Intraspecific divergence in a coastal plant, Euphorbia jolkinii, at a major biogeographic boundary in East Asia.

PREMISE: Quaternary climatic fluctuations and long-distance seed dispersal across the sea are critical factors affecting the distribution of coastal plants, but the spatiotemporal nature of population expansion and distribution change of East Asian coastal plants during this period are rarely examined. To explore this process, we investigated the genome-wide phylogenetic patterns of Euphorbia jolkinii Boiss. (Euphorbiaceae), which grows widely on littoral areas of Japan, Korea, and Taiwan. METHODS: We used plastome sequences and genome-wide single nucleotide polymorphisms in samples across the species range to reveal phylogeographic patterns and spatiotemporal distributional changes. We conducted ecological niche modeling for the present and the last glacial maximum (LGM). RESULTS: Genetic differentiation was observed between the northern and southern populations of E. jolkinii, separated by the major biogeographic boundary, the Tokara Gap. These two groups of populations differentiated during the glacial period and subsequently intermingled in the intermorainic areas of the central Ryukyu Islands after the LGM. Ecological niche models suggested that the potential range of E. jolkinii was restricted to southern Kyushu; however, it was widespread in the southern Ryukyu Islands and Taiwan during the LGM. CONCLUSIONS: This study provides evidence of genetic differentiation among coastal plant populations separated by the prominent biogeographical boundary. Although coastal plants are typically expected to maintain population connectivity through sea-drifted seed dispersal, our findings suggest that genetic differences may arise because of a combination of limited gene flow and changes in climate during the glacial period.

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 Diversity Patterns of Rare to Abundant Microbial Eukaryotes Across a Broad Range of Salinities in a Solar Saltern.

Solar salterns are excellent artificial systems for examining species diversity and succession along salinity gradients. Here, the eukaryotic community in surface water of a Korean solar saltern (30 to 380 practical salinity units) was investigated from April 2019 to October 2020 using Illumina sequencing targeting the V4 and V9 regions of 18S rDNA. A total of 926 operational taxonomic units (OTUs) and 1,999 OTUs were obtained with the V4 and V9 regions, respectively. Notably, most of the OTUs were microbial eukaryotes, and the high-abundance groups (> 5% relative abundance (RA), Alveolata, Stramenopila, Archaeplastida, and Opisthokonta) usually accounted for > 90% of the total cumulative read counts and > 80% of all OTUs. Moreover, the high-abundance Alveolata (larger forms) and Stramenopila (smaller forms) groups displayed a significant inverse relationship, probably due to predator-prey interactions. Most of the low-abundance (0.1-5% RA) and rare (< 0.1% RA) groups remained small portion during the field surveys. Taxonomic novelty (at < 90% sequence identity) was high in the Amoebozoa, Cryptista, Haptista, Rhizaria, and Stramenopila groups (69.8% of all novel OTUs), suggesting the presence of a large number of hidden species in hypersaline environments. Remarkably, the high-abundance groups had little overlap with the other groups, implying the weakness of rare-to-prevalent community dynamics. The low-abundance Discoba group alone temporarily became the high-abundance group, suggesting that it is an opportunistic group. Overall, the composition and diversity of the eukaryotic community in hypersaline environments may be persistently stabilized, despite diverse disturbance events.

A New Halophilic Heterolobosean Flagellate, Aurem hypersalina gen. n. et sp. n., Closely Related to the Pleurostomum-Tulamoeba Clade: Implications for Adaptive Radiation of Halophilic Eukaryotes.

Halophilic protozoa are independently scattered across the molecular phylogeny of eukaryotes; most of which are assigned to Heterolobosea. Here, we isolated a biflagellate from a hypersaline water of 342‰ salinity. This isolate shared several morphological features with typical halophilic heterolobosean flagellates. In addition, molecular phylogenetic trees of the 18S rRNA gene sequences clearly indicated flagellate is a heterolobosean species closely related to the halophilic Tulamoebidae. However, the flagellate was not accommodated to any described genus. Cells were ovoid-shaped, and no amoebae were observed. The two unequal flagella beat heterodynamically. An ear-like bulge at the margin of a cytostomal groove was observed. Flagellates could grow at 100-200‰ salinity, suggesting an obligately halophilic species. Currently, it appears that the new halophilic Aurem hypersalina forms a strong clade with Tulamoebidae, and is sister to the Tulamoebidae, indicating that this new clade is composed almost entirely of obligate halophilic taxa. Thus, A. hypersalina and the Tulamoebidae clade currently represent a unique adaptive radiation of halophilic eukaryotes.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

Morphology and Phylogenetic Analyses of Three Novel Naegleria Isolated from Freshwaters on Jeju Island, Korea, During the Winter Period.

