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Background and Aims Hybridization is the main driver of plant diversification, and gene flow via hybridization has multifaceted effects on plant evolution. Carex angustisquama is an extremophyte that grows on soils heavily acidified by volcanism. Despite its habitat distinct from that of other species, this species is known to form interspecific hybrids, implying interspecific gene flow. It is crucial to verify the extent and direction of interspecific gene flow between C. angustisquama and closely related species to understand the evolutionary process of an extremophyte in solfatara fields. Methods In this study, expressed sequence tag–simple sequence repeat markers were utilized to infer the extent and direction of interspecific gene flow between C. angustisquama and closely related species. Key Results Bayesian clustering and simulation analyses revealed that all individuals of the three hybrid species were classified into the first hybrid generation or first backcross to C. angustisquama; therefore, current interspecific gene flow is limited. Moreover, in the Bayesian inference of historical gene flow based on multispecies samples, the model that assumed no interspecific gene flow was the most strongly supported across all species pairs, including phylogenetically close but ecologically distinctive species pairs. Conclusions Our results revealed that interspecific gene flow between C. angustisquama and its related species has been limited both currently and historically. Moreover, our results of Bayesian inference of historical gene flow indicated that extrinsic, rather than intrinsic, factors probably act as isolating barriers between Carex species, with hybrid breakdown via microhabitat segregation being the probable potential barrier. Overall, our findings provide insights into the evolutionary process of an extremophyte in solfatara fields and offer an important example of the mechanisms of diversification of the speciose genus Carex.

Arnica mallotopus is a perennial herb endemic to the snowy regions of Japan. At the southern edge of its distribution, in Kyoto Prefecture, overgrazing by sika deer and decreased snowfall have resulted in the rapid decline of A. mallotopus populations. Therefore, there is an urgent need for a conservation genetic analysis of the remaining local populations. In this study, we first developed 13 EST-SSR markers to evaluate genetic variation in A. mallotopus. The average number of alleles per locus was 5.33. Genetic analysis using these markers showed that the investigated samples were classified into two groups corresponding to landscape structure. One group isolated from a tributary of the Yura River showed a strong population bottleneck signal, likely resulting from founder effects and subsequent drifts. On the other hand, the genetic diversity of the second group in the main distribution along the Yura River was higher and less inbred. Overall, our assessment suggested recognizing the two genetic groups as management units in conservation programs for the threatened populations.

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 (

Climate relicts hold considerable importance because they have resulted from numerous historical changes. However, there are major interspecific variations among the ways by which they survived climate changes. Therefore, investigating the factors and timing that affected population demographics can expand our understanding of how climate relicts responded to historical environmental changes. Here, we examined herbaceous hydrangeas of genus Deinanthe in East Asia, which show limited distributions and a remarkable disjunction between Japan and central China. Chloroplast genome and restriction site-associated DNA sequencing revealed that speciation event occurred in the late Miocene (ca. 7-9 Mya) in response to global climate change. Two lineages apparently remained not branched until the middle Quaternary, and afterwards started to diverge to regional population groups. The narrow endemic species in central China showed lower genetic diversity (He = 0.082), as its population size rapidly decreased during the Holocene due to isolation in montane refugia. Insular populations in the three Japanese islands (He = 0.137-0.160) showed a genetic structure that was inconsistent with sea barriers, indicating that it was shaped in the glacial period when its range retreated to coastal refugia on the exposed sea floor. Demographic modelling by stairway-plot analysis reconstructed variable responses of Japanese populations: some experienced glacial bottlenecks in refugial isolation, while post-glacial range expansion seemingly exerted founder effects on other populations. Overall, this study demonstrated the involvement of not just one, but multiple factors, such as the interplay between climate changes, geography, and other population-specific factors, that determine the demographics of climate relicts.

