Shin-Ichi Morinaga

A Genome Scan for Genes Underlying Microgeographic-Scale Local Adaptation in a Wild Arabidopsis Species.

Adaptive divergence at the microgeographic scale has been generally disregarded because high gene flow is expected to disrupt local adaptation. Yet, growing number of studies reporting adaptive divergence at a small spatial scale highlight the importance of this process in evolutionary biology. To investigate the genetic basis of microgeographic local adaptation, we conducted a genome-wide scan among sets of continuously distributed populations of Arabidopsis halleri subsp. gemmifera that show altitudinal phenotypic divergence despite gene flow. Genomic comparisons were independently conducted in two distinct mountains where similar highland ecotypes are observed, presumably as a result of convergent evolution. Here, we established a de novo reference genome and employed an individual-based resequencing for a total of 56 individuals. Among 527,225 reliable SNP loci, we focused on those showing a unidirectional allele frequency shift across altitudes. Statistical tests on the screened genes showed that our microgeographic population genomic approach successfully retrieve genes with functional annotations that are in line with the known phenotypic and environmental differences between altitudes. Furthermore, comparison between the two distinct mountains enabled us to screen out those genes that are neutral or adaptive only in either mountain, and identify the genes involved in the convergent evolution. Our study demonstrates that the genomic comparison among a set of genetically connected populations, instead of the commonly-performed comparison between two isolated populations, can also offer an effective screening for the genetic basis of local adaptation.

Day–night fluctuations in floral scent and their effects on reproductive success in Lilium auratum

We examined the contribution of diurnal and nocturnal pollination to male and female reproductive success in Lilium auratum. Plants were bagged for either 12 h during the day or at night to allow either only nocturnal or only diurnal visitors to forage throughout the flowering period. We found that there was no significant difference in the seed:ovule ratio among diurnally pollinated, nocturnally pollinated, or control flowers. However, in terms of male reproductive success, it was more advantageous for the plants to be pollinated both diurnally and nocturnally: the numbers of pollen grains remaining in diurnally pollinated or nocturnally pollinated flowers were significantly greater than those in control flowers. The total amount of floral volatiles of L. auratum was significantly higher at night than during the day. The constituents of floral scent of all time series examined were mostly monoterpenoids, many of which serve as attractants for nocturnal hawkmoths. Such nocturnally biased floral scent emission of L. auratum might achieve male reproductive success by attracting nocturnal visitors, which may suggest that the relative contribution of floral scent in this species is biased towards male reproductive success.

Consequences of differences in flowering date on seed production in Heloniopsis orientalis (Liliaceae).

We examined the consequences of differences in flowering date on seed production in the self-compatible herb Heloniopsis orientalis. The number of selfed seeds per fruit, as determined by microsatellite markers, did not depend on when the plant flowered, whereas the number of outcrossed seeds per fruit increased with later flowering dates. Consequently, the selfing rate decreased with later flowering dates. The number of seeds (including both selfed and outcrossed ones) per fruit and the seedu2009:u2009ovule ratio increased with later flowering dates. We also examined the effects of pollinators and plant size on seed production. The visitation rate of Diptera did not depend on the flowering season, whereas that of Hymenoptera markedly increased as the flowering season progressed. Diptera stayed longer than Hymenoptera on each plant and flower. Seed production per fruit did not depend on plant size. Thus, the change in selfing rate associated with later flowering dates resulted from the seasonal change in pollinators rather than plant size.

Intra- and inter-plant level correlations among floral traits in Iris gracilipes (Iridaceae)

Although the sizes of individual flowers within one plant vary, few studies to date have investigated correlations among floral traits at the intra-plant level. Variations of the pleiotropic genes or linkage disequilibrium of genes, those have been said to create larger correlations between some combinations of traits than others at the inter-plant level, cannot predict intra-plant level correlations. In this study, correlations between several combinations of floral traits, including the number and volume of pollen grains and ovules, were investigated at both intra- and inter-plant levels in Iris gracilipes. The pattern of intra-plant level correlations was similar to that of inter-plant level correlations with some exceptions; correlations between functionally related traits tended to be larger than others at both levels. For example, correlations between sepal and petal size, and between petaloid style and filament length were large at both levels. This may be explained by the genetic and the developmental relationships between some combinations of traits, rather than by (co)variations of the peculiar properties of the individual plants such as genetic variations.

