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Adaptive divergence would occur even between the insufficiently isolated populations when there is a great difference in the environments of their habitats. The individuals present in the intermediate zone of the two divergent populations are expected to have an admixed genetic structure due to gene flow. A selective pressure that acts on the genetically admixed individuals may limit the gene flow and promote the adaptive divergence. Here, we addressed a question whether the selection occurs in the genetically admixed individuals between the divergent populations and assessed its effects on the population divergence. We obtained the whole-genome sequences of a perennial montane plant, Arabidopsis halleri, which has clear phenotypic dimorphisms between altitudes, along an altitudinal gradient of 359–1,317 m with a high spatial resolution (mean altitudinal interval of 20 m). We found the zone where the highland and lowland genes were mixing. Using the FST approach, we found that 35 and 13 genes in the admixed zone had a high frequency of alleles that are accumulated in highland and lowland subpopulations, respectively, suggesting that these genes have been selected in the admixed zone. This selection might limit the gene flow and contribute to the adaptive divergence along the altitudes. In the single-nucleotide polymorphism (SNP)-based analysis, 3,000 out of 27,792 Altitude-Dependent SNPs had extremely high homozygosity in the admixed zone. In 84.7% of these SNPs, the frequency of homozygotes of highland alleles was comparable to that of lowland alleles, suggesting that these alleles are neutral but the heterozygotes were selectively eliminated. The distribution of highland and lowland alleles of these SNPs was not clearly separated between altitudes, implying that such selection did not impede the gene flow. We conclude that the selection occurring at the intermediate altitude affects the genetic structure in the admixed zone and adaptive divergence along the altitudes.

Habenaria radiata (Orchidaceae) has two whorls of perianth, comprising three greenish sepals, two white petals and one lip (labellum). By contrast, the pseudopeloric (with a decreased degree of zygomorphy) mutant cultivar of H. radiata, ‘Hishou’, has changes in the identities of the dorsal sepal to a petaloid organ and the two ventral sepals to lip‐like organs. Here, we isolated four DEFICIENS‐like and two AGL6‐like genes from H. radiata, and characterized their expression. Most of these genes revealed similar expression patterns in the wild type and in the ‘Hishou’ cultivar, except HrDEF‐C3. The HrDEF‐C3 gene was expressed in petals and lip in the wild type but was ectopically expressed in sepal, petals, lip, leaf, root and bulb in ‘Hishou’. Sequence analysis of the HrDEF‐C3 loci revealed that the ‘Hishou’ genome harbored two types of HrDEF‐C3 genes: one identical to wild‐type HrDEF‐C3 and the other carrying a retrotransposon insertion in its promoter. Genetic linkage analysis of the progeny derived from an intraspecific cross between ‘Hishou’ and the wild type demonstrated that the mutant pseudopeloric trait was dominantly inherited and was linked to the HrDEF‐C3 gene carrying the retrotransposon. These results indicate that the pseudopeloric phenotype is caused by retrotransposon insertion in the HrDEF‐C3 promoter, resulting in the ectopic expression of HrDEF‐C3. As the expression of HrAGL6‐C2 was limited to lateral sepals and lip, the overlapping expression of HrDEF‐C3 and HrAGL6‐C2 is likely to be responsible for the sepal to lip‐like identity in the lateral sepals of the ‘Hishou’ cultivar.

Trillium apetalon (4x) and T. tschonoskii (4x) hybridize commonly where both species grow sympatrically, leading to the formation of tetraploid T. miyabeanum in Hokkaido, Japan. The present study aimed to determine which isolation factor is responsible for the frequency and asymmetry of hybrid T. miyabeanum formation in a sympatric population of T. apetalon and T. tschonoskii. We examined the contributions and strengths of four reproductive isolation barriers of T. miyabeanum formation: flowering phenology, breeding system, genetic isolation, and hybrid inviability. In addition, we also investigated the effect of flowering phenology on reproductive success (i.e., seed production and outcrossing rates) and outputs (i.e., ovule production) for T. apetalon and T. tschonoskii. We calculated the absolute contribution of each isolation barrier to the total reproductive isolation and found that flowering phenology and differences in breeding systems between the two parental species were more effective when T. apetalon was the maternal parent. Furthermore, hybrids with T. apetalon as the maternal parent had lower viability than those of the reciprocal cross and did not reach the flowering stage. Particularly, absolute contribution of premating isolation, especially by flowering phenology and breeding system, was higher than that of other isolation factors for both crossing directions. For the formation of T. miyabeanum, we concluded that asymmetry of hybridization between T. apetalon and T. tschonoskii would be caused by strong premating isolations. The asymmetry of the isolating barriers may promote T. tschonoskii as the maternal parent of T. miyabeanum.

