Identification of the Plant Family Caryophyllaceae in Korea Using DNA Barcoding.

Caryophyllaceae is a large angiosperm family, with many species being utilized as ornamental or medicinal plants in Korea, in addition to several endangered species that are managed by the government. In this study, we used DNA barcoding for the accurate identification of Korean Caryophyllaceae. A total of 78 taxa (n = 215) were sequenced based on three chloroplast regions (rbcL, matK, and psbA-trnH) and nuclear ribosomal internal transcribed spacers (ITS). In the neighbor-joining tree, a higher accuracy of identification was generally observed when using ITS (>73%) rather than chloroplast regions (<62%). The highest resolution was found for rbcL + ITS (77.6%), although resolution varied according to the genus. Among the genera that included two and more species, five genera (Eremogone, Minuartia, Pseudostellaria, Sagina, and Stellaria) were successfully identified. However, the species of five other genera (Cerastium, Gypsophila, Dianthus, Silene, and Spergularia) showed relatively low resolutions (0-61.1%). In the cases of Cerastium, Dianthus, and Silene, ambiguous taxonomic relationships among unidentified species may have been a factor contributing to such low resolutions. However, in contrast to these results, Gypsophila and Spergularia have been identified well in previous studies. Our findings indicate the need of taxonomic reconsideration in Korea.

Genetic and demographic signatures accompanying the evolution of the selfing syndrome in Daphne kiusiana, an evergreen shrub.

BACKGROUND AND AIMS: The evolution of mating systems from outcrossing to self-fertilization is a common transition in flowering plants. This shift is often associated with the 'selfing syndrome', which is characterized by less visible flowers with functional changes to control outcrossing. In most cases, the evolutionary history and demographic dynamics underlying the evolution of the selfing syndrome remain poorly understood. METHODS: Here, we characterize differences in the demographic genetic consequences and associated floral-specific traits between two distinct geographical groups of a wild shrub, Daphne kiusiana, endemic to East Asia; plants in the eastern region (southeastern Korea and Kyushu, Japan) exhibit smaller and fewer flowers compared to those of plants in the western region (southwestern Korea). Genetic analyses were conducted using nuclear microsatellites and chloroplast DNA (multiplexed phylogenetic marker sequencing) datasets. KEY RESULTS: A high selfing rate with significantly increased homozygosity characterized the eastern lineage, associated with lower levels of visibility and herkogamy in the floral traits. The two lineages harboured independent phylogeographical histories. In contrast to the western lineage, the eastern lineage showed a gradual reduction in the effective population size with no signs of a severe bottleneck despite its extreme range contraction during the last glacial period. CONCLUSIONS: Our results suggest that the selfing-associated morphological changes in D. kiusiana are of relatively old origin (at least 100 000 years ago) and were driven by directional selection for efficient self-pollination. We provide evidence that the evolution of the selfing syndrome in D. kiusiana is not strongly associated with a severe population bottleneck.

The complete chloroplast genome sequence of Melampyrum koreanum (Orobanchaceae), an endemic and hemi-parasitic herb in Korea.

Melampyrum koreanum K.-J. Kim and S.-M. Yun 2012 (Orobanchaceae) is a hemi-parasitic herb, endemic to Korea. Here, the chloroplast genome of this species is reported. It was found to be 143,865 bp long, with a large single-copy region of 83,133 bp, a small single-copy region of 10,308 bp, and a pair of inverted repeat regions of 25,212 bp each. The chloroplast genome harbors 124 genes, including 79 protein-coding genes, 37 transfer RNA genes, and eight ribosomal RNA genes. Among the identified genes, rpoA and several ndh genes were determined to be pseudogenized due to the stop codon in the middle of the gene. The phylogenetic tree of the family was reconstructed based on 20 protein-coding genes, conserved across studied taxa. As a result, Melampyrum L. 1753 species were found to form a monophyletic group in the family.

Complete Plastome of Three Korean Asarum (Aristolochiaceae): Confirmation Tripartite Structure within Korean Asarum and Comparative Analyses.

The genus Asarum (Aristolochiaceae) is a well-known resource of medicinal and ornamental plants. However, the taxonomy of Korean Asarum is ambiguous due to their considerable morphological variations. Previously, a unique plastome structure has been reported from this genus. Therefore, we investigated the structural change in the plastomes within three Korean Asarum species and inferred their phylogenetic relationships. The plastome sizes of Asarum species assembled here range from 190,168 to 193,356 bp, which are longer than a typical plastome size (160 kb). This is due to the incorporation and duplication of the small single copy into the inverted repeat, which resulted in a unique tripartite structure. We first verified this unique structure using the Illumina Miseq and Oxford Nanopore MinION platforms. We also investigated the phylogeny of 26 Aristolochiaceae species based on 79 plastid protein-coding genes, which supports the monophyly of Korean Asarum species. Although the 79 plastid protein-coding gene data set showed some limitations in supporting the previous classification, it exhibits its effectiveness in delineating some sections and species. Thus, it can serve as an effective tool for resolving species-level phylogeny in Aristolochiaceae. Last, we evaluated variable sites and simple sequence repeats in the plastome as potential molecular markers for species delimitation.

