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Genus Asarum (Aristolochiaceae) shows diverse floral morphology and is hypothesized to have diversified as a result of pollinator‐mediated selection. Yet most aspects of their reproductive ecology, including pollinators, remain unclear. This study focuses on A. costatum and A. minamitanianum in Japan, a sister species pair having remarkable differences in calyx lobe length (10–20 mm and 70–180 mm, respectively). The objectives of this study are to elucidate multiple aspects of reproductive ecology of these two species and obtain evolutionary insights into floral organ elongation.We adopted combined approaches, including field observations, molecular analyses and cultivation experiments, such as pollinator observation for 3 years, fine‐scale spatial genetic analysis of 769 individuals, paternity analysis based on 566 seeds over 4 years, and control pollination experiments.Both Asarum species had strong spatial genetic structures, indicating limited seed dispersal. Pollinator observation revealed that flies and ground‐dwelling insects visited flowers of both species, but that the pollinator fauna differed between the species. The visitation rate of flies was extremely low but was more than twice as high in the species with an elongated floral appendage. Paternity analysis revealed A. minamitanianum was predominantly outcrossing, while A. costatum showed a wide range of selfing rates among fruits.These two Asarum species are likely adapted to fly pollination in the shady forest understorey, where available pollinator fauna is limited. In addition, although its function remains unclear, the elongated calyx lobe of A. minamitanianum could have evolved for effective pollen dispersal by attracting fly visitors. [ABSTRACT FROM AUTHOR]

Previous phylogeographic studies of arctic-alpine plants have partly elucidated that alpine areas at the southernmost edges of their distribution have been one of the most important areas for the survival of populations through Quaternary climatic oscillations. To reveal the genetic structure of arctic-alpine plants near the southern edge of their range in East Asia, 159 samples of Diapensia lapponica subsp. obovata (Diapensiaceae) were collected from 22 populations in Japan. Based on 1,134 bp of chloroplast DNA (cpDNA), we determined 10 haplotypes. Neighbor-joining (NJ) analysis, including D. lapponica from Sweden and Schizocodon soldanelloides, revealed that all haplotypes in Japan were monophyletic. Genetic differentiation between populations of central Honshu and northern Japan was strongly supported by the NJ tree (100%), analysis of molecular variance (AMOVA, 68%), and Monmonier’s algorithm (>95%). The most ancestral haplotype rarely occurred in Japan, which implied that the extant genetic structure was not caused by genetic divergence during colonization. Thus, these results suggest that the arctic-alpine plant D. lapponica subsp. obovata survived climatic oscillations in central Honshu. This finding supports the hypothesis that high mountains in temperate regions were important for survival of arctic-alpine plants during Quaternary climatic oscillations. [ABSTRACT FROM AUTHOR]

Phylogenetic relationships within the genus Pieris (Ericaceae) were investigated based on the rbcL and matK genes along with five spacer sequences of chloroplast DNA to address questions regarding the phylogeography of the genus in association with insular plants on the Ryukyu Islands. The most parsimonious trees indicated that P. floribunda from eastern North America is a sister taxon to the remaining taxa examined, and suggested that the East Asian taxa examined are monophyletic. A morphologically cohesive group, section Pieris, was revealed to be paraphyletic. Within the East Asian clade, insular endemics from the Ryukyu Islands, Taiwan, and mainland Japan formed a sister group to P. formosa from the Himalayas and southern China. Our data suggest that the insular endemics of the Ryukyu Islands and Taiwan arose via allopatric divergence as a result of a paleogeographical land configuration of a landbridge during the early–middle Pleistocene in the Quaternary Period. [ABSTRACT FROM AUTHOR]

Premise: Snow is an important environmental factor affecting plant distribution. Past changes in snowfall regimes may have controlled the demographies of snow-dependent plants. However, our knowledge of changes in the distribution and demographies of such plants is limited because of the lack of fossil records., Methods: Population genetic and landscape genetic analyses were used to investigate the response of population dynamics of Arnica mallotopus (Asteraceae)-a plant confined to heavy-snow areas of Japan-to changes in snowfall regimes from the Last Glacial Period to the Holocene., Results: The population genetic analysis suggested that the four geographic lineages diverged during the Last Glacial Period. The interaction between reduced snowfall and lower temperatures during this period likely triggered population isolation in separate refugia. Subpopulation differentiation in the northern group was lower than in the southern group. Our ecological niche model predicted that the current distribution was patchy in the southern region; that is, the populations were isolated by topologically flat and climatically unsuitable lowlands. The landscape genetic analysis suggested that areas with little snowfall acted as barriers to the Holocene expansion of species distribution and continued limiting gene flow between local populations., Conclusions: These findings indicate that postglacial population responses vary among regions and are controlled by environmental and geographic factors. Thus, changes in snowfall regime played a major role in shaping the distribution and genetic structure of the snow-dependent plant., (© 2024 Botanical Society of America.)