Genetic and ecological evaluation are crucial in effective management of rare and endangered species, including those exhibiting complex breeding systems such as distyly. We studied a threatened distylous herb Primula reinii in the Hakone volcano, central Japan, to obtain baseline information of reproductive and genetic status towards conservation. In two representative populations inhabiting a central cone and somma of the volcano, population size, floral morph ratio, stigmatic pollen deposition, and fruit-set were measured. Using microsatellite markers, we evaluated genetic diversity, structure and differentiation of populations. Population bottlenecks and historical changes in population size were also estimated from genotype data. We found significant deviation from equal morph ratios in the central cone population, which also exhibited skewed mating success together with a high frequency of pollination within the same morph. These trends were not detected in the somma population. From genetic insights, the central cone population showed slightly lower genetic diversity, whereas no significant deviation from Hardy-Weinberg equilibrium was found in either population. The estimated moderate genetic differentiation and admixed genetic structure suggest recent lineage divergence and/or gene flow between populations. While robust evidence for a recent bottleneck was not obtained in our analyses, a clear signature of historical population contraction was detected in the central cone population. Our findings suggest that the skewed morph ratio strongly influenced the reproduction of small and isolated populations in the short-term, highlighting the vulnerability of distylous plant populations under ongoing anthropogenic pressure.

The phylogenetic origin of endangered species extinct in the wild is not always well understood, as a consequence of limited historical records of the original populations. Magnolia pseudokobus (Ma...

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) for Mazus miquelii (Mazaceae), one of the most widespread species of the genus found in mainland China to Japan and North America, with the goal of elucidating the hidden genetic diversity and pollination ecology of the species. Of the initial 48 EST-SSR markers designed based on transcriptome data, 36 loci were successfully amplified, 16 of which were polymorphic. Polymorphisms for these markers were tested on 72 individuals from three populations in Japan. Two to eleven alleles per locus were detected, and the levels of observed and expected heterozygosity ranged from 0.181 to 0.708 and 0.154 to 0.715, respectively. Most loci were amplified successfully in five related Japanese and Taiwanese species. These markers will empower ecological and evolutionary studies in the creeping mazus and facilitate the disentanglement of phylogenetic relationships with related species.

Nuclear microsatellite markers were developed for Aphananthe aspera (Thunb.) Planch. (Cannabaceae), a deciduous canopy tree species distributed in East Asia, to evaluate the genetic diversity and genetic structure of A. aspera populations in remnant forest fragments in urbanized areas of Japan. A total of 94 primer pairs were designed based on genomic sequence data. Of the 25 primer pairs which showed clear microsatellite peaks, 20 pairs showed allelic polymorphisms in 57 individuals collected from two distant populations. The length of PCR products ranged from 120 to 482 bp, and expected heterozygosity for the 20 microsatellite markers ranged from 0.017 to 0.768. These newly developed simple sequence repeat markers will be used in population genetic studies of A. aspera to evaluate genetic diversity and the extent of genetic isolation of the fragmented populations in urban areas.

Edaphic specialization is one of the main drivers of plant diversification and has multifaceted effects on population dynamics. Carex angustisquama is a sedge plant growing only on heavily acidified soil in solfatara fields, where only extremophytes can survive. Because of the lack of closely related species in similar habitats and its disjunct distribution, the species offers ideal settings to investigate the effects of adaptation to solfatara fields and of historical biogeography on the genetic consequences of plant edaphic specialization to solfatara fields. Here, genome-wide single nucleotide polymorphisms were used to reveal the phylogenetic origin of C. angustisquama, and 16 expressed sequence tag-simple sequence repeat markers were employed to infer population demography of C angustisquama. Molecular phylogenetic analysis strongly indicated that C. angustisquama formed a monophyletic clade with Carex doenitzii, a species growing on nonacidified soil in the sympatric subalpine zone. The result of population genetic analysis showed that C. angustisquama has much lower genetic diversity than the sister species, and notably, all 16 loci were completely homozygous in most individuals of C. angustisquama. Approximate Bayesian computation analysis supported the model that assumed hierarchical declines of population size through its evolutionary sequence. We propose that the edaphic specialist in solfatara fields has newly attained the adaptation to solfatara fields in the process of speciation. Furthermore, we found evidence of a drastic reduction in genetic diversity in C. angustisquama, suggesting that the repeated founder effects associated with edaphic specialization and subsequent population demography lead to the loss of genetic diversity of this extremophyte in solfatara fields.

