Published papers

Speciation is a continuous process. Although it is known that differential adaptation can initiate divergence even in the face of gene flow, we know relatively little about the mechanisms driving complete reproductive isolation and the genomic patterns of divergence and introgression at the later stages of speciation. Sticklebacks contain many pairs of sympatric species differing in levels of reproductive isolation and divergence history. Nevertheless, most previous studies have focused on young species pairs. Here, we investigated two sympatric stickleback species, Pungitius pungitius and P. sinensis, whose habitats overlap in eastern Hokkaido; these species show hybrid male sterility, suggesting that they may be at a late stage of speciation. Our demographic analysis using whole-genome sequence data showed that these species split 1.73 Ma and came into secondary contact 37 200 years ago after a period of allopatry. This long period of allopatry might have promoted the evolution of intrinsic incompatibility. Although we detected on-going gene flow and signatures of introgression, overall genomic divergence was high, with considerable heterogeneity across the genome. The heterogeneity was significantly associated with variation in recombination rate. This sympatric pair provides new avenues to investigate the late stages of the stickleback speciation continuum. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Genetic and phenotypic analyses of independent secondary contact zones between certain pairs of divergent populations offer powerful opportunities to assess whether the consequences vary with different environmental backgrounds. Populations of the ice goby Leucopsarion petersii are distributed throughout the Japanese archipelago and comprise genetically and phenotypically divergent groups in the Japan Sea and the Pacific Ocean. In particular, populations in the Japan Sea have a larger body size and numbers of vertebrae than those in the Pacific Ocean. Herein, we performed integrated analyses of genotypes and phenotypes of two independent secondary contact zones and investigated their consequences. Population genetic analyses revealed asymmetric introgression of the mitochondrial genome of either lineage relative to little admixture of nuclear genomes in both secondary contact zones. On phenotype analyses, vertebral numbers were clearly explained by nuclear genomic ancestry in both secondary contact zones, whereas body size was not, suggesting that a little introgression of nuclear genes regulates body size. Actually, we observed biased introgression of a candidate gene, neuropeptide Y (NPY), which potentially controls body size in the ice goby. Moreover, the body size changes in the introgressed populations possibly affect the introgression patterns of mitochondrial genomes across these zones. Collectively, our results demonstrated that genomic and phenotypic consequences of secondary contact varied in marine variable environments.

Effectively using genomic information greatly accelerates conventional breeding and applying it to long-lived crops promotes the conversion to genomic breeding. Because tea plants are bred using conventional methods, we evaluated the potential of genomic predictions (GPs) and genome-wide association studies (GWASs) for the genetic breeding of tea quality-related metabolites using genome-wide single nucleotide polymorphisms (SNPs) detected from restriction site-associated DNA sequencing of 150 tea accessions. The present GP, based on genome-wide SNPs, and six models produced moderate prediction accuracy values (r) for the levels of most catechins, represented by (−)-epigallocatechin gallate (r = 0.32–0.41) and caffeine (r = 0.44–0.51), but low r values for free amino acids and chlorophylls. Integrated analysis of GWAS and GP detected potential candidate genes for each metabolite using 80–160 top-ranked SNPs that resulted in the maximum cumulative prediction value. Applying GPs and GWASs to tea accession traits will contribute to genomics-assisted tea breeding.

Grassland ecosystems worldwide have been extensively converted to other land uses and are globally imperiled. Because many grasslands have been maintained by human activities, understanding their origin and history is fundamentally important to better contemporary management. However, existing methods to reconstruct past vegetation can produce contrasting views on grassland history. Here, we inferred demographic histories of 40 populations of four grassland forb species throughout Japan using high-resolution genome sequences and model-flexible demographic simulation based on the site frequency spectrum. Although two species showed a slight decline in population size between 100 000 - 10 000 years ago, our results suggest that population sizes of studied species have been maintained within the range of 0.5 - 2.0 times the most recent estimates for at least 100 000 years across Japan. Our results suggest that greater than 90% declines in Japanese grasslands and subsequent losses of grassland species in the last 100 years are geologically and biologically important and will have substantial consequences for Japanese biota and culture. People have had critical roles in maintaining disturbance-dependent grassland ecosystems and biota in this warm and wet forested country. In these contexts, disturbances associated with forest harvesting and traditional extensive farming have the potential to maintain grassland ecosystems and can provide important opportunities to reconcile resource production and conservation of grassland biodiversity.