The Ryukyu Islands of Japan are a biodiversity hotspot due to geographical and historical factors. Tricyrtis formosana is a perennial herbaceous plant that commonly found in Taiwan. But only a few populations have been identified in a limited habitat on Iriomote Island, while populations of unknown origin occur near human settlements in an area on the main island of Okinawa. To better understand these populations of the phylogenetic uniqueness and intrinsic vulnerability, we conducted comparative analyses including (1) phylogeny and population structure with MIG-seq data, (2) photosynthesis-related traits of plants grown under common conditions and (3) transcriptome analysis to detect deleterious variations. Results revealed that T. formosana was split into two clades by the congeners and that Iriomote and Okinawa populations independently derived from ancestral Taiwanese populations in each clade. Photosynthetic efficiency was lowest in the Iriomote population, followed by Okinawa and Taiwan. Transcriptome analysis showed that the Iriomote population accumulated more deleterious variations, suggesting intrinsic vulnerability. These results indicate that each T. formosana population in Japan is phylogenetically unique and has been independently dispersed from Taiwan, and that the Iriomote population presents a high conservation difficulty with a unique photosynthesis-related characteristic and a larger amount of deleterious variations., (© 2024. The Author(s).)

Phenotypic polymorphism within a species is a notable phenomenon in evolutionary biology to understand the process of adaptive speciation and other historical events. The Saxifraga fortunei complex is a widespread herb found in East Asia. It includes several ecotypic taxa corresponding to their habitat environments. The distribution of the various ecotypes in a limited area of the Japanese Archipelago makes the species a suitable model to investigate the impact of population demographic history and natural selection on lineage diversification. Here, Sanger-based sequencing was used to estimate the divergence timeframe between populations of the Eurasian continent and Japan. Genome-wide SNPs obtained by ddRAD sequencing were used to investigate the phylogeographic origins of ecotypic taxa. The phylogenetic analyses revealed the divergence of the Japanese population from the continental population in the late Miocene. Two distinct regional clades of North and South Japan were identified; phenotypic diversification was evident only in the southern clade. The South Japan clades displayed a historical distribution expansion from north to south. The phenotypic variations appeared to have generated during the expansion. The ecotypic boundaries were incongruent with the genetic grouping. We propose that morphological and ecological specialization in Japanese populations was repeatedly generated by local natural selection., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Premise: The genetic structure between plant populations is facilitated by the spatial population arrangement and limited dispersal of seed and pollen. Saxifraga acerifolia, a local endemic species in Japan, is a habitat specialist that is confined to waterfalls in riparian environments. Its sister species, Saxifraga fortunei, is a generalist that is widely distributed along riverbanks. Here, we examined sympatric populations of the two Saxifraga species to test whether the differences in habitat preference and colonization process influenced regional and local genetic structures., Methods: To reveal genetic structures, we examined chloroplast microsatellite variations and genome-wide nucleotide polymorphisms obtained by genotyping by sequencing. We also estimated the gene flow among and within populations and performed landscape genetic analyses to evaluate seed and pollen movement and the extent of genetic isolation related to geographic distance and/or habitat differences., Results: We found strong genetic structure in the specialist S. acerifolia, even on a small spatial scale (<1 km part); each population on a different waterfall in one river system had a completely different predominant haplotype. By contrast, the generalist S. fortunei showed no clear genetic differentiation., Conclusions: Our findings suggest that the level of genetic isolation was increased in S. acerifolia by the spatially fragmented habitat and limited seed and pollen dispersal over waterfalls. Habitat differentiation between the sister taxa could have contributed to the different patterns of gene flow and then shaped the contrasting genetic structures., (© 2021 Botanical Society of America.)

Climatic perturbation during the Pleistocene era has played a major role in plant evolutionary history by altering species distribution range. However, the relative roles of climatic and geographic factors in the distribution dynamics remain poorly understood; in particular, the edaphic endemics. In this paper, we examine the evolutionary history of two ultramafic primroses, Primula hidakana and Primula takedana. These species are ecologically and morphologically distinct with disjunct distributions on Hokkaido Island, Japan. Primula hidakana is found on various rocks in southern Hokkaido and P. takedana in serpentine areas in northern Hokkaido. We performed population genetics analyses on nuclear and chloroplast data sets and tested alternative phylogenetic models of divergence using approximate Bayesian computation (ABC) analyses. Nuclear microsatellite loci clearly distinguished the two sister taxa. In contrast, chloroplast sequence variations were shared between P. takedana and P. hidakana. ABC analyses based on nuclear data supported a secondary contact scenario involving asymmetrical gene flow from P. hidakana to P. takedana. Paleodistribution modeling also supported the divergence model, and predicted their latitudinal range shifts leading to past secondary contact. Our findings highlight the importance of the distribution dynamics during the Pleistocene climatic oscillations in the evolution of serpentine plants, and demonstrate that tight species cohesion between serpentine and nonserpentine sister taxa has been maintained despite past interspecific gene flow across soil boundaries.

