The evolution of the inflorescence and number of flowers were phylogenetically conserved, associated with the historical biogeography of Heloniadeae. The inflorescences transferred from raceme to sub-umbel, and the number of flowers decreased during the dispersal process, which may be accompanied by changes in the breeding system. Besides, the anthesis period was more affected by the habitat environment than phylogenetic constraints. The flowering temperature of was below 20 °C in most species, except H. kawanoi. Such a low temperature might not be conductive to pollinator activities, but it could be compensated by sustaining seed production with long-lasting flowers.

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. AIM OF THIS STUDY: This study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. MATERIALS AND METHODS: The sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. RESULTS: The plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. CONCLUSION: The nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

Plastid genome evolution in Amazonian açaí palm (Euterpe oleracea Mart.) and Atlantic forest açaí palm (Euterpe edulis Mart.).

KEY MESSAGE: The plastomes of E. edulis and E. oleracea revealed several molecular markers useful for genetic studies in natural populations and indicate specific evolutionary features determined by vicariant speciation. Arecaceae is a large and diverse family occurring in tropical and subtropical ecosystems worldwide. E. oleracea is a hyperdominant species of the Amazon forest, while E. edulis is a keystone species of the Atlantic forest. It has reported that E. edulis arose from vicariant speciation after the emergence of the belt barrier of dry environment (Cerrado and Caatinga biomes) between Amazon and Atlantic forests, isolating the E. edulis in the Atlantic forest. We sequenced the complete plastomes of E. edulis and E. oleracea and compared them concerning plastome structure, SSRs, tandem repeats, SNPs, indels, hotspots of nucleotide polymorphism, codon Ka/Ks ratios and RNA editing sites aiming to investigate evolutionary traits possibly affected by distinct environments. Our analyses revealed 303 SNPs, 91 indels, and 82 polymorphic SSRs among both species. Curiously, the narrow correlation among localization of repetitive sequences and indels strongly suggests that replication slippage is involved in plastid DNA mutations in Euterpe. Moreover, most non-synonymous substitutions represent amino acid variants in E. edulis that evolved specifically or in a convergent manner across the palm phylogeny. Amino acid variants observed in several plastid proteins in E. edulis were also identified as positive signatures across palm phylogeny. The higher incidence of specific amino acid changes in plastid genes of E. edulis in comparison with E. oleracea probably configures adaptive genetic variations determined by vicariant speciation. Our data indicate that the environment generates a selective pressure on the plastome making it more adapted to specific conditions.

Comparative analysis of chloroplast genomes in Vasconcellea pubescens A.DC. and Carica papaya L.

The chloroplast genome is an integral part of plant genomes in a species along with nuclear and mitochondrial genomes, contributing to adaptation, diversification, and evolution of plant lineages. In the family Caricaceae, only the Carica papaya chloroplast genome and its nuclear and mitochondrial genomes were sequenced, and no chloroplast genome-wide comparison across genera was conducted. Here, we sequenced and assembled the chloroplast genome of Vasconcellea pubescens A.DC. using Oxford Nanopore Technology. The size of the genome is 158,712 bp, smaller than 160,100 bp of the C. papaya chloroplast genome. And two structural haplotypes, LSC_IRa_SSCrc_IRb and LSC_IRa_SSC_IRb, were identified in both V. pubescens and C. papaya chloroplast genomes. The insertion-deletion mutations may play an important role in Ycf1 gene evolution in family Caricaceae. Ycf2 is the only one gene positively selected in the V. pubescens chloroplast genome. In the C. papaya chloroplast genome, there are 46 RNA editing loci with an average RNA editing efficiency of 63%. These findings will improve our understanding of the genomes of these two crops in the family Caricaceae and will contribute to crop improvement.

Genetic diversity and population structure of the endangered orchid Pelatantheria scolopendrifolia (Orchidaceae) in Korea.