Aim: The Sino-Japanese Floristic Region has extremely high species diversity 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. In some plant groups, high floral trait diversity and uneven species diversity between insular systems and the continental area suggest other factors may have important roles too. The primary purpose of this study is to reveal how abiotic and biotic factors have shaped the species diversity anomaly of Heterotropa between the insular systems and the continental area. Location: The Sino-Japanese Floristic Region. Taxon: Section Heterotropa (genus Asarum; Aristolochiaceae). Methods: Using ddRAD-seq and chloroplast genome data, we built a time-calibrated phylogenetic tree including 79 species. We estimated the patterns of floral traits (flowering time and floral size) evolution using macroevolutionary modelling, and tested the correlation of speciation rate with the trait evolution rates. Finally, we estimated the isolation factors of all taxa pairs and sister-taxa pairs based on distribution range and floral traits. Results: Phylogenetic analysis indicated that Heterotropa was diverged into two clades (continental clade and insular clade) in the Miocene, and the major subclades corresponded to geographic entities. Most rate shifts accelerating floral trait's evolution occurred during the Pleistocene period. Evolution rate of floral traits showed positive correlation with the speciation rate. Large proportion of taxa in the insular clade are distributed allopatrically. Several sister pairs showed floral trait divergence with geographic overlap. Main Conclusions: The diversification of Heterotropa appears to have been triggered by geographic and climatic events, and subsequent repeated floral trait evolution with and without geographic isolation. Furthermore, the high species diversity in the insular systems would have been formed by the repeated range fragmentations and contractions. Our study demonstrates the importance of multidimensional studies to understand the diversification process of temperate plants in the Sino-Japanese Floristic Region.

Secondary metabolism is regulated by circadian rhythm based on the clock genes expression. Transcriptome analysis is an important tool to understand these mechanisms. However, sufficient genetic information for this analysis is lacking for many crops. In order to analyze the influence of independent clock genes from growth and environmental stimulation, we investigated the gene expression profiles in the roots and leaves of green perilla (Perilla frutescens), which is an important crop used as a herbal medicine. Green perilla was grown under light emitting diode and fluorescent light, after 7, 14, and 35 d. We also compared the coding DNA sequences with those in the other plants. From the transcriptomic data, green perilla was observed to have 74 contigs that did not depend on growth and cultivation condition. Some of these 74 genes might be related to the clock genes through their interactions with other genes. Some 74 genes had TATA box-like motif and a GA motif of the genes that are regulatory targets of timing of CAB expression 1 (TOC1). Thus, 74 genes that did not depend on cultivation condition and growth were considered to be under the control of TOC 1, suggesting the importance of TOC1 in circadian rhythms.

Acclimation to high temperature increases plants’ tolerance of subsequent lethal high temperatures. Although epigenetic regulation of plant gene expression is well studied, how plants maintain a memory of environmental changes over time remains unclear. Here, we show that JUMONJI (JMJ) proteins, demethylases involved in histone H3 lysine 27 trimethylation (H3K27me3), are necessary for Arabidopsis thaliana heat acclimation. Acclimation induces sustained H3K27me3 demethylation at HEAT SHOCK PROTEIN22 (HSP22) and HSP17.6C loci by JMJs, poising the HSP genes for subsequent activation. Upon sensing heat after a 3-day interval, JMJs directly reactivate these HSP genes. Finally, jmj mutants fail to maintain heat memory under fluctuating field temperature conditions. Our findings of an epigenetic memory mechanism involving histone demethylases may have implications for environmental adaptation of field plants.

Leaves within crop canopies experience variable light over the course of a day, which greatly affects photosynthesis and crop productivity. Little is known about the mechanisms of the photosynthetic response to fluctuating light and their genetic control. Here, we examined gas exchange, metabolite levels, and chlorophyll fluorescence during the photosynthetic induction response in an Oryza sativa indica cultivar with high yield (Takanari) and a japonica cultivar with lower yield (Koshihikari). Takanari had a faster induction response to sudden increases in light intensity than Koshihikari, as demonstrated by faster increases in net CO2 assimilation rate, stomatal conductance, and electron transport rate. In a simulated light regime that mimicked a typical summer day, the faster induction response in Takanari increased daily CO2 assimilation by 10%. The faster response of Takanari was explained in part by its maintenance of a larger pool of Calvin-Benson cycle metabolites. Together, the rapid responses of electron transport rate, metabolic flux, and stomatal conductance in Takanari contributed to the greater daily carbon gain under fluctuating light typical of natural environments.

