trnL-trnF Intergenic Spacer Research Articles

Indel-K2P: a modified Kimura 2 Parameters (K2P) model to incorporate insertion and deletion (Indel) information in phylogenetic analysis

ABSTRACT Evolutionary models are widely used to estimate the evolutionary relationship between the various biological taxa . Majority of the existing evolutionary models ignore insertion and deletion (indel) event, which reduces the computational difficulties, but at the same time, it causes loss of valuable phylogenetic information. In this study, we have developed a modified Kimura 2 parameters (K2P) model to utilise the indel information in phylogenetic tree construction especially in case of low substitution rate sequences Indel rich highly similar DNA sequences (trnL-trnF intergenic spacer region) of Phalaenopsis were collected from Genbank and to test the efficiency of the model in phylogenetic. Our developed Indel-K2P model and K2P model were implemented in MATLAB and calculated the bootstrap value and Robinson-Foulds (RF) distance to test the confidence and accuracy of phylogenetic tree topology. The phylogenetic trees constructed within the framework of Indel-K2P model, exhibited 92.66% accuracy and 40.9% root node confidence while the K2P model without indel shows less accuracy and less root node confidence. Our results indicate that modified Kimura 2 parameters with indel (Indel-K2P) model performed better than Kimura 2 Parameters (K2P) in phylogenetic tree construction for indel rich DNA sequences.

Rapid identification of Colchicum autumnale based on loop-mediated isothermal amplification (LAMP) assay

Colchicum autumnale (Colchicaceae) exhibits toxicity, and severe poisoning cases due to ingestion of this plant have been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of the cause of poisoning. In the present study, we developed a rapid and simple method for the detection of C. autumnale based on a loop-mediated isothermal amplification (LAMP) assay, which is 100- to 1000-fold higher than the conventional polymerase chain reaction method. Specific LAMP primers for C. autumnale were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay was performed at an isothermal reaction temperature of 63 ℃. The LAMP reaction was shown to be specific and highly sensitive to C. autumnale, given that the assay can be used for 10 pg of purified DNA. Using a simple protocol for on-site detection, the entire procedure from pretreatment to evaluation required approximately 1 h. Moreover, the LAMP method using a microfluidic device detected multiple genes, including C. autumnale-specific DNA regions, at a time. The currently proposed protocol exhibits good potential as a screening method for C. autumnale poisoning in emergency medical care.

An Elegy to Rangaeris, Including a Description of Two New Genera in the Cyrtorchis–Tridactyle Clade (Orchidaceae, Angraecinae)

The phylogenetic position of Rangaeris (Schltr.) Summerh. has been one of the most problematic issues of angraecoid orchid taxonomy. A recently published phylogenetic tree with nearly comprehensive taxon sampling of African angraecoid orchids suggested that this genus was polyphyletic, as its species appear nested within different subclades of the Cyrtorchis–Tridactyle clade. However, the lack of DNA data for the generic type, R. muscicola (Rchb. f.) Summerh., and for the little-known species R. longicaudata (Rolfe) Summerh. and R. trilobata Summerh., has precluded an integrative reappraisal of the delimitation of this genus. Here we present the results of a new phylogenetic analysis of one nuclear (ITS) and five plastid markers (matK, rps16, trnC-petN intergenic spacer, trnL-trnF intergenic spacer, ycf1) for all six species of Rangaeris, including those recently transferred to Podangis Schltr. and Ypsilopus Summerh., together with a representative sampling of 14 other species of the genera Cyrtorchis Schltr., Listrostachys Rchb. f., Plectrelminthus Raf., Summerhayesia P. J. Cribb, Tridactyle Schltr., and Ypsilopus. Based on molecular and morphological evidence, R. muscicola is transferred to Podangis, where Rangaeris is reduced to the rank of section. In addition, R. longicaudata and R. trilobata are each moved to new monotypic genera, Planetangis Stévart & Farminhão and Aziza Farminhão & D’haijère, respectively. An identification key for all genera of the Cyrtorchis–Tridactyle clade is presented, together with one for the three species of recircumscribed Podangis. Finally, taxonomic accounts of the new genera are presented, including amended descriptions, illustrations, distribution maps, and preliminary conservation status assessments of their species.