To investigate the genetic diversity and understand the process of horizontal gene transfer (HGT) in nodule bacteria associated with Lotus japonicus, we analyzed sequences of three housekeeping and five symbiotic genes using samples from a geographically wide range in Japan. A phylogenetic analysis of the housekeeping genes indicated that L. japonicus in natural environments was associated with diverse lineages of Mesorhizobium spp., whereas the sequences of symbiotic genes were highly similar between strains, resulting in remarkably low nucleotide diversity at both synonymous and nonsynonymous sites. Guanine-cytosine content values were lower in symbiotic genes, and relative frequencies of recombination between symbiotic genes were also lower than those between housekeeping genes. An analysis of molecular variance showed significant genetic differentiation among populations in both symbiotic and housekeeping genes. These results confirm that the Mesorhizobium genes required for symbiosis with L. japonicus behave as a genomic island (i.e., a symbiosis island) and suggest that this island has spread into diverse genomic backgrounds of Mesorhizobium via HGT events in natural environments. Furthermore, our data compilation revealed that the genetic diversity of symbiotic genes in L. japonicus-associated symbionts was among the lowest compared with reports of other species, which may be related to the recent population expansion proposed in Japanese populations of L. japonicus.

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.

Flowering is an important step in the life cycle of plants and indicates adaptability to external climatic cues such as temperature and photoperiod. We investigated the expression patterns of core genes related to flowering-time regulation in Japanese wild radish (Raphanus sativus var. raphanistroides) with different vernalization requirements (obligate and facultative) and further identified climatic cues that may act as natural selective forces. Specifically, we analysed flowering-time variation under different cold and photoperiod treatments in Japanese wild radish collected from the Hokkaido (northern lineage) and Okinawa (southern lineage) islands, which experience contrasting climatic cues. The cultivation experiment verified the obligate and facultative vernalization requirements of the northern and southern wild radish accessions, respectively. The expression of major genes involved in flowering time indicated that RsFLC and RsCOL5 (A&B) may interact to regulate flowering time. Notably, floral initiation in the northern lineage was strongly correlated with RsFLC expression, whereas flowering in the southern linage was correlated with induction of RsCOL5-A expression, despite high RsFLC transcript levels. These results suggested that the northern accessions are more sensitive to prolonged cold exposure, whereas the southern accessions are more sensitive to photoperiod. These different mechanisms ultimately confer an optimal flowering time in natural populations in response to locally contrasting climatic cues. This study provides new insights into the variant mechanisms underlying floral pathways in Japanese wild radish from different geographic locations., To identify climate cues that might act as natural selective forces shaping the genetic variation for flowering initiation, we conducted analyses of the flowering-time variation in response to cold treatment and day length in wild radish collected from Hokkaido and Okinawa islands with vernalization requirements. Our results collectively offer a hint that accessions with obligate vernalization requirements might exhibit a higher sensitivity to prolonged cold exposure, whereas in accessions with facultative vernalization requirements the photoperiod pathway predominates, and these mechanisms confer an optimal flowering time in natural populations, in response to their contrasting climate cues.

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.

Background Quaternary climate changes significantly impacted population demography of temperate organisms by shifting their distribution. Notably, the rear-edge populations are considered to be more prone to these changes, but empirical studies showed the southernmost fragmented populations of Japanese woody plants to harbor high genetic diversities due to their southern glacial refugia origin. Therefore, the impacts of Holocene climate warming on rear-edge populations have been rarely demonstrated. For the better interpretation of genetic backgrounds of temperate plants, the association of paleodistributions under both icy and warm climates with species-specific demographic changes is required. A perennial daylily Hemerocallis middendorffii (Asphodelaceae) is widely distributed in temperate and cool-temperate zones of East Asia. In Japan, larger populations are found in central ranges, while few small populations survive on harsh rock walls in southernmost regions. We focused on these variable populations and aimed to predict the population demography in relation to past climate changes by statistically combining population genetics with paleodistribution modeling.Results EST-SSR analysis of 737 individuals from 41 populations revealed 6 regional population groups. Four groups widely dominating the northern–central ranges harbored high genetic diversity, whereas genetic divergence within the groups was low. However, two groups at the southwest edge were geographically and genetically isolated and showed the lowest genetic diversity. Estimated paleodistributions showed a decrease of suitable range during Holocene climate optimum in comparison with LGM, and a sole variable of habitat suitability in the Holocene optimum was able to predict genetic diversity across its range. Conclusions We concluded that habitat fragmentation and population decline in relation to the climate warming during the Holocene optimum and interspecific competition with woody plants resulted in genetic isolation and impoverishment of the rear-edge populations.