Climatic changes have played major roles in plants' evolutionary history. Glacial oscillations have been particularly important, but some of their effects on plants' populations are poorly understood, including the numbers and locations of refugia in Asian warm temperate zones. In the present study, we investigated the demographic history of the broadleaved evergreen tree species Castanopsis sieboldii (Fagaceae) during the last glacial period in Japan. We used approximate Bayesian computation (ABC) for model comparison and parameter estimation for the demographic modeling using 27 EST-associated microsatellites. We also performed the species distribution modeling (SDM). The results strongly support a demographic scenario that the Ryukyu Islands and the western parts in the main islands (Kyushu and western Shikoku) were derived from separate refugia and the eastern parts in the main islands and the Japan Sea groups were diverged from the western parts prior to the coldest stage of the Last Glacial Maximum (LGM). Our data indicate that multiple refugia survived at least one in the Ryukyu Islands, and the other three regions of the western and eastern parts and around the Japan Sea of the main islands of Japan during the LGM. The SDM analysis also suggests the potential habitats under LGM climate conditions were mainly located along the Pacific Ocean side of the coastal region. Our ABC-based study helps efforts resolve the demographic history of a dominant species in warm temperate broadleaved forests during and after the last glacial period, which provides a basic model for future phylogeographical studies using this approach.

How phenotypic or genetic diversity is maintained in a natural habitat is a fundamental question in evolutionary biology. Flower color polymorphism in plants is a common polymorphism. Hepatica nobilis var. japonica on the Sea of Japan (SJ) side of the Japanese mainland exhibits within population flower color polymorphism (e.g., white, pink, and purple), while only white flowers are observed on the Pacific Ocean (PO) side. To determine the relationships between flower color polymorphism, within and among populations, and the genetic structure of H. nobilis var. japonica, we estimated the genetic variation using simple sequence repeat (SSR) markers. First, we examined whether cryptic lineages corresponding to distinct flower colors contribute to the flower color polymorphisms in H. nobilis var. japonica. In our field observations, no bias in color frequency was observed among populations on Sado Island, a region with high variation in flower color. Simple sequence repeat (SSR) analyses revealed that 18% of the genetic variance was explained by differences among populations, whereas no genetic variation was explained by flower color hue or intensity (0% for both components). These results indicate that the flower color polymorphism is likely not explained by cryptic lineages that have different flower colors. In contrast, populations in the SJ and PO regions were genetically distinguishable. As with the other plant species in these regions, refugial isolation and subsequent migration history may have caused the genetic structure as well as the spatially heterogeneous patterns of flower color polymorphisms in H. nobilis var. japonica.

Coastal plants with simple linear distribution ranges along coastlines provide a suitable system for improving our understanding of patterns of intra-specific distributional history and genetic variation. Due to the combination of high seed longevity and high dispersibility of seeds via seawater, we hypothesized that wild radish would poorly represent phylogeographic structure at the local scale. On the other hand, we also hypothesized that wild radish populations might be geographically differentiated, as has been exhibited by their considerable phenotypic variations along the islands of Japan. We conducted nuclear DNA microsatellite loci and chloroplast DNA haplotype analyses for 486 samples and 144 samples, respectively, from 18 populations to investigate the phylogeographic structure of wild radish in Japan. Cluster analysis supported the existence of differential genetic structures between the Ryukyu Islands and mainland Japan populations. A significant strong pattern of isolation by distance and significant evidence of a recent bottleneck were detected. The chloroplast marker analysis resulted in the generation of eight haplotypes, of which two haplotypes (A and B) were broadly distributed in most wild radish populations. High levels of variation in microsatellite loci were identified, whereas cpDNA displayed low levels of genetic diversity within populations. Our results indicate that the Kuroshio Current would have contributed to the sculpting of the phylogeographic structure by shaping genetic gaps between isolated populations. In addition, the Tokara Strait would have created a geographic barrier between the Ryukyu Islands and mainland Japan. Finally, extant habitat disturbances (coastal erosion), migration patterns (linear expansion), and geographic characteristics (small islands and sea currents) have influenced the expansion and historical population dynamics of wild radish. Our study is the first to record the robust phylogeographic structure in wild radish between the Ryukyu Islands and mainland Japan, and might provide new insight into the genetic differentiation of coastal plants across islands.

