Mutational effects of chronic gamma radiation throughout the life cycle of Arabidopsis thaliana: Insight into radiosensitivity in the reproductive stage.

Organisms living on Earth have always been exposed to natural sources of ionizing radiation, but following recent nuclear disasters, these background levels have often increased regionally due to the addition of man-made sources of radiation. To assess the mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life cycle of Arabidopsis thaliana grown under controlled conditions. We obtained resequencing data from 36 second generation post-mutagenesis (M2) progeny derived from 12 first generation (M1) lines grown under gamma-irradiation conditions, ranging from 0.0 to 2.0 Gray per day (Gy/day), to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and radiation dose rate from 0.0 to 2.0 Gy/day was assessed by statistical modeling. The increase in de novo mutations in response to irradiation dose fit the negative binomial model, which accounted for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, and deletions were more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation were greater during the reproductive stage. These results will provide valuable insights into practical strategies for analyzing mutational effects in wild plants growing in environments with various mutagens.

Genetic variation of the relict maple Acer miyabei: uncovering its history of disjunct occurrence and the role of mountain refugia in shaping genetic diversity.

PREMISE: Relict species provide valuable insights into the origin and formation of extant vegetation. Here, we aimed to elucidate the genetic structure and diversity of a riparian relic, Acer miyabei, in Japan. Once widely distributed, it now occurs in three isolated regions. The most northern regional group is located at low elevation on Hokkaido Island, whereas the southernmost group in central Honshu Island is at high elevation in a mountainous landscape. This contrastive distribution enables us to examine the effects of climate oscillations on genetic diversity in relation to topographic variation. METHODS: We collected 604 individuals of A. miyabei from 43 sites. Their genetic structure and diversity were analyzed using 12 microsatellite markers and cpDNA sequences. RESULTS: According to structure analyses, ∆K was lowest at K = 2; the clustering essentially separated many of the individuals in the most northern regional group from the others. In contrast, the two southern groups were not clearly differentiated from each other, despite their geographic discontinuity. The proportion of private alleles was high in populations from the mountain terrain in the southern group although the number of extant populations is limited. CONCLUSIONS: Genetic clustering of A. miyabei is not perfectly congruent with the current patterns of geographic distribution. We infer that disjunction of the two southern groups occurred more recently than that between these groups and the northern group. The mountainous landscape in the most southern region likely provided multiple refugia and contributed to the retention of distinctive genetic variation.

Development of microsatellite markers for a giant water bug, Appasus japonicus, distributed in East Asia.

We developed microsatellite markers for Appasus japonicus (Heteroptera: Belostomatidae). This belostomatid bug is distributed in East Asia (Japanese Archipelago, Korean Peninsula and mainland China) and often listed as an endangered species in the Red List or the Red Data Book at the national and local level in Japan. Here, we describe twenty novel polymorphic microsatellite loci developed for A. japonicus, and marker suitability was evaluated using 56 individuals from four A. japonicus populations (Nagano, Hiroshima and Yamaguchi prefectures, Japan, and Chungcheongnam-do, Korea). The number of alleles per locus ranged from 1 to 12 (mean = 2.5), and the average observed and expected heterozygosity and fixation index per locus were 0.270, 0.323 and 0.153, respectively. In addition, a population structure analysis was conducted using the software STRUCTURE, and its result suggested that the 20 markers described here will be useful for investigating the genetic structure of A. japonicus populations, which should contribute to population genetics studies of this species.

Experimental and Field Data Support Range Expansion in an Allopolyploid Arabidopsis Owing to Parental Legacy of Heavy Metal Hyperaccumulation.