Variation in partner quality is commonly observed in diverse cooperative relationships, despite the theoretical prediction that selection favoring high-quality partners should eliminate such variation. Here, we investigated how genetic variation in partner quality could be maintained in the nitrogen-fixing mutualism between Lotus japonicus and Mesorhizobium bacteria. We reconstructed de novo assembled full-genome sequences from nine rhizobial symbionts, finding massive variation in the core genome and the similar symbiotic islands, indicating recent horizontal gene transfer (HGT) of the symbiosis islands into diverse Mesorhizobium lineages. A cross-inoculation experiment using 9 sequenced rhizobial symbionts and 15 L. japonicus accessions revealed extensive quality variation represented by plant growth phenotypes, including genotype-by-genotype interactions. Variation in quality was not associated with the presence/absence variation in known symbiosis-related genes in the symbiosis island; rather, it showed significant correlation with the core genome variation. Given the recurrent HGT of the symbiosis islands into diverse Mesorhizobium strains, local Mesorhizobium communities could serve as a major source of variation for core genomes, which might prevent variation in partner quality from fixing, even in the presence of selection favoring high-quality partners. These findings highlight the novel role of HGT of symbiosis islands in maintaining partner quality variation in the legume–rhizobia symbiosis.

Earlier phylogenetic studies in the genusPinguicua(Lentibulariaceae) suggested that the species within a geographical region was rather monophyletic, although the sampling was limited or was restricted to specific regions. Those results conflicted with the floral morphology-based classification, which has been widely accepted to date. In the current study, one nuclear ribosomal DNA (internal transcribed spacer; ITS) and two regions of chloroplast DNA (matKandrpl32-trnL), from up to ca. 80% of the taxa in the genusPinguicula, covering all three subgenera, were sequenced to demonstrate the inconsistency and explore a possible evolutionary history of the genus. Some incongruence was observed between nuclear and chloroplast topologies and the results from each of the three DNA analyses conflicted with the morphology-based subgeneric divisions. Both the ITS tree and network, however, corresponded with the biogeographical patterns of the genus supported by life-forms (winter rosette or hibernaculum formation) and basic chromosome numbers (haploidy). The dormant strategy evolved in a specific geographical region is a phylogenetic constraint and a synapomorphic characteristic within a lineage. Therefore, the results denied the idea that the Mexican group, morphologically divided into the three subgenera, independently acquired winter rosette formations. Topological incongruence among the trees or reticulations, indicated by parallel edges in phylogenetic networks, implied that some taxa originated by introgressive hybridisation. Although there are exceptions, species within the same geographical region arose from a common ancestor. Therefore, the classification by the floral characteristics is rather unreliable. The results obtained from this study suggest that evolution within the genusPinguiculahas involved; 1) ancient expansions to geographical regions with gene flow and subsequent vicariance with genetic drift, 2) acquirement of a common dormant strategy within a specific lineage to adapt a local climate (i.e., synapomorphic characteristic), 3) recent speciation in a short time span linked to introgressive hybridisation or multiplying the ploidy level (i.e., divergence), and 4) parallel evolution in floral traits among lineages found in different geographical regions (i.e., convergence). As such, the floral morphology masks and obscures the phylogenetic relationships among species in the genus.

