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The plastid genome has proven to be an effective tool for examining deep correlations in plant phylogenetics, owing to its highly conserved structure, uniparental inheritance, and limited variation in evolutionary rates. Iridaceae, comprising more than 2,000 species, includes numerous economically significant taxa that are frequently utilized in food industries and medicines and for ornamental and horticulture purposes. Molecular studies on chloroplast DNA have confirmed the position of this family in the order Asparagales with non-asparagoids. The current subfamilial classification of Iridaceae recognizes seven subfamilies—Isophysioideae, Nivenioideae, Iridoideae, Crocoideae, Geosiridaceae, Aristeoideae, and Patersonioideae—which are supported by limited plastid DNA regions. To date, no comparative phylogenomic studies have been conducted on the family Iridaceae. We assembled and annotated (de novo) the plastid genomes of 24 taxa together with seven published species representing all the seven subfamilies of Iridaceae and performed comparative genomics using the Illumina MiSeq platform. The plastomes of the autotrophic Iridaceae represent 79 protein-coding, 30 tRNA, and four rRNA genes, with lengths ranging from 150,062 to 164,622 bp. The phylogenetic analysis of the plastome sequences based on maximum parsimony, maximum likelihood, and Bayesian inference analyses suggested that Watsonia and Gladiolus were closely related, supported by strong support values, which differed considerably from recent phylogenetic studies. In addition, we identified genomic events, such as sequence inversions, deletions, mutations, and pseudogenization, in some species. Furthermore, the largest nucleotide variability was found in the seven plastome regions, which can be used in future phylogenetic studies. Notably, three subfamilies—Crocoideae, Nivenioideae, and Aristeoideae—shared a common ycf2 gene locus deletion. Our study is a preliminary report of a comparative study of the complete plastid genomes of 7/7 subfamilies and 9/10 tribes, elucidating the structural characteristics and shedding light on plastome evolution and phylogenetic relationships within Iridaceae. Additionally, further research is required to update the relative position of Watsonia within the tribal classification of the subfamily Crocoideae.

Endemic plants are important for understanding phylogenetic relationships, biogeographical history, and genetic variation because of their restricted distribution and their role in conserving biodiversity. Here, we investigated the phylogenetic relationships of the Korean endemic Fraxinus chiisanensis by reconstructing the molecular phylogeny of Fraxinus based on two nuclear DNA (nrITS and phantastica) and two chloroplast DNA (psbA-trnH and rpl32-trnL) regions. Within our fossil-calibrated phylogenetic framework, we also inferred the biogeographical history of F. chiisanensis. To provide a scientific basis for the conservation of F. chiisanensis, we determined the levels of genetic diversity and genetic differentiation in this species. Combining information from nuclear and chloroplast DNA sequence data, our molecular phylogenetic analyses identified F. chiisanensis as a genetically distinct unit from its sister group, Fraxinus platypoda from Japan. Our molecular dating analyses using nuclear and chloroplast DNA data sets show F. chiisanensis diverged from its sister F. platypoda in the Early or Middle Miocene and differentiated in the Late Miocene on the Korean Peninsula. Our results suggest that the divergence of F. chiisanensis was associated with the submergence of the East China Sea land bridge and enhanced monsoons in East Asia. When compared to F. platypoda, F. chiisanensis exhibits low genetic diversity within populations and high genetic differentiation among populations. These results help us to understand the evolutionary history of F. chiisanensis and to develop a conservation strategy for this species.

Understanding of intercontinental distribution in the Northern Hemisphere has attracted a lot of attention from botanists. However, although Orchidaceae is the largest group of angiosperms, biogeographical studies on the disjunctive pattern have not been sufficient for this family. Goodyera R. Br. (tribe Cranichideae, subfamily Orchidoideae, family Orchidaceae) is widely distributed in temperate and tropical regions. Although the phylogenetic relationship of Goodyera inferred from both morphological and molecular data has been conducted, the sampled taxa were mainly distributed in Asia regions that resulted in non-monophyly of this genus. In this study, the complete plastid genomes of Goodyera, generated by next-generation sequencing (NGS) technique and sampled in East Asia and North America, were used to reconstruct phylogeny and explore the historical biogeography. A total of 18 Goodyera species including seven newly sequenced species were analyzed. Based on 79 protein-coding genes, the phylogenetic analysis revealed that Goodyera could be subdivided into four subclades with high support values. The polyphyletic relationships among Goodyera taxa were confirmed, and the unclear position of G. foliosa was also resolved. The datasets that are composed of the 14 coding sequences (CDS) (matK, atpF, ndhK, accD, cemA, clpP, rpoA, rpl22, ndhF, ccsA, ndhD, ndhI, ndhA, and ycf 1) showed the same topology derived from 79 protein-coding genes. Molecular dating analyses revealed the origin of Goodyera in the mid-Miocene (15.75 Mya). Nearctic clade of Goodyera was diverged at 10.88 Mya from their most recent common ancestor (MRCA). The biogeographical reconstruction suggests that subtropical or tropical Asia is the origin of Goodyera and it has subsequently spread to temperate Asia during the Miocene. In addition, Nearctic clade is derived from East Asian species through Bering Land Bridge (BLB) during the Miocene. The speciation of Goodyera is most likely to have occurred during Miocene, and climatic and geological changes are thought to have had a part in this diversification. Our findings propose both origin and vicariance events of Goodyera for the first time and add an example for the biogeographical history of the Northern Hemisphere.

Scientists have long been captivated by biogeographic disjunctions, and disjunctions between East Asia and North America have been particularly well-studied at the genus and family levels. By contrast, disjunctions between eastern and western Asia have received less attention. Euonymus L. is taxonomically divided into two sections based on the number of cells in anthers as follows: E. sect. Uniloculares has one-celled anthers and occurs mainly in Asia, whereas E. sect. Biloculares has two-celled anthers and is distributed globally. We used Illumina sequencing to investigate the genomes of four species in sect. Uniloculares. The chloroplast (cp) genomes are highly conserved (157,290–158,094 bp). Pseudogenisation of ndhF and intron loss in rps16 was detected. Based on the cp genomes of the four species of E. sect. Uniloculares, we propose a novel hypothesis of disjunction between eastern and western Asia. Biogeographic reconstruction and molecular dating revealed that sect. Uniloculares separated from its sect. Biloculares forebears 4.0 Mya during the Pliocene era. The radial diversification of sect. Uniloculares from East Asia and the establishment of the western Asian clade during the Pleistocene era (1.9 Mya) were the results of both dispersal and vicariance, making the section the youngest diverged clade conforming to age estimation. The centre of origin of sect. Uniloculares was determined to be in East Asia. Disjunctions and diversification between eastern and western Asia in sect. Uniloculares are thought to have been caused by changes in monsoon patterns, temperature variations, and the emergence of the Gobi Desert.

We found an unrecorded species, Carex molestiformis Reznicek & Rothrock, in Byeokparyeong, Daehwa-myeon, Pyeongchang-gun, Gangwon-do, South Korea. This species is native to southern parts of the United States (ca. 11 states, including Arkansas, Missouri, and Oklahoma). These plants are usually distributed densely in stream flood plains, bottomlands, stream banks, and along roadsides. Belonging to section Ovales Kunth, perennial herbs with gynecandrous spikes, C. molestiformis is closely related to C. maackii Maxim. and C. scoparia Willd. and is distinguished from them by the number (2-4) of spikes in each inflorescence and the width (2.6-3.4 mm) and number (6-9) of veins of perigynia. A new Korean name of C. molestiformis ‘Ga- Neun-Ta-Rae-Sa-Cho’ was given, as it has slender overall appearance compared with C. maackii (Ta-Rae-Sa- Cho). We also provide a description, illustrations, photographs and a key of related taxa in Korea. We compared three DNA barcode region (chloroplast DNA matK, ndhF and nuclear ribosomal DNA internal transcribed spacer) sequences from C. molestiformis with those of C. maackii and C. scoparia, determining eight speciesspecific single nucleotide polymorphism sites for C. molestiformis.

