Revised molecular phylogeny, global biogeography, and diversification of palms subfamily Coryphoideae (Arecaceae) based on low copy nuclear and plastid regions.

Coryphoideae are palmate-leaved palms from the family Arecaceae consisting of 46 genera representing 421 species. Although several phylogenetic analyses based on different genomic regions have been carried out on Coryphoideae, a fully resolved molecular phylogenetic tree has not been reported yet. To achieve this, we applied two phylogenetic reconstruction methods: Maximum Likelihood and Bayesian Inference, using amplified sampling by retrieving chloroplast and nuclear DNA sequences from NCBI and adding newly produced sequences from Indian accession into the dataset. The same dataset (chloroplast + nuclear DNA sequences) was used to estimate divergence times and the evolutionary history of Coryphoideae with a Bayesian uncorrelated, lognormal relaxed-clock approach and a Statistical Divergence-Vicariance Analysis method, respectively. The phylogenetic analyses based on a combined chloroplast and nuclear DNA sequence dataset showed well-resolved relationships within the subfamily. Both phylogenetic trees divide Coryphoideae into two main groups: CSPT (Crysophileae, Sabaleae, Phoeniceae, and Trachycarpeae) and the Syncarpous group. These main groups are segregated into eight tribes (Trachycarpeae, Phoeniceae, Sabaleae, Crysophileae, Borasseae, Corypheae, Caryoteae, and Chuniophoeniceae) and four subtribes (Rhapidine, Livistoninae, Hyphaeninae, and Lataniinae) with strong support-values. Most previously unresolved and doubtful relationships within tribes Trachycarpeae and Crysophilieae are now resolved and well-supported. The reconstructed phylogenetic trees support all previous systematic revisions of the subfamily. All Indian sampled species of Arenga, Bentinckia, Hyphaene, and Trachycarpus show close relation with their respective congeneric species. Molecular dating results and integration of biogeography suggest that Coryphoideae originated in Laurasia at ~95.12 Ma and then diverged into the tropical and subtropical regions of the whole world. This study offers the correct combination of nuclear and plastid regions to test the current and future systematic revisions.

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data.

A group of temperate grassland plant species termed the "Mansen elements" occurs in Japan and is widely distributed in the grasslands of continental East Asia. It has been hypothesized that these species are continental grassland relicts in Japan that stretch back to a colder age, but their migration history has not been elucidated. To assess the migration history of the Mansen elements, we performed phylogeographic analyses of Tephroseris kirilowii, a member of this group, using single-nucleotide polymorphisms (SNPs) obtained from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq). It was estimated that the Japanese populations of T. kirilowii were divided from those of continental East Asia at 25.2 thousand years ago (ka) with 95% highest probability density interval (HPD) of 15.3-40.0 ka and that Japanese clades first diverged at 20.2 ka with 95% HPD of 10.4-30.1 ka. As the climatically suitable range during the last glacial maximum (LGM) estimated using ecological niche modeling (ENM) was limited in Japan and there was a slight genetic differentiation among Japanese populations, a post-glacial expansion of T. kirilowii in the Japanese Archipelago was indicated.

Characterization and Comparison of Two Complete Plastomes of Rosaceae Species (Potentilla dickinsii var. glabrata and Spiraea insularis) Endemic to Ulleung Island, Korea.

Potentilla dickinsii var. glabrata and Spiraea insularis in the family Rosaceae are species endemic to Ulleung Island, Korea, the latter of which is listed as endangered. In this study, we characterized the complete plastomes of these two species and compared these with previously reported plastomes of other Ulleung Island endemic species of Rosaceae (Cotoneaster wilsonii, Prunus takesimensis, Rubus takesimensis, and Sorbus ulleungensis). The highly conserved complete plastomes of P. dickinsii var. glabrata and S. insularis are 158,637 and 155,524 base pairs with GC contents of 37% and 36.9%, respectively. Comparative phylogenomic analysis identified three highly variable intergenic regions (trnT-UGU/trnL-UAA, rpl32/trnL-UAG, and ndhF/rpl32) and one variable genic region (ycf1). Only 14 of the 75 protein-coding genes have been subject to strong purifying selection. Phylogenetic analysis of 23 representative plastomes within the Rosaceae supported the monophyly of Potentilla and the sister relationship between Potentilla and Fragaria and indicated that S. insularis is sister to a clade containing Cotoneaster, Malus, Pyrus, and Sorbus. The plastome resources generated in this study will contribute to elucidating the plastome evolution of insular endemic Rosaceae on Ulleung Island and also in assessing the genetic consequences of anagenetic speciation for various endemic lineages on the island.

Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, Camaenidae) in South Korea.

Aegista Albers, 1850 is a large genus of the land snail family Camaenidae Pilsbry, 1895 and distributed in south, southeast and east Asian countries (from India and Nepal to Korea and Japan). Fourteen species and subspecies of Aegista are known from South Korea. They were described, based only on shell morphology during 1887-1943 and our knowledge on diversity of Korean Aegista has seldom been updated since then. In this study, we provide the report on the first molecular investigation of diversity of Aegista in South Korea, which unmasked some of overlooked diversity of this group.

Cytogenetics, Typification, Molecular Phylogeny and Biogeography of Bentinckia (Arecoideae, Arecaceae), an Unplaced Indian Endemic Palm from Areceae.

Bentinckia is a genus of flowering plants which is an unplaced member of the tribe Areceae (Arecaceae). Two species are recognized in the genus, viz. B. condapanna Berry ex Roxb. from the Western Ghats, India, and B. nicobarica (Kurz) Becc. from the Nicobar Islands. This work constitutes taxonomic revision, cytogenetics, molecular phylogeny, and biogeography of the Indian endemic palm genus Bentinckia. The present study discusses the ecology, morphology, taxonomic history, distribution, conservation status, and uses of Bentinckia. A neotype was designated for the name B. condapanna. Cytogenetical studies revealed a new cytotype of B. condapanna representing 2n = 30 chromosomes. Although many phylogenetic reports of the tribe Areceae are available, the relationship within the tribe is still ambiguous. To resolve this, we carried out Bayesian Inference (BI) and Maximum Likelihood (ML) analysis using an appropriate combination of chloroplast and nuclear DNA regions. The same phylogeny was used to study the evolutionary history of Areceae. Phylogenetic analysis revealed that Bentinckia forms a clade with other unplaced members, Clinostigma and Cyrostachys, and together they show a sister relationship with the subtribe Arecinae. Biogeographic analysis shows Bentinckia might have originated in Eurasia and India.

Isolation and characterization of microsatellite markers from Tiarella polyphylla (Saxifragaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Tiarella polyphylla in Korea and Japan to study the genetic diversity and genetic structure of the species. METHODS AND RESULTS: We successfully isolated a total of nine polymorphic simple sequence repeat loci. These nine loci contained from two to eight alleles per locus. The observed and expected heterozygosities ranged from 0.000 to 0.250 and 0.000 to 0.974, respectively. CONCLUSIONS: The markers developed in this study may be used to estimate genetic diversity, genetic structure, and historical and recent gene flow in T. polyphylla, which requires conservation and management.

Immunocytochemical localization of the AMPA glutamate receptor subtype GluR2/3 in the squid optic lobe.

As a major excitatory neurotransmitter in the cephalopod visual system, glutamate signaling is facilitated by ionotropic receptors, such as α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR). In cephalopods with large and well-developed brains, the optic lobes (OL) mainly process visual inputs and are involved in learning and memory. Although the presence of AMPAR in squid OL has been reported, the organization of specific AMPAR-containing neurons remains unknown. This study aimed to investigate the immunocytochemical localization of the AMPA glutamate receptor subtype 2/3-immunoreactive (GluR2/3-IR) neurons in the OL of Pacific flying squid (Tordarodes pacificus). Morphologically diverse GluR2/3-IR neurons were predominantly located in the tangential zone of the medulla. Medium-to-large GluR2/3-IR neurons were also detected. The distribution patterns and cell morphologies of calcium-binding protein (CBP)-IR neurons, specifically calbindin-D28K (CB)-, calretinin (CR)-, and parvalbumin (PV)-IR neurons, were similar to those of GluR2/3-IR neurons. However, two-color immunofluorescence revealed that GluR2/3-IR neurons did not colocalize with the CBP-IR neurons. Furthermore, the specific localizations and diverse types of GluR2/3-IR neurons that do not express CB, CR, or PV in squid OL were determined. These findings further contribute to the existing data on glutamatergic visual systems and provide new insights for understanding the visual processing mechanisms in cephalopods.