Due to substantial population decline, the Korean orchid P. scolopendrifolia is considered endangered and highly threatened. Like many endangered species, it is vulnerable to biological and anthropogenic threats that can lead to the loss of genetic diversity and, ultimately, extinction. Therefore, the assessment of genetic diversity and population genetic structure is imperative for conservation. In this study, we newly developed 15 polymorphic microsatellite markers. Analyses of genetic diversity and population genetic structure that included 182 samples from 11 populations were conducted using microsatellite markers and four noncoding regions of chloroplast DNA. Our study revealed a relatively low level of genetic diversity (Ho = 0.529, He = 0.356), albeit harboring with private alleles based on microsatellite genotyping data, and high haplotype diversities based on chloroplast DNA sequences data. The results of STRUCTURE and PCoA based on microsatellite genotyping data showed population differentiations. An AMOVA based on chloroplast DNA sequence data further corroborated these conclusions, indicating about 70% of variations found among populations. Low genetic diversity and divergence among the population might have been caused by factors, such as asexual reproduction, demographic events (bottleneck and population expansion), geographic isolation, and low gene flow. The development and implication of conservation strategies and management of P. scolopendrifolia are proposed based on these results.

Extensive Genetic Connectivity and Historical Persistence Are Features of Two Widespread Tree Species in the Ancient Pilbara Region of Western Australia.

Phylogeographic studies can be used as a tool to understand the evolutionary history of a landscape, including the major drivers of species distributions and diversity. Extensive research has been conducted on phylogeographic patterns of species found in northern hemisphere landscapes that were affected by glaciations, yet the body of literature for older, unaffected landscapes is still underrepresented. The Pilbara region of north-western Australia is an ancient and vast landscape that is topographically complex, consisting of plateaus, gorges, valleys, and ranges, and experiences extreme meteorological phenomena including seasonal cyclonic activity. These features are expected to influence patterns of genetic structuring throughout the landscape either by promoting or restricting the movement of pollen and seed. Whilst a growing body of literature exists for the fauna endemic to this region, less is known about the forces shaping the evolution of plant taxa. In this study we investigate the phylogeography of two iconic Pilbara tree species, the Hamersley Bloodwood (Corymbia hamersleyana) and Western Gidgee (Acacia pruinocarpa), by assessing patterns of variation and structure in several chloroplast DNA regions and nuclear microsatellite loci developed for each species. Gene flow was found to be extensive in both taxa and there was evidence of long-distance seed dispersal across the region (pollen to seed ratios of 6.67 and 2.96 for C. hamersleyana and A. pruinocarpa, respectively), which may result from flooding and strong wind gusts associated with extreme cyclonic activity. Both species possessed high levels of cpDNA genetic diversity in comparison to those from formerly glaciated landscapes (C. hamersleyana = 14 haplotypes, A. pruinocarpa = 37 haplotypes) and showed evidence of deep lineage diversification occurring from the late Miocene, a time of intensifying aridity in this landscape that appears to be a critical driver of evolution in Pilbara taxa. In contrast to another study, we did not find evidence for topographic features acting as refugia for the widely sampled C. hamersleyana.

The chloroplast genome sequence of the green macroalga Caulerpa okamurae (Ulvophyceae, Chlorophyta): Its structural features, organization and phylogenetic analysis.

To clarify evolutionary characteristics, phylogenetic relationships as well as species identification of C. okamurae, we determined the cpDNA sequence of Caulerpa okamurae using de novo sequencing in the present study. The cpDNA of C. okamurae was 148,274 bp in length, and it lacked the inverted repeat commonly found in vascular green plants. The cpDNA of C. okamurae was highly compact with a gene density of 71.7%. Moreover, it was an AT-rich genome (65.5%) consisting 76 protein-coding genes (PCGs), 27 transfer RNA (tRNA) genes, three ribosomal RNA (rRNA) genes, 32 putative open reading frames (ORFs) and six introns. Additionally, the six introns were annotated in six genes as follows: psbA, rpoB, ftsH, psbD, atpF and cysA. The overall base composition of its cpDNA was 65.46% for AT. A total of 56 genes were encoded on the light strand, while all the other 50 chloroplast genes were encoded on the heavy strand. All of the PCGs had ATG as their start codon and employed TAA, TGA or TAG as their termination codon. Phylogenetic analyses suggested that the complete cpDNA sequence of C. okamurae fell in the Chlorophyta, Ulvophyceae, Bryopsidales, and Caulerpaceae and more resembled the cpDNAs of C. racemosa, C. cliftonii voucher and Tydemania expeditionis. Taken together, our data offered useful information for the studies of C.okamurae on evolutionary characteristics, phylogenetic relationships as well as species identification.

