Hepatica insularis Nakai, a perennial herb endemic to warm-temperate regions on the Korean Peninsula, has been a subject of taxonomic debate due to its morphological similarities with Hepatica asiatica Nakai. To address the taxonomic ambiguity and provide evolutionary insights into H. insularis, we developed 15 polymorphic microsatellite markers. Of these, eight markers were successfully cross-amplified in H. asiatica, demonstrating their broader applicability. The markers exhibited high levels of polymorphism, with the number of alleles per locus ranging from two to eleven. The expected heterozygosity (H) and observed heterozygosity (H ) values ranged from 0.180 to 0.802 and from 0.000 to 0.933, respectively. The developed markers will serve as a valuable tool for future phylogeographic studies aiming to understand the genetic structure and diversity of H. insularis and related taxa.

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.

Ligularia stenocephala (Maxim.) Matsum. & Koidz is a widely known edible plant species in Korea. It contains various useful antioxidant compounds and has been developed as a horticultural cultivar blooming showy inflorescence. We report the complete plastid genome (plastome) of Ligularia stenocephala, a collection from Korea. The plastome of L. stenocephala is 151,158 base pairs (bp) long and includes a pair of inverted repeat (IR) regions (24,830 bp each) that are separated by a large single copy (LSC) region (83,265 bp) and a small single copy (SSC) region (18,233 bp). The phylogenetic tree shows that L. stenocephala is closely related to L. fischeri with strong bootstrap support.

Pyropia yezoensis is the most widely cultivated and economically important marine red alga. However, due to its simple morphology, it is not easy to identify P. yezoensis cultivars by phenotype. In the present study, simple sequence repeats (SSRs) were identified in the scaffold-genome sequence of P. yezoensis cultivar SG104. These SSRs were used to develop SSR markers that could be used to study genetic diversity of P. yezoensis and identify P. yezoensis cultivars. A total of 23,120 SSRs that contained di-, tri-, tetra-, penta-, or hexa-nucleotide repeat motifs were identified. Of the 353 SSR markers that were selected for further analysis, 12 SSRs were selected for their ability to distinguish cultivars and assess cultivar genetic diversity. The 12 SSR loci yielded a total of 30 alleles, ranging from two to three alleles per locus. With the exception of cultivars HP2 and HG, all cultivars generated a single allele for each SSR locus. As such, these results demonstrate that these cultivars are double haploid lines. Cluster and STRUCTURE analyses of the SSR marker data indicated that the studied cultivars could be separated into two main clusters, which have different blade types. These SSR markers will be useful for assessing the genetic diversity of P. yezoensis germplasm collections and can be used for the identification and future registration of new cultivars. The findings of the present study will facilitate the fine genetic mapping and quantitative trait loci analysis of P. yezoensis , and, as such, may provide a framework for the molecular breeding of new P. yezoensis cultivars.

The dynamic evolution of mitochondrial gene and intron content has been reported across the angiosperms. However, a reference mitochondrial genome (mitogenome) is not available in Rubiaceae. The phylogenetic utility of mitogenome data at a species level is rarely assessed. Here, we assembled mitogenomes of six Damnacanthus indicus (Rubiaceae, Rubioideae) representing two varieties (var. indicus and var. microphyllus). The gene and intron content of D. indicus was compared with mitogenomes from representative angiosperm species and mitochondrial contigs from the other Rubiaceae species. Mitogenome structural rearrangement and sequence divergence in D. indicus were analyzed in six individuals. The size of the mitogenome in D. indicus varied from 417,661 to 419,435 bp. Comparing the number of intact mitochondrial protein-coding genes in other Gentianales taxa (38), D. indicus included 32 genes representing several losses. The intron analysis revealed a shift from cis to trans splicing of a nad1 intron (nad1i728) in D. indicus and it is a shared character with the other four Rubioideae taxa. Two distinct mitogenome structures (type A and B) were identified. Two-step direct repeat-mediated recombination was proposed to explain structural changes between type A and B mitogenomes. The five individuals from two varieties in D. indicus diverged well in the whole mitogenome-level comparison with one exception. Collectively, our study elucidated the mitogenome evolution in Rubiaceae along with D. indicus and showed the reliable phylogenetic utility of the whole mitogenome data at a species-level evolution.