Arctic-alpine plants have expanded and contracted their ranges in response to the Pleistocene climate oscillations. Today, many arctic-alpine plants have vast distributions in the circumarctic region as well as marginal, isolated occurrences in high mountains at lower latitudes. These marginal populations may represent relict, long-standing populations that have persisted for several cycles of cold and warm climate during the Pleistocene, or recent occurrences that either result from southward step-wise migration during the last glacial period or from recent long-distance dispersal. In light of these hypotheses, we investigated the biogeographic history of the marginal Japanese populations of the widespread arctic-alpine plant Vaccinium vitis-idaea (Ericaceae), which is bird-dispersed, potentially over long distances. We sequenced three nuclear loci and one plastid DNA region in 130 individuals from 65 localities covering its entire geographic range, with a focus on its marginal populations in Japan. We found a homogenous genetic pattern across its enormous range based on the loci analysed, in contrast to the geographically structured variation found in a previous study of amplified fragment length polymorphisms in this species. However, we found several unique haplotypes in the Japanese populations, excluding the possibility that these marginal populations result from recent southward migration. Thus, even though V. vitis-idaea is efficiently dispersed via berries, our study suggests that its isolated populations in Japan have persisted during several cycles of cold and warm climate during the Pleistocene.

The broadleaved evergreen forests of the East Asian warm temperate zone are characterised by their high biodiversity and endemism, and there is therefore a need to extend our understanding of its genetic diversity and phylogeographic patterns. Castanopsis (Fagaceae) is one of the dominant tree species in the broadleaved evergreen forests of Japan. In this study we investigate the genetic diversity, genetic structure and leaf epidermal morphology of 63 natural populations of C. sieboldii and C. cuspidata, using 32 Expressed Sequence Tag associated microsatellites. The overall genetic differentiation between populations was low (GST = 0.069 in C. sieboldii and GST = 0.057 in C. cuspidata). Neighbor-joining tree and Bayesian clustering analyses revealed that the populations of C. sieboldii and C. cuspidata were genetically clearly differentiated, a result which is consistent with the morphology of their epidermal cell layers. This suggests that C. sieboldii and C. cuspidata should be treated as independent species, although intermediate morphologies are often observed, especially at sites where the two species coexist. The higher level of genetic diversity observed in the Kyushu region (for both species) and the Ryukyu Islands (for C. sieboldii) is consistent with the available fossil pollen data for Castanopsis-type broadleaved evergreen trees during the Last Glacial Maximum and suggests the existence of refugia for Castanopsis forests in southern Japan. Within the C. sieboldii populations, Bayesian clustering analyses detected three clusters, in the western and eastern parts of the main islands and in the Ryukyu Islands. The west-east genetic differentiation observed for this species in the main islands, a pattern which is also found in several plant and animal species inhabiting Castanopsis forests in Japan, suggests that they have been isolated from each other in the western and eastern populations for an extended period of time, and may imply the existence of eastern refugia.

Background: Understanding demographic histories, such as divergence time, patterns of gene flow, and population size changes, in ecologically diverging lineages provide implications for the process and maintenance of population differentiation by ecological adaptation. This study addressed the demographic histories in two independently derived lineages of flood-resistant riparian plants and their non-riparian relatives [Ainsliaea linearis (riparian) and A. apiculata (non-riparian); A. oblonga (riparian) and A. macroclinidioides (non-riparian); Asteraceae] using an isolation-with-migration (IM) model based on variation at 10 nuclear DNA loci., Results: The highest posterior probabilities of the divergence time parameters were estimated to be ca. 25,000 years ago for A. linearis and A. apiculata and ca. 9000 years ago for A. oblonga and A. macroclinidioides, although the confidence intervals of the parameters had broad ranges. The likelihood ratio tests detected evidence of historical gene flow between both riparian/non-riparian species pairs. The riparian populations showed lower levels of genetic diversity and a significant reduction in effective population sizes compared to the non-riparian populations and their ancestral populations., Conclusions: This study showed the recent origins of flood-resistant riparian plants, which are remarkable examples of plant ecological adaptation. The recent divergence and genetic signatures of historical gene flow among riparian/non-riparian species implied that they underwent morphological and ecological differentiation within short evolutionary timescales and have maintained their species boundaries in the face of gene flow. Comparative analyses of adaptive divergence in two sets of riparian/non-riparian lineages suggested that strong natural selection by flooding had frequently reduced the genetic diversity and size of riparian populations through genetic drift, possibly leading to fixation of adaptive traits in riparian populations. The two sets of riparian/non-riparian lineages showed contrasting patterns of gene flow and genetic differentiation, implying that each lineage showed different degrees of reproductive isolation and that they had experienced unique evolutionary and demographic histories in the process of adaptive divergence.

