Comparative Analyses of Complete Chloroplast Genomes and Karyotypes of Allotetraploid Iris koreana and Its Putative Diploid Parental Species (Iris Series Chinenses, Iridaceae).

The Iris series Chinenses in Korea comprises four species (I. minutoaurea, I. odaesanensis, I. koreana, and I. rossii), and the group includes some endangered species, owing to their high ornamental, economic, and conservation values. Among them, the putative allotetraploid, Iris koreana (2n = 4x = 50), is hypothesized to have originated from the hybridization of the diploids I. minutoaurea (2n = 2x = 22) and I. odaesanensis (2n = 2x = 28) based on morphological characters, chromosome numbers, and genome size additivity. Despite extensive morphological and molecular phylogenetical studies on the genus Iris, little is known about Korean irises in terms of their complete chloroplast (cp) genomes and molecular cytogenetics that involve rDNA loci evolution based on fluorescence in situ hybridization (FISH). This study reports comparative analyses of the karyotypes of the three Iris species (I. koreana, I. odaesanensis, and I. minutoaurea), with an emphasis on the 5S and 35S rDNA loci number and localization using FISH together with the genome size and chromosome number. Moreover, the cp genomes of the same individuals were sequenced and assembled for comparative analysis. The rDNA loci numbers, which were localized consistently at the same position in all species, and the chromosome numbers and genome size values of tetraploid Iris koreana (four 5S and 35S loci; 2n = 50; 1C = 7.35 pg) were additively compared to its putative diploid progenitors, I. minutoaurea (two 5S and 35S loci; 2n = 22; 1C = 3.71 pg) and I. odaesanensis (two 5S and 35S loci; 2n = 28; 1C = 3.68 pg). The chloroplast genomes were 152,259-155,145 bp in length, and exhibited a conserved quadripartite structure. The Iris cp genomes were highly conserved and similar to other Iridaceae cp genomes. Nucleotide diversity analysis indicated that all three species had similar levels of genetic variation, but the cp genomes of I. koreana and I. minutoaurea were more similar to each other than to I. odaesanensis. Positive selection was inferred for psbK and ycf2 genes of the three Iris species. Phylogenetic analyses consistently recovered I. odaesanensis as a sister to a clade containing I. koreana and I. minutoaurea. Although the phylogenetic relationship, rDNA loci number, and localization, together with the genome size and chromosome number of the three species, allowed for the inference of I. minutoaurea as a putative maternal taxon and I. odaesanensis as a paternal taxon, further analyses involving species-specific molecular cytogenetic markers and genomic in situ hybridization are required to interpret the mechanisms involved in the origin of the chromosomal variation in Iris series Chinenses. This study contributes towards the genomic and chromosomal evolution of the genus Iris.

Comparative analysis of two Korean irises (Iris ruthenica and I. uniflora, Iridaceae) based on plastome sequencing and micromorphology.

Iris ruthenica Ker Gawl. and I. uniflora Pall. ex Link, which are rare and endangered species in Korea, possess considerable horticultural and medicinal value among Korean irises. However, discrimination of the species is hindered by extensive morphological similarity. Thus, the aim of the present study was to identify discriminating features by comparing the species' complete plastid genome (i.e., plastome) sequences and micromorphological features, including leaf margins, stomatal complex distribution (hypostomatic vs. amphistomatic leaves), anther stomata density, and tepal epidermal cell patterns. Plastome comparison revealed slightly divergent regions within intergenic spacer regions, and the most variable sequences, which were distributed in non-coding regions, could be used as molecular markers for the discrimination of I. ruthenica and I. uniflora. Phylogenetic analysis of the Iris species revealed that I. ruthenica and I. uniflora formed a well-supported clade. The comparison of plastomes and micromorphological features performed in this study provides useful information for elucidating taxonomic, phylogenetic, and evolutionary relationships in Iridaceae. Further studies, including those based on molecular cytogenetic approaches using species specific markers, will offer insights into species delimitation of the two closely related Iris species.

Cytological Study of Cypripedium japonicum Thunb. (Orchidaceae Juss.): An Endangered Species from Korea.

Changes in chromosome number and karyotype evolution are important to plant diversification, as they are both major drivers of speciation processes. Herein, chromosome number, karyotype, and genome size of the Korean lady's slipper orchid Cypripedium japonicum Thunb., an endangered species, were investigated in natural populations. Furthermore, all cytological data from this species are reported herein for the first time. The chromosome number of all investigated C. japonicum plants was diploid (2n = 2x = 22), with x = 11 as base chromosome number, whereby the species can now be clearly distinguished from the Japanese lady's slipper orchid. The karyotypes of all studied individuals were of similar length, symmetrical, and rather unimodal. Flow cytometry of the C. japonicum revealed that the genome size ranged from 28.38 to 30.14 pg/1C. Data on chromosome number and karyotypes were largely consistent with previous results indicating that Korean (x = 11) populations of C. japonicum are more closely related to Chinese populations (x = 11) compared to Japanese (x = 10) populations. These comprehensive cytological results will benefit the efforts to discriminate the geographically isolated and endangered Eastern Asian (China, Japan, and Korea) lady's slipper orchid species.

Rear-edge, low-diversity, and haplotypic uniformity in cold-adapted Bupleurum euphorbioides interglacial refugia populations.

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.

Genome Size and Chromosome Number Evolution in Korean Iris L. Species (Iridaceae Juss.).

Chromosome numbers, karyotypes, and genome sizes of 14 Iris L. (Iridaceae Juss.) species in Korea and their closely related taxon, Sisyrinchium rosulatum, are presented and analyzed in a phylogenetic framework. To date, understanding the chromosomal evolution of Korean irises has been hampered by their high chromosome numbers. Here, we report analyses of chromosome numbers and karyotypes obtained via classic Feulgen staining and genome sizes measured using flow cytometry in Korean irises. More than a two-fold variation in chromosome numbers (2n = 22 to 2n = 50) and over a three-fold genome size variation (2.39 pg to 7.86 pg/1 C) suggest the putative polyploid and/or dysploid origin of some taxa. Our study demonstrates that the patterns of genome size variation and chromosome number changes in Korean irises do not correlate with the phylogenetic relationships and could have been affected by different evolutionary processes involving polyploidy or dysploidy. This study presents the first comprehensive chromosomal and genome size data for Korean Iris species. Further studies involving molecular cytogenetic and phylogenomic analyses are needed to interpret the mechanisms involved in the origin of chromosomal variation in the Iris.

Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells.

Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) transcription factor expressed in neuronal precursors in the developing nervous system. The function of Ngn1 in neurogenesis has been shown in various aspects. In this study, we investigated the neurogenic potential of Ngn1 using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To investigate the expression of Ngn1, expression vectors for the full-length and the C- terminal deletion mutant of Ngn1 were constructed and their transactivation potential was verified using reporter gene containing the E-box sequence. Overexpression of the full-length Ngn1 induced neurite outgrowth in F11 cells in the absence of cAMP. A C-terminal deletion mutant, Ngn1(1-197), inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that the Ngn1 plays an important role in differentiation of neuroblastoma cells and the C terminus of Ngn1 is essential for the efficient differentiation.