Taxillus yadoriki (Loranthaceae) is a hemiparasitic evergreen shrub distributed in Korea and Japan. We report the complete chloroplast genome of T. yadoriki to provide insight into the phylogenetic relationship of Loranthaceae. This genome is 122,192 bp long, with two IR regions (22,756 bp each) that separate a large single-copy (LSC) region (70,628 bp) and a small single-copy (SSC) region (6052 bp). It contains 109 genes that encode 68 proteins, 8 rRNAs, and 33 tRNAs. All of ndh genes have been lost and the SSC region consists of only four genes similar to other Taxillus species. In ML phylogenetic, monophyly of Taxillus was strongly supported with high bootstrap value and formed a sister group with Scurrula.

Polygala tenuifolia Willd. is an Oriental plant that is widely distributed in East Asia; but the medicinal plant is a critically endangered species in Korea, where only a few individuals exist. In spite of that, there have been no molecular conservation studies on P. tenuifolia. In this study, we elucidated the complete chloroplast (cp) genome of P. tenuifolia and investigated its phylogenetic position based on the cp genomes of related taxa. Results showed the cp genome was 165,423 bp in length, consisting of a large single-copy region of 83,699 bp, a small single-copy region of 8,044 bp, and two inverted repeat regions of 36,840 bp. In addition, the plastid genome contained 123 genes, including 85 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes. The present study is the first to report the complete cp genome of genus Polygala, information which may be valuable for future molecular phylogenetic and conservation studies on P. tenuifolia.

Jeju Island is located at a marginal edge of the distributional range of East Asian evergreen broad-leaved forests. The low genetic diversity of such edge populations is predicted to have resulted from genetic drift and reduced gene flow when compared to core populations. To test this hypothesis, we examined the levels of genetic diversity of marginal-edge populations of Quercus gilva, restricted to a few habitats on Jeju Island, and compared them with the southern Kyushu populations. We also evaluated their evolutionary potential and conservation value. The genetic diversity and structure were analyzed using 40 polymorphic microsatellite markers developed in this study. Ecological Niche Modeling (ENM) has been employed to develop our insights, which can be inferred from historical distribution changes. Contrary to our expectations, we detected a similar level of genetic diversity in the Jeju populations, comparable to that of the southern Kyushu populations, which have been regarded as long-term glacial refugia with a high genetic variability of East Asian evergreen trees. We found no signatures of recent bottlenecks in the Jeju populations. The results of STRUCTURE, neighbor-joining phylogeny, and Principal Coordinate Analysis (PCoA) with a significant barrier clearly demonstrated that the Jeju and Kyushu regions are genetically distinct. However, ENM showed that the probability value for the distribution of the trees on Jeju Island during the Last Glacial Maximum (LGM) converge was zero. In consideration of these results, we hypothesize that independent massive postglacial colonization from a separate large genetic source, other than Kyushu, could have led to the current genetic diversity of Jeju Island. Therefore, we suggest that the Jeju populations deserve to be separately managed and designated as a level of management unit (MU). These findings improve our understanding of the paleovegetation of East Asian evergreen forests, and the microevolution of oaks.

Microsatellite markers were newly isolated for Sarcandra glabra (Chloranthaceae), an ecologically important species in the evergreen broad-leaved forests of East Asia. On the Korean Peninsula, S. glabra is endangered, being extremely restricted to a few habitats on Jeju Island, which is the northern edge of their distribution range with evolutionary potential. Recently, populations have been rapidly declining internationally, increasing the urgency for the conservation of Jeju populations. For long-term conservation, we designed 24 polymorphic microsatellite markers in three multiplex sets. S. glabra frequently breed through vegetative reproduction. Therefore, the ability to detect clones was also tested. From two natural populations, the number of alleles per locus ranged from 2 to 14 (average, 4.292). Values for expected heterozygosity ranged from 0.000 to 0.891 (mean, 0.263). As a result of the genotype accumulation curve, the complete recognition (100%) of all multilocus genotypes can be achieved using only 21 loci, indicating that markers showed a high ability to accurately identify individuals in natural populations. These markers will be beneficial for establishing suitable conservation strategies for S. glabra in East Asia as well as Jeju Island.