The genus Naegleria is one of the best known heterolobosean groups, and is the causative agent of primary amoebic meningoencephalitis. This group is rarely studied in temperate regions during winter. Here, three novel Naegleria were isolated from freshwaters on Jeju Island, Korea, during winter. Two isolates were amoeboflagellates, and one of the three amoebae did not undergo enflagellation. All amoebae had eruptive pseudopodia, and the layer of refractile granules around a large nucleus. They formed a cyst with ~2 pores in the cyst stage. The amoeboflagellate form had two flagella and no division in the flagellate stage, and no cytostome. These features are very similar to typical Naegleria. Furthermore, our isolates were able to grow at > 30 °C, suggesting that they had different thermophilicity from Naegleria in polar regions. All amoebae were largely encysted at 5 or 10 °C, indicating that they were likely encysted during winter. Based on the 18S rRNA gene and the ITS1-5.8S rRNA gene-ITS2 sequences, the phylogenetic analyses consistently revealed that the isolates are members of the Naegleria group. However, the isolates differ from other species in both phylogenetic trees. Thus, Naegleria in cold habitats appeared to have a high degree of novelty, but their thermophilicity may be dependent on locality.

A New Heterolobosean Amoeboflagellate, Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea, Korea.

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5-28.0 µm and 5.4-12.6 µm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 µm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo- and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1-5.8S rRNA gene-ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.

Characterization of a Deep-Branching Heterolobosean, Pharyngomonas turkanaensis n. sp., Isolated from a Non-Hypersaline Habitat, and Ultrastructural Comparison of Cysts and Amoebae Among Pharyngomonas Strains.

An unusual heterolobosean amoeba, isolate LO, was isolated recently from a sample with a salinity of ~4‰, from Lake Turkana in East Africa. 18S rDNA phylogenies confirm that isolate LO branches among halophilic amoeboflagellates assigned to Pharyngomonas. We examined the ultrastructure of the amoeba and cyst stages of isolate LO, as well as the amoebae and cysts of Pharyngomonas kirbyi (isolates AS12B and SD1A). The amoebae of all three isolates lacked discrete dictyosomes and had discoidal/flattened mitochondrial cristae, but the mitochondria were not enrobed by rough endoplasmic reticulum. The cysts of all three isolates showed a thick, bipartite cyst wall, and lacked cyst pores. The cysts of isolate LO were distinct in that the ectocyst was very loose-fitting, and could contain "crypts". No flagellate form of isolate LO has been observed to date, and a salinity-for-growth experiment showed that isolate LO can grow at 15-100‰ salinity, indicating that it is halotolerant. By contrast, other studied Pharyngomonas isolates are amoeboflagellates and true halophiles. Therefore, we propose isolate LO as a new species, Pharyngomonas turkanaensis n. sp. It is possible that P. turkanaensis descended from halophilic ancestors, and represents a secondary reestablishment of a physiology adapted for moderate salinity.

The kinetoplastid parasite Azumiobodo hoyamushi, the causative agent of soft tunic syndrome of the sea squirt Halocynthia roretzi, resides in the East Sea of Korea.

Mass mortality of the edible sea squirt Halocynthia roretzi since the 1990s in the southern and eastern seas of Korea has caused large economic losses. The disease is characterized by symptoms of initially softened and thinned tunics that eventually rupture. Thus, the disease is called soft tunic syndrome (STS); however, the causative agent in these regions is unknown. In the present study, two kinetoplastid organisms were isolated from STS sea squirts collected from culture farms in Tongyeong located in the East Sea of Korea. Phylogenetic analysis of 18S rRNA sequences identified these organisms as Azumiobodo hoyamushi and Procryptobia sorokini. These kinetoplastids were injected into healthy sea squirts and cultured at 15°C for 13days. Sea squirts injected with A. hoyamushi showed 100% STS whereas, P. sorokini did not induce disease, thereby confirming A. hoyamushi as the causative agent of STS. A. hoyamushi flourishes in vitro at 10-15°C, and dies at temperatures below 5°C or above 20°C. The optimum salinity level for growth is 30-35psu, and death occurs below 25psu. These optima coincide with marine temperature and salinity levels between March and June on the southern coasts of Korea, the period when the syndrome occurs at the highest frequency. The identification here of A. hoyamushi as the causative agent of STS and our findings regarding its optimum growth conditions should lead to methods for reducing the incidence of STS.