Biogeography, phylogenetic relationships and morphological analyses of the South American genus Mutisia L.f. (Asteraceae) shows early connections of two disjunct biodiversity hotspots

The Andes is recognized as one of the most biodiverse places on Earth, promoting in its uplift process a series of recent rapid diversification events in different biotic groups like birds, mammals, insects and vascular plants. The uplift of the Andes during the Cenozoic acted as a barrier for many biotic groups, as a scenario for radiation processes due to occupancy of different niches and as a corridor for others. Connections between the Andes and the Atlantic Forest showed intermittent phases along the Cenozoic, affecting the distribution patterns and diversification of different biotic groups. Nowadays, the Andes and the Atlantic Forest are both considered globally relevant biodiversity hotspots. Floristic groups thriving in both hotspots are crucial for a better understanding of their biogeographic history, as well as for informing future conservation actions. Mutisia (Asteraceae), a genus comprising 63 perennial shrubs and vines endemic to South America, shows a marked West-East disjunction: Most species occupy almost the whole Andean chain from Colombia to Patagonia, while a second group encompasses four species distributed in eastern Brazil and the surrounding areas of Paraguay, Uruguay and Argentina. We reconstructed the phylogeny of the genus to assess its possible biogeographic history. We analysed three DNA regions, i.e. the chloroplast trnL-trnF intergenic spacer and the nuclear ribosomal internal and external transcribed spacers, ITS and ETS. Using maximum likelihood and Bayesian inference, gene trees were reconstructed, and a concatenated phylogenetic tree was inferred. Divergence times were estimated by means of BEAST, and the ancestral areas were inferred using BioGeoBEARS. An ancestral reconstruction of morphological traits was also performed, as well as maps representing current richness hotspots within the genus. Phylogenetic analyses strongly support the monophyly of Mutisia, with two well-supported main clades: clade A, with presence of Atlantic-central-northern Andes species, and clade B, with central/southern Andes species. Dating analyses suggest that a main clade separation occurred at the early Miocene, followed by the separation of the Atlantic clade A2 by the late Miocene, and more recent radiations occurred in the central, northern and southern Andes during the Pliocene. Results are in tune with other angiosperm taxa that also underwent rapid radiations, possibly related to environmental and pollinator changes. The biogeographic history of Mutisia is related to morphological adaptations, history and geographic factors acting since the Miocene along the Andes and adjacent areas. Threat assessments and conservation actions for the genus shall include the whole distribution range, including low-range northern and southern Andes species, as well as the distinctive Atlantic Forest clade.

Molecular identification and analysis of Psidium guajava L. from indigenous tribes of Taiwan

Psidium guajava L. is a perennial fruit tree in subtropical and tropical areas. In Taiwan, P. guajava has been used as anthro-pharmacological plants by aboriginal people to treat acute diarrhea, cough and intestinal spasmodic diseases. The classification and functional identification of P. guajava remains unsolved these days. In this study, molecular markers 18S rRNA, internal transcribe spacer (ITS) region of ribosomal DNA, trnL intron and trnL-trnF intergenic spacer (IGS) of chloroplast DNA (cpDNA), and random amplified polymorphic DNA (RAPD) were used for the molecular identification of 18 P. guajava samples from different indigenous tribes, 2 from non-indigenous tribe, and 12 commercial cultivars from markets in Taiwan. Molecular methods restricted fragment length polymorphism (RFLP) and denatured gradient gel electrophoresis (DGGE) are found time-consuming and less efficient as compared to RAPD, thus are not suitable for samples of high homology. In this study, 18S rDNA, ITS and cpDNA trnL intron and trnL-trnK intergenic spacer were also tested molecular marker; however, results analyzed by molecular algorithm UPGMA, Neighbor-Joining, Parsimony or Maximum likelihood showed no discriminations (data not shown). On the other side, ten 10-mer oligonucleotide primers were used in RAPD to amplify the specific genes from 32 guava samples. Four primers, OPB 17, OPG 6, OPY 15 and OPY 18, were able to direct the amplification and yielded a total of 82 polymorphic RAPD patterns. Thirty-two genotypes on the dendrogram were identified and were divided into two major groups, the uncultivated and commercial cultivars. Based on the cluster analysis, the red-flesh Psidium samples that were believed to have high medical function were grouped independently. The results suggest that RAPD is useful for the discrimination of uncultivated, cultivars and potential Psidium of high economy.