Abstract The high genetic diversity of rear‐edge refugia populations is predicted to have resulted from species repeatedly migrating to low latitudes during glacial periods over the course of Quaternary climate change. However, several recent empirical studies of cold‐tolerant plants revealed the opposite pattern. We investigated whether current habitats of the cold‐adapted and range‐restricted Bupleurum euphorbioides in the Baekdudaegan, South Korea, and North Korea could be interglacial refugia, and documented how their rear‐edge populations differ genetically from those of typical temperate species. Phylogeographic analysis and ecological niche modeling (ENM) were used. The genetic structure was analyzed using microsatellite markers and chloroplast DNA sequences. The congener B. longiradiatum was included as a typical temperate plant species. Despite having almost identical life history traits, these congeneric species exhibited contrasting patterns of genetic diversity. ENM revealed an apparent maximum range contraction during the last interglacial. In contrast, its range expanded northward to the Russian Far East (Primorsky) during the Last Glacial Maximum. Thus, we hypothesize that B. euphorbioides retreated to its current refugia during interglacial periods. Unlike populations in the central region, the rear‐edge populations were genetically impoverished and uniform, both within populations and in pooled regional populations. The rear‐edge B. euphorbioides survived at least one past interglacial, contributing to the species’ genetic diversity. We believe that such genetic variation in the cold‐adapted B. euphorbioides gives the species the necessary adaptations to survive an upcoming favorable environment (the next glacial), unless there is artificial environmental change.

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.

Ceratopteris thalictroides is a semi-aquatic fern with a circumtropical distribution. Because this species is designated internationally on the IUCN Red List as requiring at least some concern, Korean populations are of great concern for the species’ long-term survival, as they are at the northern limit of the species distribution. To establish an effective conservation strategy for those populations at the genetic level, we used the Mi-Seq platform to develop three sets of 25 polymorphic microsatellite markers for C. thalictroides, which is endangered in Korea. In populations sampled from Busan and Gochang, the number of alleles ranged from 2 to 13 (average of 5.64), and plants presented an expected heterozygosity of 0.000 to 0.860. These markers will be useful for evaluating the genetic status and conserving Korean populations of C. thalictroides more effectively. Keywords: Ceratopteris thalictroides , conservation, microsatellite markers

Plants of Daphne kiusiana on Jeju Island exhibit morphologically and genetically unique traits. Its taxonomic entity is still unclear and populations are decreasing in size because of human activity. Taxonomic delimitation and conservation efforts are required at the genomic level. We determined the complete chloroplast (cp) genome of this endangered species on Jeju Island, South Korea. Its sequence is 171,491 bp long, and comprises a pair of 41,891 bp inverted repeats, a large-single-copy (LSC) region (85,028 bp), and a diminished small-single-copy region (2681 bp). The cp genome has 135 genes, including 89 that encode proteins, plus 38 tRNAs, and eight rRNAs. Five genes (rpl32, ndhF, accD, clpP, and ndhK) are pseudogenized or lost in its cpDNA. When compared with that of a related species, Aquilaria sinensis, its LSC region contains a large inversion event between rbcL and rps12 that caused pseudogenization of clpP and accD.

