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.
Number of flowers per plant
We set 14, 4, and 5 quadrats of the size 0.5 m × 0.5 m at the ridges, slopes, and valleys, respectively. During 2009 and 2010, we counted the number of flowering plants, flowers per plant, and total number of flowers within each quadrat, discriminating between morphs (pin and thrum).
Number of plants
We set 14, 4, and 5 quadrats of the size 0.5 m × 0.5 m at the ridges, slopes, and valleys, respectively. During 2009 and 2010, we counted the number of plants within each quadrat.
Pollinator visitation
We observed pollinator visits to the flowers of P. modesta during peak flowering at each site: early June, early July, and late July at the ridge, slope, and valley sites, respectively. Field observations were conducted during calm and fine periods during daylight hours (9:00 am–3:00 pm). The total observation times were 25 hours per 5 days, 12 hours per 3 days, and 14 hours per 4 days, at the ridge, slope, and valley sites, respectively. On each observation day, we arbitrary set 2 m × 2 m plot and recorded the pollinators and the number of flowers of P. modesta. Pollinators were classified into Diptera, Hymenoptera, Lepidoptera, and others during field observations, and major pollinators were collected and identified at the family or species level if possible.
Seed-set by experimental crosses
To estimate the potential ability to produce seeds at each site (ridge, slope, and valley), we conducted artificial pollination experiments within and nearby the quadrats over 2 years (2010 and 2011). The total numbers of pin and thrum plants pollinated were 78 and 77 during 2010 and 59 and 81 during 2011, respectively. The plants were covered with fine-meshed nylon nets to exclude insect visitors. For each plant, one target flower bud was emasculated just before opening, and other flowers were removed. After opening of the target flowers, hand pollination was conducted by legitimate pollen grains collected from a single pollen donor growing at least 5 m away from the recipient plant. All fruits derived from the pollination treatments were harvested just before dehiscence, and the number of seeds and undeveloped seeds or ovules were counted using a microscope.
Seed-set under natural conditions
To estimate the number of ovules and the seeds per fruit under natural conditions at each site (ridge, slope, and valley), we examined the plants growing within and nearby the quadrats over 2 years (2010 and 2011). The total number of pin and thrum plants examined was 78 and 79 during 2010 and 170 and 157 during 2011, respectively. We collected several fruits from each plant (2.8 on average) during 2010, and one fruit per plant during 2011. The total number of fruits collected from the pin and thrum plants was 233 and 204 during 2010 and 170 and 157 during 2011, respectively. The fruits were collected just before dehiscence, and the number of seeds and undeveloped seeds or ovules were counted using a microscope.
