Biogeographical patterns and speciation of the genus Pinguicula (Lentibulariaceae) inferred by phylogenetic analyses.

Earlier phylogenetic studies in the genus Pinguicua (Lentibulariaceae) suggested that the species within a geographical region was rather monophyletic, although the sampling was limited or was restricted to specific regions. Those results conflicted with the floral morphology-based classification, which has been widely accepted to date. In the current study, one nuclear ribosomal DNA (internal transcribed spacer; ITS) and two regions of chloroplast DNA (matK and rpl32-trnL), from up to ca. 80% of the taxa in the genus Pinguicula, covering all three subgenera, were sequenced to demonstrate the inconsistency and explore a possible evolutionary history of the genus. Some incongruence was observed between nuclear and chloroplast topologies and the results from each of the three DNA analyses conflicted with the morphology-based subgeneric divisions. Both the ITS tree and network, however, corresponded with the biogeographical patterns of the genus supported by life-forms (winter rosette or hibernaculum formation) and basic chromosome numbers (haploidy). The dormant strategy evolved in a specific geographical region is a phylogenetic constraint and a synapomorphic characteristic within a lineage. Therefore, the results denied the idea that the Mexican group, morphologically divided into the three subgenera, independently acquired winter rosette formations. Topological incongruence among the trees or reticulations, indicated by parallel edges in phylogenetic networks, implied that some taxa originated by introgressive hybridisation. Although there are exceptions, species within the same geographical region arose from a common ancestor. Therefore, the classification by the floral characteristics is rather unreliable. The results obtained from this study suggest that evolution within the genus Pinguicula has involved; 1) ancient expansions to geographical regions with gene flow and subsequent vicariance with genetic drift, 2) acquirement of a common dormant strategy within a specific lineage to adapt a local climate (i.e., synapomorphic characteristic), 3) recent speciation in a short time span linked to introgressive hybridisation or multiplying the ploidy level (i.e., divergence), and 4) parallel evolution in floral traits among lineages found in different geographical regions (i.e., convergence). As such, the floral morphology masks and obscures the phylogenetic relationships among species in the genus.

Herbarium collection-based phylogenetics of the ragweeds (Ambrosia, Asteraceae).

Ambrosia (Asteraceae) is a taxonomically difficult genus of weedy, wind-pollinated plants with an apparent center of diversity in the Sonoran Desert of North America. Determining Ambrosia's evolutionary relationships has been the subject of much interest, with numerous studies using morphological characters, cytology, comparative phytochemistry, and chloroplast restriction site variation to produce conflicting accounts the relationships between Ambrosia species, as well as the classification of their close relatives in Franseria and Hymenoclea. To resolve undetermined intra-generic relationships within Ambrosia, we used DNA extracted from tissues obtained from seed banks and herbarium collections to generate multi-locus genetic data representing nearly all putative species, including four from South America. We performed Bayesian and Maximum-Likelihood phylogenetic analyses of six chloroplast-genome and two nuclear-genome markers, enabling us to infer monophyly for the genus, resolve major infra-generic species clusters, as well as to resolve open questions about the evolutionary relationships of several Ambrosia species and former members of Franseria. We also provide molecular data supporting the hypothesis that A. sandersonii formed through the hybridization of A. eriocentra and A. salsola. The topology of our chloroplast DNA phylogeny is almost entirely congruent with the most recent molecular work based on chloroplast restriction site variation of a much more limited sampling of 14 North American species of Ambrosia, although our improved sampling of global Ambrosia diversity enables us to draw additional conclusions. As our study is the first direct DNA sequence-based phylogenetic analyses of Ambrosia, we analyze the data in relation to previous taxonomic studies and discuss several instances of chloroplast/nuclear incongruence that leave the precise geographic center of origin of Ambrosia in question.

Phylogenetic relationships among low-ploidy species of Poa using chloroplast sequences.

Species of the genus Poa are taxonomically and genetically difficult to delineate owing to high and variable polyploidy, aneuploidy, and challenging breeding systems. Approximately 5% of the proposed species in Poa are considered to include or comprise diploids, but very few of those diploids are represented in seed collections. Recent phylogenetic studies of Poa have included some diploid species to elucidate Poa genome relationships. In this study, we build upon that foundation of diploid Poa relationships with additional confirmed diploid species and accessions, and with additional chloroplast sequences. We also include samples of P. pratensis and P. arachnifera to hone in on possible ancestral genomes in these two agronomic and highly polyploidy species. Relative to most species of Poa, Poa section Dioicopoa (P. ligularis, P. iridifolia, and P. arachnifera) contained relatively large chromosomes. Phylogenies were constructed using the TLF gene region and five additional chloroplast genes, and the placement of new species and accessions fit within chloroplast lineages previously reported better than by taxonomic subgenera and sections. Low-ploidy species in the P chloroplast lineage, such as P. iberica and P. remota, grouped closest to P. pratensis.

Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

Pleistocene glaciations, demographic expansion and subsequent isolation promoted morphological heterogeneity: A phylogeographic study of the alpine Rosa sericea complex (Rosaceae).

While most temperate plants probably underwent glacial constriction to refugia and interglacial expansion, another type of interglacial refugia might have existed to maintain alpine plants during warm periods. To test this hypothesis, we applied phylogeographic methods to 763 individuals (62 populations) which belong to 7 taxonomically difficult species of the Rosa sericea complex distributed in alpine regions of the temperate and subtropical zones in eastern Asia. We used three chloroplast (cp) DNA fragments (trnL-trnF, ndhF-rpl32 and ndhJ-trnF) approximately 3,100 bp and nuclear microsatellite (nSSR) on eight sites to determine whether cold tolerant plants experienced expansion during the Pleistocene. The neutral test and mismatch distribution analysis (MDA) indicated that whole populations and major lineages of the Qinghai-Tibet Plateau (QTP) underwent expansion during the middle to late Pleistocene. Environmental niche modeling (ENM) indicates more suitable habitats during the Last Glacial Maximum (LGM) than at present. We concluded that the demographic history of R. sericea, which diverged in the middle Pleistocene, was mostly affected by climatic oscillations instead of by geographical barriers. The low genetic divergence, as well as the weak phylogenetic structure in the R. sericea complex both support treating this complex as a single taxon.

Tertiary origin and pleistocene diversification of dragon blood tree (Dracaena cambodiana-Asparagaceae) populations in the Asian tropical forests.

BACKGROUND: The origin of extraordinarily rich biodiversity in tropical forests is often attributed to evolution under stable climatic conditions over a long period or to climatic fluctuations during the recent Quaternary period. Here, we test these two hypotheses using Dracaena cambodiana, a plant species distributed in paleotropical forests. METHODS: WE ANALYZED NUCLEOTIDE SEQUENCE DATA OF TWO CHLOROPLAST DNA (CPDNA: atpB-rbcL and trnD-trnT) regions and genotype data of six nuclear microsatellites from 15 populations (140 and 363 individuals, respectively) distributed in Indochina Peninsular and Hainan Island to infer the patterns of genetic diversity and phylogeographic structure. The population bottleneck and genetic drift were estimated based upon nuclear microsatellites data using the software programs BOTTLENECK and 2MOD. The lineage divergence times and past population dynamics based on cpDNA data were estimated using coalescent-based isolation-with-migration (IMa) and BEAST software programs. RESULTS: A significant phylogeographic structure (N ST = 0.876, G ST = 0.796, F ST-SSR = 0.329, R ST = 0.449; N ST>G ST, R ST>F ST-SSR, P<0.05) and genetic differentiation among populations were detected. Bottleneck analyses and Bayesian skyline plot suggested recent population reduction. The cpDNA haplotype network revealed the ancestral populations from the southern Indochina region expanded to northward. The most recent ancestor divergence time of D. cambodiana dated back to the Tertiary era and rapid diversification of terminal lineages corresponded to the Quaternary period. CONCLUSIONS: The results indicated that the present distribution of genetic diversity in D. cambodiana was an outcome of Tertiary dispersal and rapid divergence during the Quaternary period under limited gene flow influenced by the uplift of Himalayan-Tibetan Plateau and Quaternary climatic fluctuations respectively. Evolutionary processes, such as extinction-recolonization during the Pleistocene may have contributed to the fast diversification in D. cambodiana.

Motif analysis unveils the possible co-regulation of chloroplast genes and nuclear genes encoding chloroplast proteins.