Differentiation of medicinal Dendrobium species (Orchidaceae) using molecular markers and scanning electron microscopy

Dendrobium species have long been used as functional food supplements and herbal medicines in Asia. However, inappropriate usage of the Dendrobium species variants is rampant because of the considerable differences in the cost of the variants. Furthermore, the similar appearance of the dried plants of the Dendrobium species makes it difficult to discriminate among the individual members. In this study, simple and sensitive methods based on molecular and morphological studies were developed to verify authenticity of the Dendrobium species used in the preparation of medicines, particularly that of the most expensive variety, D. huoshanense. Molecular and anatomical differences among the 8 commonly used Dendrobium species (6 used for medicinal purposes and 2 for ornamental purposes) were studied. The ribosomal DNA internal transcribed spacer (ITS), chloroplast DNA trnL intron, and the trnL-trnF intergenic spacer (IGS) of the DNA of the 8 species were sequenced and compared. The comparison results highlighted considerable differences between the IGS region of D. huoshanense and that of other Dendrobium species to enable a clear distinction between them. A novel primer set was designed to specifically amplify the DNA of D. huoshanense. The leaf and stem morphologies of the 8 Dendrobium species were also studied by scanning electron microscopy (SEM). Granular mucilage and acicular grains in the vascular bundles were present only in the medicinal Dendrobium species but not in the ornamental ones.

Lapaea (Plantaginaceae, Gratioleae), a new genus endemic to the Espinhaço Range (Brazil) with a remarkable red-flowered new species

Stemodia sensu lato is one of the largest genera of the Gratioleae (Plantaginaceae). Recent phylogenetic studies corroborated the paraphyly of Stemodia in its traditional circumscription and suggested the segregation of S. stellata from this genus. This species belongs to a morphologically cohesive group along with S. cipoensis, S. harleyi and S. lobata, all endemic to the Espinhaço Range, eastern Brazil. In this study, we tested the monophyly and interspecific relationships of this group by reconstructing its phylogeny, based on sequences of three cpDNA regions (rps16 and trnL introns and the trnL–trnF intergenic spacer), and one nrDNA region (ITS1 spacer), using Maximum parsimony and Bayesian inference. The monophyly of the 'S. stellata group' was confirmed, and its segregation from Stemodia corroborated. We propose Lapaea as a new genus to accommodate its species, with subsequent four new combinations: L. cipoensis, L. harleyi, L. lobata, and L. stellata. Additionally, we describe and illustrate L. rubriflora, a remarkable red-flowered new species, based on morphological and micromorphological evidence. Lastly, a taxonomic revision of the species recognized in Lapaea is presented, providing an identification key, photos, descriptions, distribution map and notes on their morphology, geographic distribution, habitat, ecology, and conservation status. Lapaea is restricted to the Espinhaço Range comprising five threatened species, each of them with a narrow distribution and phylogenetic relationships consistent with biogeographic provinces.

<p><strong>The non-monophyly of <em>Dasymaschalon dasymaschalum</em> (Annonaceae) revealed by a plastid DNA phylogeny, with <em>D. halabalanum</em> sp. nov. from Thailand and <em>D. argenteum</em> comb. nov.</strong></p>

An extended molecular phylogeny of the genus Dasymaschalon (Annonaceae) has been reconstructed using up to six plastid DNA regions (matK, ndhF, rbcL exons; trnL intron; psbA-trnH, trnL-trnF intergenic spacers). The results unraveled the non-monophyly of a widely distributed D. dasymaschalum. A lineage of D. dasymaschalum native to Java and cultivated at Bogor Botanical Garden represents the true D. dasymaschalum, whereas the name Pelticalyx argentea is applicable to a distantly related clade of D. dasymaschalum from mainland Asia. Dasymaschalon argenteum comb. nov. is accordingly made. Additionally, the true D. dasymaschalum has been retrieved as the sister group of D. halabalanum, a new species from Narathiwat Province, southern Thailand herein described. Pedicel length, petal size and color, and the number of stamens per flower principally distinguish the new species from its sister group.