Multiple origins and the population genetic structure of Rubus takesimensis (Rosaceae) on Ulleung Island: Implications for the genetic consequences of anagenetic speciation.

To determine the origin and genetic consequences of anagenesis in Rubus takesimensis on Ulleung Island, Korea, we compared the genetic diversity and population structure of R. takesimensis with those of its continental progenitor R. crataegifolius. We broadly sampled a total of 315 accessions in 35 populations and sequenced five noncoding regions of chloroplast DNA. Rubus takesimensis emerged as nonmonophyletic and several geographically diverse continental populations were likely responsible for the origin of R. takesimensis; the majority of R. takesimensis accessions were sisters to the clade containing accessions of R. crataegifolius, primarily from the Korean peninsula, while rare accessions from three populations shared common ancestors with the ones from the southern part of the Korean peninsula, Jeju Island, and Japan. A few accessions from the Chusan population originated independently from the Korean peninsula. Of 129 haplotypes, 81 and 48 were found exclusively in R. crataegifolius and R. takesimensis, respectively. We found unusually high genetic diversity in two regions on Ulleung Island and no geographic population structure. For R. crataegifolius, two major haplotype groups were found; one for the northern mainland Korean peninsula, and the other for the southern Korean peninsula and the Japanese archipelago. Compared with populations of R. crataegifolius sampled from Japan, much higher haplotype diversity was found in populations from the Korean peninsula. The patterns of genetic consequences in R. takesimensis need to be verified for other endemic species based on chloroplast DNA and independent nuclear markers to synthesize emerging patterns of anagenetic speciation on Ulleung Island.

Population genetic structure and demography of Magnolia kobus: variety borealis is not supported genetically.

Species delimitations by morphological and by genetic markers are not always congruent. Magnolia kobus consists of two morphologically different varieties, kobus and borealis. The latter variety is characterized by larger leaves than the former. For the conservation of M. kobus genetic resources in natural forests, the relationships between morphological and genetic variation should be clarified. We investigated variations in nuclear microsatellites, chloroplast DNA (cpDNA) sequences and leaf morphological traits in 23 populations of M. kobus over the range of species. Two genetically divergent lineages, northern and southern were detected and their geographical boundary was estimated to be at 39°N. The northern lineage consisted of two genetic clusters and a single cpDNA haplotype, while the southern one had multiple genetic clusters and cpDNA haplotypes. The northern lineage showed significantly lower genetic diversity than the southern. Approximate Bayesian computation indicated that the northern and southern lineages had experienced, respectively, population expansion and long-term stable population size. The divergence time between the two lineages was estimated to be 565,000 years ago and no signature of migration between the two lineages after divergence was detected. Ecological niche modeling showed that the potential distribution area in northern Japan at the last glacial maximum was very small. It is thus considered that the two lineages have experienced different population histories over several glacial-inter-glacial cycles. Individuals of populations in the central to northern part of Honshu on the Sea of Japan side and in Hokkaido had large leaf width and area. These leaf characteristics corresponded with those of variety borealis. However, the delimitation of the northern and southern lineages detected by genetic markers (39°N) was not congruent with that detected by leaf morphologies (36°N). It is therefore suggested that variety borealis is not supported genetically and the northern and southern lineages should be considered separately when identifying conservation units based not on morphology but on genetic markers.