Complex flowers containing large numbers of petals are especially prized as ornamental plants. Double-flowered Tricyrtis macranthopsis , in which stamens and carpels are converted to petaloid organs, is related to AGAMOUS ( AG ) gene expression. So we isolated AGAMOUS gene from T. macranthopsis , named as TrimAG . Since TrimAG expression was limited to whorls 3 and 4 and ectopic expression of TrimAG in Arabidopsis thaliana caused the same phenotype as overexpression of other AG orthologs in transgenic Arabidopsis , this gene has similar function as its AG orthologs. In the double-flowered T. macranthopsis cultivar, the level of TrimAG expression was reduced in the two inner reproductive whorls (stamens and carpels) and caused reproductive organ replacement with petaloid tepals and floral indeterminacy. Southern blot analysis of genomic DNA indicated that there were no significant differences between the double-flowered cultivar and wild-type plants. Analysis of promoter and AG intron II sequences did not identify any critically mutated sequences in the doubled-flowered cultivar. These results suggest that reduced TrimAG expression in whorls 3 and 4 might be caused by mutation in one of the genes in regulatory network which controls AGAMOUS expression or transcriptional silencing by methylation of TrimAG promoter/intron sequences.

We examined the edge structure of graphite formed by a cathodic electrochemical reaction in acid solutions associated with water electrolysis. Pole figures of the X-ray diffraction show that the edge orientation is parallel to the zigzag edge direction on a macroscopic scale. The polarization dependence of the G-band and D-band of the Raman spectra is consistent with the zigzag edge formation on a microscopic scale. It was found that the etched carbon atoms are converted to CH4 in the evolving gas and molecular species in the solution.

Garden asparagus (Asparagus officinalis L.) is an economically important plant. This species is dioecious, and male plants are considered to be more desirable than females due to their higher yields. To reduce the time required for asparagus breeding, molecular marker techniques have been employed to identify sex-linked DNA markers. In the present study, we converted the male-specific random amplified polymorphic DNA marker T35R54-1600 into a sequence tagged site marker. We cloned a male-specific DNA fragment amplified with the T35R54 primer and determined the sequence of the fragment. The size of T35R54-1600 is 1,586 bp, and this fragment is not homologous to known sex-linked BAC sequences, indicating that this fragment is a new sex-linked region. Within this fragment, we designed the primer pair ‘MSSTS710’ to amplify a 710 bp region. This marker could be used to identify the sex of eight cultivars of A. officinalis: ‘Mary Washington 500W’, ‘UC157’, ‘Harumachi Green’, ‘Super Welcome’, ‘F4’, ‘Pacific 2000’, ‘F2’ and ‘Backlim’. We also analyzed the applicability of this marker to two dioecious Asparagus species, A. schoberioides and A. kiusianus, which are cross-compatible with A. officinalis. Although male-specific DNA fragments of two dioecious Asparagus species, A. schoberioides and A. kiusianus, were generated using the existing male-specific marker Asp1T7sp, no amplicon was obtained using the MSSTS710 marker. Since MSSTS710 can be employed for sex identification only in A. officinalis and not in closely related Asparagus species, the DNA region around the MSSTS710 marker must be variable among Asparagus species.

B class MADS-box genes play important roles in petal and stamen development. Some monocotyledonous species, including liliaceous ones, produce flowers with petaloid tepals in whorls 1 and 2. A modified ABCE model has been proposed to explain the molecular mechanism of development of two-layered petaloid tepals. However, direct evidence for this modified ABCE model has not been reported to date. To clarify the molecular mechanism determining the organ identity of two-layered petaloid tepals, we used chimeric repressor gene-silencing technology (CRES-T) to examine the suppression of B function in the liliaceous ornamental Tricyrtis sp. Transgenic plants with suppressed B class genes produced sepaloid tepals in whorls 1 and 2 instead of the petaloid tepals as expected. In addition, the stamens of transgenic plants converted into pistil-like organs with ovule- and stigma-like structures. This report is the first to describe the successful suppression of B function in monocotyledonous species with two-layered petaloid tepals, and the results strongly support the modified ABCE model.