From the laboratory to the field: assaying histone methylation at FLOWERING LOCUS C in naturally growing Arabidopsis halleri

Gene regulatory mechanisms are often defined in studies performed in the laboratory but are seldom validated for natural habitat conditions, i.e., in natura. Vernalization, the promotion of flowering by winter cold, is a prominent naturally occurring phenomenon, so far best characterized using artificial warm and cold treatments. The floral inhibitor FLOWERING LOCUS C (FLC) gene of Arabidopsis thaliana has been identified as the central regulator of vernalization. FLC shows an idiosyncratic pattern of histone modification at different stages of cold exposure, believed to regulate transcriptional responses of FLC. Chromatin modifications, including H3K4me3 and H3K27me3, are routinely quantified using chromatin immunoprecipitation (ChIP), standardized for laboratory samples. In this report, we modified a ChIP protocol to make it suitable for analysis of field samples. We first validated candidate normalization control genes at two stages of cold exposure in the laboratory and two seasons in the field, also taking into account nucleosome density. We further describe experimental conditions for performing sampling and sample preservation in the field and demonstrate that these conditions give robust results, comparable with those from laboratory samples. The ChIP protocol incorporating these modifications, Field ChIP, was used to initiate in natura chromatin analysis of AhgFLC, an FLC orthologue in A. halleri, of which a natural population is already under investigation. Here, we report results on levels of H3K4me3 and H3K27me3 at three representative regions of AhgFLC in controlled cold and field samples, before and during cold exposure. We directly compared the results in the field with those from laboratory samples. These data revealed largely similar trends in histone modification dynamics between laboratory and field samples at AhgFLC, but also identified some possible differences. The Field ChIP method described here will facilitate comprehensive chromatin analysis of AhgFLC in the future to contribute to our understanding of gene regulation in fluctuating natural environments.

Fine-scale flower-visiting behavior revealed by using a high-speed camera

Most estimations of the pollination efficiency of insects have been based on observation by the naked human eye. However, insect behaviors are often too rapid to analyze sufficiently this way. Here we demonstrate the use of high-speed cameras to analyze the fine-scale behaviors of Macroglossum pyrrhosticta, Xylocopa appendiculata, and Papilio dehaanii when visiting Clerodendrum trichotomum. The fine-scale nectar drinking time, number of contacts with anthers and/or stigmas, and frequencies of body part contact with anthers and/or stigmas differed significantly among pollinator species. Pollination efficiency was not equal among pollinators. In addition, M. pyrrhosticta made the least number of contacts with anthers and/or stigmas even though it showed the highest visitation frequency. These results demonstrate that when examined from the viewpoint of rapid visitation behaviors, pollination dynamics differ among pollinator species, and flower visits and pollination rates are not equal.

Poor correlation between the removal or deposition of pollen grains and frequency of pollinator contact with sex organs

Pollinators deposit pollen grains on stigmas and remove pollen grains from anthers. The mechanics of these transfers can now be quantified with the use of high-speed video. We videoed hawkmoths, carpenter bees, and swallowtail butterflies pollinating Clerodendrum trichotomum. The number of grains deposited on stigmas did not vary significantly with the number of times pollinators contacted stigmas. In contrast, pollen removal from the anthers increased significantly with the number of contacts to anthers. Pollen removal varied among the three types of pollinators. Also, the three types carried pollen on different parts of their bodies. In hawkmoths and carpenter bees, a large number of contacted body part with anthers differed significantly from the body part that attached a large number of pollen grains. Our results indicate that a large number of contacts by pollinators does not increase either the male or female reproductive success of plants compared to a small number of contacts during a visit.