Generic status of Winitia (Annonaceae, Miliuseae) reaffirmed by molecular phylogenetic analysis, including a new species and a new combination from Thailand

The generic status of Winitia Chaowasku (Annonaceae Juss., Miliuseae Hook.f. & Thomson) is reaffirmed by an extensive phylogenetic reconstruction using seven plastome regions (matK, ndhF, rbcL, ycf1 exons; trnL intron; psbA-trnH, trnL-trnF intergenic spacers) and including, among others, seven accessions of Winitia plus two accessions of its sister group, Stelechocarpus Hook.f. & Thomson. The results disclosed a maximally supported clade of Winitia, as well as of Stelechocarpus. The sister relationship of the two genera is still rather poorly supported and the branch uniting them is very short, whereas the branches leading to Winitia and to Stelechocarpus are relatively long, corresponding to their considerable morphological differences. Additionally, in Miliuseae there is a particular indel of eight continuous base pairs in the trnL-trnF intergenic spacer potentially diagnostic for generic discrimination, i.e., members in the same genus possess the same indel structure (absence or presence of a gap), and this indel differentiates Winitia from Stelechocarpus. Winitia cauliflora (Scheff.) Chaowasku appears polyphyletic and Winitia longipes (Craib) Chaowasku & Aongyong comb. nov. based on Stelechocarpus longipes Craib is consequently proposed. Furthermore, our phylogenetic data support a new species, Winitia thailandana Chaowasku & Aongyong sp. nov. from southern Thailand, which is described and illustrated. A key to genera in the sageraeoid clade (Sageraea-Winitia-Stelechocarpus) and a key to the four species of Winitia are provided.

Contradictory between morphology and phylogenetic trees of Orthosiphon spp. (Lamiaceae) from Indonesia

The nuclear ribosomal Internal Transcribed Spacer region (ITS), and three chloroplast loci (trnL-trnF, rps16 and trnL) were also carried out in this study. Morphological analysis of leaves, stems, and flowers is used to identify visual differences. This study was the purpose of analyzed morphological and phylogenetic relationships among ten taxa of Orthosiphon from the analysis of DNA sequences. The results that the difference in leaves, flowers and stems is very striking in seven taxa of Orthosiphon aristatus (OGP and OGW) and O. endanghidayatae (OWW, OG + W and OW + W). The phylogenetic trees constructed from cpDNA (trnL gene, trnL-trnF intergenic spacer region, and rps16 region) of using NJ methods among taxa of Orthosiphon aristatus and O. endanghidayatae collected from Indonesia showed constant topologies with high bootstrap values (BS 86% and 100%, respectively), but were different from those ITS of nrDNA that the phylogenetic trees supported low bootstrap values (65% by NJ). Although differing from morphology to species of Orthosiphon spp. but still similar in DNA analysis. The results of this study indicate that the speciation process of O. endanghidayatae (OWW, OG + W and OW + W) is as a process of morphological changes faster than the process of genetic change.

The phylogenetic position of Aglaodorum Schott (Araceae – Aroideae – Aglaonemateae)

Analysis of chloroplast DNA sequences (trnL intron and trnLtrnF intergenic spacer) of the helophytic colony-forming Aglaodorum griffithii (Schott) Schott and other representative species of Araceae supported its placement within Aglaonema Schott. It is therefore proposed that Aglaodorum Schott should be recognised as a generic synonym of Aglaonema.