Historical migration and taxonomic entity of Korean endemic shrub Lespedeza maritima (Fabaceae) based on microsatellite loci.

Various plant species are endemic to the Korean Peninsula, but their evolutionary divergence and establishment are poorly understood. One of these, Lespedeza maritima, has been proposed as either a hybrid (L. cyrtobotrya × L. maximowiczii) or a synonym of L. thunbergii. A distinct taxon, L. uekii, has been proposed for inland populations. We investigated genetic diversity and structure in L. maritima and related taxa to resolve this. Genotypes of L. maritima (n = 244, including L. uekii) were determined using 12 microsatellite loci, then compared with those of related species. Genetic diversity within L. maritima was estimated, and Bayesian clustering analysis was used to represent its genetic structure and that of related taxa. Its distribution during the last glacial maximum (LGM) was predicted using ecological niche modelling (ENM). Neighbour-joining (NJ) analysis and principal coordinate analysis (PCoA) were used to investigate relationships among species. Bayesian tree based on nuclear ribosomal internal transcribed spacers (nrITS) was also reconstructed to show relationships and divergence time among species. Morphological features were examined using flower characteristics. In result, expected heterozygosity (H E) and allelic richness (A R) within L. maritima were higher in southern than northern populations. Bayesian clustering analysis largely assigned populations to two clusters (K = 2) (south vs. north). The ENM showed that L. maritima occurred around the East China Sea and Korean Strait land bridge during the LGM. Compared with other Lespedeza species, L. maritima was assigned to an independent cluster (K = 2-5), supported by the NJ, PCoA, Bayesian tree and morphological examination results. Lespedeza maritima and L. uekii were clustered to one clade on Bayesian tree. Given results, current L. maritima populations derive from post-LGM colonization away from southern refugia. The type L. uekii, which grows inland, is thought synonym of L. maritima. In addition, L. maritima is considered a distinct species, compared with related taxa.

The complete plastid genome of Rhamnus taquetii, an endemic shrub on the Jeju Island of Korea.

Rhamnus taquetii (family Rhamnaceae) is a shrub , endemic to Korea. Here, the R. taquetii plastid genome was found to be 161,205 bp long and consists of quadripartite structures; a large single-copy region of 89,373 bp, a small single-copy region of 18,936 bp, and a pair of inverted repeat regions of 26,448 bp each. The GC content of the sequence was found to be 37.1%. The plastid genome was found to harbor 129 genes, including 84 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. On the phylogenetic tree of Rosales (based on 63 protein-coding genes), Rhamnaceaewas found to be monophyletic.

Insights into genomic structure and evolutionary processes of coastal Suaeda species in East Asia using cpDNA, nDNA, and genome-wide SNPs.

Species in the genus Suaeda have few diagnostic characters and substantial morphological plasticity. Hence, regional floras do not provide clear taxonomic information for Suaeda spp. in East Asia. In order to assess the taxonomy of four species in the genus Suaeda (S. australis, S. maritima, S. japonica, and S. heteroptera), cpDNA (rpl32-trnL and trnH-psbA), nDNA (ITS), and MIG-seq analyses were carried out. Genome-wide SNP results indicated three lineages: (1) S. australis in Korea and S. maritima in Japan, (2) S. maritima in Korea and S. heteroptera in China, and (3) S. japionica. In phylogenetic trees and genotype analyses, cpDNA and nDNA results showed discrepancies, while S. japonica and S. maritima in Korea, and S. heteroptera in China shared the same haplotype and ribotype. We suggest that the shared haplotype may be due to chloroplast capture. Based on our results, we assume that S. japonica was formed by homoploid hybrid speciation between the two lineages.

Plastid genome evolution in tribe Desmodieae (Fabaceae: Papilionoideae).