Accelerated premature stress-induced senescence of young annulus fibrosus cells of rats by high glucose-induced oxidative stress.

PURPOSES: Diabetes mellitus (DM) is thought to be an important aetiological factor in intervertebral disc degeneration. A glucose-mediated increase of oxidative stress is a major causative factor in development of diseases associated with DM. The aim of this study was to investigate the effect of high glucose on mitochondrial damage, oxidative stress and senescence of young annulus fibrosus (AF) cells. METHODS: AF cells were isolated from four-week-old young rats, cultured, and placed in either 10 % FBS (normal control) or 10 % FBS plus two different high glucose concentrations (0.1 M and 0.2 M) (experimental conditions) for one and three days. We identified and quantified the mitochondrial damage and reactive oxygen species (ROS) (oxidative stress). We also identified and quantified the occurrence of senescence and telomerase activity. Finally, the expressions of proteins were determined related to replicative senescence (p53-p21-pRB) and stress-induced senescence (p16-pRB). RESULTS: Two high glucoses enhanced the mitochondrial damage in young rat AF cells, which resulted in an excessive generation of ROS in a dose- and time-dependent manner for one and three days compared to normal control. Two high glucose concentrations increased the occurrence of senescence of young AF cells in a dose- and time-dependent manner. Telomerase activity declined in a dose- and time-dependent manner. Both high glucose treatments increased the expressions of p16 and pRB proteins in young rat AF cells for one and three days. However, compared to normal control, the expressions of p53 and p21 proteins were decreased in young rat AF cells treated with both high glucoses for one and three days. CONCLUSIONS: The present study demonstrated that high glucose-induced oxidative stress accelerates premature stress-induced senescence in young rat AF cells in a dose- and time-dependent manner rather than replicative senescence. These results suggest that prevention of excessive generation of oxidative stress by strict blood glucose control could be important to prevent or to delay premature intervertebral disc degeneration in young patients with DM.

Survivorship of implanted bone marrow-derived mesenchymal stem cells in acute rotator cuff tear.

BACKGROUND: This study examined whether a mesenchymal stem cells (MSCs)-seeded 3-dimensional construct into a tendon defect would promote cellular differentiation and matrix healing. MATERIALS AND METHODS: Bone marrow was harvested from the iliac crests of 2 male New Zealand White rabbits. The MSCs were cultured, and an open-cell polylactic acid (OPLA) scaffold was encapsulated with these cells. The injury model was a 5-mm × 5-mm-sized full-thickness window defect in the central part of each rotator cuff tendon. The defects on the right side were grafted with the autologous MSCs-seeded OPLA scaffold implant and a biodegradable suture. The same procedure was done on the left side, except a cell-free OPLA scaffold was used. Three rabbits were used as controls, without treatment of the tendon defect. Samples were harvested at 2, 4, and 6 weeks for analysis, which included evaluation of gross morphology, fluorescent analysis, histologic assessment, and immunohistochemistry studies. RESULTS: The expression of immunohistochemical stainings for collagen I was higher in the scaffold with MSCs than in the scaffold without MSCs. The expression of collagen II, however, was not different between the scaffolds with and without MSCs. CONCLUSIONS: Even though this is a short-term study, we demonstrated that many MSCs in the scaffold survived after implantation in an acute rabbit rotator cuff defect. Furthermore, the generation of type I collagen increased more in the scaffold with MSCs than it did in the scaffold without MSCs. MSCs are thought to promote tendon healing by producing type I collagen when they are applied at the tendon defect.

The complete chloroplast genome of Porella gracillima Mitt. (Porellaceae) and phylogenetic relationships to other bryophytes.