Evidence of hybridization between Galatella villosa and G. linosyris, and a taxonomic reappraisal of the hybrid G. ×subvillosa

At the westernmost distribution of the steppe herbaceous plant, Galatella villosa, in Hungary, Serbia and Ukraine, we recently observed intermediate specimens between this species and its close relative, G. linosyris. We were able to demonstrate the hybrid origin of these individuals by sequencing the biparentally inherited nuclear ribosomal internal transcribed spacer (nrITS) region and checking additive polymorphism in the hybrids. In addition, examination of the maternally inherited plastid regions (trnH-psbA and trnL-trnF intergenic spacers) revealed that G. villosa is likely to be the maternal parent in the Hungarian and Ukrainian populations and G. linosyris in the Serbian population. The intermediate forms produced only sterile seeds. The alleged hybrid between the above two species has already been described as G. ×subvillosa based on a very brief diagnosis. Still, the analysis of the morphological characters using linear discriminant analyses clearly separated the holotype of G. ×subvillosa from individuals of G. linosyris × G. villosa. The latter appeared to be morphologically intermediate between populations of G. villosa and G. linosyris. Contrary to the originally stated hybrid origin of the type plants of G. ×subvillosa, morphological evidence indicates the involvement of G. divaricata not G. linosyris. The hybrid G. linosyris × G. villosa is thus described here, as a new nothospecies G. ×feketegaborii. This study highlights the power of easily available molecular phylogenetic tools for demonstrating the hybrid origin of plants and illustrates how additive polymorphism can be distinguished from other types of intraindividual polymorphism in nuclear DNA sequences.

Genetic Diversity, Phylogeography and Population gene flow of Tunisian Pistaciavera L.

The aim of this paper was to determine how the pistachio trees evolve, and the evolving factors that influence pistachio population establishment. For that, we investigated pistachio genetic structure from some regions of Tunisia, by sequencing two noncoding chloroplastic regions (trnL (UAA) and trnL-trnF intergenic spacer). We found strong genetic diversity among groups, with the absence of high differentiation between population pairs. A deep phylogeographical break separated two major clusters: "El-Guetar and "Gafsa/Sidi-Bouzid. This conclusion is proved by the haplotype networks, the phylogenetic trees, and the molecular variance analysis. Different interpretations were proposed to explain this cytoplasm dimorphism, based on the molecular evolution and demographic history analysis: (1) the domestication events, which are very important to understand the variability between the prospective areas, (2) the gene flow between them, a process that occurs both in time and in space through pollen and seeds, and strongly interacts with the local farming systems, (3) the geographical barriers that exist, which limit gene flow transfer and make particular climatic conditions of the El-Guetar oasis. Given that, the genetic diversity study within Tunisian pistachio cultivars is very useful to contribute to the national management effort for the improvement and conservation of pistachio genetic resources. Moreover, in 2017, Tunisia ranked among the top 10 countries in global pistachio production. Thus, studying the diversity of Tunisian pistachio can make an important impact on global production.

Analysis of matK, rbcL and trnL-trnF Intergenic Spacer Sequences on Durik-Durik (Syzygium sp)

Syzygium sp. with the local name of durik-durik, that grows in Riau Province, Indonesia, is one of many adapted plants to flooding stress in floodplain ecosystems, and plays a role for survival of the ecosystems. This study reports analysis of three DNA barcodes, i.e. matK, rbcL and trnL-trnF intergenic spacer on this plant. The fresh leaves of this plant were picked up from floodplain ecosystem in Pelalawan, Riau Province for DNA extraction requiring. In this study had been obtained DNA sequences of matK, rbcL and trnL-trnF intergenic spacer with the size of 686 bp, 641 bp and 828 bp, respectively. The BLASTn analysis based on those sequences showed that none of the accessions of the Syzygium genus were 100% similar to Syzygium sp. The phylogenetic tree showed that Syzygium sp. formed one group with the same species of Syzygium and separated from other genera in Myrtaceae family. These results confirmed that durik-durik was a member Syzygium. Those sequences had been deposited in GenBank and could be used for molecular identification of this plant.

On the Issue of Hybridogenic Origin of Bidens × decipiens Warnst.

The hybrid origin of the Bidens × decipiens species, which was earlier ascribed to the North American alien species B. connata, was confirmed by analyzing the nucleotide sequences of the ITS nuclear site. The name “B. connata” is indicated as invalid for a taxon that does not grow in North America. Earlier, we tested the hypothesis of hybrid origin of B. × decipiens using ISSR analysis and revealed that B. × decipiens can be considered as a complex of hybrids and backcrosses of B. cernua and B. frondosa. Analysis of the Internal transcribed spacer (ITS) and the trnL-trnF intergenic spacer confirmed the hybrid origin of this taxon and made it possible to establish that B. cernua is the maternal species and B. frondosa with a high probability is the paternal species of the taxon.

