Recent advances in adaptation genomics in fumarole fields, an overlooked extreme environment.

Extreme environments and plants thriving in them, known as extremophytes, offer promising platforms for studying the diverse adaptive mechanisms that have evolved in plants. However, research on adaptation to extreme environments is still limited to those environments where model species or their relative can survive. Fumarole fields, an extreme environment often overlooked, are characterized by multi-hazardous abiotic stressors, including atmospheric contamination (high concentration of H2S, SO2, and CO2), high soil temperature (~60℃), and strong soil acidification (pH=2-3). These conditions make fumarole fields a rich source for studying stress tolerance mechanisms in plants. In this review, we highlight the recent ecological, physiological, and genomic advances involved in fumarole field adaptation, and discuss the forward avenues. The studies outlined in this paper demonstrate that the extreme levels of abiotic stressors found in fumarole fields make them unparalleled field laboratories for studying the unknown stress tolerance mechanisms, warranting further genomic assessments. Some studies succeeded in identifying genes associated with fumarole field adaptation and shedding light on evolutionary implications; however, they have also encountered challenges such as limited genome resources and high genetic differentiation from related species and/or neighboring populations. To overcome such difficulties, we propose integrating ecophysiological and genomic approaches, drawing from the recent studies in other extreme environments. We expect that further studies in the fumarole fields will contribute to broadening our general knowledge of the limits of life.

High-intensity light promotes adaptive divergence of photosynthetic traits between sun-exposed and shaded populations in Saxifraga fortunei.

PREMISE: Light is essential for plants, and local populations exhibit adaptive photosynthetic traits depending on their habitats. Although plastic responses in morphological and/or physiological characteristics to different light intensities are well known, adaptive divergence with genetic variation remains to be explored. This study focused on Saxifraga fortunei (Saxifragaceae) growing in sun-exposed and shaded habitats. METHODS: We measured the leaf anatomical structure and photosynthetic rate of plants grown in their natural habitats and in a common greenhouse (high- and low-intensity light experimental sites). To assess differences in ecophysiological tolerance to high-intensity light between the sun and shade types, we evaluated the level of photoinhibition of photosystem II and the leaf mortality rate under high-intensity light conditions. In addition, population genetic analysis was conducted to investigate phylogenetic origins. RESULTS: Clear phenotypic differences were found between the sun and shade types despite their recent phylogenetic origin. The leaf anatomical structure and photosynthetic rate showed plastic changes in response to growing conditions. Moreover, the sun type had a well-developed palisade parenchyma and a higher photosynthetic rate, which were genetically fixed, and a lower level of photoinhibition under high-intensity light. CONCLUSIONS: Our findings demonstrate that light intensity is a selective pressure that can rapidly promote phenotypic divergence between the sun and shade types. While phenotypic changes in multiple photosynthetic traits were plastic, genetic divergence in specific traits related to adaptation to high-intensity light would be fundamental for ecotypic divergence to different light regimes.

Genetic consequences of Last Glacial-Holocene changes in snowfall regime in Arnica mallotopus populations: A plant confined to heavy-snow areas of Japan.

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.

Phylogeographic analysis of Saxifraga fortunei complex (Saxifragaceae) reveals multiple origins of morphological and ecological variations in the Japanese Archipelago.

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.

Inferring historical survivals of climate relicts: the effects of climate changes, geography, and population-specific factors on herbaceous hydrangeas.

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.

Species cohesion of an extremophyte (Carex angustisquama, Cyperaceae) in solfatara fields maintained under interspecific natural hybridization.

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.

Comparative analysis of spatial genetic structures in sympatric populations of two riparian plants, Saxifraga acerifolia and Saxifraga fortunei.

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.

Genetic consequences of plant edaphic specialization to solfatara fields: Phylogenetic and population genetic analysis of Carex angustisquama (Cyperaceae).

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.

Speciation and subsequent secondary contact in two edaphic endemic primroses driven by Pleistocene climatic oscillation.

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.

Exploring Genetic Diversity and Signatures of Horizontal Gene Transfer in Nodule Bacteria Associated with Lotus japonicus in Natural Environments.

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.

Genetic data reveals a complex history of multiple admixture events in presently allopatric wild gingers (Asarum spp.) showing intertaxonomic clinal variation in calyx lobe length.

Clinal variation is a major pattern of observed phenotypic diversity and identifying underlying demographic processes is a necessary step to understand the establishment of clinal variation. The wild ginger series Sakawanum (genus Asarum) comprises four taxa, which exhibit intertaxonomic clinal variation in calyx lobe length across two continental islands isolated by a sea strait. To test alternative hypotheses of the evolutionary history and to determine the implications for the formation of clinal variation, we conducted approximate Bayesian computation (ABC) analysis and ecological niche modeling (ENM). ABC analysis indicated that the scenario assuming multiple admixture events was strongly supported. This scenario assumed two admixture events occurred between morphologically distinct taxa, likely leading to the generation of intermediate taxa. One of the admixture events was estimated to have occurred during the last glacial maximum (LGM), during which the taxa were estimated to have formed a common refugia in southern areas by ENM analysis. Although four taxa are currently distributed allopatrically on different islands and trans-oceanic dispersal appears unlikely, the formation of a land bridge and the geographic range shift to refugia would have allowed secondary contact between previously isolated taxa. This study suggests that clinal variation can be shaped by demographic history including multiple admixtures due to climatic oscillations.