Background: Soybean is one of the most important crop sources of tocopherols (Toc). However, the content of α-Toc, an isoform with the highest vitamin E activity in humans, is low in most cultivars. With the aim of broadening genetic variability, we performed quantitative trait locus (QTL) analysis for a high seed α-Toc trait detected in a wild soybean and characterized the sequence polymorphisms and expression profiles of γ-tocopherol methyltransferase (γ-TMT) genes as potential candidates. Results: A recombinant inbred line population was developed from a cross between the low α-Toc breeding line TK780 and the high α-Toc wild accession B04009. The α-Toc content in seeds correlated strongly with the ratio of α-Toc to γ-Toc contents. QTL analysis using a high-density map constructed with 7710 single nucleotide polymorphisms (SNPs) generated by restriction site-associated DNA sequencing detected six QTLs involved in α-Toc biosynthesis. Of these, three in chromosomes (Chr) 9, 11, and 12 produced consistent effects during a 2-year trial. B04009 allele at QTLs in Chr9 and Chr12 and TK780 allele at the QTL in Chr11 each promoted the conversion of γ-Toc to α-Toc, which elevated the seed α-Toc content. SNPs and indels were detected between the parents in three γ-TMT genes (γ-TMT1, γ-TMT2, and γ-TMT3) co-located in the QTLs in Chr9 and Chr12, of which some existed in the cis-regulatory elements associated with seed development and functions. In immature cotyledons, γ-TMT3 was expressed at higher levels in B04009 than TK780, irrespective of two thermal conditions tested, whereas the expression of γ-TMT2 was markedly upregulated under higher temperatures, particularly in B04009. Conclusions: We identified QTLs consistently controlling α-Toc biosynthesis in wild soybean seeds in 2-year trials. The QTL on Chr9 had been previously identified in soybean, whereas the QTLs on Chr11 and Chr12 were novel. Further molecular dissections and characterization of the QTLs may facilitate the use of high α-Toc alleles from wild soybean in soybean breeding and an understanding of the molecular mechanisms underlying α-Toc biosynthesis in soybean seeds.

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.

Hybridization between divergent lineages generates new allelic combinations. One mechanism that can hinder the formation of hybrid populations is mitonuclear incompatibility, that is, dysfunctional interactions between proteins encoded in the nuclear and mitochondrial genomes (mitogenomes) of diverged lineages. Theoretically, selective pressure due to mitonuclear incompatibility can affect genotypes in a hybrid population in which nuclear genomes and mitogenomes from divergent lineages admix. To directly and thoroughly observe this key process, we de novo sequenced the 747-Mb genome of the coastal goby, Chaenogobius annularis, and investigated its integrative genomic phylogeographics using RNA-sequencing, RAD-sequencing, genome resequencing, whole mitogenome sequencing, amplicon sequencing, and small RNA-sequencing. Chaenogobius annularis populations have been geographically separated into Pacific Ocean (PO) and Sea of Japan (SJ) lineages by past isolation events around the Japanese archipelago. Despite the divergence history and potential mitonuclear incompatibility between these lineages, the mitogenomes of the PO and SJ lineages have coexisted for generations in a hybrid population on the Sanriku Coast. Our analyses revealed accumulation of nonsynonymous substitutions in the PO-lineage mitogenomes, including two convergent substitutions, as well as signals of mitochondrial lineage-specific selection on mitochondria-related nuclear genes. Finally, our data implied that a microRNA gene was involved in resolving mitonuclear incompatibility. Our integrative genomic phylogeographic approach revealed that mitonuclear incompatibility can affect genome evolution in a natural hybrid population.

Under low iron (Fe) availability, plants transcriptionally induce various genes responsible for Fe uptake and translocation to obtain adequate amounts of Fe. Although transcription factors and ubiquitin ligases involved in these Fe deficiency responses have been identified, the mechanisms coordinating these pathways have not been clarified in rice. Recently identified Fe-deficiency-inducible IRON MAN (IMA)/FE UPTAKE-INDUCING PEPTIDE (FEP) positively regulates many Fe-deficiency-inducible genes for Fe uptake in Arabidopsis. Here, we report that the expression of two IMA/FEP genes in rice, OsIMA1 and OsIMA2, is strongly induced under Fe deficiency, positively regulated by the transcription factors IDEF1, OsbHLH058, and OsbHLH059, as well as OsIMA1 and OsIMA2 themselves, and negatively regulated by HRZ ubiquitin ligases. Overexpression of OsIMA1 or OsIMA2 in rice conferred tolerance to Fe deficiency and accumulation of Fe in leaves and seeds. These OsIMA-overexpressing rice exhibited enhanced expression of all of the known Fe-deficiency-inducible genes involved in Fe uptake and translocation, except for OsYSL2, a Fe-nicotianamine transporter gene, in roots but not in leaves. Knockdown of OsIMA1 or OsIMA2 caused minor effects, including repression of some Fe uptake-and translocation-related genes in OsIMA1 knockdown roots. These results indicate that OsIMA1 and OsIMA2 play key roles in enhancing the major pathway of the Fe deficiency response in rice.

RNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions but its use in large-scale experiments is limited by its high cost and labour requirements. In this study, we have established a high-throughput and cost-effective RNA-Seq library preparation method that does not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature (≥62 °C) to suppress RT of A-rich sequences in rRNA, and then pools all samples into a single tube. Both single-read and paired-end sequencing of libraries is enabled. We found that the pooled RT products contained large amounts of RNA, mainly rRNA, causing over-estimations of the quantity of DNA and unstable tagmentation results. Degradation of RNA before tagmentation was found to be necessary for the stable preparation of libraries. We named this protocol low-cost and easy RNA-Seq (Lasy-Seq) and used it to investigate temperature responses in Arabidopsis thaliana. We analysed how sub-ambient temperatures (10–30 °C) affected the plant transcriptomes using time-courses of RNA-Seq from plants grown in randomly fluctuating temperature conditions. Our results suggest that there are diverse mechanisms behind plant temperature responses at different time scales.

Leaf photosynthetic rate changes across the growing season as crop plants age. Most studies of leaf photosynthesis focus on a specific growth stage, leaving the question of which pattern of photosynthetic dynamics maximizes crop productivity unanswered. Here we obtained high-frequency data of canopy leaf CO2 assimilation rate (A) of two elite rice (Oryza sativa) cultivars and 76 inbred lines across the whole growing season. The integrated A value after heading was positively associated with crop growth rate (CGR) from heading to harvest, but that before heading was not. A curve-smoothing analysis of A after heading showed that accumulated A at > 80% of its maximum (A80) was positively correlated with CGR in analyses of all lines mixed and of lines grouped by genetic background, while the maximum A and accumulated A at ≤ 80% were less strongly correlated with CGR. We also found a genomic region (~ 12.2 Mb) that may enhance both A80 and aboveground biomass at harvest. We propose that maintaining a high A after heading, rather than having high maximum A, is a potential target for enhancing rice biomass accumulation.

Epicotyl length (ECL) of adzuki bean (Vigna angularis) affects the efficiency of mechanized weeding and harvest. The present study investigated the genetic factors controlling ECL. An F2 population derived from a cross between the breeding line ‘Tokei1121’ (T1121, long epicotyls) and the cultivar ‘Erimo167’ (common epicotyls) was phenotyped for ECL and genotyped using simple sequence repeats (SSRs) and single-nucleotide polymorphism (SNP) markers. A molecular linkage map was generated and fifty-two segregating markers, including 27 SSRs and 25 SNPs, were located on seven linkage groups (LGs) at a LOD threshold value of 3.0. Four quantitative trait loci (QTLs) for ECL, with LOD scores of 4.0, 3.4, 4.8 and 6.4, were identified on LGs 2, 4, 7 and 10, respectively; together, these four QTLs accounted for 49.3% of the phenotypic variance. The segregation patterns observed in F5 residual heterozygous lines at qECL10 revealed that a single recessive gene derived from T1121 contributed to the longer ECL phenotype. Using five insertion and deletion markers, this gene was fine mapped to a ~255 kb region near the end of LG10. These findings will facilitate marker-assisted selection for breeding in the adzuki bean and contribute to an understanding of the mechanisms associated with epicotyl elongation.

This research was conducted to identify quantitative trait loci (QTL) associated with egg-related traits by constructing a genetic linkage map based on single nucleotide polymorphism (SNP) markers using restriction-site associated DNA sequencing (RAD-seq) in Japanese quail. A total of 138 F2 females were produced by full-sib mating of F1 birds derived from an intercross between a male of the large-sized strain with three females of the normal-sized strain. Eggs were investigated at two different stages: the beginning stage of egg-laying and at 12 weeks of age (second stage). Five eggs were analyzed for egg weight, lengths of the long and short axes, egg shell strength and weight, yolk weight and diameter, albumen weight, egg equator thickness, and yolk color (L*, a*, and b* values) at each stage. Moreover, the age at first egg, the cumulative number of eggs laid, and egg production rate were recorded. RAD-seq developed 118 SNP markers and mapped them to 13 linkage groups using the Map Manager QTX b20 software. Markers were spanned on 776.1 cM with an average spacing of 7.4 cM. Nine QTL were identified on chromosomes 2, 4, 6, 10, 12, and Z using the simple interval mapping method in the R/qtl package. The QTL detected affected 10 egg traits of egg weight, lengths of the long and short axes of egg, egg shell strength, yolk diameter and weight, albumen weight, and egg shell weight at the beginning stage, yellowness of the yolk color at the second stage, and age at first egg. This is the first report to perform a quail QTL analysis of egg-related traits using RAD-seq. These results highlight the effectiveness of RAD-seq associated with targeted QTL and the application of marker-assisted selection in the poultry industry, particularly in the Japanese quail.

