Yoshiaki Kameyama

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.

Intrinsic and extrinsic factors acting on the reproductive process in alpine‐snowbed plants: roles of phenology, biological interaction, and breeding system

Pollinator activity and competition for pollinators lead to quantitative and qualitative pollen limitations on seed production and affect the reproductive success of plant species, depending on their breeding system (e.g., self-compatibility and heterospecific compatibility) and genetic load (e.g., inbreeding depression and hybrid inviability). In alpine ecosystems, snowmelt regimes determine the distribution and phenology of plant communities. Plant species growing widely along a snowmelt gradient often grow with different species among local populations. Their pollinators also vary in their abundance, activity, and behavior during the season. These variations may modify plant–pollinator and plant–plant interactions. We integrated a series of our studies on the alpine dwarf shrub, Phyllodoce aleutica (Ericaceae), to elucidate the full set of intrinsic (species-specific breeding system) and extrinsic factors (snow condition, pollinator activity, and interspecific competition) acting on their reproductive process. Seasonality of pollinator activity led to quantitative pollen limitation in the early-blooming populations, whereas in the late-blooming populations, high pollinator activity ensured pollination service, but interspecific competition for pollinators led to qualitative and quantitative pollen limitation in less competitive species. However, negative effects of illegitimate pollen receipt on seed-set success might be reduced when cryptic incompatibility systems (i.e., outcross pollen grains took priority over self- and heterospecific pollen grains) could effectively prevent ovule and seed discounting. Our studies highlight the importance of species-specific responses of plant reproduction to changing pollinator availability along environmental gradients to understand the general features of pollination networks in alpine ecosystems.

Natural genetic differentiation and human-mediated gene flow: the spatiotemporal tendency observed in a long-lived Cinnamomum camphora (Lauraceae) tree

The camphor tree (Cinnamomum camphora) is an important natural resource in East Asia. From the early eighteenth to mid-twentieth century, C. camphora was widely cultivated to obtain camphor, a white crystalline substance used as a repellent, a component of medicine, and an ingredient in the production of smokeless gunpowder and celluloid. The vast utilization and cultivation of C. camphora have obscured its natural distribution, and the genetic structure of this species was likely affected by both natural factors and human activities in different areas and at different times. To estimate this process, we collected 817 samples from Japan, China, and Taiwan, including samples from trees estimated to be hundreds to thousands of years old. Population genetic analyses using 11 microsatellite markers detected the footprints of ancient genetic differentiation and recent human-mediated gene flow. The strong genetic differentiation between areas (Japan vs. China and Taiwan) and the decreased genetic diversity in Japan can be ascribed to the long-term geographical isolation during and after the glacial period. Within each area, the genetic composition of the cultivated or planted populations was often inconsistent with the surrounding natural and/or older specimens. This finding may be due to the regional transfer of plant materials with no regard to their genetic origin. We also detected rare but evident immigration from different areas; furthermore, these non-native genets seemed to locally hybridize with the native genets. We suggest that the intentional transfer and/or artificial hybridization between areas should be prohibited in order to preserve the genetic composition of C. camphora.

Male-biased hermaphrodites in a gynodioecious shrub, Daphne jezoensis

Gynodioecy, a state where female and hermaphrodite plants coexist in populations, has been widely proposed an intermediate stage in the evolutionary pathway from hermaphroditism to dioecy. In the gynodioecy-dioecy pathway, hermaphrodites may gain most of their fitness through male function once females invade populations. To test this prediction, comprehensive studies on sex ratio variation across populations and reproductive characteristics of hermaphrodite and female phenotypes are necessary. This study examined the variation in sex ratio, sex expression, flower and fruit production and sexual dimorphism of morphological traits in a gynodioecious shrub, Daphne jezoensis, over multiple populations and years. Population sex ratio (hermaphrodite:female) was close to 1:1 or slightly hermaphrodite-biased. Sex type of individual plants was largely fixed, but 15% of plants changed their sex during a 6-year census. Hermaphrodite plants produced larger flowers and invested 2.5 times more resources in flower production than female plants, but they exhibited remarkably low fruit set (proportion of flowers setting fruits). Female plants produced six times more fruits than hermaphrodite plants. Low fruiting ability of hermaphrodite plants was retained even when hand-pollination was performed. Fruit production of female plants was restricted by pollen limitation under natural conditions, irrespective of high potential fecundity, and this minimised the difference in resources allocated to reproduction between the sexes. Negative effects of previous flower and fruit production on current reproduction were not apparent in both sexes. This study suggests that gynodioecy in this species is functionally close to a dioecious mating system: smaller flower production with larger fruiting ability in female plants, and larger flower production with little fruiting ability in hermaphrodite plants.

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.