Relative contributions of neutral and non-neutral processes to clinal variation in calyx lobe length in the series Sakawanum (Asarum: Aristolochiaceae).

Background and Aims: Clines, the gradual variation in measurable traits along a geographical axis, play a major role in evolution and can contribute to our understanding of the relative roles of selective and neutral process in trait variation. Using genetic and morphological analyses, the relative contributions of neutral and non-neutral processes were explored to infer the evolutionary history of species of the series Sakawanum (genus Asarum), which shows significant clinal variation in calyx lobe length. Methods: A total of 27 populations covering the natural geographical distribution of the series Sakawanum were sampled. Six nuclear microsatellite markers were used to investigate genetic structure and genetic diversity. The lengths of calyx lobes of multiple populations were measured to quantify their geographical and taxonomic differentiation. To detect the potential impact of selective pressure, morphological differentiation was compared with genetic differentiation (QCT-FST comparison). Key Results: Average calyx lobe length of A. minamitanianum was 124.11 mm, while that of A. costatum was 13.80 mm. Though gradually changing along the geographical axis within series, calyx lobe lengths were significantly differentiated among the taxa. Genetic differentiation between taxa was low (FST = 0.099), but a significant geographical structure along the morphological cline was detected. Except for one taxon pair, pairwise QCT values were significantly higher than the neutral genetic measures of FST and G'ST. Conclusions: Divergent selection may have driven the calyx lobe length variation in series Sakawanum taxa, although the underlying mechanism is still not clear. The low genetic differentiation indicates recent divergence and/or gene flows between geographically close taxa. These neutral processes would also affect the clinal variation in calyx lobe lengths. Overall, this study implies the roles of population history and divergent selection in shaping the current cline of a flower trait in the series Sakawanum.

Late Pleistocene origin of the entire circumarctic range of the arctic-alpine plant Kalmia procumbens.

The circumarctic ranges of arctic-alpine plants are thought to have been established in the late Pliocene/early Pleistocene, when the modern arctic tundra was formed in response to climate cooling. Previous findings of range-wide genetic structure in arctic-alpine plants have been thought to support this hypothesis, but few studies have explicitly addressed the temporal framework of the genetic structure. Here, we estimated the demographic history of the genetic structure in the circumarctic Kalmia procumbens using sequences of multiple nuclear loci and examined whether its genetic structure reflects prolonged isolation throughout the Pleistocene. Both Bayesian clustering and phylogenetic analyses revealed genetic distinction between alpine and arctic regions, whereas detailed groupings were somewhat discordant between the analyses. By assuming a population grouping based on the phylogenetic analyses, which likely reflects a deeper intraspecific divergence, we conducted model-based analyses and demonstrated that the intraspecific genetic divergence in K. procumbens likely originated during the last glacial period. Thus, there is no need to postulate range separation throughout the Pleistocene to explain the current genetic structure in this species. This study demonstrates that range-wide genetic structure in arctic-alpine plants does not necessarily result from the late Pliocene/early Pleistocene origin of their circumarctic ranges and emphasizes the importance of a temporal framework of the current genetic structure for understanding the biogeographic history of the arctic flora.

Contrasting evolutionary processes during Quaternary climatic changes and historical orogenies: a case study of the Japanese endemic primroses Primula sect. Reinii.

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.

Genetic structure of Hepatica nobilis var. japonica, focusing on within population flower color polymorphism.

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.

Lineage isolation in the face of active gene flow in the coastal plant wild radish is reinforced by differentiated vernalisation responses.