Recent plastid genome (plastome) studies of legumes (family Fabaceae) have shown that this family has undergone multiple atypical plastome evolutions from each of the major clades. The tribe Desmodieae belongs to the Phaseoloids, an important but systematically puzzling clade within Fabaceae. In this study, we investigated the plastome evolution of Desmodieae and analyzed its phylogenetic signaling. We sequenced six complete plastomes from representative members of Desmodieae and from its putative sister Phaseoloid genus Mucuna. Those genomes contain 128 genes and range in size from 148,450 to 153,826 bp. Analyses of gene and intron content revealed similar characters among the members of Desmodieae and Mucuna. However, there were also several distinct characters identified. The loss of the rpl2 intron was a feature shared between Desmodieae and Mucuna, whereas the loss of the rps12 intron was specific to Desmodieae. Likewise, gene loss of rps16 was observed in Mucuna but not in Desmodieae. Substantial sequence variation of ycf4 was detected from all the sequenced plastomes, but pseudogenization was restricted to the genus Desmodium. Comparative analysis of gene order revealed a distinct plastome conformation of Desmodieae compared with other Phaseoloid legumes, i.e., an inversion of an approximately 1.5-kb gene cluster (trnD-GUC, trnY-GUA, and trnE-UUC). The inversion breakpoint suggests that this event was mediated by the recombination of an 11-bp repeat motif. A phylogenetic analysis based on the plastome-scale data set found the tribe Desmodieae is a highly supported monophyletic group nested within the paraphyletic Phaseoleae, as has been found in previous phylogenetic studies. Two subtribes (Desmodiinae and Lespedezinae) of Desmodieae were also supported as monophyletic groups. Within the subtribe Lespedezinae, Lespedeza is closer to Kummerowia than Campylotropis.

ITS non-concerted evolution and rampant hybridization in the legume genus Lespedeza (Fabaceae).

The internal transcribed spacer (ITS) as one part of nuclear ribosomal DNA is one of the most extensively sequenced molecular markers in plant systematics. The ITS repeats generally exhibit high-level within-individual homogeneity, while relatively small-scale polymorphism of ITS copies within individuals has often been reported in literature. Here, we identified large-scale polymorphism of ITS copies within individuals in the legume genus Lespedeza (Fabaceae). Divergent paralogs of ITS sequences, including putative pseudogenes, recombinants, and multiple functional ITS copies were sometimes detected in the same individual. Thirty-seven ITS pseudogenes could be easily detected according to nucleotide changes in conserved 5.8S motives, the significantly lower GC contents in at least one of three regions, and the lost ability of 5.8S rDNA sequence to fold into a conserved secondary structure. The distribution patterns of the putative functional clones were highly different between the traditionally recognized two subgenera, suggesting different rates of concerted evolution in two subgenera which could be attributable to their different extents/frequencies of hybridization, confirmed by our analysis of the single-copy nuclear gene PGK. These findings have significant implications in using ITS marker for reconstructing phylogeny and studying hybridization.

Phylogeography of East Asian Lespedeza buergeri (Fabaceae) based on chloroplast and nuclear ribosomal DNA sequence variations.

The dynamic changes in land configuration during the Quaternary that were accompanied by climatic oscillations have significantly influenced the current distribution and genetic structure of warm-temperate forests in East Asia. Although recent surveys have been conducted, the historical migration of forest species via land bridges and, especially, the origins of Korean populations remains conjectural. Here, we reveal the genetic structure of Lespedeza buergeri, a warm-temperate shrub that is disjunctively distributed around the East China Sea (ECS) at China, Korea, and Japan. Two non-coding regions (rpl32-trnL, psbA-trnH) of chloroplast DNA (cpDNA) and the internal transcribed spacer of nuclear ribosomal DNA (nrITS) were analyzed for 188 individuals from 16 populations, which covered almost all of its distribution. The nrITS data demonstrated a genetic structure that followed geographic boundaries. This examination utilized AMOVA, comparisons of genetic differentiation based on haplotype frequency/genetic mutations among haplotypes, and Mantel tests. However, the cpDNA data showed contrasting genetic pattern, implying that this difference was due to a slower mutation rate in cpDNA than in nrITS. These results indicated frequent migration by this species via an ECS land bridge during the early Pleistocene that then tapered gradually toward the late Pleistocene. A genetic isolation between western and eastern Japan coincided with broad consensus that was suggested by the presence of other warm-temperate plants in that country. For Korean populations, high genetic diversity indicated the existence of refugia during the Last Glacial Maximum on the Korean Peninsula. However, their closeness with western Japanese populations at the level of haplotype clade implied that gene flow from western Japanese refugia was possible until post-glacial processing occurred through the Korea/Tsushima Strait land bridge.

Isolation and characterization of 28 microsatellite loci for a Korean endemic, Lespedeza maritima (Fabaceae).

PREMISE OF THE STUDY: We developed microsatellite primers for Lespedeza maritima (Fabaceae), a Korean endemic shrub, and conducted cross-amplifications for closely related species. METHODS AND RESULTS: We produced 28 polymorphic microsatellite markers through reference mapping of 300-bp paired-end reads obtained from Illumina MiSeq data. For 47 individual plants from two populations, the total alleles numbered two to 13, and observed and expected heterozygosities ranged from 0.067 to 0.867 and from 0.064 to 0.848, respectively. Most of these markers were well amplified in closely related species. CONCLUSIONS: In future research, the microsatellite markers described here will help reveal the taxonomic entity of this species.