The Sino-Japanese Floristic Region is highly diverse with respect to temperate plants. However, the reasons for this diversity are poorly understood because most studies have only considered geographic isolation caused by climatic oscillations.Heterotropa(genusAsarum; Aristolochiaceae) diverges here and shows high species diversity in insular systems (63 species) compared to continental areas (25 species).Heterotropashows low dispersal ability with small distribution ranges, implying diversification by geographic events, and high floral diversity, implying pollinator-mediated diversification. To reveal how abiotic and biotic factors have shaped the diversity anomaly ofHeterotropa, we conducted phylogenetic analysis using ddRAD-seq and chloroplast genome datasets including 79 species, estimation of floral trait evolution, and comparison of isolation factors within clades based on distribution range and floral trait analysis. Phylogenetic analysis indicates thatHeterotropaoriginated in mainland China and expanded to the Japanese Archipelago in the Miocene, and the major clades almost correspond to geographic distributions. Floral traits evolved repeatedly in the tip nodes within the clades. Although the major clades include a high proportion of species pairs showing isolation by floral traits, there are no conditional relationships between two isolation factors, indicating that most species pairs with floral trait isolation are distributed allopatrically. The repeated exposure and submergence of land-bridges caused by climatic oscillations would have led to significant population fragmentation in insular systems. Thus, the diversity anomaly ofHeterotropawould have resulted from geographic and climatic events during the Miocene, while subsequent repeated floral trait evolution would have followed geographic isolation during the Pleistocene.

In diverse mutualistic relationships, genetic variations in impact on the growth of interacting partners—variations in partner quality—are common, despite the theoretical prediction that selection favoring high-quality partners should eliminate such variations. Here, we investigated how variations in partner quality could be maintained in the nitrogen-fixing mutualism between Lotus japonicus and Mesorhizobium bacteria. We reconstructed de novo assembled full-genome sequences from nine rhizobial symbionts, finding massive variations in the core genome and the contrastingly similar symbiotic islands, indicating recent horizontal gene transfer (HGT) of the symbiosis islands into diverse Mesorhizobium lineages. A cross-inoculation experiment using nine sequenced rhizobial symbionts and 15 L. japonicus accessions revealed extensive quality variations represented by plant growth phenotypes, including genotype-by-genotype interactions. Quality variations were not associated with the presence/absence variations of known symbiosis-related genes in the symbiosis island, but rather, showed significant correlations with the core genome variations, supported by SNP- and kinship matrix-based association analyses. These findings highlight the novel role of HGT of symbiosis islands, which indirectly supply mutations of core genomes into L. japonicus-associated bacteria, thereby contributing to the maintenance of variations in partner quality.

Series Sakawanum (genus Asarum, Aristolochiaceae) comprises four taxa of forest understory herbaceous plants, which exhibits a geographical cline of calyx lobe length across taxonomic units...

Primula reinii (Primulaceae), a perennial herb belonging to the Primula section Reinii , occurs on wet, shaded rocky cliffs in the mountains of Japan. This threatened species comprises four varieties; these plants are very localized and rare in the wild. In this study, 43 microsatellite markers were developed using MiSeq sequencing to facilitate conservation genetics of these critically endangered primroses. We developed novel microsatellite markers for three varieties of P. reinii , and tested its polymorphism and genetic diversity using natural populations. These novel markers displayed relatively high polymorphism; the number of alleles and expected heterozygosities ranged from 2 to 6 (mean = 3.2) and 0.13 to 0.82 (mean = 0.45), respectively. All loci were in Hardy–Weinberg equilibrium. These microsatellite markers will be powerful tools to assess P. reinii genetic diversity and develop effective conservation and management strategies.

Background and aims Recent biogeographic studies have illustrated that Quaternary climatic changes and historical orogenies have driven the development of high levels of biodiversity. In this context, phylogenetic niche conservatism may play a role as a major precursor of allopatric speciation. However, the effects of niche evolution on the diversification patterns of plant species under rapid habitat changes are still unknown. Here, Primula section Reinii, one of the few primroses endemic to the Japanese Archipelago, was investigated. This study aimed to clarify the phylogenetic position and relationships of section Reinii, interpret the biogeographic and diversification patterns of this group and gain a better understanding of the role of climatic niche evolution in the Japanese endemic primroses. Methods Dated phylogeny for Primula section Reinii is presented based on the sequences of six chloroplast genes and one nuclear gene. Biogeographic history was reconstructed using statistical dispersal-vicariance analysis. Macro-evolutionary modelling of the climatic niche was combined with biogeographic inferences. Key results Section Reinii was shown to be monophyletic based on chloroplast and nuclear sequences. Fossil-calibrated dating analysis estimated that this section diverged from its sister taxon, sect. Cortusoides, around 1.82 million years ago, and intraspecific diversification occurred within the last million years. This time frame was characterized by Quaternary climatic oscillations and uplift of high mountains in Japan. Biogeographic inference suggested that this section originated at the northern end of the Japanese Archipelago and then dispersed southward to other islands. Models of climatic niche evolution indicated that the closely related species P. reinii and P. tosanensis have contrasting niche optima and rates of niche evolution. Conclusion Our results highlight that spatiotemporal heterogeneity in the Japanese islands may play a significant role in the biogeographic history of Japanese endemic primroses. Contrasting evolutionary processes found in closely related species illustrate the effects of climatic niche evolution on species' diversification patterns.

