Manual crosses and pre and post-pollination isolation index

To determine the level of compatibility between species, experimental crosses were carried out in the sympatric area of Botton. Fresh flowers with intact pollinaria were randomly selected. Interspecific hand-pollinations were performed by removing pollinaria through touching the viscidia with a plastic toothpick and placing them on the stigmas of plants of the other species. Crossing combinations were performed bi-directionally (P. bifolia/P. chlorantha and P. chlorantha/P. bifolia) with each plant providing and receiving pollen, and included control-treatments (Table 1).

To prevent the potential negative effects of over-pollination on fruit set and seed viability, a maximum of three flowers per individual were hand-pollinated. This experiment is based on Xu et al. (2011). To prevent insect visits after experimental crossings, each inflorescence was covered with a pollination bag (to prevent pollination by insects) before and after the cross-pollination. Fruit initiation and development were monitored until fruits were mature (about one month after pollination). All crossed capsules were collected from the two investigated sympatric species and stored in silica gel. In addition, eight allopatric and 12 sympatric individuals of P. bifolia and intermediate morphotypes (Table 1) were also covered with a pollination bag before anthesis to determine the degree of autonomous self-pollination.

Seeds produced by interspecific (hand pollinations), intraspecific crosses and also in the autonomous self-pollination treatment were harvested and brought to the laboratory. Seeds were observed under a microscope (100× magnification) to distinguish seeds containing one large viable embryo from non-viable seeds (i.e., small or aborted embryos or no embryo). Samples of 300 seeds per fruit were scored in order to estimate the percentage of viable seeds for each fruit (Xu et al., 2011). The significance of different seed viability among interspecies and intraspecies crosses was assessed using Student’s t-test, after normality testing of data distribution by the Shapiro test (Royston, 1982).

We also examined and quantified the effect of post-pollination barriers using indices of reproductive isolation (RI) (Kay, 2006). Based on the methods proposed in Scopece et al. (2007) and Marques et al. (2014), we estimated two measures of post-pollination reproductive isolation. We firstly estimated the post-pollination pre-zygotic isolation index as the proportion of fruits formed after interspecific crosses in relation to the proportion of fruits formed after intraspecific crosses:

Then, we calculated post-zygotic isolation index as the percentage of viable seeds from interspecific crosses in relation to the proportion of viable seeds obtained from intraspecific crosses, describing the embryo mortality:

In addition, since flowering time is known to contribute to the maintenance of phenotypic polymorphism, we estimated the strength of RI value, which corresponds to flowering phenology. The overall flowering period was recorded for both Platanthera species only at Botton site. Plants were checked every three days during one flowering season (2015). For the investigation of flowering phenology we examined: the beginning of blooming (first flower opened), the end of the flowering period (when the last flower opened). The RI phenology index was calculated as: RI_phenology = 1 − (overlapping flowering period between species (number of days)/flowering period (number of days)) (Ma et al., 2016).