Eco-Evolutionary Genomic Observation for Local and Global Environmental Changes

Local and global environmental changes produce shifts in species distribution as well as adaptive evolution in wild species. Recent advances in genomic analysis tools, such as the next-generation DNA sequencing technique, have allowed genetic observation of ecological and evolutionary responses to the environmental changes. In this review, we propose three approaches for ‘eco-evolutionary genomic observation’ to assess and predict the effect of environmental changes on biodiversity. First, paleo-ecogenomics, the genomic and metagenomic analyses of ancient pollen grains or sediments, can be employed to identify long-term evolutionary changes, thereby facilitating the reconstruction of ancient biological communities and interactions. Second, chronological genomics, which uses museum or herbarium specimen samples to study population genomics, can be used to elucidate short-term evolutionary changes and detect the adaptive genes favored by natural or human-induced selection. Third, ecological genome niche modeling, based on the distribution data of adaptive variants detected by genome-wide scans and the geographic patterns of various environmental factors, will be useful for prediction of evolutionary responses to future environmental changes. These new approaches provide a point of view for observation of evolution caused by environmental changes in chronological order.

Functional divergence of duplicate genes several million years after gene duplication in Arabidopsis

Abstract Lineage-specific duplicated genes likely contribute to the phenotypic divergence in closely related species. However, neither the frequency of duplication events nor the degree of selection pressures immediately after gene duplication is clear in the speciation process. Here, using Illumina DNA-sequencing reads from Arabidopsis halleri, which has multiple closely related species with high-quality genome assemblies (A. thaliana and A. lyrata), we succeeded in generating orthologous gene groups in Brassicaceae. The duplication frequency of retained genes in the Arabidopsis lineage was ∼10 times higher than the duplication frequency inferred by comparative genomics of Arabidopsis, poplar, rice and moss (Physcomitrella patens). The difference of duplication frequencies can be explained by a rapid decay of anciently duplicated genes. To examine the degree of selection pressure on genes duplicated in either the A. halleri-lyrata or the A. halleri lineage, we examined positive and purifying selection in the A. halleri-lyrata and A. halleri lineages throughout the ratios of nonsynonymous to synonymous substitution rates (KA/KS). Duplicate genes tended to have a higher proportion of positive selection compared with non-duplicated genes. Interestingly, we found that functional divergence of duplicated genes was accelerated several million years after gene duplication compared with immediately after gene duplication.

Enhanced functional divergence of duplicate genes several million years after gene duplication in the Arabidopsis lineage

Lineage-specifically duplicated genes likely contribute to the phenotypic divergence in closely related species. However, neither the frequency of duplication events nor the degree of selective pressures immediately after gene duplication is clear in the speciation process. Plants have substantially higher gene duplication rates than most other eukaryotes. Here, using Illumina short reads from Arabidopsis halleri, which has highly qualified plant genomes in close species (Brassica rapa, A. thaliana and A. lyrata), we succeeded in generating orthologous gene groups among B. rapa, A. thaliana, A. lyrata and A. halleri. The frequency of duplication events in the Arabidopsis lineage was approximately 10 times higher than the frequency inferred by comparative genomics of Arabidopsis, poplar, rice and moss. Of the currently retained genes in A. halleri, 11–24% had undergone gene duplication in the Arabidopsis lineage. To examine the degree of selective pressure for duplicated genes, we calculated the ratios of nonsynonymous to synonymous substitution rates (KA/KS) in the A. halleri-lyrata and A. halleri lineages. Using a maximum-likelihood framework, we examined positive (KA/KS > 1) and purifying selection (KA/KS < 1) at a significant level (P < 0.01). Duplicate genes tended to have a higher proportion of positive selection compared with non-duplicated genes. More interestingly, we found that functional divergence of duplicated genes was accelerated several million years after gene duplication at a higher proportion than immediately after gene duplication.