First molecular phylogenetic insights into the evolution of Eriocaulon (Eriocaulaceae, Poales).

Eriocaulon is a genus of c. 470 aquatic and wetland species of the monocot plant family Eriocaulaceae. It is widely distributed in Africa, Asia and America, with centres of species richness in the tropics. Most species of Eriocaulon grow in wetlands although some inhabit shallow rivers and streams with an apparent adaptive morphology of elongated submerged stems. In a previous molecular phylogenetic hypothesis, Eriocaulon was recovered as sister of the African endemic genus Mesanthemum. Several regional infrageneric classifications have been proposed for Eriocaulon. This study aims to critically assess the existing infrageneric classifications through phylogenetic reconstruction of infrageneric relationships, based on DNA sequence data of four chloroplast markers and one nuclear marker. There is little congruence between our molecular results and previous morphology-based infrageneric classifications. However, some similarities can be found, including Fyson's sect. Leucantherae and Zhang's sect. Apoda. Further phylogenetic studies, particularly focusing on less well sampled regions such as the Neotropics, will help provide a more global overview of the relationships in Eriocaulon and may enable suggesting the first global infrageneric classification.

Detection of an invasive aquatic plant in natural water bodies using environmental DNA.

The ability to detect founding populations of invasive species or rare species with low number of individuals is important for aquatic ecosystem management. Traditional approaches use historical data, knowledge of the species' ecology and time-consuming surveys. Within the past decade, environmental DNA (eDNA) has emerged as a powerful additional tracking tool. While much work has been done with animals, comparatively very little has been done with aquatic plants. Here we investigated the transportation and seasonal changes in eDNA concentrations for an invasive aquatic species, Elodea canadensis, in Norway. A specific probe assay was developed using chloroplast DNA to study the fate of the targeted eDNA through space and time. The spatial study used a known source of Elodea canadensis within Lake Nordbytjern 400 m away from the lake outlet flowing into the stream Tveia. The rate of disappearance of E. canadensis eDNA was an order of magnitude loss over about 230 m in the lake and 1550 m in the stream. The time series study was performed monthly from May to October in lake Steinsfjorden harbouring E. canadensis, showing that eDNA concentrations varied by up to three orders of magnitude, peaking during fall. In both studies, the presence of suspended clay or turbidity for some samples did not hamper eDNA analysis. This study shows how efficient eDNA tools may be for tracking aquatic plants in the environment and provides key spatial and temporal information on the fate of eDNA.

Ecotypic divergences of the alpine herb Potentilla matsumurae adapted to fellfield-snowbed habitats across a series of mountain sky islands.

PREMISE: Divergent selection due to environmental heterogeneity can lead to local adaptation. However, the ecological and evolutionary processes of local adaptation that occurs across multiple regions are often unknown. Our previous studies reported on the ecotypic divergence within a local area of variation of Potentilla matsumurae, an alpine herb adapted to the fellfield-snowbed environment. Here we investigated large-scale geographic patterns of ecotypic differentiation in this species to infer local adaptation and selective forces across multiple regions. METHODS: We compiled information on the overall distributions of fellfield and snowbed habitats on the mountains in Japan across the distribution of the species. Next, we conducted common garden experiments to test the adaptive divergence of the fellfield-snowbed plants derived from multiple regions. Finally, we evaluated phylogeographic structures based on cpDNA and allozyme variations and inferred the evolutionary history of ecotype differentiation. RESULTS: The mosaic distribution of the fellfield-snowbed ecotypes across isolated mountaintops constitutes indirect evidence for habitat-specific natural selection. The significant difference in survivorship between the ecotypes observed in a controlled snow environment provides more substantial evidence of local selection. Phylogeographic structures support the hypothesis that ecotypic divergence events from fellfield to snowbed populations occurred independently in at least two distinct regions. CONCLUSIONS: Ecotypic divergence of P. matsumurae has occurred across a series of mountain sky islands. Local selection in snowy environments is a driving force that maintains the divergent ecotypes across multiple mountain regions and can contribute to the diversification of plants in heavy-snow regions.