Chloroplasts play critical roles in land plant cells. Despite their importance and the availability of at least 200 sequenced chloroplast genomes, the number of known DNA regulatory sequences in chloroplast genomes are limited. In this paper, we designed computational methods to systematically study putative DNA regulatory sequences in intergenic regions near chloroplast genes in seven plant species and in promoter sequences of nuclear genes in Arabidopsis and rice. We found that -35/-10 elements alone cannot explain the transcriptional regulation of chloroplast genes. We also concluded that there are unlikely motifs shared by intergenic sequences of most of chloroplast genes, indicating that these genes are regulated differently. Finally and surprisingly, we found five conserved motifs, each of which occurs in no more than six chloroplast intergenic sequences, are significantly shared by promoters of nuclear-genes encoding chloroplast proteins. By integrating information from gene function annotation, protein subcellular localization analyses, protein-protein interaction data, and gene expression data, we further showed support of the functionality of these conserved motifs. Our study implies the existence of unknown nuclear-encoded transcription factors that regulate both chloroplast genes and nuclear genes encoding chloroplast protein, which sheds light on the understanding of the transcriptional regulation of chloroplast genes.

Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants.

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH-psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1-92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9-79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

Preliminary survey of taxonomical problems, pharmacognostical characteristics, and chloroplast DNA polymorphisms of the folk medicinal herb Artemisia campestris from the Ryukyu Islands, Japan.

Artemisia campestris L. (Compositae) occurs naturally along the coastline of the Ryukyu Islands and has been traditionally used as a folk medicine for the treatment of liver and kidney disorders. The authors obtained specimens from the Ishigaki and Kume Islands of the Ryukyu Islands, Japan, and from the USA. A survey of the literature revealed that the Japanese name for A. campestris is Niitaka-yomogi or Riukiu-yomogi. Two distinct overall plant-form phenotypes were identified: an erect phenotype with long, upright, and straight main axis and assurgent branches; and a prostrate phenotype, having branches that are longer than the main axis and which grow along the ground. Except for the number of ray flowers, most of the flower head characters in the erect phenotypes were significantly larger than those in the prostrate phenotypes. In this experiment, the flower heads contained only small amounts of either capillarisin (<0.01-0.11 of the dry weight, % DW) and 6,7-dimethylesculetin (<0.01-0.30% DW), or none at all. DNA polymorphisms at two sites of the rpl16-rpl14 spacer region (nucleotide position 181-189 and 291-300 from the 5' end) revealed the existence of four different haplotypes. The number of adenines at nucleotide positions 291-300 appeared to be polymorphic within A. campestris from the Ryukyu Islands. Conversely, geographic differences between specimens from the Ryukyu Islands and USA manifested as a nine-base deletion at nucleotide positions 181-189. From a pharmacognostical context, the use of A. campestris flower heads as a substitute for Artemisiae capillaris Flos is not effective.

Multilevel control of organelle DNA sequence length in plants.

We have compared the length of noncoding organelle DNA spacers in a broad sample of plant species characterized by different life history traits to test hypotheses regarding the nature of the mechanisms driving changes in their size. We first demonstrate that the spacers do not evolve at random in size but have experienced directional evolutionary trends during plant diversification. We then study the relationships between spacer lengths and other molecular features and various species attributes by taking into account population genetic processes acting within cell lineages. Comparative techniques are used to test these relationships while controlling for species phylogenetic relatedness. The results indicate that spacer length depends on mode of organelle transmission, on population genetic structure, on nucleotide content, on rates of molecular evolution, and on life history traits, in conformity with predictions based on a model of intracellular competition among replicating organelle genomes.

Prokaryote phylogeny meets taxonomy: an exhaustive comparison of composition vector trees with systematic bacteriology.

We perform an exhaustive, taxon by taxon, comparison of the branchings in the composition vector trees (CVTrees) inferred from 432 prokaryotic genomes available on 31 December 2006, with the bacteriologists' taxonomy--primarily the latest online Outline of the Bergey's Manual of Systematic Bacteriology. The CVTree phylogeny agrees very well with the Bergey's taxonomy in majority of fine branchings and overall structures. At the same time most of the differences between the trees and the Manual have been known to biologists to some extent and may hint at taxonomic revisions. Instead of demonstrating the overwhelming agreement this paper puts emphasis on the biological implications of the differences.

Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii.

Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA RPL16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA RPL16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.

Phylogenetic relationships in Elymus (Poaceae: Triticeae) based on the nuclear ribosomal internal transcribed spacer and chloroplast trnL-F sequences.

To estimate the phylogenetic relationship of polyploid Elymus in Triticeae, nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL-F sequences of 45 Elymus accessions containing various genomes were analysed with those of five Pseudoroegneria (St), two Hordeum (H), three Agropyron (P) and two Australopyrum (W) accessions. The ITS sequences revealed a close phylogenetic relationship between the polyploid Elymus and species from the other genera. The ITS and trnL-F trees indicated considerable differentiation of the StY genome species. The trnL-F sequences revealed an especially close relationship of Pseudoroegneria to all Elymus species included. Both the ITS and trnL-F trees suggested multiple origins and recurrent hybridization of Elymus species. The results suggested that: the St, H, P, and W genomes in polyploid Elymus were donated by Pseudoroegneria, Hordeum, Agropyron and Australopyrum, respectively, and the St and Y genomes may have originated from the same ancestor; Pseudoroegneria was the maternal donor of the polyploid Elymus; and some Elymus species showed multiple origin and experienced recurrent hybridization.