Bauhinia coccinea is a tropical woody plant widely distributed in Vietnam and Unnan in southern China. Although many studies have shown the biological activities of extracts from various other species in the genus, no studies have investigated the effects of B. coccinea extracts on biological systems. In the present study, a quantitative analysis of four marker compounds of ethanol extracts of B. coccinea branches (EEBC) was performed using the high performance liquid chromatography (HPLC)-photodiode array (PDA) method. Among gallic acid, (+)-catechin, ellagic acid, and quercitrin contained in EEBC, the most abundant compound was (+)-catechin (18.736 mg/g). In addition, we investigated the EEBC on neuroprotection, antioxidation, and Alzheimer’s disease (AD) marker molecules, acetylcholinesterase (AChE), and amyloid-β (Aβ). EEBC significantly inhibited hydrogen peroxide (H2O2)-induced cell death in a HT22 neuronal cell line and increased 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl scavenging activity markedly. EEBC also inhibited AChE and Aβ aggregation. Among the four compounds, gallic acid exhibited strong inhibitory effects against AChE activation. In the Aβ aggregation assay, the four marker compounds exhibited inhibitory effects lower than 30%. According to the results, EEBC could exert anti-AChE activation and Aβ aggregation activities based on the interactive effects of the marker compounds. Our findings suggest that EEBC are sources of therapeutic candidates for application in the development of AD medication based on AChE and Aβ dual targeting.

Prunus × nudiflora is an endemic species to Jeju Island, South Korea and economically important as ornamentals. According to recent molecular phylogenetic and genomic studies, this species is revealed as a F1 hybrid originating from a cross between maternal Prunus pendula f. ascendens and paternal Prunus jamasakura. However, the plate specimen designated as type materials of P. × nudiflora does not permit a precise application of the name because of its poor condition. The herbarium specimens related with that illustration were examined to clarify the identity of P. × nudiflora, and an epitype is selected. Our epitypification proposed here will ensure nomenclatural accuracy for most material from cultivation, where the name P. × nudiflora is widely used. Keywords: Epitypification, Endemic species, Prunus × nudiflora, Rosaceae, South Korea

During the bloom events of the harmful dinoflagellate Cochlodinium polykrikoides in August and October, 2012, infections by two different Amoebophrya species were observed in Korean coastal waters. To investigate the dynamics of the two parasites and their relative impact on the host populations, a quantitative real-time PCR (qPCR) method was applied to detect and quantify the parasites in the free-living and parasitic stages. Each specific primer set of the target species, Amoebophrya sp. 1 and sp. 2 was designed on the large subunit (LSU) and the first internal transcribed spacer (ITS1) of ribosomal RNA (rRNA) gene, respectively. Dynamics of the two Amoebophrya species via qPCR assay showed distinct patterns during the C. polykrikoides bloom events. Amoebophrya sp. 1 showed peaks during both bloom events in August and October with relatively low copies (106 to 107 copies L−1), while Amoebophrya sp. 2 appeared only during the bloom event in October with very high copies (109 to 1010 copies L−1). Overall, the qPCR measurements for the dynamics of two Amoebophrya species in the parasitic stage (> 5 μm fractions) were consistent with parasite prevalence through microscopic observations. Amoebophrya sp. 1 infections were observed during both bloom events in August and October with relatively low parasite prevalence (0.1–1.5%), while Amoebophrya sp. 2 infections were detected only during the bloom event in October with high prevalence (up to 45%). Taken together, Amoebophrya sp. 1 may be a generalist and C. polykrikoides may not be its primary host, while Amoebophrya sp. 2 may be a specialist which can substantially impact host population dynamics.

Neuroinflammation plays a pivotal role in the pathogenesis of neurodegenerative diseases and is characterized by microglial dysregulation. Here, we explored the beneficial effects of a leaf extract of Elaeagnus glabra f. oxyphylla (EGFO), a native medicinal plant to Korea, South China, Japan, and Taiwan, on neuroinflammation using lipopolysaccharide (LPS)-stimulated BV-2 microglia. Levels of the inflammatory mediators were determined by enzyme-linked immunosorbent assays and reverse transcription–polymerase chain reaction. The phospho levels of mitogen-activated protein kinases, which are key kinase molecules in the inflammatory signaling pathway in microglia, were analyzed by Western blotting. Treatment with EGFO significantly suppressed the LPS-mediated induction of nitric oxide and prostaglandin E2. Consistently, EGFO treatment in LPS-stimulated BV-2 cells markedly reduced the inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels. The best concentration of EGFO that could reduce TNF-α and IL-6 was 100 μg/mL. EGFO relatively reduced the messenger RNA expression of TNF-α and IL-6 by 0.36 and 0.32-fold ratio, respectively, compared to LPS treatment. Moreover, EGFO markedly reduced the phospho levels of p38 and the c-jun N-terminal kinase. Furthermore, antibody microarray and immunoblotting data revealed that the pharmacological mechanisms driving the antineuroinflammatory action of EGFO involve prevention of the cyclin D3/cyclin-dependent kinase 11p58 (CDK11p58) interaction. In conclusion, our results demonstrate that EGFO alleviates the inflammatory response through the suppression of cyclin D3/CDK11p58 coupling in LPS-activated BV-2 microglia. We propose the potential of EGFO as a novel drug candidate for neurodegenerative diseases by targeting neuroinflammation.

Background Carduus, commonly known as plumeless thistles, is a genus in the Asteraceae family that exhibits both medicinal value and invasive tendencies. However, the genomic data of Carduus (i.e., complete chloroplast genomes) have not been sequenced. Methods We sequenced and assembled the chloroplast genome (cpDNA) sequences of three Carduus species using the Illumina Miseq sequencing system and Geneious Prime. Phylogenetic relationships between Carduus and related taxa were reconstructed using Maximum Likelihood and Bayesian Inference analyses. In addition, we used a single nucleotide polymorphism (SNP) in the protein coding region of the matK gene to develop molecular markers to distinguish C. crispus from C. acanthoides and C. tenuiflorus. Results The cpDNA sequences of C. crispus, C. acanthoides, and C. tenuiflorus ranged from 152,342 bp to 152,617 bp in length. Comparative genomic analysis revealed high conservation in terms of gene content (including 80 protein-coding, 30 tRNA, and four rRNA genes) and gene order within the three focal species and members of subfamily Carduoideae. Despite their high similarity, the three species differed with respect to the number and content of repeats in the chloroplast genome. Additionally, eight hotspot regions, including psbI-trnS_GCU, trnE_UUC-rpoB, trnR_UCU-trnG_UCC, psbC-trnS_UGA, trnT_UGU-trnL_UAA, psbT-psbN, petD-rpoA, and rpl16-rps3, were identified in the study species. Phylogenetic analyses inferred from 78 protein-coding and non-coding regions indicated that Carduus is polyphyletic, suggesting the need for additional studies to reconstruct relationships between thistles and related taxa. Based on a SNP in matK, we successfully developed a molecular marker and protocol for distinguishing C. crispus from the other two focal species. Our study provides preliminary chloroplast genome data for further studies on plastid genome evolution, phylogeny, and development of species-level markers in Carduus.