Both cultivated soybean and its wild relative Glycine soja exhibit strong photoperiodic sensitivity at different latitudes. Recent studies have demonstrated that the blue light-absorbing cryptochrome gene, CRY1a, is involved in the photoperiodic flowering of soybeans. However, no sequence variation was found in the cDNA among cultivars at different latitudinal clines. In the present study, we examined whether positive selection due to polymorphisms in the cryptochrome genes of G. soja occurs. Partial DNA sequences, mainly exons, of cryptochrome genes CRY1a-1d and CRY2a-2c were analyzed for 18 accessions in the Japanese archipelago. The neutral evolutionary pattern of the polymorphisms for all cryptochrome genes except for CRY1a was summarized using Tajima's D test and low nucleotide diversity was shown for all genes. Although CRY1a did not show neutral evolution, balancing selection was recognized in the intron while not in the exon. No geographical pattern of polymorphisms was observed in the cryptochrome genes. These results reject the possibility of cryptochrome genes being involved in the photoperiodic flowering of wild soybeans along a latitudinal cline.

Even in cases in which geographic isolation appears to have driven the speciation of regional endemics, range shifts during the Pleistocene climatic oscillations may also have influenced their evolutionary history. Elucidating speciation history can provide novel insights into evolutionary dynamics following climatic oscillations. We demonstrated a sister relationship between the Japanese alpine endemic Cardamine nipponica and the currently allopatric, widespread arctic-alpine Cardamine bellidifolia (Brassicaceae) based on internal transcribed spacer (ITS) sequences and 10 other nuclear genes. Speciation history was inferred using demographic parameters under the isolation with migration model. The estimated demographic parameters showed that the population size of C. nipponica was similar to that of C. bellidifolia and that gene flow occurred exclusively from C. nipponica to C. bellidifolia after speciation. The inferred speciation history, which included gene flow, suggests that geographic barriers between the peripheral C. nipponica and the widespread C. bellidifolia were reduced during the Pleistocene. The asymmetric introgression implies that genetic isolation may have been involved in the speciation of C. nipponica. Our results suggest that even currently allopatric species may not have diverged solely under geographic isolation, and that their evolutionary history may have been influenced by Pleistocene range dynamics., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Populations of arctic alpine plants likely disappeared and re-colonised several times at the southern edge of their distributions during glacial and interglacial cycles throughout the Quaternary. Range shift and population fragmentation after a glacial period would affect the genetic structure of such plants in southernmost populations. We aimed to elucidate how climatic oscillations influenced the population subsistence of alpine plants in the Japanese Archipelago as one of the southernmost populations, by inferring the genetic structure of Arabidopsis kamchatica subsp. kamchatica and the intraspecific littoral taxon, subsp. kawasakiana. We identified genotypes based on the haplotypes of five nuclear genes and two chloroplast DNA spacers for 164 individuals from 24 populations. Most populations harboured only one private genotype, whereas few polymorphisms were found in each population. Two genetic genealogies were found, suggesting that northern Japanese populations of alpine subsp. kamchatica, subsp. kawasakiana and the northerly subsp. kamchatica in eastern Russia and Alaska clustered and differentiated from populations in central Honshu, western Japan and Taiwan. During climatic oscillations, the genetic structure of extant southernmost populations would have been shaped by strong genetic drift under population fragmentation and randomly fixed to a single genotype among their ancestral polymorphisms.

Premise of the Study: Microsatellite loci were developed for Lathyrus japonicus (Fabaceae) to evaluate genetic diversity, population genetic structure, and demographic patterns of this species in Japan., Methods and Results: In total, eight primer sets were identified in Japanese populations of L. japonicus and polymorphisms were evaluated in 83 individuals from three populations, including the northern and southern geographic range in Japan. The number of alleles per locus ranged from four to 20, and gene diversity per locus ranged from 0.636 to 0.935 among three populations. In addition, six of the eight loci could be successfully amplified in L. pratensis., Conclusions: These markers will be useful for studies of genetic variations, genetic structure, and gene flow of L. japonicus, which is important to investigate the demographic patterns of this coastal plant. In addition, the primers are useful for further genetic structure analysis of L. pratensis.