BACKGROUND: The respective role and relative importance of natural selection and gene flow in the process of population divergence has been a central theme in the speciation literature. A previous study presented conclusive evidence that wild radish on Japanese islands comprises two genetically isolated lineages: the southern and northern groups. However, a general understanding of the lineage isolation with frequent seed flow of the coastal plant species is still unclear. We surveyed nucleotide polymorphisms over 14 nuclear loci in 72 individuals across the Japan-Ryukyu Islands Arc to address the demographic history of wild radish utilising the isolation-with-migration (IM) model. In addition, we investigated the flowering times of individuals in different wild radish lineages, with and without cold exposure, to assess their respective vernalisation responses. RESULTS: Coalescent simulations suggested that divergence between the southern and northern lineages of wild radish began ~18,000 years ago, initially during the Last Glacial Maximum (LGM) period. The gene flow from the southern to northern groups was considerably higher than that in the opposite direction, indicating effective dispersal of viable seeds via the northward Kuroshio Current. Our greenhouse experiments indicated that cold exposure was not required for flowering in the southern group, but could advance the date of flowering, suggesting that vernalisation would be facultative in the southern group. In contrast, the northern group was either unable to flower or flowered later without prior cold exposure, and thus had an obligate requirement for cold treatment. CONCLUSIONS: The south-north lineage divergence in wild radish could be triggered by a directional change in the sea current during the ice age, despite gene flow due to the high dispersability and longevity of seeds. We also found that temperature profoundly affected the vernalisation responses of wild radish, which may repress reproductive success and ultimately drive and reinforce intra-specific differentiation between the two lineages of wild radish. This study provides new insights into the maintenance of lineage differentiation with on-going gene flow in coastal plants.

Persistent history of the bird-dispersed arctic-alpine plant Vaccinium vitis-idaea L. (Ericaceae) in Japan.

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.

Molecular evidence for ancient relicts of arctic-alpine plants in East Asia.

Following climate cooling at the end of the Tertiary, arctic-alpine plants attained most of their extant species diversity. Because East Asia was not heavily glaciated, the importance of this region as a location for the long-term persistence of these species and their subsequent endemism during the Pleistocene was proposed in early discussions of phytogeography. However, this hypothesis remains to be fully tested. Here, we address this hypothesis by elucidating the phylogenetic history of Phyllodoce (Ericaceae). A phylogenetic tree based on multiple nuclear loci revealed that Phyllodoce nipponica was not derived from widespread species such as the arctic-alpine Phyllodoce caerulea, but rather represented an independent lineage sister to the clade of widespread relatives. Molecular dating indicated a mid-Pleistocene divergence of P. nipponica. These findings exclude the hypothesis that P. nipponica was derived from an arctic-alpine species that extended its range southwards during recent glacial periods. Instead, our results support the hypothesis that P. nipponica is an ancestral species which persisted in the Japanese archipelago during the mid- and late Pleistocene. Our findings demonstrate support for the early proposal and shed light on the importance of the Japanese archipelago for the evolution and persistence of arctic-alpine species.

Polymorphisms of E1 and GIGANTEA in wild populations of Lotus japonicus.

In plants, timing of flowering is an essential factor that controls the survival rates of descendants. The circadian clock genes E1 and GIGANTEA (GI) play a central role in transmitting signals to flowering locus T (FT) in leguminous plants. Lotus japonicus is a wild Japanese species that ranges from northern Hokkaido to the southern Ryukyus and exhibits a wide range in terms of the time between seeding and first flowering. In this study, we first identified LjGI and analyzed polymorphisms of LjE1 and LjGI among wild populations covering the entire distribution range of this species in Japan. LjGI had a coding sequence (CDS) length of 3495 bp and included 14 exons. The homologies of DNA and amino acid sequences between LjGI and GmGI were 89 and 88% (positive rate was 92%), respectively. LjE1 harbored five nucleic acid changes in a 552 bp CDS, all of which were nonsynonymous; four of the changes were located in the core function area. LjE1 alleles exhibited partial north-south differentiation and non-neutrality. In contrast, the LjGI harbored one synonymous and one nonsynonymous change. Thus, our study suggests that LjE1 may be involved in the control of flowering times, whereas LjGI may be under strong purifying selection.

First Record of Comparative Plastid Genome Analysis and Phylogenetic Relationships among Corylopsis Siebold & Zucc. (Hamamelidaceae).

Corylopsis Siebold & Zucc. (Hamamelidaceae) is widely used as a horticultural plant and comprises approximately 25 species in East Asia. Molecular research is essential to distinguish Corylopsis species, which are morphologically similar. Molecular research has been conducted using a small number of genes but not in Corylopsis. Plastid genomes of Corylopsis species (Corylopsis gotoana, Corylopsis pauciflora, and Corylopsis sinensis) were sequenced using next-generation sequencing techniques. Repeats and nucleotide diversity that could be used as DNA markers were also investigated. A phylogenetic investigation was carried out using 79 protein-coding genes to infer the evolutionary relationships within the genus Corylopsis. By including new plastomes, the overall plastid genome structure of Corylopsis was similar. Simple sequence repeats of 73-106 SSRs were identified in the protein-coding genes of the plastid genomes, and 33-40 long repeat sequences were identified in the plastomes. The Pi value of the rpl33_rps18 region, an intergenic spacer, was the highest. Phylogenetic analysis demonstrated that Corylopsis is a monophyletic group and Loropetalum is closely related to Corylopsis. C. pauciflora, C. gotoana, and C. spicata formed a clade distributed in Japan, whereas C. sinensis, C. glandulifera, and C. velutina formed a clade that was distributed in China.