The species within Orobanchaceae cover all forms of parasitism from full autotrophy to holoparasitism. Although parasitic species have gone through dramatic genome changes, the plastomes of land plants generally contain highly conserved structures. We investigated the complete plastome sequence of hemiparasitic Pedicularis ishidoyana, belonging to the largest genus in Orobanchaceae, and compared it with the highly rearranged genomes of other taxa in that family. This plastome comprises a total of 152 571 bp, with two inverted repeats (IRs) of 35 281 bp, a large single-copy region (LSC) of 81 982 bp, and an extremely reduced small single-copy (SSC) region of 27 bp. Although IR expansion and SSC contraction have been reported in the plastomes of other hemiparasitic species in Orobanchaceae, the IR region of P. ishidoyana is extremely expanded, resulting in a very small SSC region. This genome contains 101 genes that encode 67 proteins, four rRNAs, 30 tRNAs, and 22 duplicated genes in the IR region. In contrast to other hemi- or holoparasitic members of Orobanchaceae, genes in the ndh family in P. ishidoyana (except ndhE) have lost their function through pseudogenization (six genes) or deletion (four genes). Therefore, it seems that the degradation of this gene family in P. ishidoyana has occurred independently. Phylogenetic analysis, including all parasitic types, strongly supports the conclusion that P. ishidoyana diverged early from autotrophs and is sister to holoparasitic taxa. Our results suggest that these hemiparasitic Pedicularis plants might represent an early stage of parasitism that has developed stepwise in this family from non-parasites to holoparasites.

Genetic sub-populations (clades) of cosmopolitan marine diatom Pseudo-nitzschia pungens might have distinct habitats, and their hybrid zone is suspected in higher latitude area of the West Pacific area, however, it is still unrevealed because of technical difficulties and lack of evidences in natural environments. The aim of this study is to investigate the habitat characteristics of each clade of P. pungens on geographical distribution with the habitat temperature ranges of each clade and to reveal their hybrid zone in the West Pacific area. We employed the 137 number of nucleotide sequences of P. pungens and its sampling data (spatial and temporal scale) originated from the West Pacific area, and used field application of qPCR assay for intra-specific level of P. pungens. Only two genotypes, clade I and III, were identified in the West Pacific area. Clade I was distributed from 39 to 32.3°N, and clade III were from 1.4 to 34.4°N. The estimated habitat temperature for the clade I and clade III ranges were 8.1–26.9 °C and 24.2–31.2 °C, respectively. The latitudinal distributions and temperature ranges of each clade were significantly different. The qPCR assay employed, and results suggested that the hybrid zone for clade I and III has been observed in the southern Korean coasts, and clade III might be introduced from the Southern Pacific area. The cell abundances of clade III were strongly related with the higher seawater temperature and warm current force. This study has defined distinct habitat characteristics of genetically different sub-populations of P. pungens, and revealed its hybrid zone in natural environment for the first time. We also provided strong evidences about dispersion of the population of clade III to higher latitude in the West Pacific area.

Although there have been extensive studies on dinoflagellate blooms in recent decades, the mechanism that allows the maintenance of blooms over long periods remains uncertain, and studies on genetically differentiated subpopulations may provide insights into this mechanism. In this study, the influence of two genetically distinct subpopulations of the dinoflagellate Cochlodinium polykrikoides, referred to as Group I and IV, on bloom duration in Korean coastal waters (KCW) was examined using a quantitative PCR (qPCR) assay. In this study, a C. polykrikoides bloom occurred over a longer period in 2009 (49 days), whereas the bloom period was shorter in 2010 (35 days). The qPCR results indicate that intraspecific bloom succession between Groups I and IV occurred in 2009, whereas only a single subpopulation (Group I) was responsible for the bloom in 2010. Based on the statistical analysis, the Group I and Group IV blooms occurred under significantly different environmental conditions (p ≤ 0.05) in terms of water temperature, pH, and phosphate concentration, and these subpopulations exhibited significantly different relationships with environmental factors, particularly water temperature (p 0.01). This variability may allow blooms to continue through intraspecific bloom succession even after environmental conditions change. Southern KCW are affected by outer regions via the Tsushima Warm Current (TWC) every summer. Group IV (≤1108 ± 69 cells L

Prunus yedoensis Matsumura is one of the popular ornamental flowering cherry trees native to northeastern Asia, and its wild populations have only been found on Jeju Island, Korea. Previous studies suggested that wild P. yedoensis (P. yedoensis var. nudiflora) is a hybrid species; however, there is no solid evidence on its exact parental origin and genomic organization. In this study, we developed a total of 38 nuclear gene-based DNA markers that can be universally amplifiable in the Prunus species using 586 Prunus Conserved Orthologous Gene Set (Prunus COS). Using the Prunus COS markers, we investigated the genetic structure of wild P. yedoensis populations and evaluated the putative parental species of wild P. yedoensis. Population structure and phylogenetic analysis of 73 wild P. yedoensis accessions and 54 accessions of other Prunus species revealed that the wild P. yedoensis on Jeju Island is a natural homoploid hybrid. Sequence-level comparison of Prunus COS markers between species suggested that wild P. yedoensis might originate from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura. Moreover, approximately 81% of the wild P. yedoensis accessions examined were likely F1 hybrids, whereas the remaining 19% were backcross hybrids resulting from additional asymmetric introgression of parental genotypes. These findings suggest that complex hybridization of the Prunus species on Jeju Island can produce a range of variable hybrid offspring. Overall, this study makes a significant contribution to address issues of the origin, nomenclature, and genetic relationship of ornamental P. yedoensis.

To date, plastome studies of mycoheterotrophic orchids have focused on nongreen mycoheterotrophic or partially mycoheterotrophic species. Cymbidium macrorhizon is a fully mycoheterotrophic orchid that lacks leaves and roots, although its inflorescence rachis is pale green. It has degraded stomata, specific fungal partners, and high concentrations of heavy stable nitrogen and carbon isotopes. Therefore, the plastome of this species is expected to represent an early stage of a fully mycoheterotrophic plastome. In this study, we sequenced the plastomes of C. macrorhizon and closely related species (C. ensifolium, C. kanran, and C. lancifolium). Plastomes of the four Cymbidium species were almost identical structurally, but differed somewhat from those of previously studied species. The genes for the photosynthetic subunits of NADH dehydrogenase, ndhF and ndhH, were absent from all four newly sequenced plastomes, whereas only ndhJ was absent from C. ensifolium. In section Pachyrhizanthe (C. lancifolium and C. macrorhizon), ndhE, ndhI, and ndhJ were pseudogenized. With the exception of ndh and ycf, 64 protein-coding genes in C. macrorhizon were apparently functional. Most of them were highly conserved and under purifying selection. Therefore, no direct evidence is available to suggest that genes related to photosynthesis have lost their functions in C. macrorhizon. This discordance between molecular and physiological features for the trophic status of C. macrorhizon might result from a lag between photosynthetic function loss and relaxed purifying selection.

Premise of the study Understanding the underlying mechanisms for the evolution of intercontinental disjunct patterns has long fascinated botanists. We present a molecular phylogenetic reconstruction of Maianthemum s.l. (including Smilacina) with a focus on Maianthemum s.s. species, which have a disjunct distribution between Eurasia and North America. Within this evolutionary framework, we clarify the systematic classification and biogeographical history of Maianthemum s.s. Method Data from two nuclear and five chloroplast DNA regions were analyzed using the programs PAUP*, RAxML, MrBayes, BEAST, and RASP. Key results Our molecular phylogeny supports Maianthemum s.s. as monophyletic. Maianthemum bifolium and M. canadense are grouped according to their taxonomic species, but the accessions of M. dilatatum are divided into two well-defined groups, i.e., one comprising western North American accessions, and the other composed of northeast Asian accessions. Molecular dating and biogeographic reconstructions suggest a northeast Asian origin for Maianthemum s.s. and that a complicated pattern of divergent evolution began approximately in the late Miocene. Intercontinental disjunctions of Maianthemum s.s. in the Northern Hemisphere appear to have occurred two times during the Pliocene. Conclusions Based on our results, two possible ways to treat Maianthemum s.s. species can be envisioned: (1) elevate Asian populations of M. dilatatum to specific rank; or (2) combine the three Maianthemum s.s. species into a broadly defined single species. We recommend treatment of Maianthemum s.s. as a single species. The biogeographic patterns of Maianthemum s.s. coupled with molecular dating suggest both vicariance and long-distance dispersal events as key mechanisms for diversification of the clade.