Porella gracillima Mitt. (Jungermanniidae, Porellaceae), a bryophyte is widespread in temperate Asia and North America. In Korea, P. gracillima is mainly observed in shaded and dried rocks or tree trunks on mountains. Here, we determined the complete chloroplast (cp) genome sequence of P. gracillima to provide useful genetic information in the phylogenetic relationship, phylogeographic history, and conservation of the species. The complete cp genome of P. gracillima was assembled using NGS Illumina HiSeqX platform. The cp genome was 121,867 bp in length (GC contents, 33.7%) and showed a typical quadripartite structure, consisting of a large single copy (LSC) of 83,406 bp, a small single copy (SSC) of 19,692 bp, and two inverted repeats (IRs) of 9,385 bp. Phylogenetic analysis shows that Porellaceae was a sister group of Radulaceae, which agrees with the findings of the previous phylogenetic studies. Our cp genome data of P. gracillima may contribute to a better understanding of the evolution of the Porella in Porellaceae and will help to infer its molecular identification, thereby providing a guideline for conservation.

Phytochemical profiling of Symplocos tanakana Nakai and S. sawafutagi Nagam. leaf and identification of their antioxidant and anti-diabetic potential.

Symplocos sp. contains various phytochemicals and is used as a folk remedy for treatment of diseases such as enteritis, malaria, and leprosy. In this study, we discovered that 70% ethanol extracts of Symplocos sawafutagi Nagam. and S. tanakana Nakai leaves have antioxidant and anti-diabetic effects. The components in the extracts were profiled using high-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry; quercetin-3-O-(6''-O-galloyl)-ß-d-galactopyranoside (6) and tellimagrandin II (7) were the main phenolic compounds. They acted as strong antioxidants with excellent radical scavenging activity and as inhibitors of non-enzymatic advanced glycation end-products (AGEs) formation. Mass fragmentation analysis demonstrated that compounds 6 and 7 could form mono- or di-methylglyoxal adducts via reaction with methylglyoxal, which is a reactive carbonyl intermediate and an important precursor of AGEs. In addition, compound 7 effectively inhibited the binding between AGE2 and receptor for AGEs as well as the activity of α-glucosidase. Enzyme kinetic study revealed that compound 7 acts as a competitive inhibitor of α-glucosidase, through interaction with the active site of the enzyme. Therefore, compounds 6 and 7, the major constituents of S. sawafutagi and S. tanakana leaves, are promising for developing drugs for preventing or treating diseases caused by aging and excessive sugar consumption.

The genetic structure and demographic history of Zabelia tyaihyonii, endemic to Korean limestone karst forests, based on genome-wide SNP markers.

Similar to the global phenomenon, many plant species endemic to Korean limestone karst forests are at risk of extinction due to human intervention. Zabelia tyaihyonii is a familiar shrub, called "Hardy abelia" and "Fragrant abelia" growing in the karst forests of Korea, where it is one of the most threatened species. We investigated the genetic structure and demographic history of Z. tyaihyonii, which allow us to develop appropriate conservation and management strategies. The genetic structure was evaluated using a total of 187 samples from 14 populations, covering the entire distribution of Z. tyaihyonii in South Korea. We utilized 254 and 1753 SNP loci obtained via MIG-seq (Multiplexed ISSR Genotyping by sequencing) for structure and demographic analyses, respectively. The population demographic modeling was performed with site frequency spectrum. To gain further historical insights, we also employed ENM (Ecological Niche Modeling). We found two distinct clusters (CLI and CLII) of ancient origin (ca. 490 ka). Despite CLII experiencing a more severe bottleneck, both clusters showed similar levels of genetic diversity, indicating mutual historical gene flow. Their historical distribution range seems to have changed very little. We proposed a historical distribution scenario for Z. tyaihyonii, taking into account its intrinsic factors, and emphasized a more complex response to Quaternary climate change beyond simple allopatric speciation models. These findings provide valuable insights for conservation and management strategies for Z. tyaihyonii.

Effects of different ion compositions on growth of obligately halophilic protozoan Halocafeteria seosinensis.

Substantial halophilic organisms have been found in 100-200‰ salinities. These ranges represent a highly specialized halophilic environment to which only a few halotolerant species have adapted. Recent studies have underlined the existence of diverse obligately halophilic protozoa in the salinity ranges of 100-200‰. The ranges of salinity under which these organisms can grow have been examined to some extent, but the balance of specific ions that will support growth has not been investigated. The heterotrophic nanoflagellate Halocafeteria, the type strain of which grows optimally at 150‰ salinity and 35°C, is a commonly encountered obligate halophile found in very hypersaline environments. These extreme environments can vary in their Mg:Ca ratios (i.e. weight ratios) and sulfate concentrations. To examine growth response of Halocafeteria to the different chemical compositions, densities of Halocafeteria seosinensis strain EHF34 were monitored in seven different ion composition media for 9 days at 1- to 2-day intervals (at 150‰ salinity and 35°C, with no prey limitation). Halocafeteria does not grow at Mg:Ca ratios of 35 and 100 and at high sulfate concentrations of 11.6 and 31.6 g l(-1). It grows well in 0.6 g l(-1) sulfate media at Mg:Ca ratios of 2, 10 or 35, but not 100. The present study demonstrates that the growth of the obligate halophile Halocafeteria can be affected by different ion compositions in hypersaline environments. Therefore, Halocafeteria may not be ubiquitous in hypersaline environments due to its ionic requirements.