Dual colonization of European high-altitude areas from Asia by Callianthemum (Ranunculaceae)

Floristic similarities between European and Asian mountain ranges have long been recognized, and the hypothesis that European mountain plant taxa immigrated from Asian mountain areas has been confirmed by several molecular phylogenetic analyses. Callianthemum contains ca. 14 species, of which ca. 11 are distributed in Asia and three in Europe. A molecular phylogenetic analysis of the genus using ITS and four plastid DNA sequences (matK, rpL32–trnL intergenic spacer, trnL–trnF intergenic spacer, trnV–ndhC intergenic spacer) revealed that the genus reached Europe twice, with C. coriandrifolium representing one, and C. anemonoides and C. kernerianum a second lineage. Support for C. anemonoides and C. kernerianum as sister species is weak. The crown group ages of the C. coriandrifolium lineage (median 2.2 million years) and of the C. anemonoides/C. kernerianum lineage (median 1.62 million years) are similar and place their immigration to Europe in the Quaternary. Analysis of climatic data shows that C. coriandrifolium grows in colder climate than C. anemonoides/C. kernerianum and that the climatic niches of the three European species are different considering all climatic variables analyzed.

The invasion of an annual exotic plant species affects the above- and belowground plant diversity in deciduous forests to a different extent

Invasive plant species can significantly affect native biodiversity and ecosystem functioning. Even though the majority of ecosystems have more than 50% of the plant biomass belowground, most studies investigating the effects of invasive species on plant diversity focus on the aboveground vegetation. DNA-based methods allow the determination of belowground plant structures. Using these techniques, we examined potential effects of the annual invasive plant Impatiens glandulifera on both the above- and belowground plant species richness and composition in mixed deciduous forests in Northwestern Switzerland. We established 24 plots in three forest areas invaded by I. glandulifera and in three adjacent forest areas, which were not yet invaded. In each plot, we determined plant species richness and abundance in the aboveground vegetation, and collected soil samples at depths of 0–10 cm and 11–20 cm to determine belowground plant species richness. We extracted DNA from fine roots in the soil samples and applied the FAFLP technique (fluorescent amplified fragment length polymorphism) for two different regions of the chloroplast DNA (trnL-trnF intergenic spacer and P6 loop). We established a reference library for all plant species occurring in the study areas to identify the species present in mixed-root samples. Our results showed that I. glandulifera caused shifts in both the above- and belowground plant species composition. Plant species richness was reduced by 30% aboveground in invaded plots, but not belowground in the same plots. Many geophytes and woody species were found belowground but not aboveground in invaded plots. Root biomass was reduced by 35–55% in invaded plots, most probably due to allelopathic compounds released by the invasive plant into the soil. Our field survey shows that above- and belowground plant communities respond differently to the invasion of an annual plant species, and that the invasive species can negatively affect forest ecosystem functions, by reducing root biomass and altering plant species richness and composition.

A Useful Technical Application of the Identification of Nucleotide Sequence Polymorphisms and Gene Resources for Cinnamomum osmophloeum Kaneh. (Lauraceae)