Microsatellite markers were isolated for Daphne kiusiana var. kiusiana (Thymelaeaceae), an evergreen broad-leaved shrub endemic to Korea and Japan. Because its populations in Jeju Island are morphologically controversial, and consistently threatened by anthropogenic pressures, taxonomic delimitation and conservation effort are required at the genetic level. We developed 21 polymorphic microsatellite loci from Next Generation Sequencing data. The primer set included di-, tri-, and tetra-nucleotide repeats. Variability in the markers was tested for 80 individuals of D. kiusiana from three natural populations in Jeju Island and Japan. Among the 21 loci, three were unavailable for population JKJU of Japan. The Neighbor-Joining tree based on microsatellite markers described here classified the three populations into two groups according to geographical or morphological traits. These will be a powerful genetics tool for determining the taxonomic boundary and establishing suitable conservation strategies for D. kiusiana in Jeju Island. Keywords: conservation, Daphne kiusiana, Jeju Island, microsatellite markers, taxonomic entity

Historical migration patterns of boreal and alpine plants have had a narrow focus on the Korean Peninsula, generally being characterized as southern relics from northern-sourced populations. Here, we present another hypothesis for the pattern associated with Korean alpine species. A genetic analysis was conducted with Leontopodium japonicum Miq., which grows in high mountain ranges with a disjunct distribution across Korea, China, and Japan. We inferred its phylogeography using sequences of nuclear (ITS) and chloroplast DNA (trnL–F, rps16, and rpl16) for 11 populations from Korea and adjacent regions. Our molecular data (SAMOVA and network analyses) revealed a distinct genetic isolation of Korean populations with the highest genetic differentiation (Korea vs. China and Japan). By comparison, a nonsignificant level of differentiation, but a high degree of genetic diversity, was detected between Chinese and Japanese populations, resembling that of warm-temperate species. These findings demonstrate that, rather than migrating southward from more northern latitudes, current populations in Korea are distributed due to colonization via East China Sea land bridges, similar to movement by warm-temperate species. Furthermore, geographical isolation because of an oceanic barrier has led to allopatric speciation for Korean populations. This specific scenario for L. japonicum is a meaningful example that will enhance our understanding of the history of plants growing in alpine (or subalpine) zones of Korea.

Premise of the study: Microsatellite primers were developed for Pedicularis ishidoyana (Orobanchaceae), an endangered and hemiparasitic plant that is narrowly endemic to Korea. Because its populations are threatened by loss of suitable habitat, conservation efforts are required.Methods and Results: We developed polymorphic microsatellite loci through reference mapping of 300-bp paired-end reads obtained from Illumina MiSeq data. In all, 74 primer pairs were designed and 32 were amplified. Of these, 18 pairs were polymorphic, with two to six alleles each occurring in 26 individual plants. Expected and observed heterozygosities ranged from 0.142 to 0.703 and from 0.077 to 0.615, respectively.Conclusions: These microsatellite markers are expected to be useful for studies of the population genetics of P. ishidoyana

Based on specimens examined, a distribution map of Trachelospermum asiaticum in Korea is provided. The species is distributed on Jeju Island and over the southern and western coastal areas in Korea. The northern limit of distribution is the Deokjeok Islands of Incheon. The distributional range of this species is consistent with that of the Jeju and South coastal floristic subregions of Korea. On the other hand, we investigated the anatomical characteristics of root, stem, leaf and ovary, in addition to the ultrastructure of pollen grains and leaf surfaces of the species, as these have been scarcely examined.

Pedicularis growing at Mt. Halla of Jeju Island is known as an endemic species of P. hallaisanensis Hurusawa. On the other hand, the plant is morphologically similar to P. amoena, P. spicata, and P. verticillata in gross morphology, so the taxonomic treatment of the taxon remains controversial. To clarify the taxonomic position of the plants, we examined external morphological characters and nuclear ribosomal DNA ITS sequences for P. hallaisanensis and its related species. The plants of Mt. Halla are clearly different from P. amoena and P. verticillata in the morphology of calyx lobes, the length of galea and lower lip, density of glandular hairs on plants, presences of the radical leaves after anthesis and molecular data. However, P. hallaisanensis is not clearly separated from P. spicata distributed in N. E. Asia on external morphological characters and DNA sequences of internal transcribed spacers. In this study, the morphological and molecular data suggested that P. hallaisanensis should be merged into the former species.