In the present study, quantitative trait loci (QTLs) analysis was performed to identify the chromosomal positions of growth andcarcass-relatedtrait QTLs using 319 F2 chickens obtainedfrom intercrosses of an Oh-Shamo male and four White Plymouth Rock females. Body weight was measured weekly until the birds were 7 weeks old. Carcassrelatedtraits were also measuredat this timepoint. A genetic linkage map was constructedusing 545 single nucleotide polymorphism (SNP) markers that were developedusing a restriction-site associatedDNA sequencing method. The linkage map includedthe 23 autosomes andthe Z chromosome. Using simple interval QTL mapping, we were able to identify 10 significant andsuggestive main-effect QTLs for growth andcarcass-relatedtraits present on chromosomes 1, 2, 3, 5, 8, 19, 24, andZ. These loci explained 5.60 -16.52% of the phenotypic variances. The chromosomal positions of the 10 QTLs overlappedwith those of previously reportedQTLs, whereas the targetedtraits varied. Our QTLs will aidfuture breeding programs in improving growth andmeat yieldof chickens (e.g., via marker-assisted selection), particularly in the Japanese brandchicken industry.

Evolution of mating systems has become one of the most important research areas in evolutionary biology. Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. littorale. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

Sulawesi, an island located in a biogeographical transition zone between Indomalaya and Australasia, is famous for its high levels of endemism. Ricefishes (family Adrianichthyidae) are an example of taxa that have uniquely diversified on this island. It was demonstrated that habitat fragmentation due to the Pliocene juxtaposition among tectonic subdivisions of this island was the primary factor that promoted their divergence; however, it is also equally probable that habitat fusions and resultant admixtures between phylogenetically distant species may have frequently occurred. Previous studies revealed that some individuals of Oryzias sarasinorum endemic to a tectonic lake in central Sulawesi have mitochondrial haplotypes that are similar to the haplotypes of O. eversi, which is a phylogenetically related but geologically distant (ca. 190 km apart) adrianichthyid endemic to a small fountain. In this study, we tested if this reflects ancient admixture of O. eversi and O. sarasinorum. Population genomic analyses of genome-wide single-nucleotide polymorphisms revealed that O. eversi and O. sarasinorum are substantially reproductively isolated from each other. Comparison of demographic models revealed that the models assuming ancient admixture from O. eversi to O. sarasinorum was more supported than the models assuming no admixture; this supported the idea that the O. eversi-like mitochondrial haplotype in O. sarasinorum was introgressed from O. eversi. This study is the first to demonstrate ancient admixture of lacustrine or pond organisms in Sulawesi beyond 100 km. The complex geological history of this island enabled such island-wide admixture of lacustrine organisms, which usually experience limited migration.

Background: The three-spined stickleback (Gasterosteus aculeatus) is a remarkable system to study the genetic mechanisms underlying parallel evolution during the transition from marine to freshwater habitats. Although the majority of previous studies on the parallel evolution of sticklebacks have mainly focused on postglacial freshwater populations in the Pacific Northwest of North America and northern Europe, we recently use Japanese stickleback populations for investigating shared and unique features of adaptation and speciation between geographically distant populations. However, we currently lack a comprehensive phylogeny of the Japanese three-spined sticklebacks, despite the fact that a good phylogeny is essential for any evolutionary and ecological studies. Here, we conducted a phylogenomic analysis of the three-spined stickleback in the Japanese Archipelago. Results: We found that freshwater colonization occurred in multiple waves, each of which may reflect different interglacial isolations. Some of the oldest freshwater populations from the central regions of the mainland of Japan (hariyo populations) were estimated to colonize freshwater approximately 170,000 years ago. The next wave of colonization likely occurred approximately 100,000 years ago. The inferred origins of several human-introduced populations showed that introduction occurred mainly from nearby habitats. We also found a new habitat of the three-spined stickleback sympatric with the Japan Sea stickleback (Gasterosteus nipponicus). Conclusions: These Japanese stickleback systems differ from those in the Pacific Northwest of North America and northern Europe in terms of divergence time and history. Stickleback populations in the Japanese Archipelago offer valuable opportunities to study diverse evolutionary processes in historical and contemporary timescales.