B class MADS-box genes play important roles in petal and stamen development. Some monocotyledonous species, including liliaceous ones, produce flowers with petaloid tepals in whorls 1 and 2. A modified ABCE model has been proposed to explain the molecular mechanism of development of two-layered petaloid tepals. However, direct evidence for this modified ABCE model has not been reported to date. To clarify the molecular mechanism determining the organ identity of two-layered petaloid tepals, we used chimeric repressor gene-silencing technology (CRES-T) to examine the suppression of B function in the liliaceous ornamental Tricyrtis sp. Transgenic plants with suppressed B class genes produced sepaloid tepals in whorls 1 and 2 instead of the petaloid tepals as expected. In addition, the stamens of transgenic plants converted into pistil-like organs with ovule- and stigma-like structures. This report is the first to describe the successful suppression of B function in monocotyledonous species with two-layered petaloid tepals, and the results strongly support the modified ABCE model.

The sex type of Trillium camschatcense has been considered to be strictly monomorphic, composed of only hermaphroditic individuals. However, field observations have detected anther-degenerated (AD) plants coexisting with hermaphrodites. The present study aimed to elucidate whether AD plants could be regarded as females and, if so, how their loss of male function is compensated. Because T. camschatcense retains both self-compatible (SC) and self-incompatible (SI) populations, the frequencies of AD plants in multiple populations (22 SC and eight SI) were examined to predict the fate of these individuals under contrasting breeding systems. Morphological and genetic analyses in a SC population demonstrated that AD plants were completely male sterile, but female fertile. Although the quantity of seed produced was similar, hermaphrodites produced seeds predominantly via selfing, whereas seeds of AD plants were entirely outcrossed. Because inbreeding depression was severe, AD plants achieved a fitness advantage through inbreeding avoidance. However, the frequency of AD plants varied among SC populations (0–42%), suggesting that environmental variance can modify the relative fitness. Conversely, the frequency was suppressed among SI populations (0–2%). Because SI hermaphrodites are free from inbreeding, AD plants would not benefit from inbreeding avoidance and would find it difficult to invade a SI population.

The evolution of selfing from outcrossing ancestors is known to have occurred repeatedly in angiosperms. Theoretical studies have argued that the transition from outcrossing to selfing is accomplished more easily than the reverse case, and phylogenetic analyses involving self-compatible (SC) and self-incompatible (SI) species has basically supported this assumption. The evolutionary direction of self-compatibility and self-incompatibility was examined in Trillium camschatcense, which contains geographically widespread SC populations, and restricted SI populations. Ecological surveys have revealed that the SC populations were suitable for outcrossing, and selfing in these populations did not confer any fitness advantage. Since reproductive fitness indicates the possibility of an evolutionary shift from self-compatibility to self-incompatibility, the phylogenetic relationships of SI and SC populations of T. camschatcense were investigated based on cpDNA variations and nuclear DNA microsatellite polymorphisms. Although phylogenetic analyses did not provide credible evidence to determine evolutionary direction, the SI populations turned out to be monophyletic with extremely low genetic differentiation. Based on these results, we proposed two possible scenarios for the evolutionary backgrounds of SI and SC populations in T. camschatcense. The plausibility of each scenario was evaluated based on the reproductive and geographical features of the mating systems.

The evolution of self-fertilization from primarily outcrossing ancestors is one of the most common evolutionary transitions in plants; however, the ecological mechanisms that maintain self-fertilization have remained controversial. Theoretical studies suggest that selfing is advantageous over outcrossing in terms of genetic transmission and assurance of seed production under pollen-limited circumstances. Trillium camschatcense is a herbaceous perennial distributed in Hokkaido and northern Honshu, Japan. Geographical variation in the breeding system (self-compatible, SC; or self-incompatible, SI) has been reported in populations in Hokkaido. Here, we used several SC and SI populations of T. camschatcense to investigate the adaptive significance and the evolutionary basis of self-fertilization. Pollination experiments and genetic analyses demonstrated that the potential availability of outcross pollen in SC populations was sufficient and that the number of pollen donors was equal to that of SI populations. However, despite the high availability of outcross pollen, the SC populations produced seeds predominantly by selfing and so underwent severe inbreeding depression. Although none of the suggested advantages for self-fertilization were supported by our analyses, we propose two possible scenarios for the evolution of self-fertilization in T. camschatcense.