Extensive allopolyploidy in the neotropical genus Lachemilla (Rosaceae) revealed by PCR-based target enrichment of the nuclear ribosomal DNA cistron and plastid phylogenomics.

PREMISE OF THE STUDY: Polyploidy has been long recognized as an important force in plant evolution. Previous studies had suggested widespread occurrence of polyploidy and the allopolyploid origin of several species in the diverse neotropical genus Lachemilla (Rosaceae). Nonetheless, this evidence has relied mostly on patterns of cytonuclear discordance, and direct evidence from nuclear allelic markers is still needed. METHODS: Here we used PCR target enrichment in combination with high throughput sequencing to obtain multiple copies of the nuclear ribosomal (nr) DNA cistron and 45 regions of the plastid genome (cpDNA) from 219 accessions representing 48 species of Lachemilla and to explore the allopolyploid origin of species in this group. KEY RESULTS: We were able to identify multiple nrDNA ribotypes and establish clear evidence of allopolyploidy in 33 species of Lachemilla, showing that this condition is common and widespread in the genus. Additionally, we found evidence for three autopolyploid species. We also established multiple, independent origins of several allopolyploid species. Finally, based solely on the cpDNA phylogeny, we identified that the monotypic genus Farinopsis is the sister group of Lachemilla and allied genera within subtribe Fragariinae. CONCLUSIONS: Our study demonstrates the utility of the nuclear ribosomal DNA cistron to detect allopolyploidy when concerted evolution of this region is not complete. Additionally, with a robust chloroplast phylogeny in place, the direction of hybridization events can be established, and multiple, independent origins of allopolyploid species can be identified.

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

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

Unmasking cryptic biodiversity in polyploids: origin and diversification of Aster amellus aggregate.

Background and Aims: The origin of different cytotypes by autopolyploidy may be an important mechanism in plant diversification. Although cryptic autopolyploids probably comprise the largest fraction of overlooked plant diversity, our knowledge of their origin and evolution is still rather limited. Here we study the presumed autopolyploid aggregate of Aster amellus, which encompasses diploid and hexaploid cytotypes. Although the cytotypes of A. amellus are not morphologically distinguishable, previous studies showed spatial segregation and limited gene flow between them, which could result in different evolutionary trajectories for each cytotype. Methods: We combine macroevolutionary, microevolutionary and niche modelling tools to disentangle the origin and the demographic history of the cytotypes, using chloroplast and nuclear markers in a dense population sampling in central Europe. Key Results: Our results revealed a segregation between diploid and hexaploid cytotypes in the nuclear genome, where each cytotype represents a monophyletic lineage probably homogenized by concerted evolution. In contrast, the chloroplast genome showed intermixed connections between the cytotypes, which may correspond to shared ancestral relationships. Phylogeny, demographic analyses and ecological niche modelling supported an ongoing differentiation of the cytotypes, where the hexaploid cytotype is experiencing a demographic expansion and niche differentiation with respect to its diploid relative. Conclusions: The two cytotypes may be considered as two different lineages at the onset of their evolutionary diversification. Polyploidization led to the occurrence of hexaploids, which expanded and changed their ecological niche.

Organellar phylogenomics inform systematics in the green algal family Hydrodictyaceae (Chlorophyceae) and provide clues to the complex evolutionary history of plastid genomes in the green algal tree of life.