Empirical evidence is limited on whether allopolyploid species combine or merge parental adaptations to broaden habitats. The allopolyploid Arabidopsis kamchatica is a hybrid of the two diploid parents Arabidopsis halleri and Arabidopsis lyrata. A. halleri is a facultative heavy metal hyperaccumulator, and may be found in cadmium (Cd) and zinc (Zn) contaminated environments, as well as non-contaminated environments. A. lyrata is considered non-tolerant to these metals, but can be found in serpentine habitats. Therefore, the parents have adaptation to different environments. Here, we measured heavy metals in soils from native populations of A. kamchatica. We found that soil Zn concentration of nearly half of the sampled 40 sites was higher than the critical toxicity level. Many of the sites were near human construction, suggesting adaptation of A. kamchatica to artificially contaminated soils. Over half of the A. kamchatica populations had >1,000 µg g-1 Zn in leaf tissues. Using hydroponic treatments, most genotypes accumulated >3,000 µg g-1 Zn, with high variability among them, indicating substantial genetic variation in heavy metal accumulation. Genes involved in heavy metal hyperaccumulation showed an expression bias in the A. halleri-derived homeolog in widely distributed plant genotypes. We also found that two populations were found growing on serpentine soils. These data suggest that A. kamchatica can inhabit a range of both natural and artificial soil environments with high levels of ions that either of the parents specializes and that it can accumulate varying amount of heavy metals. Our field and experimental data provide a compelling example of combining genetic toolkits for soil adaptations to expand the habitat of an allopolyploid species.

Draft Genome Sequence of Novel Metschnikowia sp. Strain JCM 33374, a Nectar Yeast Isolated from a Bumblebee.

Here, we report the draft genome sequence of Metschnikowia sp. strain JCM 33374, a nectar yeast isolated from a bumblebee (Bombus diversus). The genome of 20.1 Mb is a naturally heterozygous diploid. Phylogenetic analysis with related taxa demonstrated that the strain is very likely a novel species.

Ecotypic divergences of the alpine herb Potentilla matsumurae adapted to fellfield-snowbed habitats across a series of mountain sky islands.

PREMISE: Divergent selection due to environmental heterogeneity can lead to local adaptation. However, the ecological and evolutionary processes of local adaptation that occurs across multiple regions are often unknown. Our previous studies reported on the ecotypic divergence within a local area of variation of Potentilla matsumurae, an alpine herb adapted to the fellfield-snowbed environment. Here we investigated large-scale geographic patterns of ecotypic differentiation in this species to infer local adaptation and selective forces across multiple regions. METHODS: We compiled information on the overall distributions of fellfield and snowbed habitats on the mountains in Japan across the distribution of the species. Next, we conducted common garden experiments to test the adaptive divergence of the fellfield-snowbed plants derived from multiple regions. Finally, we evaluated phylogeographic structures based on cpDNA and allozyme variations and inferred the evolutionary history of ecotype differentiation. RESULTS: The mosaic distribution of the fellfield-snowbed ecotypes across isolated mountaintops constitutes indirect evidence for habitat-specific natural selection. The significant difference in survivorship between the ecotypes observed in a controlled snow environment provides more substantial evidence of local selection. Phylogeographic structures support the hypothesis that ecotypic divergence events from fellfield to snowbed populations occurred independently in at least two distinct regions. CONCLUSIONS: Ecotypic divergence of P. matsumurae has occurred across a series of mountain sky islands. Local selection in snowy environments is a driving force that maintains the divergent ecotypes across multiple mountain regions and can contribute to the diversification of plants in heavy-snow regions.

Development and evaluation of microsatellite markers for Acer miyabei (Sapindaceae), a threatened maple species in East Asia.

PREMISE OF THE STUDY: Twelve microsatellite markers were developed and characterized in a threatened maple species, Acer miyabei (Sapindaceae), for use in population genetic analyses. METHODS AND RESULTS: Using Ion Personal Genome Machine (PGM) sequencing, we developed microsatellite markers with perfect di- and trinucleotide repeats. These markers were tested on a total of 44 individuals from two natural populations of A. miyabei subsp. miyabei f. miyabei in Hokkaido Island, Japan. The number of alleles per locus ranged from two to eight. The observed and expected heterozygosities per locus ranged from 0.05 to 0.75 and from 0.05 to 0.79, respectively. Some of the markers were successfully transferred to the closely related species A. campestre, A. platanoides, and A. pictum. CONCLUSIONS: The developed markers will be useful in characterizing the genetic structure and diversity of A. miyabei and will help to understand its spatial genetic variation, levels of inbreeding, and patterns of gene flow, thereby providing a basis for conservation.

Changes in pollinator fauna affect altitudinal variation of floral size in a bumblebee-pollinated herb.