The evolution of greenish sepals from petaloid outer tepals has occurred repeatedly in various lineages of non-grass monocots. Studies in distinct monocot species showed that the evolution of sepals could be explained by the ABC model; for example, the defect of B-class function in the outermost whorl was linked to the evolution of sepals. Here, floral MADS-box genes from three sepal-bearing monocotyledonous Trilliaceae species, Trillium camschatcense, Paris verticillata, and Kinugasa japonica were examined. Unexpectedly, expression of not only A- but also B-class genes was detected in the sepals of all three species. Although the E-class gene is generally expressed across all floral whorls, no expression was detected in sepals in the three species examined here. Overexpression of the E-class SEPALLATA3-like gene from T. camschatcense (TcamSEP) in Arabidopsis thaliana produced phenotypes identical to those reported for orthologs in other monocots. Additionally, yeast hybrid experiments indicated that TcamSEP could form a higher-order complex with an endogenous heterodimer of B-class APETALA3/DEFICIENS-like (TcamDEF) and PISTILLATA/GLOBOSA-like (TcamGLO) proteins. These results suggest a conserved role for Trilliaceae SEPALLATA3-like genes in functionalization of the B-class genes, and that a lack of SEPALLATA3-like gene expression in the outermost whorl may be related to the formation of greenish sepals.

Trillium camschatcense is a herbaceous perennial plant distributed in Hokkaido and northern Honshu, Japan. Geographical variations in the breeding system (partial selfing or obligate outcrossing) are reported in the populations of Hokkaido. We isolated six polymorphic microsatellite loci from this species. The number of allele per locus ranged from four to 12, whereas the expected heterozygosities ranged from 0.69 to 0.83. These markers may allow further investigations to reveal the evolutionary and ecological processes of mating system in T. camschatcense .

Interspecific hybridization is a useful technique to introduce characteristics from wild species into crops. Garden asparagus (Asparagus officinalis, 2n = 2x = 20) is an economically important vegetable that is native to the Mediterranean region but widely cultivated in many countries. The genus Asparagus is comprised of over 100 species. Asparagus kiusianus (2n = 2x = 20) is a wild asparagus species endemic to Japan. This species occurs on the coast and is likely to be a salt-resistant species. Although the geographic distribution of these two species is not close, molecular phylogenetic analysis demonstrated that these two species are closely related. In this study, a reciprocal cross between A. officinalis and A. kiusianus was carried out by hand pollination, and progeny were obtained from both crossings. These progeny exhibited a morphologically intermediate phenotype in terms of flower shape, and restriction fragment length polymorphism analysis confirmed that these were indeed interspecific hybrids. The interspecific hybrids were fertile, and backcross progeny with garden asparagus was also generated. These interspecific hybrids are expected to supply novel traits to garden asparagus.

The genus Asparagus comprises approximately 200 species, some of which are commercially cultivated, such as the garden asparagus (A. officinalis). Many Asparagus species, including A. officinalis, are dioecious and have been grouped into a subgenus distinct from that of hermaphroditic species. Although many interspecific crossings have been attempted to introduce useful traits into A. officinalis, only some of the dioecious species were found to be cross-compatible with A. officinalis. Here, molecular phylogenetic analyses were conducted to determine whether interspecific crossability is proportional to the genetic distance between the crossing pairs and to further clarify the evolutionary history of the Asparagus genus. A clade with all cross-compatible species and no cross-incompatible species was recovered in the phylogenetic tree based on analyses of non-coding cpDNA regions. In addition, a sex-linked marker developed for A. officinalis amplified a male-specific region in all cross-compatible species. The phylogenetic analyses also provided some insights about the evolutionary history of Asparagus; for example, by indicating that the genus had its origin in southern Africa, subsequently spreading throughout the old world through intensive speciation and dispersal. The results also suggest that dioecious species were derived from a single evolutionary transition from hermaphroditism in Asparagus. These findings not only contribute towards the understanding of the evolutionary history of the genus but may also facilitate future interspecific hybridization programs involving Asparagus species.

We developed 11 polymorphic microsatellite [simple sequence repeat (SSR)] loci from genomic DNA of Veratrum album ssp. oxysepalum using a dual-suppression polymerase chain reaction technique and an improved method. These markers, with four to 17 alleles per locus, identified 47 genotypes in 48 samples collected from a population in Hokkaido, Japan. The observed and expected heterozygosities ranged from 0.042 to 0.938 and from 0.448 to 0.909, respectively. These SSR markers will be available to identify genets and evaluate genetic diversity of V. album ssp. oxysepalum.

We found that graphite surfaces can be etched by electrochemical reduction in sulfuric acid. Terraces with straight edges crossing with 60o-multiple angles were found on surfaces by AFM observation. It suggests that crystallographic edges can be formed at room temperature in a controlled manner. Layer-by-layer etching of graphene sheets was possible by tuning the etching condition.