PREMISE OF THE STUDY: Phylogenomic analyses across the green algae are resolving relationships at the class, order, and family levels and highlighting dynamic patterns of evolution in organellar genomes. Here we present a within-family phylogenomic study to resolve genera and species relationships in the family Hydrodictyaceae (Chlorophyceae), for which poor resolution in previous phylogenetic studies, along with divergent morphological traits, have precluded taxonomic revisions. METHODS: Complete plastome sequences and mitochondrial protein-coding gene sequences were acquired from representatives of the Hydrodictyaceae using next-generation sequencing methods. Plastomes were characterized, and gene order and content were compared with plastomes spanning the Sphaeropleales. Single-gene and concatenated-gene phylogenetic analyses of plastid and mitochondrial genes were performed. KEY RESULTS: The Hydrodictyaceae contain the largest sphaeroplealean plastomes thus far fully sequenced. Conservation of plastome gene order within Hydrodictyaceae is striking compared with more dynamic patterns revealed across Sphaeropleales. Phylogenetic analyses resolve Hydrodictyon sister to a monophyletic Pediastrum, though the morphologically distinct P. angulosum and P. duplex continue to be polyphyletic. Analyses of plastid data supported the neochloridacean genus Chlorotetraëdron as sister to Hydrodictyaceae, while conflicting signal was found in the mitochondrial data. CONCLUSIONS: A phylogenomic approach resolved within-family relationships not obtainable with previous phylogenetic analyses. Denser taxon sampling across Sphaeropleales is necessary to capture patterns in plastome evolution, and further taxa and studies are needed to fully resolve the sister lineage to Hydrodictyaceae and polyphyly of Pediastrum angulosum and P. duplex.

Complete chloroplast genome of seven Fritillaria species, variable DNA markers identification and phylogenetic relationships within the genus.

Fritillaria spp. constitute important traditional Chinese medicinal plants. Xinjiang is one of two diversity hotspots in China in which eight Fritillaria species occur, two of which are endemic to the region. Furthermore, the phylogenetic relationships of Xinjiang Fritillaria species (including F. yuminensis) within the genus are unclear. In the present study, we sequenced the chloroplast (cp) genomes of seven Fritillaria species in Xinjiang using the Illumina HiSeq platform, with the aim of assessing the global structural patterns of the seven cp genomes and identifying highly variable cp DNA sequences. These were compared to previously sequenced Fritillaria cp genomes. Phylogenetic analysis was then used to evaluate the relationships of the Xinjiang species and assess the evolution of an undivided stigma. The seven cp genomes ranged from 151,764 to 152,112 bp, presenting a traditional quadripartite structure. The gene order and gene content of the seven cp genomes were identical. A comparison of the 13 cp genomes indicated that the structure is highly conserved. Ten highly divergent regions were identified that could be valuable in phylogenetic and population genetic studies. The phylogenetic relationships of the 13 Fritillaria species inferred from the protein-coding genes, large single-copy, small single-copy, and inverted repeat regions were identical and highly resolved. The phylogenetic relationships of the species corresponded with their geographic distribution patterns, in that the north group (consisting of eight species from Xinjiang and Heilongjiang in North China) and the south group (including six species from South China) were basically divided at 40°N. Species with an undivided stigma were not monophyletic, suggesting that this trait might have evolved several times in the genus.

An analysis of Echinacea chloroplast genomes: Implications for future botanical identification.

Echinacea is a common botanical used in dietary supplements, primarily to treat upper respiratory tract infections and to support immune function. There are currently thought to be nine species in the genus Echinacea. Due to very low molecular divergence among sister species, traditional DNA barcoding has not been successful for differentiation of Echinacea species. Here, we present the use of full chloroplast genomes to distinguish between all 9 reported species. Total DNA was extracted from specimens stored at the National Museum of Natural History, Smithsonian Institution, which had been collected from the wild with species identification documented by experts in the field. We used Next Generation Sequencing (NGS) and CLC Genomics Workbench to assemble complete chloroplast genomes for all nine species. Full chloroplasts unambiguously differentiated all nine species, compared with the very few single nucleotide polymorphisms (SNPs) available with core DNA barcoding markers. SNPs for any two Echinacea chloroplast genomes ranged from 181 to 910, and provided robust data for unambiguous species delimitation. Implications for DNA-based species identification assays derived from chloroplast genome sequences are discussed in light of product safety, adulteration and quality issues.