A set of 11 polymorphic microsatellite markers has been developed and characterized for the critically endangered species Pieris amamioshimensis. Fifty-nine individuals of an ex-situ population were used to identify these markers. The total number of alleles for each locus ranged from 3 to 9, with an average of 5.4. The expected heterozygosities (HS) and observed heterozygosities (HO) ranged from 0.47 to 0.77 and 0.22 to 0.88, respectively. In total, four loci exhibited significant deviations from Hardy–Weinberg equilibrium: two loci showed significant heterozygosity excess and the other two loci showed significant heterozygosity deficit. The polymorphism information content (0.43 ≤ PIC ≤ 0.73), the probability of exclusions (PE1 = 0.9565, PE2 = 0.9969 and PE3 = 0.9999) and probabilities for identity (PI = 3.78 × 10−9 and PI-Sib = 2.35 × 10−4) suggest that these markers are useful for estimating not only genetic diversity but also parentage, for the ex-situ conservation management of populations.

The genus Asarum (Aristolochiaceae) encompasses approximately 120 species from five sections. Taxonomic controversies concerning the genus Asarum and/or its intrageneric classification remain unresolved. In particular, sect. Heterotropa accounts for a large percentage of the genus (80 of 120 species) and is well diverged in the Sino–Japanese Forest subkingdom. Reconstruction of Heterotropa phylogeny and estimation of its divergence times would provide significant insight into the process of species diversity in the Sino–Japanese floristic region. This study encompassed 106 operational taxonomic units (OTUs), and phylogenetic analyses were conducted based on internal transcribed spacer (ITS) and matK sequences. Although the matK sequences provided informative results solely for section Geotaenium, phylogenetic trees based on ITS regions yielded a clear result for several sections. Three sections, Asarum, Geotaenium and Asiasarum, were supported as robust monophyletic groups, whereas Heterotropa had low support. Sect. Hexastylis was revealed to be polyphyletic, suggesting taxonomic reconstruction would be needed. Sect. Heterotropa comprises two clades, which correspond to species distribution ranges: mainland China and the island arc from Taiwan to mainland Japan via the Ryukyu Islands. It is notable that the common ancestry of the latter clade in the eastern Asian islands was highly supported, suggesting that the present species diversity of Heterotropa was initially caused by allopatric range fragmentation in East Asia.

Based on organelle DNA phylogeographic analyses and ecological niche modeling (ENM), we investigated the range-shift history of the Japanese wingnut (Pterocarya rhoifolia) during the Quaternary climatic oscillations with particular emphasis on the Last Glacial Maximum (LGM). Phylogeographic patterns of this species were determined using 376 individuals from 53 populations for chloroplast DNA sequencing of three spacers. Spatial analysis of molecular variance revealed that the current phylogeographic structure would be sculptured by multiple range shifts from each glacial refugium, which would have been repeated several times during the Quaternary climatic oscillations. High haplotype diversity and private haplotypes were detected in southwestern Japan, where wingnut is currently infrequent and found mainly in high mountains, whereas in northernmost Japan, haplotype diversity was low though this plant is quite common at present. According to ENM approach, during the LGM, the climatically suitable d...