The plant genus Cinnamomum contains economically important evergreen aromatic trees and shrubs belonging to the laurel family, Lauraceae. Our study tree species Cinnamomum osmophloeum Kaneh. (CO) has high economic value in Taiwan. The present study attempts to identify the gene resources of Cinnamomum osmophloeum Kaneh. by analyzing the nucleotide sequences of the partial noncoding internal transcribed spacer 2 (pITS2) of the ribosomal DNA and the trnL-trnF chloroplast genome. Seventy-three geographical strains of Cinnamomum osmophloeum, preserved in the Lien Hua-Chin Research Center of the Forestry Research Institute and the Hua-Lin Forestry Center of Chinese Culture University, were collected and analyzed by PCR amplification and DNA sequencing to study the genetic diversity and nucleotide sequence polymorphisms of the tested specimens. Our results allowed us to accurately identify the lineage of Cinnamomum osmophloeum and to conclude that the strains belonging to the Lien Hua-Chin Research Center had much higher genetic diversity than those preserved in the Hua-Lin Forestry Center. Multiple sequence alignments demonstrated that the variability of the nucleotide sequence polymorphisms for the pITS2 region was higher than those of the trnL intron and trnL-trnF intergenic spacer (IGS) regions among the 73 tested specimens of Cinnamomum osmophloeum. Cluster analyses, using the neighbor-joining and maximum parsimony methods, for the 73 tested geographical strains of Cinnamomum osmophloeum and species of Cinnamomum registered in the GenBank and EMBL databases were performed to demonstrate the genus and species distribution of the samples. Here, we describe the use of pITS2 polymorphisms as a genetic classifier and report the establishment of a DNA sequence database for CO gene resource identification. The sequence database described in this study can be used to identify CO specimens at the inter- or intraspecies level using pITS2 DNA sequences, which illustrates its value in gene resource identification. Our study results can be used further for correctly identifying the true Cinnamomum osmophloeum Kaneh.

Molecular Phylogeography and Ecological Niche Modeling of Sibbaldia procumbens s.l. (Rosaceae).

The phylogeographical analysis and ecological niche modeling (ENM) of the widely distributed Northern Hemisphere Sibbaldia procumbens s.l. can help evaluate how tectonic motion and climate change helped shape the current distribution patterns of this species. Three chloroplast regions (the atpI-atpH and trnL-trnF intergenic spacers and the trnL intron) were obtained from 332 (156 from present study and 176 from the previous study) individuals of S. procumbens s.l. An unrooted haplotype network was constructed using the software NETWORK, while BEAST was used to estimate the divergence times among haplotypes. ENM was performed by MAXENT to explore the historical dynamic distribution of S. procumbens s.l. The haplotype distribution demonstrates significant phylogeographical structure (NST > GST; P < 0.01). The best partitioning of genetic diversity by SAMOVA produced three groups, while the time to the most recent common ancestor of all haplotypes was estimated to originate during the Miocene, with most of the haplotype diversity having occurred during the Quaternary. The MAXENT analysis showed S. procumbens s.l. had a wider distribution range during the last glacial maximum and a narrower distribution range during the last interglacial, with predictions into the future showing the distribution range of S. procumbens s.l. shrinking.

Ecological, phylogenetical, and pharmacognostical characteristics of Aconitum kiyomiense endemic to Hida highlands, Takayama city, Gifu Prefecture, Japan.

Aconitum kiyomiense Kadota (Ranunculaceae) is endemic to Takayama city, Gifu Prefecture, central Japan. We collected specimens from marshes and flood plains at altitudes ranging from 852 to 1085m and from a new habitat consisting of a mesic meadow in the subalpine belt (1681m). Glabrous pedicels and flowering sequence of inflorescence were used for identification, but intra-species variations in the pilus of pedicels (glabrous, pilose, and chimeric types) were observed. Although the flowering sequence has been reported as both indeterminate and determinate, all specimens in the present study were determinate. No intra-species variation was detected via partial nuclear internal transcribed space, and sequences did not match another 17 East Eurasian continent subgenus Aconitum species. The chloroplast trnL-trnF intergenic spacer region (trnL-trnF) showed three different haplotypes. The trnL-trnF dominant haplotype sequence was identical to that of A. kusnezoffii growing on the Eurasian continent, suggesting that A. kiyomiense is more primitive than other Japanese aconitum and a relic species of the Eurasian continent. We report the first detection of aconitine alkaloids in the tuberous roots, which exhibited aconitine alkaloid contents varying from 0.32 to 4.05mg/g dry weight (mg/g) for aconitine, 0.02 to 4.12mg/g for hypaconitine, undetectable to 0.05mg/g for jesaconitine, and 0.42 to 3.76mg/g for mesaconitine. The variation of aconitine alkaloid components and contents appeared to be random and did not vary with inflorescence phenotype, trnL-trnF haplotype, environmental habitat conditions, or the geographic region of the collection sites. Since most populations showed no genetic intra-variation, it will be necessary to maintain the continuity of habitats and designate areas for conservation of genetic diversity at the population level.