Adaptive evolution of chloroplast genome structure inferred using a parametric bootstrap approach.

BACKGROUND: Genome rearrangements influence gene order and configuration of gene clusters in all genomes. Most land plant chloroplast DNAs (cpDNAs) share a highly conserved gene content and with notable exceptions, a largely co-linear gene order. Conserved gene orders may reflect a slow intrinsic rate of neutral chromosomal rearrangements, or selective constraint. It is unknown to what extent observed changes in gene order are random or adaptive. We investigate the influence of natural selection on gene order in association with increased rate of chromosomal rearrangement. We use a novel parametric bootstrap approach to test if directional selection is responsible for the clustering of functionally related genes observed in the highly rearranged chloroplast genome of the unicellular green alga Chlamydomonas reinhardtii, relative to ancestral chloroplast genomes. RESULTS: Ancestral gene orders were inferred and then subjected to simulated rearrangement events under the random breakage model with varying ratios of inversions and transpositions. We found that adjacent chloroplast genes in C. reinhardtii were located on the same strand much more frequently than in simulated genomes that were generated under a random rearrangement processes (increased sidedness; p < 0.0001). In addition, functionally related genes were found to be more clustered than those evolved under random rearrangements (p < 0.0001). We report evidence of co-transcription of neighboring genes, which may be responsible for the observed gene clusters in C. reinhardtii cpDNA. CONCLUSION: Simulations and experimental evidence suggest that both selective maintenance and directional selection for gene clusters are determinants of chloroplast gene order.

CVTree: a phylogenetic tree reconstruction tool based on whole genomes.

Composition Vector Tree (CVTree) implements a systematic method of inferring evolutionary relatedness of microbial organisms from the oligopeptide content of their complete proteomes (http://cvtree.cbi.pku.edu.cn). Since the first bacterial genomes were sequenced in 1995 there have been several attempts to infer prokaryote phylogeny from complete genomes. Most of them depend on sequence alignment directly or indirectly and, in some cases, need fine-tuning and adjustment. The composition vector method circumvents the ambiguity of choosing the genes for phylogenetic reconstruction and avoids the necessity of aligning sequences of essentially different length and gene content. This new method does not contain 'free' parameter and 'fine-tuning'. A bootstrap test for a phylogenetic tree of 139 organisms has shown the stability of the branchings, which support the small subunit ribosomal RNA (SSU rRNA) tree of life in its overall structure and in many details. It may provide a quick reference in prokaryote phylogenetics whenever the proteome of an organism is available, a situation that will become commonplace in the near future.

Assessing hybridization in natural populations of Penstemon (Scrophulariaceae) using hypervariable intersimple sequence repeat (ISSR) bands.

Inferences regarding hybridization rely on genetic markers to differentiate parental taxa from one another. Intersimple sequence repeat (ISSR) markers are based on single-primer PCR reactions where the primer sequence is derived from di- and trinucleotide repeats. These markers have successfully been used to assay genetic variability among cultivated plants, but have not yet been tested in natural populations. We used genetic markers generated from eight ISSR primers to examine patterns of hybridization and purported examples of hybrid speciation in Penstemon (Scrophulariaceae) in a hybrid complex involving P. centranthifolius, P. grinnellii, P. spectabilis and P. clevelandii. This hybrid complex has previously been studied using three molecular data sets (allozymes, and restriction-site variation of nuclear rDNA and chloroplast DNA). These studies revealed patterns of introgression involving P. centranthifolius, but were unsuccessful in determining whether gene flow occurs among the other species, and support for hypotheses of diploid hybrid speciation was also lacking. In this study, we were able to fingerprint each DNA accession sampled with one to three ISSR primers and most accessions could be identified with a single primer. We found population- and species-specific markers for each taxon surveyed. Our results: (i) do not support the hybrid origin of P. spectabilis; (ii) do support the hypothesis that P. clevelandii is a diploid hybrid species derived from P. centranthifolius and P. spectabilis; and (iii) demonstrate that pollen-mediated gene flow via hummingbird vectors is prevalent in the hybrid complex.