Crinum asiaticum var. japonicum Baker is a Korean herbal medicine that is traditionally prescribed for reducing fever and inflammation. In the present study, we investigated if the ethanol extract of C. asiaticum seeds (ECAS) influences the hallmarks of Alzheimer’s disease (AD) pathogenesis. ECAS markedly inhibited the activity of acetylcholinesterase (AChE). Concurrent treatment with hydrogen peroxide (H2O2) and ECAS significantly prevented the neuronal cell death by regulating phosphorylation of cyclic adenosine monophosphate response element-binding protein and p38 mitogen-activated protein kinase. ECAS revealed antineuroinflammatory effects by inhibiting nitric oxide production and suppressing inducible nitric oxide expression in lipopolysaccharide-stimulated BV-2 microglia. Furthermore, the high-performance liquid chromatographic analysis determined lycorine as a standard compound of ECAS. Our data suggest that ECAS has inhibitory effects on AD pathogenesis such as AChE activation, neuronal damage, and neuroinflammation.

Gnetum (Gnetaceae, Gnetales) is a gymnosperm genus with ca. 35 species distributed in tropical forests around the world. Due to its dioecious habit and lack of diagnostic characters from vegetative tissue, the identification of Gnetum species is not easy without seeds or reproductive structures. To identify and verify their phylogenetic positions, we applied DNA barcoding to Cambodian Gnetum collections gathered between 2010 and 2015, with previously designed cp matK gene primers. We newly sequenced partial matK sequences from 72 Gnetum collections, 43 out of 72 from Cambodia, and analyzed 115 Gnetum accessions using the neighbor-joining method. The resulting neighbor-joining tree categorized Cambodian Gnetum samples into three clades of species: G. macrostachyum, G. montanum, and G. aff. gracilipes. The recognition of G. aff. gracilipes in Cambodia is reported here for the first time. Taxonomic information for the three recognized Cambodian Gnetum species is provided and the benefits of the taxonomic reevaluation assisted by DNA barcoding are emphasized in this work.

Diospyros lotus L. (Ebenaceae) is a deciduous plant that was traditionally used to treat various diseases such as sedation, constipation, and hypertension. However, its effects on Alzheimer’s diseases (AD) have not been reported. We investigated the potent biological effects of D. lotus on AD and performed quantitative analysis of 7 standard compounds in D. lotus leaves. First, two parts leaf and branch of D. lotus were compared to examine the effects on amyloid-β (Aβ) aggregation and oxidative stress. Ethanol extract of D. lotus leaves (EDLL) had higher activities on the Aβ disaggregation and antioxidation compared with ethanol extract of D. lotus branches (EDLB). Second, we have focused on the biological activities of EDLL for neurocellular analyses. In HT22 neuronal cells, EDLL reversed hydrogen peroxide (H2O2)-damaged cell death. In BV-2 microglia, EDLL suppressed lipopolysaccharide-stimulated productions of nitric oxide (NO) and prostaglandin E2. Third, we performed quantitative analyses of 7 standard compounds in D. lotus using high-performance liquid chromatography. Among 7 compounds, myricitrin (44.189 mg/g) was the most abundant compound in EDLL. Myricetin presented the marked inhibitory effect on Aβ aggregation, indicating its potential as a bioactive compound to control Aβ aggregation. Overall, this study suggests that EDLL may be a promising therapeutic agent for AD via Aβ disaggregation, and inhibition of neuronal cell damage and inflammation.

Background Genomic data play an important role in plant research because of its implications in studying genomic evolution, phylogeny, and developing molecular markers. Although the information of invasive alien plants was collected, the genomic data of those species have not been intensively studied. Methods We employ the next generation sequencing and PCR methods to explore the genomic data as well as to develop and test the molecular markers. Results In this study, we characterize the chloroplast genomes (cpDNA) of Cenchrus longispinus and C. echinatus, of which the lengths are 137,144 and 137,131 bp, respectively. These two newly sequenced genomes include 78 protein-coding genes, 30 tRNA, and four rRNA. There are 56 simple single repeats and 17 forward repeats in the chloroplast genome of C. longispinus. Most of the repeats locate in non-coding regions. However, repeats can be found in infA, ndhD, ndhH, ndhK, psbC, rpl22, rpoC2, rps14, trnA-UGC, trnC-GCA, trnF-GAA, trnQ-UUG, trnS-UGA, trnS-GCU, and ycf15. The phylogenomic analysis revealed the monophyly of Cenchrus but not Panicum species in tribe Paniceae. The single nucleotide polymorphism sites in atpB, matK, and ndhD were successfully used for developing molecular markers to distinguish C. longispinus and related taxa. The simple PCR protocol for using the newly developed molecular markers was also provided.

Intercontinental floristic disjunction between East Asia and North America in the Northern Hemisphere has received much attention during the past decades, but few studies have focused on the family level. Melanthiaceae, containing 196 species and 17 genera circumscribed in five tribes, is disjunctly distributed in Eurasia and North America. It is one of the foremost models for studying the evolution of biogeographic patterns in this region. Here, we present a fossil-calibrated, molecular phylogeny of Melanthiaceae based on two chloroplast DNA datasets: one dataset includes extensive sampling (94 species representing all 17 genera of Melanthiaceae) of four chloroplast DNA regions (atpB, rbcL, matK, and ndhF) and the other includes six species representing all tribes of the family for 78 coding genes of the chloroplast genome. Within this framework, we infer the historical biogeography of Melanthiaceae. Both datasets produce well-resolved phylogenies of Melanthiaceae showing the monophyly of the family and the relationships among the five tribes. Melanthieae is found to be sister to the rest of the tribes of the family and the remaining taxa are divided into two major clades consisting of the Chionographideae + Heloniadeae clade and the Parideae + Xerophylleae clade. The molecular dating and the ancestral area analyses suggest that Melanthiaceae most likely originated in North America with its crown group dated at 92.1 mya in the late Cretaceous. The favored ancestral areas at the crown lineages of tribes are also in North America. In the family, seven independent migrations into East Asia from North America are inferred to have occurred in the Oligocene and the Miocene-Pliocene via historical paleo-land bridge connections. Cooling trends during the Oligocene resulted in the present East Asia-North America disjunct distribution, while the warm period during the middle Miocene and habitat heterogeneity stimulated diversification in East Asia. Our study provides the phylogenetic and biogeographical history of the Melanthiaceae and adds an example of “out of North America” migration in the biogeographic history of the Northern Hemisphere.

Tribe Forsythieae (Oleaceae), containing two genera (Abeliophyllum and Forsythia) and 13 species, is economically important plants used as ornamentals and in traditional medicine. This tribe species occur primarily in mountainous regions of Eurasia with the highest species diversity in East Asia. Here, we examine 11 complete chloroplast genome and nuclear cycloidea2 (cyc2) DNA sequences of 10 Forsythia species and Abeliophyllum distichum using Illumina platform to provide the phylogeny and biogeographic history of the tribe. The chloroplast genomes of the 11 Forsythieae species are highly conserved, except for a deletion of about 400 bp in the accD–psaI region detected only in Abeliophyllum. Within Forsythieae species, analysis of repetitive sequences revealed a total of 51 repeats comprising 26 forward repeats, 22 palindromic repeats, and 3 reverse repeats. Of those, 19 repeats were common and 32 were unique to one or more Forsythieae species. Our phylogenetic analyses supported the monophyly of Forsythia and its sister group is Abeliophyllum using the concatenated dataset of 78 chloroplast genes. Within Forsythia, Forsythia likiangensis and F. giraldiana were basal lineages followed by F. europaea; the three species are characterized by minutely serrate or entire leaf margins. The remaining species, which are distributed in East Asia, formed two major clades. One clade included F. ovata, F. velutina, and F. japonica; they are morphologically supported by broadly ovate leaves. Another clade of F. suspensa, F. saxatilis, F. viridissima, and F. koreana characterized by lanceolate leaves (except F. suspensa which have broad ovate leaves). Although cyc2 phylogeny is largely congruent to chloroplast genome phylogeny, we find the discordance between two phylogenies in the position of F. ovata suggesting that introgression of the chloroplast genome from one species into the nuclear background of another by interspecific hybridization in East Asian Forsythia species. Molecular dating and biogeographic reconstructions suggest an origin of the Forsythieae species in East China in the Miocene. Distribution patterns in Forsythia indicated that the species were radially differentiated from East China, and the speciation of the European F. europaea was the result of both vicariance and dispersal in the late Miocene to Pliocene.