Characterization of Selenaion koniopes n. gen., n. sp., an amoeba that represents a new major lineage within heterolobosea, isolated from the Wieliczka salt mine.

A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73‰ saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater-like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm--these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep-branching position being moderate (i.e. maximum likelihood bootstrap support--67%; posterior probability--0.98). Cells grew at 15-150‰ salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.

Transcriptomic analysis of brine shrimp Artemia franciscana across a wide range of salinities.

Brine shrimp Artemia franciscana, a commercially important species, can thrive in a wide range of salinities and is commonly found in hypersaline lakes and solar salterns. Transcriptome analysis can enhance the understanding of the adaptative mechanisms of brine shrimp in aquaculture. RNA sequencing (RNAseq) data was generated from A. franciscana adults that were salt-adapted for 2-4 weeks at five salinities: 35, 50, 100, 150, and 230 psu. Long-read isoform sequencing (IsoSeq) data was used to construct a high-quality transcriptome assembly. Also, the gene expression patterns in A. franciscana adults were examined. Notably, the transcriptional response of A. franciscana's acclimation to intermediate salinities (50-150 psu) displayed frequently and differentially U-shaped or inverted U-shaped expression patterns. In addition, the types of genes showing two nonmonotonic expression patterns were distinct from each other. The coordinated shifts in gene expression suggest different homeostatic strategies of A. franciscana at specific salinities; such strategies may enhance population fitness at extreme salinities. Our study should promote a scientific concept for the gene expression patterns of A. franciscana along a broad salinity gradient, and a variety of salinity and prey should be monitored for testing the gene expression pattern of this important aquaculture species.

Accumulation patterns of intracellular salts in a new halophilic amoeboflagellate, Euplaesiobystra salpumilio sp. nov., (Heterolobosea; Discoba) under hypersaline conditions.

Halophilic microbial eukaryotes are present in many eukaryotic lineages and major groups; however, our knowledge of their diversity is still limited. Furthermore, almost nothing is known about the intracellular accumulation of salts in most halophilic eukaryotes. Here, we isolate a novel halophilic microbial eukaryote from hypersaline water of 134 practical salinity units (PSU) in a solar saltern. This species is an amoeboflagellate (capable of the amoeba-flagellate-cyst transformation) in the heterolobosean group and belongs to the genus Euplaesiobystra based on morphological data and 18S rDNA sequences. However, the isolate is distinct from any of the described Euplaesiobystra species. Especially, it is the smallest Euplaesiobystra to date, has a distinct cytostome, and grows optimally at 75-100 PSU. Furthermore, the phylogenetic tree of the 18S rDNA sequences demonstrates that the isolate forms a strongly supported group, sister to Euplaesiobystra hypersalinica. Thus, we propose that the isolate, Euplaesiobystra salpumilio, is a novel species. E. salpumilio displays a significantly increased influx of the intracellular Na+ and K+ at 50, 100, and 150 PSU, compared to freshwater species. However, the intracellular retention of the Na+ and K+ at 150 PSU does not significantly differ from 100 PSU, suggesting that E. salpumilio can extrude the Na+ and K+ from cells under high-salinity conditions. Interestingly, actively growing E. salpumilio at 100 and 150 PSU may require more intracellular accumulation of Na+ than the no-growth but-viable state at 50 PSU. It seems that our isolate displays two salt metabolisms depending on the tested salinities. E. salpumilio shows a salt-in strategy for Na+ at lower salinity of 100 PSU, while it displays a salt-out strategy for Na+ at higher salinity of 150 PSU. Our results suggest that the novel halophilic E. salpumilio fundamentally uses a salt-out strategy at higher salinities, and the accumulation patterns of intracellular salts in this species are different from those in other halophilic microbial eukaryotes.

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.