Geographic trait variations are often caused by locally different selection regimes. As a steep environmental cline along altitude strongly influences adaptive traits, mountain ecosystems are ideal for exploring adaptive differentiation over short distances. We investigated altitudinal floral size variation of Campanula punctata var. hondoensis in 12 populations in three mountain regions of central Japan to test whether the altitudinal floral size variation was correlated with the size of the local bumblebee pollinator and to assess whether floral size was selected for by pollinator size. We found apparent geographic variations in pollinator assemblages along altitude, which consequently produced a geographic change in pollinator size. Similarly, we found altitudinal changes in floral size, which proved to be correlated with the local pollinator size, but not with altitude itself. Furthermore, pollen removal from flower styles onto bees (plant's male fitness) was strongly influenced by the size match between flower style length and pollinator mouthpart length. These results strongly suggest that C. punctata floral size is under pollinator-mediated selection and that a geographic mosaic of locally adapted C. punctata exists at fine spatial scale.

Development and evaluation of microsatellite markers for the gynodioecious shrub Daphne jezoensis (Thymelaeaceae).

PREMISE OF THE STUDY: Ten microsatellite markers were developed and characterized in a gynodioecious summer-deciduous shrub, Daphne jezoensis, to facilitate studies of the evolution of gynodioecy in the species. • METHODS AND RESULTS: We used a next-generation sequencing approach with the Ion Personal Genome Machine (PGM) system to identify and develop microsatellite markers with perfect di- and trinucleotide repeats. These markers were tested with 47 samples from two natural populations. The mean observed and expected heterozygosities per population ranged from 0.40 to 0.46 and 0.60 to 0.66, respectively. • CONCLUSIONS: The developed markers will be useful to study the mating system, gene flow, and population genetic structure of D. jezoensis.

Kinship between parents reduces offspring fitness in a natural population of Rhododendron brachycarpum.

BACKGROUND AND AIMS: A reduction in offspring fitness resulting from mating between neighbours is interpreted as biparental inbreeding depression. However, little is known about the relationship between the parents' genetic relatedness and biparental inbreeding depression in their progeny in natural populations. This study assesses the effect of kinship between parents on the fitness of their progeny and the extent of spatial genetic structure in a natural population of Rhododendron brachycarpum. METHODS: Kinship coefficients between 11,858 pairs of plants among a natural population of 154 R. brachycarpum plants were estimated a priori using six microsatellite markers. Plants were genotyped, and pairs were selected from among 60 plants to vary the kinship from full-sib to unrelated. After a hand-pollination experiment among the 60 plants, offspring fitness was measured at the stages of seed maturation (i.e. ripening) under natural conditions, and seed germination and seedling survival under greenhouse conditions. In addition, spatial autocorrelation was used to assess the population's genetic structure. KEY RESULTS: Offspring fitness decreased significantly with increasing kinship between parents. However, the magnitude and timing of this effect differed among the life-cycle stages. Measures of inbreeding depression were 0.891 at seed maturation, 0.122 (but not significant) at seed germination and 0.506 at seedling survival. The local population spatial structure was significant, and the physical distance between parents mediated the level of inbreeding between them. CONCLUSIONS: The level of inbreeding between individuals determines offspring fitness in R. brachycarpum, especially during seed maturation. Genetic relatedness between parents caused inbreeding depression in their progeny. Therefore, biparental inbreeding contributes little to reproduction and instead acts as a selection force that promotes outcrossing, as offspring of more distant (less related) parents survive better.

Morphological and genetic variations of Potentilla matsumurae (Rosaceae) between fellfield and snowbed populations.

Identifying ecological factors associated with local differentiation of populations is important for understanding microevolutionary processes. Alpine environments offer a unique opportunity to investigate the effects of habitat-specific selective forces and gene flow limitations among populations at a microscale on local adaptation because the heterogeneous snowmelt patterns in alpine ecosystems provide steep environmental changes. We investigated the variation in morphological traits and enzyme loci between fellfield and snowbed populations of Potentilla matsumurae, a common alpine herb with a wide distribution along snowmelt gradients in northern Japan. We found significant differences in morphological traits between fellfield and snowbed habitats in a northern distribution region. These differences were maintained when plants were grown under uniform conditions in a greenhouse. Allozyme variations among 15 populations from geographically separated regions with different historical backgrounds showed that the populations are more genetically differentiated between the fellfield and snowbed habitats within a region than between populations occupying the same habitat type in different regions. These results suggest that variation in snowmelt regimes could be a driving force creating local adaptation and genetic differentiation of alpine plant populations.

Adaptive significance of self-fertilization in a hermaphroditic perennial, Trillium camschatcense (Melanthiaceae).

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.