Variations in tree architecture and in the genetic structure of Larix kaempferi on Mt. Fuji were surveyed along altitudinal gradients using 11 nSSR loci. In total, 249 individuals from six populations along three trails at altitudes ranging from approximately 1,300 to 2,700 m were investigated. Gradual changes in tree architecture with increasing elevation, from erect trees to flag trees and krummholz mats, were observed in the high-altitude populations (> 2,000 m) on all trails. These findings suggest that tree architecture is correlated with the severe environmental conditions associated with increasing elevation, such as strong winds. In contrast to obvious variations in tree architecture, the genetic diversity of populations along the trails was almost uniform (H (E) = 0.717-0.762) across the altitudinal range. The results of the AMOVA and STRUCTURE analyses, and the analysis for isolation by distance pattern, suggest homogeneous genetic structuring across all populations on Mt. Fuji, while the pairwise F (ST) showed barriers to gene flow between altitudinal populations that were demarcated as high- or low-altitude populations by Abies-Tsuga forest. Although the evergreen coniferous forests on the mountainside may hinder gene flow, this may be explained by the long-distance seed dispersal of the Japanese larch and/or a short population history resulting from eruptions or slush avalanches, although evergreen coniferous forests on the mountainside may hinder gene flow.

Japanese horse chestnut (Aesculus turbinata: Hippocastanaceae) is one of the typical woody plants that grow in temperate riparian forests in the Japanese Archipelago. To analyze the phylogeography of this plant in the Japanese Archipelago, we determined cpDNA haplotypes for 337 samples from 55 populations covering the entire distribution range. Based on 1,313 bp of two spacers, we determined ten haplotypes that are distinguished from adjacent haplotypes by one or two steps. Most of the populations had a single haplotype, suggesting low diversity. Spatial analysis of molecular variance suggested three obvious phylogeographic structures in western Japan, where Japanese horse chestnut is scattered and isolated in mountainous areas. Conversely, no clear phylogeographic structure was observed from the northern to the southern limit of this species, including eastern Japan, where this plant is more common. Rare and private haplotypes were also found in southwestern Japan, where Japanese horse chestnuts are distributed sparsely. These findings imply that western Japan might have maintained a relatively large habitat for A. turbinata during the Quaternary climatic oscillations, while northerly regions could not.

Various photoreceptors in plants are used to monitor important environmental light signals and regulate plant development. Despite their functional importance, recent studies have demonstrated that red/far-red absorbing phytochromes or blue/UV-A absorbing cryptochromes are involved in local adaptation within a species' range. In the present study, to exemplify the intraspecific photoreceptor evolutionary pattern, the genetic structures of cryptochrome genes (CRY1 and CRY2) in Cardamine nipponica (Brassicaceae), of which PHYE, a gene coding one of the phytochromes, was found to be involved in local adaptation between central and northern Japanese populations. Although clear genetic differentiations between central and northern Japan were detected (CRY1: F (ST) = 0.63, CRY2: F (ST) = 0.53), overall nucleotide diversity was very low (CRY1: π (Total) = 0.0014, CRY2: π (Total) = 0.0013), and the polymorphism patterns were neutral (CRY1: Tajima's D = 0.084, P = 0.32, CRY2: D = -0.014, P = 0.39). Therefore, the involvement of cryptochromes in the adaptation to local environments is difficult to postulate. Consequently, this study along with our previous findings suggest that intraspecific photoreceptor gene polymorphisms in C. nipponica were mostly suppressed by purifying selection due to their functional importance as photoreceptors, while some of the photoreceptors may play substantial roles in adaptation to local environments.

Phytochromes play a key role in allowing plants to monitor their surrounding environment and, conversely, adaptation to local environments has driven the evolutionary history of phytochromes. As a result of natural selection, polymorphisms in phytochrome genes would thus be expected to exhibit locus-specific phylogeographic structure. To evaluate this hypothesis, we conducted a phylogeographic investigation based on four nuclear genes, including two phytochrome genes (PHYB and PHYE) using 155 samples of Arcterica nana from the entire range of the Japanese archipelago. Bayesian clustering revealed geographic differentiation between northern and southern Japan when all four genes were included. However, this geographic differentiation is inconsistent with previously reported genetic structure of genome-wide polymorphisms based on amplified fragment length polymorphisms, as these did not show geographic differentiation throughout the Japanese archipelago. In contrast, the north-south differentiation was not apparent when PHYE was excluded. This indicates that PHYE alone could be responsible for the north-south differentiation (F(CT) = 0.15, P < 0.001). Furthermore, a single nonsynonymous polymorphism (C360T) strongly contributed to geographic differentiation (F(CT) = 0.57, P < 0.001) and its corresponding amino acid replacement (P120L) was significantly under positive selection based on maximum likelihood analysis (P = 0.98). Consequently, the locus-specific geographic differentiation in PHYE could be caused by natural selection, suggesting the involvement of PHYE in local adaptation between populations of A. nana in northern and southern Japan. This finding is consistent with a previous study on Cardamine nipponica, indicating the importance of PHYE for local adaptation in Japanese alpine plants.