Infrageneric Plastid Genomes of Cotoneaster (Rosaceae): Implications for the Plastome Evolution and Origin of C. wilsonii on Ulleung Island.

Cotoneaster is a taxonomically and ornamentally important genus in the family Rosaceae; however, phylogenetic relationships among its species are complicated owing to insufficient morphological diagnostic characteristics and hybridization associated with polyploidy and apomixis. In this study, we sequenced the complete plastomes of seven Cotoneaster species (C. dielsianus, C. hebephyllus, C. integerrimus, C. mongolicus, C. multiflorus, C. submultiflorus, and C. tenuipes) and included the available complete plastomes in a phylogenetic analysis to determine the origin of C. wilsonii, which is endemic to Ulleung Island, Korea. Furthermore, based on 15 representative lineages within the genus, we carried out the first comparative analysis of Cotoneaster plastid genomes to gain an insight into their molecular evolution. The plastomes were highly conserved, with sizes ranging from 159,595 bp (C. tenuipes) to 160,016 bp (C. hebephyllus), and had a GC content of 36.6%. The frequency of codon usage showed similar patterns among the 15 Cotoneaster species, and 24 of the 35 protein-coding genes were predicted to undergo RNA editing. Eight of the 76 common protein-coding genes, including ccsA, matK, ndhD, ndhF, ndhK, petA, rbcL, and rpl16, were positively selected, implying their potential roles in adaptation and speciation. Of the 35 protein-coding genes, 24 genes (15 photosynthesis-related, seven self-replications, and three others) were found to harbor RNA editing sites. Furthermore, several mutation hotspots were identified, including trnG-UCC/trnR-UCU/atpA and trnT-UGU/trnL-UAA. Maximum likelihood analysis based on 57 representative plastomes of Cotoneaster and two Heteromeles plastomes as outgroups revealed two major lineages within the genus, which roughly correspond to two subgenera, Chaenopetalum and Cotoneaster. The Ulleung Island endemic, C. wilsonii, shared its most recent common ancestor with two species, C. schantungensis and C. zabelii, suggesting its potential origin from geographically close members of the subgenus Cotoneaster, section Integerrimi.

The Organization of Somatostatin-Immunoreactive Cells in the Visual Cortex of the Gerbil.

Somatostatin (SST) is widely expressed in the brain and plays various, vital roles involved in neuromodulation. The purpose of this study is to characterize the organization of SST neurons in the Mongolian gerbil visual cortex (VC) using immunocytochemistry, quantitative analysis, and confocal microscopy. As a diurnal animal, the Mongolian gerbil provides us with a different perspective to other commonly used nocturnal rodent models. In this study, SST neurons were located in all layers of the VC except in layer I; they were most common in layer V. Most SST neurons were multipolar round/oval or stellate cells. No pyramidal neurons were found. Moreover, 2-color immunofluorescence revealed that only 33.50%, 24.05%, 16.73%, 0%, and 64.57% of SST neurons contained gamma-aminobutyric acid, calbindin-D28K, calretinin, parvalbumin, and calcium/calmodulin-dependent protein kinase II, respectively. In contrast, neuropeptide Y and nitric oxide synthase were abundantly expressed, with 80.07% and 75.41% in SST neurons, respectively. Our immunocytochemical analyses of SST with D1 and D2 dopamine receptors and choline acetyltransferase, α7 and ß2 nicotinic acetylcholine receptors suggest that dopaminergic and cholinergic fibers contact some SST neurons. The results showed some distinguishable features of SST neurons and provided some insight into their afferent circuitry in the gerbil VC. These findings may support future studies investigating the role of SST neurons in visual processing.