Primula reinii var. rhodotricha is a perennial herb endemic to the limestone slope of Mt. Buko, located approximately 50 km northwest of central Tokyo, Japan. In recent years, its natural population size has decreased markedly due to limestone mining, and this species has been assigned to the ‘Critically Endangered (CR)’ category on the latest Japanese Red List. Although a remnant population of this species has been protected by a mining company outside their historical distribution range on Mt. Buko, the ex situ conservation of this endangered plant has been difficult because of insufficient low seed production. The genetic status of ex situ P. reinii var. rhodotricha and related species were investigated to develop an effective conservation plan for this species. Microsatellite analysis indicated that the ex situ population harbors lower genetic diversity than sister taxa, providing molecular evidence for the recent critical status designation of the ex situ population, whereas the presence of rare alleles may imply further potential for seed reproduction by outcrossing. Therefore, an appropriate propagation strategy that considers genetic diversity is needed for restoration and recovery of this critically endangered ex situ primrose population.

Premise Saxifraga fortunei (Saxifragaceae) includes several infraspecific taxa that are ecologically and morphologically distinct. To investigate the evolutionary history of phenotypic polymorphisms in this species, we developed expressed sequence tag-simple sequence repeat (EST-SSR) markers for S. fortunei. Methods and results We developed 26 polymorphic markers based on transcriptome data obtained from Illumina HiSeq 2000. Within three populations of S. fortunei var. incisolobata, the number of alleles ranged from four to 25, and the levels of observed and expected heterozygosity ranged from 0.200 to 0.847 and from 0.209 to 0.930, respectively. Furthermore, all 26 loci showed transferability for S. fortunei var. obtusocuneata and S. fortunei var. suwoensis, and 18 loci were also successfully amplified in S. acerifolia. Conclusions These newly developed EST-SSR markers will prove useful to infer the evolutionary history of S. fortunei var. incisolobata and its relatives in population genetic studies.

EST-SSR markers were developed for Asarum sakawanum var. stellatum to evaluate the genetic diversity and genetic structure of series Sakawanum and assess their utility in other series in the genus Asarum. A total of 1864 primer pairs, including 1864 tri-nucleotide SSR motifs, were designed based on transcriptome data. Of the 96 primers tested, 17 loci were successfully amplified and polymorphic in series Sakawanum taxa. Furthermore, all these loci were successfully amplified in A. takaoi, whereas only 10 loci were amplified in the more phylogenetically distant A. pellucidum. For populations of A. sakawanum var. stellatum, the values of observed and expected heterozygosity ranged from 0.261 to 0.957 and 0.317 to 0.881, respectively. These newly developed EST-SSR markers will be used in population genetic studies of series Sakawanum to infer the evolutionary history and genetic diversity of these series, many of which are endangered in Japan.

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.

Aim The Aleutian Islands, one of the southern margins of Beringia, were covered with ice caps during the Last Glacial Maximum (LGM). Although insular refugia for animal species have been hypothesized in this region, phylogeographical studies have never explored the biogeographical history of plant species. We aim to assess the population history of the flora of the Aleutian Islands and its biogeographical relevance to East Asia. Location The Aleutian Islands, Kamchatka, the Japanese Archipelago. Methods We sequenced seven nuclear loci from the alpine shrub Therorhodion camtschaticum (Pall.) Small (Ericaceae) and elucidated the geographical distribution of genetic variation. The divergence history among populations from the Aleutian Islands, Kamchatka and northern Japan was inferred by analyses based on coalescent simulations. Results Populations were genetically distinct between northern Japan and Beringia. In spite of the genetic differentiation, the divergence between Beringia and northern Japan occurred during or after the last glacial period [maximum-likelihood estimates (MLEs) = 10.6–15.5 ka with 95% highest posterior densities (HPDs): 1.7–43.5 ka]. The latter region has a larger population size than the former region. In contrast, widespread genetic similarity was revealed among the geographically separated populations in the Aleutian Islands and southern Kamchatka. Among these populations, a model assuming population decline fits better than a model that assumes either constant population size or population growth. Main conclusions Our study suggests that insular refugia were unlikely for T. camtschaticum in the Aleutian Islands. Instead, T. camtschaticum may have had a wider range during LGM, encompassing both the Aleutian Islands and southern Kamchatka, or have colonized the Aleutian Islands from a LGM refugium that was the source of colonization into southern Kamchatka as well or encompassed the latter region. Our study provides novel insight into the biogeographical history of terrestrial biota in the Aleutian Islands.