The Pleistocene was characterized by a cyclic pattern of cold and warm climatic periods, or climatic oscillations, which caused fluctuations in the distributions of organisms. This resulted in drastic changes in demography, thereby accelerating the genetic divergence of populations. Phylogeographic studies have elucidated the history of populations during the Pleistocene. However, given the lack of model-based analysis of population histories, previous phylogeographic studies could not adequately evaluate the effect of these Pleistocene climatic oscillations on the genetic divergence and migration events between populations. Populations of Japanese alpine plants in central and northern Japan are highly differentiated, and a history of isolation between regions during the Pleistocene was inferred. Using sequences of 10 nuclear genes (ca. approximately 7,000 bp in total) from Cardamine nipponica (Brassicaceae), we applied an isolation with migration (IM) model to test the significance of the isolation history between central and northern Japan and to assess whether range shifts during the Pleistocene climatic oscillations were involved in the genetic differentiation between regions. The estimated divergence time indicates that the two regions were separated about 100,000-110,000 years ago. The exclusive occurrence of closely related haplotypes within each region (parsimony network) and the high level of genetic differentiation between the regions (mean F(ST) = 0.417) indicate that genetic divergence occurred following the isolation of the two regions. Therefore, the genetic differentiation between regions was shaped during the Pleistocene, especially during the last glacial and inter and postglacial periods. In addition, our multilocus analysis showed that populations in central and northern Japan were completely isolated after they split. Geographic separation and subsequent restricted migration events among mountains could explain this isolation history between regions. Furthermore, genetic drift in the reduced populations would remove evidence of occasional migration, emphasizing the isolation history. Therefore, our application of a demographic model demonstrated the Pleistocene origin of geographic differentiation statistically and provided a plausible migration history for C. nipponica.

Previous phylogeographic studies of alpine plants in Japan have inferred that populations in central Honshu persisted during the Pleistocene climatic oscillations and suggested interglacial survival in high mountains. However, Arcterica nana (Maxim.) Makino (Ericaceae) exhibits a homogenous genetic structure throughout Japan and may therefore have a unique phylogeographic history. This inconsistency could have resulted from insufficient resolution of previously analyzed chloroplast DNA sequences. Therefore, we conducted a phylogeographic investigation based on amplified fragment length polymorphisms. Using 176 individuals from 21 populations, the relationships among individuals and populations were determined by principal coordinate analysis and a neighbor-joining tree, respectively. In addition, genetic differentiation was estimated using analysis of molecular variance and spatial autocorrelation analysis. These analyses demonstrate a homogenous structure throughout the entire Japanese range, supporting the previous cpDNA phylogeography. Although this genetic structure is inconsistent with those of other alpine plants, it is difficult to postulate that pre-existing genetic differentiation was swamped exclusively within A. nana. Therefore, this homogenous genetic structure may have been caused by the distinct history of populations of A. nana. Specifically, the southern-ward migration and the subsequent continuous populations enabled gene flow throughout the Japanese archipelago during the last glacial period. Thus, our data suggest that alpine plants in the Japanese archipelago did not always experience a shared distribution change following climatic oscillations.

Molecular phylogeography has inferred the history of differentiation between regions and/or among populations following the Pleistocene climatic oscillations, mostly based on the genetic structure of organelle DNA. However, such genetic structure only reflects the history of a single gene, and studies based on single-copy genes of nuclear DNA (nDNA) are required for phylogeography, although their efficiency remains unclear. To examine the utility of nDNA loci, the genetic structures of three genes from Cardamine nipponica, which is closely related to the model species Arabidopsis thaliana, were elucidated: the nDNA genes DET1, PHYA, PHYE, as well as chloroplast DNA (cpDNA). In 279 individuals collected from throughout the range of the species, strong genetic differentiation between northern and central Japan was found for all loci. This result suggested that populations in central Japan experienced a different history from those in northern Japan during the Pleistocene climatic oscillations. In addition, the evidence of refugia at the edges of the distribution, where the genetic structure was less influenced by colonization following range expansion, was shown for several loci. The specific genetic structure within the southernmost populations of northern Japan suggested that this region was also isolated during range expansion. Hence, the consistent history among loci and a more detailed history from several loci indicated that cpDNA can represent the history of vicariance and demonstrated the efficiency of single-copy nuclear genes in phylogeography.