Further understanding of the life history of Heterosigma akashiwo (Raphidophyceae) requires identification and isolation of cysts. In this study, we provide a more detailed description of the cyst wall, the germination pore, the structure underneath the lid of germination pore (SULG) and the germination process. The SULG is associated with cyst germination and may be a useful diagnostic feature of H. akashiwo cysts. Our refined description of cysts of H. akashiwo may allow for accurate identification in environmental samples.

Reliable and accurate species identification is essential to establish a strategy for monitoring and manipulation of plant populations for conservation biology. However, identification of nonflowering plants is difficult. Based on recent advances in molecular biological techniques, various molecular identification methodologies have been proposed and applied in plant science, with a focus on medicinally valuable species. Lady’s slipper orchids (Cypripedium) are a representative terrestrial orchid group that includes many endangered species. Of 46 species, 3 (Cypripedium japonicum Thunb., Cypripedium macranthos Sw., and Cypripedium guttatum var. koreanum Nakai) were reported from Korea and categorised as endangered species or at risk of becoming endangered because of the rapidly decreasing population. We generated an accurate molecular identification system for these species using the sequence variation and species-specific SNPs of two plastid loci, rpoC2 and the IGS region between atpF and atpH. We selected the atpF-H region for molecular identification of the Korean Cypripedium and related Taiwan-endemic taxon Cypripedium formosanum Hayata. Cypripedium guttatum var. koreanum contained large deletions of over 300 bp, and could be distinguished by electrophoresis. For the other three species, we designed new primers based on a specific insertion (C. macranthos) and SNPs (C. japonicum and C. formosanum). We confirmed that molecular identification enables detection of each species using species-specific primers.

The phylogenetic positions of the families Campynemataceae and Corsiaceae within the order Liliales remains unclear. To date, molecular data from the plastid genome of Corsiaceae has been obtained exclusively from Arachnitis, for which alignment and phylogenetic inference has proved difficult. The extent of gene conservation among mycoheterotrophic species within Corsiaceae remains unknown. To clarify the phylogenetic position of Campynemataceae and Corsiaceae within Liliales, functional plastid-coding genes of species representing both families have been analyzed. Examination of two phylogenetic data sets of plastid genes employing parsimony, maximum-likelihood, and Bayesian inference methods strongly supported both families forming a basal clade to the remaining taxa of Liliales. The first data set consists of five functional plastid-encoded genes (matK, rps7, rps2, rps19, and rpl2) sequenced from Corsia dispar (Corsiaceae). The data set included 31 species representing all families within Liliales, as well as selected orders that are related closely to Liliales (10 outgroup species from Asparagales, Dioscoreales, and Pandanales). The second phylogenetic analysis was based on 75 plastid genes. This data set included 18 species from Liliales, representing major clades within the order, and 10 outgroup species from Asparagales, Dioscoreales, and Pandanales. In this latter data set, Campynemataceae was represented by 60 plastid-encoded genes sequenced from herbarium material of Campynema lineare. A large proportion of the plastid genome of C. dispar was also sequenced and compared to the plastid genomes of photosynthetic plants within Liliales and mycoheterotrophic plants within Asparagales to explore plastid genome reduction. The plastid genome of C. dispar is in the advanced stages of reduction, which signifies its high dependency on mycorrhizal fungi and is suggestive of a loss in photosynthetic ability. Functional plastid genes found in C. dispar may be applicable to other species in Corsiaceae, which will provide a basis for in-depth molecular analyses of interspecies relationships within the family, once molecular data from other members become available.

Main conclusion The complete chloroplast genome of two colchicine medicinal plants is reported for the first time. Deletion ofycf15 gene occurred only inColchicumbut not inGloriosaand suggests this as a potential marker for delineating the two species. Colchicum autumnale L. and Gloriosa superba L. are well-known sources of colchicine, a type of alkaloid and an ancient anti-inflammatory drug used to prevent gout. Accordingly, this alkaloid has been used as a chemical marker for identifying the expanded Colchicaceae family. In the present study, we report the complete chloroplast genome (cpDNA) sequence of two colchicine medicinal plants (G. superba and C. autumnale) that belong to the tribe Colchiceae of the Colchicaceae family. In C. autumnale, the circular double-stranded cpDNA sequence of 156,462 bp consists of two inverted repeat (IR) regions of 27,741 bp each, a large single-copy region (LSC) of 84,246 bp, and a small single-copy region (SSC) of 16,734 bp. The cpDNA sequence of G. superba is longer than that of C. autumnale (157,924 bp), which consists of two IRs (28,063 bp), an SSC (16,786 bp), and an LSC (85,012 bp). Significant structural differences between them were observed in the ycf15 gene. ycf15 gene was absent from C. autumnale cpDNA and affected the length of the chloroplast genome between the species. Furthermore, this gene loss event was specific to the expanded genus of Colchicum sensu Vinnersten and Manning. Therefore, this gene may be an effective and powerful molecular marker for identifying the Colchicum genus within the family.

Plastid genome studies on Orchidaceae, one of the largest families of flowering plants, have been limited to the subfamily Epidendroideae. To increase understanding of the plastid genome evolution in Orchidaceae, we performed a complete plastid genome analysis of the slipper orchid Cypripedium japonicum positioned in the basal clade of the family. No significant gene deletions were observed in the plastid genome of the slipper orchid composed of 85 coding genes including members of the ndh gene family, which are mostly deleted or show pseudogenisation in orchids, although matK was identified as a pseudogene due to a frameshift mutation. Results also revealed that C. japonicum contains the largest plastid genome (174,417 bp) within monocots and the third largest one in Magnoliophyta. This is a new type of plastid genome extended due to abnormally frequent AT residue repeats within noncoding regions without inverted repeat (IR) expansion. In addition, we detected 25 plastid microsatellites and compared them among seven populations from Korea and Japan. These microsatellites may be applicable to population genetics and conservation biology studies on slipper orchids, which have become rare and are nearing extinction. AT-rich regions in the introns may also play a role in effective splicing of the genome after matK lost its function. As AT-rich regions are difficult to amplify and sequence using normal sequencing technologies, we propose a revised methodology for sequencing these regions.

Chloroplast genomes (cpDNA) are highly valuable resources for evolutionary studies of angiosperms, since they are highly conserved, are small in size, and play critical roles in plants. Slipped-strand mispairing (SSM) was assumed to be a mechanism for generating repeat units in cpDNA. However, research on the employment of different small repeated sequences through SSM events, which may induce the accumulation of distinct types of repeats within the same region in cpDNA, has not been documented. Here, we sequenced two chloroplast genomes from the endemic species Heloniopsis tubiflora (Korea) and Xerophyllum tenax (USA) to cover the gap between molecular data and explore ‘hot spots’ for genomic events in Melanthiaceae. Comparative analysis of 23 complete cpDNA sequences revealed that there were different stages of deletion in the rps16 region across the Melanthiaceae. Based on the partial or complete loss of rps16 gene in cpDNA, we have firstly reported potential molecular markers for recognizing two sections (Veratrum and Fuscoveratrum) of Veratrum. Melathiaceae exhibits a significant change in the junction between large single copy and inverted repeat regions, ranging from trnH_GUG to a part of rps3. Our results show an accumulation of tandem repeats in the rpl23-ycf2 regions of cpDNAs. Small conserved sequences exist and flank tandem repeats in further observation of this region across most of the examined taxa of Liliales. Therefore, we propose three scenarios in which different small repeated sequences were used during slipped-strand mispairing (SSM) events to generate newly distinct types of repeats. Occasionally, prior to the SSM process, point mutation event and double strand break repair occurred and induced the formation of initial repeat units which are indispensable in the SSM process. SSM may have likely occurred more frequently for short repeats than for long repeat sequences in tribe Parideae (Melanthiaceae, Liliales). Collectively, these findings add new evidence of dynamic results from SSM in chloroplast genomes which can be useful for further evolutionary studies in angiosperms. Additionally, genomics events in cpDNA are potential resources for mining molecular markers in Liliales.