Construction and implementation of floating wetpark as effective constructed wetland for industrial textile wastewater treatment.

Fimbristylis dichotoma, Ipomoea aquatica, Pluchea tomentosa and their co-plantation (consortium FIP) autonomously degrade Orange 3R. Consortium FIP showed 84% removal of Orange 3R within 48 h, which is a higher dye elimination rate than individual plant systems. Oxidoreductase enzymes like tyrosinase (76%), varatryal alcohol oxidase (85%), lignin peroxidase (150%), riboflavin reductase (151%), laccase (171%), NADH-DCIP reductase (11%) and azo reductase (241%) were expressed in consortia FIP during Orange 3R degradation. UV-vis spectroscopy, enzyme activities, HPTLC, FTIR and GC-MS confirmed mineralization of Orange 3R into its metabolites. Microscopic investigation of root tissue revealed the harsh effect of dye on root tissues. Toxicity assessment on the HepG2 cell line demonstrated the toxic nature of Orange 3R, which gets reduced after phyto-treatment with consortia FIP. Floating wetpark of consortia FIP was found more efficient for the treatment of industrial textile waste and accomplished 87%, 86%, 75%, 49% and 46% removal of COD, BOD, color, TSS and TDS of effluent.

The complete chloroplast genome sequence of a narrow alpine endemic, Taraxacum hallaisanense (Asteraceae), on Jeju Island, Korea.

The first complete chloroplast genome sequence of Jeju Island endemic diploid dandelion, Taraxacum hallaisanense, is reported in this study. The plastome size is 151,554 bp in total length, with one large single copy (LSC; 84,066 bp), one small single copy (SSC; 18,524 bp), and two inverted repeat (IR) regions (IRa and IRb, each with 24,482 bp). The overall GC content is 37.7% and the genome contained 129 genes, including 84 protein-coding with 2 pseudogenes (ycf1 and accD), 37 transfer RNA, and 8 ribosomal RNA genes. Phylogenetic analysis of 19 representative plastomes of the Asteraceae suggests that Taraxacum is monophyletic with strong bootstrap support and also that T. hallaisanense is closely related to T. mongolicum.

The complete chloroplast genome sequence of coastal psammophyte, Ixeris repens (Asteraceae, subtribe Crepidinae), in Korea.

We report the first complete chloroplast genome sequence of psammophyte, Ixeris repens, on the coastal dunes in Korea. The complete plastid genome is 153,017 bp in total length, with one large single copy (LSC; 84,242 bp), one small single copy (SSC; 18,495 bp), and two inverted repeat (IR) regions (IRa and IRb, each with 25,140 bp). The overall GC content is 37.6% and the genome contains 130 genes, including 85 protein-coding, 37 transfer RNA and 8 ribosomal RNA genes. Phylogenetic analysis based on 17 representative plastomes of the family Asteraceae suggests that Ixeris repens is sister to congeneric species I. polycephala with strong bootstrap support (100%) and also that monophyletic Ixeris is sister to the clade containing Taraxacum, Youngia, Lapsanastrum, and Crepidiastrum.

Characterization and Dynamics of Intracellular Gene Transfer in Plastid Genomes of Viola (Violaceae) and Order Malpighiales.

Functional gene transfer from organelles to the nucleus, known as intracellular gene transfer (IGT), is an ongoing process in flowering plants. The complete plastid genomes (plastomes) of two Ulleung island endemic violets, Viola ulleungdoensis and V. woosanensis, were characterized, revealing a lack of the plastid-encoded infA, rpl32, and rps16 genes. In addition, functional replacement of the three plastid-encoded genes in the nucleus was confirmed within the genus Viola and the order Malpighiales. Three strategies for the acquisition of a novel transit peptide for successful IGT were identified in the genus Viola. Nuclear INFA acquired a novel transit peptide with very low identity between these proteins, whereas the nuclear RPL32 gene acquired an existing transit peptide via fusion with the nuclear-encoded plastid-targeted SOD gene (Cu-Zn superoxide dismutase superfamily) as one exon, and translated both proteins in the cytosol using alternative mRNA splicing. Nuclear RPS16 contains an internal transit peptide without an N-terminal extension. Gene loss or pseudogenization of the plastid-borne rpl32 and rps16 loci was inferred to occur in the common ancestor of the genus Viola based on an infrageneric phylogenetic framework in Korea. Although infA was lost in the common ancestor of the order Malpighiales, the rpl32 and rps16 genes were lost multiple times independently within the order. Our current study sheds additional light on plastid genome composition and IGT mechanisms in the violet genus and in the order Malpighiales.