Restricted female function of hermaphrodites in a gynodioecious shrub, Daphne jezoensis (Thymelaeaceae)

Gynodioecy is the coexistence of hermaphrodites and females in a population. It is supposed to be an intermediate stage in the evolutionary pathway from hermaphroditism to dioecy in angiosperm. Hermaphrodites gain fitness through both seed and pollen production whereas females gain fitness only through seed production. As females spread in a gynodioecious population, sexual selection prompts hermaphrodites to invest in male function and male-biased hermaphrodites prevail. In the gynodioecious shrub Daphne jezoensis (Thymelaeaceae), female frequency is stably around 50% in most populations, and fruit-set rate of hermaphrodites is commonly low. Therefore, D. jezoensis is likely at a later stage in the evolutionary pathway. Female function of hermaphrodites (fruit-set rate, selfing rate, seed size, and germination rate) was assessed in three populations under natural conditions. In order to evaluate the potential seed fertility and inbreeding depression by selfing in hermaphrodites, hand pollination treatments were also performed. Over a 2-year period under natural conditions, 18–29% of hermaphrodites and 69–81% of females set fruit. Across all three populations, the mean fruit-set rate ranged 9.5–49.2% in females and only 3.9–10.2% in hermaphrodites. Even with artificial outcross-pollination, 59–91% of hermaphrodites failed to set any fruit. When self-pollination was performed in hermaphrodites, both of fruit-set and germination rates were decreased, indicating early-acting inbreeding depression. In addition, more than half of the hermaphrodite seeds were produced by selfing under natural pollination, but pollinator service was still required. Totally, hermaphrodites performed poorly as seed producers because of the intrinsically-low fruiting ability and a combination of autogamous selfing and strong inbreeding depression, indicating the absence of reproductive assurance. These results indicate that the mating system of D. jezoensis is functionally close to dioecy.

Seasonal changes in pollen limitation and femaleness along the snowmelt gradient in a distylous alpine herb, Primula modesta

Abstract Flowering phenology of alpine plants is strongly determined by the timing of snowmelt, and the conditions of pollination of widely distributed plants vary greatly during their flowering season. We examined the reproductive success of the distylous alpine herb, Primula modesta, along the snowmelt gradient under natural conditions, and compared it with the result of artificial pollination experiments. In addition, the compositions and visit frequencies of pollinators to the flower of P. modesta were examined during the flowering period. The pin and thrum plants of P. modesta growing at the same site have an equal ability to produce seeds if a sufficient amount of legitimate pollen grains are deposited on the stigma surface. However, under natural conditions, their seed‐set success was often (even if not always) restricted by pollen limitation, and the functional gender of the pin and thrum plants biased to the female and male, respectively, associated with their growing sites. These variations were not ascribed to resource limitation nor biased morph ratio but to the seasonal changes in pollination situations, a replacement of pollinator types from long‐ to short‐tongued pollinators resulted in unidirectional pollen transfer from long stamens (thrum plants) to long styles (pin plants). The functional gender specialization may enhance the evolution of dioecy from heterostyly, but the severe pollen limitation may cause the breakdown of heterostyly into homostyly. To consider the evolutionary pathway of heterostylous plants, an accumulation of the empirical data is required demonstrating how phenological synchrony between plants and pollinators is decided and to what degree this relationship is stable over years, along with estimates of selection and gene flow in individual plants.

Environmental conditions for seed germination and seedling growth of Cinnamomum camphora (Lauraceae): the possibility of regeneration in an abandoned deciduous broad-leaved forest, eastern Japan

ABSTRACT Cinnamomum camphora is an evergreen broad-leaved tree distributed throughout southeastern China, Taiwan, and southern Japan. This tree has been widely cultivated and utilized by humans since ancient times; therefore, its natural distribution and regeneration are ambiguous. We established a mowing zone and a control zone in an abandoned deciduous broad-leaved forest in eastern Japan. In the mowing zone, an evergreen dwarf bamboo, Pleioblastus chino, and evergreen broad-leaved trees such as Quercus myrsinifolia and Eurya japonica were cut at ground level. We set nine study sites (1 × 1 m quadrats) on the forest floor of these zones, and measured the fraction of absorbed photosynthetically active radiation (fAPAR), soil moisture content, and soil temperature. Overall, 120 seeds of C. camphora were sown at a depth of 1 cm on each site, and seed germination and the survival and growth of seedlings were monitored over 4 years. Seed germination ratios at the sites were negatively affected by fAPAR and soil moisture content and positively affected by daily fluctuation of soil temperature. Seedling survival ratios and heights were negatively affected by fAPAR and positively affected by soil moisture content; no seedlings survived at the control site, and the height of seedlings was significantly higher under the canopy gap. Seeds of C. camphora could germinate in an abandoned secondary forest in eastern Japan, but the dominance of P. chino largely prevented the establishment of seedlings; the presence of a canopy gap, along with consecutive mowing, was considered to be indispensable for regeneration.