The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium, known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense, all four strains of A. poporum produced a new azaspiracid toxin, based on LC–MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized.

While the toxic dinoflagellate Cochlodinium polykrikoides is known to form blooms that are maintained for extended periods, the genetic differentiation of these blooms are currently unknown. To assess this, we developed a real-time PCR assay to quantify C. polykrikoides at the intra-specific level, and applied this assay to field samples collected in Korean coastal waters from summer through fall. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides : Philippines, East Asian, and American/Malaysian. Significant linear relationships ( r 2 ≥ 0.995) were established between C t and the log of the copy number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms in Korean coastal waters were found to be comprised of Philippines and East Asian ribotypes but not the American/Malaysian ribotype. The Philippines ribotype was found to be highly abundant during summer bloom initiation and peak, whereas the East Asian ribotype became the dominant ribotype in the fall. As such, this newly developed qPCR assay can be used to quantify the cryptic ecological succession of sub-populations of C. polykrikoides during blooms that light microscopy and previously developed qPCR assays cannot resolve.

This study aims to define the phylogenetic relationship within Korean Pinus L. and to find themolecular markers which resolve the phylogenetic relationship in genus Pinus. cpDNA atpF-H and psbA-trnHregions were used as molecular markers. We performed the molecular phylogenetic study on 17 taxa of Pinus inKorea. The combined analyses of two gene loci showed that Korean Pinus was a monophyletic group supportedby 100% BP. According to the results of separate analyses, psbA-trnH region seems to work better resolvingpower to clarify the phylogenetic ambiguity in Korean Pinus than those of atpF-H region. Also, we tried tochecked the value and resolution of two chloroplast DNA loci on phylogenetic implications. Keywords: Pinus, subgenus Diploxylon & Haploxylon, atpF-H, psbA-trnH 적 DNA 요: 엽록체 atpF-H, psbA-trnH region을 마커로활용하여국내에분포하는 소나무속 식물들 중 17분류군에 대한 분자계통학적 연구를 수행하여 한국산 소나무속의 계통학적 유연관계를 규명하고, 소나무속의유연관계를 잘 나타낼 수 있는 분자마커를 찾아내고자 연구가 수행되었다. atpF-H, psbA-trnH region의 조합분석결과 한국산 소나무속은 100%의 BP로 지지되는 단계통군으로 확인되었으며, 소나무아속과 잣나무아속으로 명확히 구분되어졌다. 본 연구에서 이용된 두 개의 분자마커 중 psbA-trnH region이 atpF-H region보다한국산 소나무속의 계통 및 유연관계를 규명하는데 다소 높은 해상력을 나타내었다.

Copyright © 2014 The Korean Society of Phycology 57 http://e-algae.kr pISSN: 1226-2617 eISSN: 2093-0860 Phylogenetic position of eight Amphora sensu lato (Bacillariophyceae) species and comparative analysis of morphological characteristics Pengbin Wang, Bum Soo Park, Jin Ho Kim, Joo-Hwan Kim, Hae-Ok Lee and Myung-Soo Han* Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Korea Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, Korea

During the floristic survey on Phnom Bokor National Park, Kampot, Cambodia, we encountered Balanophorafungosa var. indica, which is a tropical holoparasitic plant. To identify its host species, we collected host roots and trees nearbyand tried to identify them using DNA barcoding approach. We applied plastid rbcL and matK gene regions as DNA barcodemarkers, and successfully amplified and sequenced the markers from 15 host roots and seven tree samples. Obtained host rootsequences were identified as Primulaceae, Celastraceae, Myrtaceae, and Oleaceae, while trees nearby are Oleaceae, Myrta-ceae, Sapindaceae, Rosaceae, Clusiaceae, Ericaceae, and Lauraceae. At genus level, host species are identified as Myrsine,Euonymus , Syzygium, and Olea, but failed in species discrimination. Myrsine (Primulaceae) and Olea (Oleaceae) are reportedhere as host species of B. fungosa var. indica for the first time. Further sampling and comparative work, and DNA barcodingwill help recognize the biodiversity of the area and host species of Balanophora, together with their evolution.

The complete chloroplast genome of Chionographis japonica (Willd.) Maxim. (Melanthiaceae, Liliales) was mapped using polymerase chain reaction and the Sanger method. The circular double-stranded DNA was a typical quadripartite structure consisting of two inverted repeated regions (27,397 bp), a small single copy region (18,205 bp), and a large single-copy region (81,646 bp), with a total length of 154,645 bp. The genome consisted of 137 coding genes, including 91 protein-coding genes, 38 distinct tRNA, and 8 rRNA genes. The ycf15 and ycf68 genes had several internal stop codons interpreted as pseudogenes. The inverted repeat (IR) region expanded to part of the rps3 gene in the junction between large single-copy and IRA regions in C. japonica. We designed 785 primers, of which 481 were used to map the entire chloroplast genome of C. japonica. Primers were compared with the complete chloroplast sequence of Smilax china (Smilacaceae) to identify primers that could be used for other Liliales members and whole chloroplast genome sequencing. Of the primers used for C. japonica, 398 could be used with other smaller species within the order.

Colchicaceae (Liliales) comprise c. 250 species and 15–21 genera of rhizomatous or cormous perennials and are distributed through temperate and tropical Africa, Europe, Asia and North America. They have been traditionally divided into two subfamilies, but this division is still unclear and the phylogenetic relationships among members of the family and the sister relationships of the family have been debated. Moreover, it is uncertain whether the expanded circumscription of Colchicum, Gloriosa and Wurmbea to include Androcymbium, Littonia and Onixotis plus Neodregea, respectively, is unavoidable. In this study, sequences of three plastid genes, atpB, matK and rbcL, were analysed to reconstruct phylogenetic relationships in Colchicaceae. The matK region was the most variable and provided the most potentially parsimony-informative sites. We also conducted molecular phylogenetic analyses using the combined data with sequences for three non-coding regions generated by Vinnersten and Reeves. Our results strongly support the monophyly of Colchicaceae and the redefined circumscription to include two subfamilies, Wurmbeoideae and Uvularioideae. The Uvularia-Disporum clade, i.e. Uvularioideae, is sister to the rest of Colchicaceae and differentiated from Burchardia, previously suggested to be sister to the rest of Colchicaceae. In our analysis, Burchardia is placed in Wurmbeoideae. A close relationship between Tripladenia and Schelhammera was strongly supported and these are transferred to Wurmbeoideae from Uvularioideae. The six tribes sensu Vinnersten and Manning were shown to be monophyletic. Our results support the expanded circumscription of Colchicum (including Androcymbium) and Gloriosa (including Littonia), but the circumscription of Wurmbea and related genera requires further study. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 255–269.