Characterization and comparative analysis among plastome sequences of eight endemic Rubus (Rosaceae) species in Taiwan.

Genus Rubus represents the second largest genus of the family Rosaceae in Taiwan, with 41 currently recognized species across three subgenera (Chamaebatus, Idaoeobatus, and Malochobatus). Despite previous morphological and cytological studies, little is known regarding the overall phylogenetic relationships among the Rubus species in Taiwan, and their relationships to congeneric species in continental China. We characterized eight complete plastomes of Taiwan endemic Rubus species: subg. Idaeobatus (R. glandulosopunctatus, R. incanus, R. parviaraliifolius, R rubroangustifolius, R. taitoensis, and R. taiwanicolus) and subg. Malachobatus (R. kawakamii and R. laciniastostipulatus) to determine their phylogenetic relationships. The plastomes were highly conserved and the size of the complete plastome sequences ranged from 155,566 to 156,236 bp. The overall GC content ranged from 37.0 to 37.3%. The frequency of codon usage showed similar patterns among species, and 29 of the 73 common protein-coding genes were positively selected. The comparative phylogenomic analysis identified four highly variable intergenic regions (rps16/trnQ, petA/psbJ, rpl32/trnL-UAG, and trnT-UGU/trnL-UAA). Phylogenetic analysis of 31 representative complete plastomes within the family Rosaceae revealed three major lineages within Rubus in Taiwan. However, overall phylogenetic relationships among endemic species require broader taxon sampling to gain new insights into infrageneric relationships and their plastome evolution.

Cytotype diversity and genome size variation in eastern Asian polyploid Cardamine (Brassicaceae) species.

BACKGROUND AND AIMS: Intraspecific ploidy-level variation is an important aspect of a species' genetic make-up, which may lend insight into its evolutionary history and future potential. The present study explores this phenomenon in a group of eastern Asian Cardamine species. METHODS: Plant material was sampled from 59 localities in Japan and Korea, which were used in karyological (chromosome counting) and flow cytometric analyses. The absolute nuclear DNA content (in pg) was measured using propidium iodide and the relative nuclear DNA content (in arbitrary units) was measured using 4,6-diamidino-2-phenylindole fluorochrome. KEY RESULTS: Substantial cytotype diversity was found, with strikingly different distribution patterns between the species. Two cytotypes were found in C. torrentis sensu lato (4x and 8x, in C. valida and C. torrentis sensu stricto, respectively), which displays a north-south geographical pattern in Japan. Hypotheses regarding their origin and colonization history in the Japanese archipelago are discussed. In Korean C. amaraeiformis, only tetraploids were found, and these populations may in fact belong to C. valida. C. yezoensis was found to harbour as many as six cytotypes in Japan, ranging from hexa- to dodecaploids. Ploidy levels do not show any obvious geographical pattern; populations with mixed ploidy levels, containing two to four cytotypes, are frequently observed throughout the range. C. schinziana, an endemic of Hokkaido, has hexa- and octoploid populations. Previous chromosome records are also revised, showing that they are largely based on misidentified material or misinterpreted names. CONCLUSIONS: Sampling of multiple populations and utilization of the efficient flow cytometric approach allowed the detection of large-scale variation in ploidy levels and genome size variation attributable to aneuploidy. These data will be essential in further phylogenetic and evolutionary studies.

The complete chloroplast genome sequence of Thymus quinquecostatus var. japonicus (Lamiaceae), an endemic to Ullenung Island of Korea.