Ranunculus nipponicus (Makino) Nakai is a vulnerable aquatic macrophyte in the Kinki district, which is the southernmost distribution of this species in Japan. The genetic diversity and structure within and among eleven extant populations were assessed using the inter-simple sequence repeats (ISSR) polymerase chain reaction in association with combinations of propagation pattern (clonal and/or seeds) and genotypic geographical structure. In total, 53 bands were amplified, of which 18 (34%) were polymorphic. Analysis of the ISSR bands identified 46 genotypes among 81 individuals from one stream population and 72 distinct genotypes among 147 individuals in the Kinki district. An unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed some unity among upstream and downstream subpopulations within one stream and eleven populations. The Shannon index of genetic diversity was 0.109 for one stream population and 0.313 for total genetic diversity, suggesting relatively high genetic diversity. Analysis of molecular variance (AMOVA) revealed that 84.1% of the total genetic diversity occurred among populations and the remaining diversity (15.9%) occurred within populations. Significant genetic differentiation occurred among populations in the Kinki district. These results suggest that conservation of each population is important for maintaining genetic diversity of R. nipponicus in this district.

Molecular phylogeographic studies have revealed the genetic patterns and glacial-interglacial history of many plant and animal species. To infer the Quaternary history of alpine plants in the Japanese archipelago, which is poorly known, we investigated 203 individuals of Potentilla matsumurae and its varieties from 22 populations. We found 11 haplotypes based on approximately 1400 bp of two intergenetic spacers in chloroplast DNA (trnT-L and rpl20-rps20). The distribution of these haplotypes was geographically structured, which was supported by haplotype composition, principal component analysis, and unweighted pair group method with arithmetic mean (UPGMA), and N(ST) (0.71) was significantly greater than G(ST) (0.68). In addition to the positive correlation between genetic and geographic distance (Mantel test, r = 0.497, P < 0.001), an abrupt genetic change was detected between mountains in central Honshu and the Tohoku region. This genetic boundary was further supported by analysis of molecular variance (AMOVA), and high variation (54.0%) was explained by differences on either side of this boundary. Moreover, haplotypes in central Honshu were thought to have diverged, based on an outgroup comparison. These results suggest that mountains in central Honshu served as refugia during the Quaternary climatic oscillation, although the results could not reveal the history of most mountains in the Tohoku region and Hokkaido. Nevertheless, following floristic studies, our results indicate that alpine plants in Japan experienced a history different from that in Europe; i.e. they retreated into refugia during warm periods to avoid forest development, rather than glaciers.

To evaluate the trend of elderly patients visiting the emergency department of a Japanese University Hospital, out patient-based records were reviewed of the emergency department of Kyushu University Hospital from 2000 to 2004. A total number of 7610 emergency patients visited the department during the five year period. The median (25%, 75%) of age was 32 (22, 56). Patients aged 65 years and over accounted for 16% of all attendances. All the patients were classified into 6 groups according to the diagnosis, (1) Respiratory, (2) Circulatory, (3) Central nervous system, (4) Abdominal, (5) Trauma, and (6) Others. The median age in each group was (1) 27 (15, 49), (2) 66 (53, 76), (3) 51 (27, 67), (4) 33 (22, 56), and (5) 26 (20, 46), respectively. There was a statistically significant difference observed, reciprocally except between (1) and (5) (P < 0.05). The patients showed statistically significant difference in the annual transition of the disease (P < 0.0001). In the elderly, the annual transition of the disease showed statistically significant decreases in Circulatory (P = 0.0015) and in Central nervous (P < 0.0001), and an increase in Abdominal (P < 0.0001), respectively. Death rate at the outpatient clinic in the elderly showed much higher than in the younger (P < 0.0001). Admission rate was also much higher in the elderly than in the younger (P < 0.0001). Elderly emergency patients have both internal and external intrinsic factors. They have to be treated carefully since their condition easily deteriorates. Provisions for the problems surrounding the elderly should be made as a nationwide effort.

We conducted a phylogeographic study on the alpine plant Arcterica nana based on haplotypes of chloroplast DNA. Using a sequence of approximately 1,071 bp of intergenic spacers of chloroplast DNA (trnT-L, psbB-psbF), we detected 13 haplotypes among 193 individuals sampled from 22 populations. Two dominant haplotypes were distributed over the entire range of this species in Japan, and we found several local private haplotypes. An analysis of molecular variance (AMOVA) indicated no geographic structure within the haplotype distribution. In addition, the genetic distance was not related to its corresponding geographic distance (Mantel test: r=-0.049, P=0.66), indicating a homogeneous geographic structure throughout the entire distribution range in the Japanese archipelago. The most parsimonious explanation for this geographic structure is that A. nana spread across its extant distribution range in the Japanese archipelago through a recent range expansion event. However, this pattern is inconsistent with the previous phylogeography of Japanese alpine plants, which reveals that haplotypes in central Honshu are differentiated from those in more northern regions. Arcterica nana may have experienced a different history from other alpine plants, suggesting that the history of Japanese alpine flora may include at least two different geographic radiation patterns.