Many molecular studies have shown the monocot order Liliales to be well supported; morphologically, it is defined by synapomorphies of tepalar nectaries and extrorse anthers, in contrast with septal nectaries and introrse anthers commonly found in other monocots, especially Asparagales, with which it was often confused in the past. It comprises c. 1500 species, 67 genera and 9–11 families. Although monophyly is clear, the phylogenetic relationships among some of the families are still unclear. In this study, we examine the inter- and infrafamilial relationships among Liliales in phylogenetic analyses based on four plastid loci (matK, rbcL, atpB and atpF-H). We performed phylogenetic analyses and constructed maximum parsimony and Bayesian trees for 49 genera and 148 taxa in ten families of Liliales sensu Angiosperm Phylogeny Group (APG) III using the combined DNA data. The monophyly of Liliales, except for Corsiaceae (Arachnitis), was strongly supported by both analyses. Campynemataceae were sister to the rest of the order, excluding Corsiaceae. The other families formed two well-defined clades, (Colchicaceae + Alstroemeriaceae) and (Liliaceae, Smilacaceae, (Rhipogonaceae + Philesiaceae)), and one weakly supported clade with Melanthiaceae and Petermanniaceae. Subfamilial and tribal circumscriptions for the three larger families, Colchicaceae, Melanthiaceae and Liliaceae, agreed well with the results of this study, except for the subfamily Calochortoideae of Liliaceae, which was split into two separate clades of Calochortus and Tricyrtis. In addition, we found several taxa with a 10-bp inversion in matK, which could contribute additional homoplasy to these analyses if included without re-coding. Phylogenetic relationships among families of Liliales were better defined here than in a previous molecular analysis, although the placement of Corsiaceae with plastid data remains problematic. Based on these results, reconsideration of the circumscriptions of Rhipogonaceae + Philesiaceae and the subfamilial circumscription for Calochortoideae of Liliaceae is suggested. © 2013 The Linnean Society of London

To clarify the significant genomic events of the tribe Parideae, we analysed the complete chloroplast genome sequences of two Trillium species representing two subgenera: Trillium and Phyllantherum. The results showed that the cpDNAs of Parideae are highly conserved across genome structure, gene order and contents. However, the chloroplast genome of T. maculatum contained a 3.4kb inverted sequence between ndhC - rbcL in the LSC region. In addition, we found three different types of trnI-CAU duplication. These genomic features provide informative molecular markers for identifying the infrageneric taxa of Trillium and improve our understanding of the evolution patterns of Parideae., Genomic events including gene loss, duplication, pseudogenization and rearrangement in plant genomes are valuable sources for exploring and understanding the process of evolution in angiosperms. The family Melanthiaceae is distributed in temperate regions of the Northern Hemisphere and divided into five tribes (Heloniadeae, Chionographideae, Xerophylleae, Melanthieae and Parideae) based on the molecular phylogenetic analyses. At present, complete chloroplast genomes of the Melanthiaceae have been reported from three species. In the previous genomic study of Liliales, a trnI-CAU gene duplication event was reported from Paris verticillata, a member of Parideae. To clarify the significant genomic events of the tribe Parideae, we analysed the complete chloroplast genome sequences of two Trillium species representing two subgenera: Trillium and Phyllantherum. In Trillium tschonoskii (subgenus Trillium), the circular double-stranded cpDNA sequence of 156 852 bp consists of two inverted repeat (IR) regions of 26 501 bp each, a large single-copy (LSC) region of 83 981 bp and a small single-copy (SSC) region of 19 869 bp. The chloroplast genome sequence of T. maculatum (subgenus Phyllantherum) is 157 359 bp in length, consisting of two IRs (25 535 bp), one SSC (19 949 bp) and one LSC (86 340 bp), and is longer than that of T. tschonoskii. The results showed that the cpDNAs of Parideae are highly conserved across genome structure, gene order and contents. However, the chloroplast genome of T. maculatum contained a 3.4-kb inverted sequence between ndhC and rbcL in the LSC region, and it was a unique feature for subgenera Phyllantherum. In addition, we found three different types of gene duplication in the intergenic spacer between rpl23 and ycf2 containing trnI-CAU, which were in agreement with the circumscription of subgenera and sections in Parideae excluding T. govanianum. These genomic features provide informative molecular markers for identifying the infrageneric taxa of Trillium and improve our understanding of the evolution patterns of Parideae in Melanthiaceae.

We performed a phylogenetic analysis of 13 families of Asparagales sensu APG III, including 59 Korean taxa representing 10 families based on five plastid regions sequences (matK, rbcL, rpoC1, rps3, and atpF-H intergenic spacer). Parsimony and Bayesian analyses were conducted to clarify the relationships among Korean Asparagales. Among the five regions, matK and atpF-H were the most informative. The most congruent phylogenetic trees were obtained from the combinations of four regions excluding atpF-H. Although both the matK and rbcL gene trees implied that the family system of Asparagales sensu APG III should be modified to include three expanded families, our combined four-regions data set resulted in a highly resolved topology with strong support for the following: (1) Asparagaceae s.l.; (2) Amaryllidaceae s.l.; (3) Hemerocallidoideae of Xanthorrhoeaceae s.l.; (4) Iridaceae; (5) Hypoxidaceae; and (6) Orchidaceae. However, the position of Scilla (Scilloideae of Asparagaceae s.l.) was ambiguous. Hemerocallidoideae was a sister group of both the expanded families of Asparagaceae s.l. and Amaryllidaceae s.l., which are considered the core asparagoids, while Orchidaceae was sister to all other Asparagales. We discuss differentiation of the morphological character within the Korean Asparagales, which clearly suggests that the subfamily Nolinoideae of Asparagaceae s.l. exhibits different character states than the other subfamilies. We compared 10 representative morphological characters to the molecular phylogenetic relationships, and propose that the circumscription within Asparagales sensu APG III needs to be reconsidered.

A new species, Hoya ignorata T. B. Tran, Rodda, Simonsson & Joongku Lee (Apocynaceae, Asclepiadoideae) is described and illustrated from Vietnam. The new taxon, widely distributed in Southeast Asia, does not seem to be related to any previously described Hoya R. Br., due to its small cupular coronas lacking lobes, and pendulous pollinia. These morphological characters, new for the genus Hoya, are discussed in light of the recent broadening of the circumscription of the genus following the results of molecular phylogenies and morphological studies.

Hoya sapaensis T.B. Tran & Rodda sp. nova (Apocynaceae) from Vietnam is described, illustrated and compared with the morphologically similar H. carnosa and H. bonii. Hoya sapaensis distinctly differs in the length of the pollinium and corpusculum, number of flowers per inflorescence, and the shape of the corolla.

† Background Previous phylogenetics studies of Asparagales, although extensive and generally well supported, have left several sets of taxa unclearly placed and have not addressed all relationships within certain clades thoroughly (some clades were relatively sparsely sampled). One of the most important of these is sampling within and placement of Nolinoideae (Ruscaceae s.l.) of Asparagaceae sensu Angiosperm Phylogeny Group (APG) III, which subfamily includes taxa previously referred to Convallariaceae, Dracaenaaceae, Eriospermaceae, Nolinaceae and Ruscaceae. † Methods A phylogenetic analysis of a combined data set for 126 taxa of Ruscaceae s.l. and related groups in Asparagales based on three nuclear and plastid DNA coding genes, 18S rDNA (1796 bp), rbcL (1338 bp) and matK (1668 bp), representing a total of approx. 4.8 kb is presented. Parsimony and Bayesian inference analyses were conducted to elucidate relationships of Ruscaceae s.l. and related groups, and parsimony bootstrap analysis was performed to assess support of clades. † Key Results The combination of the three genes results in the most highly resolved and strongly supported topology yet obtained for Asparagales including Ruscaceae s.l. Asparagales relationships are nearly congruent with previous combined gene analyses, which were reflected in the APG III classification. Parsimony and Bayesian analyses yield identical relationships except for some slight variation among the core asparagoid families, which nevertheless form a strongly supported group in both types of analyses. In core asparagoids, five major clades are identified: (1) Alliaceae s.l. (sensu APG III, Amarylidaceae ‐Agapanthaceae ‐Alliaceae); (2) Asparagaceae ‐Laxmanniaceae ‐Ruscaceae s.l.; (3) Themidaceae; (4) Hyacinthaceae; (5) Anemarrhenaceae ‐ Behniaceae ‐Herreriaceae ‐Agavaceae (clades 2 ‐5 collectively Asparagaceae s.l. sensu APG III). The position of Aphyllanthes is labile, but it is sister to Themidaceae in the combined maximum-parsimony tree and sister to Anemarrhenaceae in the Bayesian analysis. The highly supported clade of Xanthorrhoeaceae s.l. (sensu APG III, including Asphodelaceae and Hemerocallidaceae) is sister to the core asparagoids. Ruscaceae s.l. are a well-supported group. Asparagaceae s.s. are sister to Ruscaceae s.l., even though the clade of the two families is weakly supported; Laxmanniaceae are strongly supported as sister to Ruscaceae s.l. and Asparagaceae. Ruscaceae s.l. include six principal clades that often reflect previously named groups: (1) tribe Polygonateae (excluding Disporopsis); (2) tribe Ophiopogoneae; (3) tribe Convallarieae (excluding Theropogon); (4) Ruscaceae s.s. + Dracaenaceae + Theropogon + Disporopsis + Comospermum; (5) Nolinaceae, (6) Eriospermum. † Conclusions The analyses here were largely conducted with new data collected for the same loci as in previous studies, but in this case from different species/DNA accessions and greater sampling in many cases than in previously published analyses; nonetheless, the results largely mirror those of previously conducted studies. This demonstrates the robustness of these results and answers questions often raised about reproducibility of DNA results, given the often sparse sampling of taxa in some studies, particularly the earliest ones. The results also provide a clear set of patterns on which to base a new classification of the subfamilies of Asparagaceae s.l., particularly Ruscaceae s.l. (¼ Nolinoideae of Asparagaceae s.l.), and examine other putatively important characters of Asparagales.