The complete chloroplast (cp) genome sequence of Thymus quinquecostatus var. japonicus, an endemic species to Ulleung Island of Korea and used as plant material for folk remedies, was firstly analyzed in this study. It showed a typical circular structure composed of 151,782 bp in length and comprised of a large single-copy region (82,903bp) and a small single-copy region (17,667 bp) which were separated by two inverted repeat regions (25,606 bp). From the phylogenetic analyses of related taxa using the complete chloroplast genome sequences, it was proved that T. quinquecostatus var. japonicus is sister to the member of genus Mentha within the subfamily Nepetoideae.

Plastome Characterization and Phylogenomics of East Asian Beeches with a Special Emphasis on Fagus multinervis on Ulleung Island, Korea.

Beech trees of the genus Fagus (Fagaceae) are monoecious and distributed in the Northern Hemisphere. They represent an important component of mixed broad-leaved evergreen-deciduous forests and are an economically important source of timber. Despite their ecological and economical importance, however, little is known regarding the overall plastome evolution among Fagus species in East Asia. In particular, the taxonomic position and status of F. multinervis, a beech species endemic to Ulleung Island of Korea, remains unclear even today. Therefore, in this study, we characterized four newly completed plastomes of East Asian Fagus species (one accession each of F. crenata and F. multinervis and two accessions of F. japonica). Moreover, we performed phylogenomic analyses comparing these four plastomes with F. sylvatica (European beech) plastome. The four plastomes were highly conserved, and their size ranged from 158,163 to 158,348 base pair (bp). The overall GC content was 37.1%, and the sequence similarity ranged from 99.8% to 99.99%. Codon usage patterns were similar among species, and 7 of 77 common protein-coding genes were under positive selection. Furthermore, we identified five highly variable hotspot regions of the Fagus plastomes (ccsA/ndhD, ndhD/psaC, ndhF/rpl32, trnS-GCU/trnG-UCC, and ycf1). Phylogenetic analysis revealed the monophyly of Fagus as well as early divergence of the subgenus Fagus and monophyletic Engleriana. Finally, phylogenetic results supported the taxonomic distinction of F. multinervis from its close relatives F. engleriana and F. japonica. However, the sister species and geographic origin of F. multinervis on Ulleung Island could not be determined.

First record of the land gastropod genus Otesiopsis from South Korea (Helicarionoidea Bourguignat, 1877).

Otesiopsis Habe, 1946 is a land snail genus, which is known so far from Japan and Taiwan. Individuals of this genus were collected from Gageodo Island, South Korea. This is the first record of the genus Otesiopsis from this country. The individuals from Gageodo Island appear to be a new species because of their unique shell morphologies, while the details of their anatomy are still lacking. Further studies are encouraged to investigate the taxonomic and conservation status of the genus Otesiopsis in South Korea.

Comparison of the Whole-Plastome Sequence between the Bonin Islands Endemic Rubus boninensis and Its Close Relative, Rubus trifidus (Rosaceae), in the Southern Korean Peninsula.

Rubus boninensis is a rare endemic species found on the Bonin Islands with a very restricted distribution. It is morphologically most closely related to Rubus trifidus, occurring widely in the southern Korean peninsula and Japan. This species pair provides a good example of anagenetic speciation on an oceanic island in the northwestern Pacific Ocean-R. trifidus as a continental progenitor and R. boninensis as an insular derivative species. In this study, we firstly characterized the complete plastome of R. boninensis and R. trifidus and compared this species pair to another anagenetically derived species pair (R. takesimensis-R. crataegifolius). The complete plastome of R. trifidus was 155,823 base pairs (bp) long, slightly longer (16 bp) than that of R. boninensis (155,807 bp). No structural or content rearrangements were found between the species pair. Eleven hotspot regions, including trnH/psbA, were identified between R. trifidus and R. boninensis. Phylogenetic analysis of 19 representative plastomes within the family Rosaceae suggested sister relationships between R. trifidus and R. boninensis, and between R. crataegifolius and R. takesimensis. The plastome resources generated by the present study will help elucidate plastome evolution and resolve phylogenetic relationships within highly complex and reticulated lineages of the genus Rubus.