Premise of the Study To enhance our understanding of evolutionary consequences and to establish a suitable conservation strategy, we isolated microsatellite markers for the endangered Bupleurum latissimum (Apiaceae), which is endemic to the oceanic Ulleung Island. We also attempted cross-amplification in B. euphorbioides and B. longeradiatum to investigate its continental progenitors. Methods and Results Using high-throughput sequencing data, we developed 26 polymorphic microsatellite loci in three multiplexes, of which 13 loci were polymorphic in the two related species. For B. latissimum, alleles numbered two to four and the observed and expected heterozygosity ranged from 0.000 to 0.500 and 0.061 to 0.529, respectively. Conclusions These developed markers will be useful for understanding evolutionary patterns of B. latissimum in an oceanic island system and for establishing suitable conservation strategies at the genetic level.

We report a newly found natural habitat of Abeliophyllum distichum in mountainous slope range of Yeongdong-gun, Chungbuk Province. Abeliophyllum distichum Nakai is one of the Korean monotypic endemic species. Natural growth habitats of this species have been recorded from seven sites up to now, and all of the natural habitats are located in middle (Chungbuk Prov.) and middle west (Jeonbuk Prov.) parts of South Korea. Among the previously recorded seven natural habitats, six sites have been designated as Korean national monuments and protected with in situ conservation. New natural habitat of A. distichum is located on northwest slope of stiff hillock area beside the small stream, Seolgye-ri, Yeongdong-eup, Yeongdong-gun, Chungbuk Province. Total growing area is nearly 3,000 ㎡. It is 10-25 cm in soil depth and pH 5.0-6.5 in soil acidity in that area. And many of A. distichum are clustered with 2-5 individuals extended by stoloniferous asexual reproduction. And the total numbers of A. distichum are about 700 individuals with only typical white flowers, and the ratio between pin type and thrum type is 37% and 63%, respectively. The huge population of A. distichum is growing with Quercus mongolica-Fraxinus rhynchophylla association in a mixed forest, and it shows high affinity with Stephanandra incisa, Ligustrum obtusifolium, Euonymus alatus for. ciliatodentatus, and Smilax sieboldi.

A recently published Korean Flora, "The genera of vascular plants of Korea (GFK)", includes the descriptions and keys for 217 families, 1,044 genera, and 3,209 species of Korean vascular plants. We reclassified these taxa according to the recent APG classification system, which resulted in 64 orders, 204 families, 1,044 genera and 3,209 species. Twenty-two families from the GFK were abandoned because of changes to the familial delimitations in the APG system. In contrast, the number of families in the Liliaceous group was increased. The Liliaceae in the GFK included 31 genera and 109 species. These taxa are now assigned to 10 families in four different orders including Liliales, Asparagales, Alismatales, and Dioscoreales because of the drastic changes to the monocot classification system in the past 20 years. In addition, the family name of the Aucubaceae was changed to Garryaceae. As a result, the number of families in the GFK has been reduced to 204. The results were summarized in four tables and two figures at the levels of unofficial higher taxonomic hierarchies, orders, families and genera. This new information can provide a guidelines for selecting the phylogenetic analysis unit for the Korean tree of life (KTOL) project. Futhermore, the updated classification system also provides an important summarization for the systematic community for placing the Korean flora in a modern phylogenetic context.

Two transgenic lines, of Nicotiana benthamiana expressing Turnip crinkle virus (TCV)-coat protein (CP) gene with contrasting phenotype, the highest (#3) and the lowest (#18) CP expressers, were selected and challenged with the homologous TCV. The former, the highest expresser, showed nearly five times more CP expression than the latter. Progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. The infected progenies of #3 line showed mild and delayed symptom with TCV. This is a coat protein-mediated resistance (CP-MR), and its resistance level is directly proportional to CP transgene expression. However, CP-MR of the transgenic plants was specific only for TCV but not for heterologous viruses. Newly growing leaves of those infected progenies of #3 line did not show any visible symptoms at 4-week post-inoculation (wpi) with TCV, suggesting a reversal from infection. This was confirmed by RT-PCR analysis with the disappearance of the target at 4 wpi. This is a case of RNA-mediated resistance, and a threshold level of transgene expression may be needed to achieve the silent state. To confirm the RNA silencing, we infiltrated Agrobacterium carrying TCV-CP into leaves of progenies of #3 and performed RT-PCR analysis. The results indicate that TCV-CP's suppressor activity against RNA silencing itself can be silenced by the homologous expression of TCV-CP in the transgenic plants. The transgenic plants containing TCV-CP seem to be a model system to study viral protection mediated by a combination of protein and RNA silencing.

Phylogenetic relationships were examined for 17 taxa of Lycoris by RAPD analysis. The length of the amplified DNA fragments ranged from 300 bp to 1,700 bp. 57 scorable RAPD markers were observed from PCR reactions with five random oligoprimers. The analysis by UPGMA sepatated the examined taxa of Lycoris into were clusters. First group was comprised of ten taxa of L. chinensis var. sinuolata, L. sanguinea var. koreana, L. uydoensis, L. flavescens, L. radiata var. pumila, L. radiata, L. squamigera, L. chejuensis, L. aurea and L. guangxiensis, second group of L. haywardii, L. sprengeri, L. rosea, L. straminea and L. houdyshii, third group of outgroup of Narcissus tazetta var. chinensis and Crinum asiaticum var. japonicum. From the viewpoint of cytological characters such as polyploidy and karyotype, the RAPD analysis was very useful to show the relationship among the intraspecific taxa of Lycoris.

We performed the numerical analyses of thirty two morphological characters for twenty four populations of eight Korean Ilex L. taxa. Principal component analyses showed that the first three principal components were related to the total covariance by 67.0%, and the proportions of PCl, PC2 and PC3 were 31.5%, 21.1%, and 14.4%, respectively. And the closely related characteristics to the PCl, PC2 and PC3 were some reproductive characters such as the morphology of sepal, petal, anther, pistil and fruits and vegetative characters such as the morphology of petiol and leaf margin, the trichomes on the twigs, the leaf duration. From the two dimensional plottings by the eigenvalues of PCl, PC2 and PC3, six grouped were clustered as Ilex integra, I cornuta, I x wandoensis, I. rotunda, I. macropoda and I. macropoda for. pseudomacropoda, I. crenata and I. crenata var. microphilla. The numerical analysis was useful for the taxonomy of Korean Ilex because it clearly seperated the populations of taxa included in this study. The identification key was provided with the diagnostic characters.