2.4 Pollination efficiency
Pollination efficiency was estimated by counting pollen tubes on pistil.
This approach is commonly employed for this purpose since it provides a
good proxy of conspecific pollen deposition (Biella et al., 2019 b,
Akter, Biella, Batáry, & Klečka, 2020) and seed production (Alonso,
Herrera, & Ashman, 2012). Twenty pistils of the more abundant flowering
species were collected at each sampling site, stored in a solution of
EtOH and CH2O (66.5/33.5/2
EtOH/H2O/CH2O v/v/v) and preserved at
room temperature until further analysis. Once in laboratory, pistils
were softened in 4 M NaOH and stained with 0.1% aniline blue in 0.1 M
K2HPO4 for 12 hours. After being washed with distilled water, pistils
were mounted on slides with glycerine and observed with a fluorescence
microscope. When pollen tubes were not visible, we counted the number of
pollen grains on stigmas considering that only pollen with tubes still
attached to the stigma should remain after sample preparation (Biella et
al., 2019 b) and that this number is expected to be correlated to the
amount of germinated tubes (Stavert, Bailey, Kirkland, & Rader 2020).
Pollen tube counts were performed for those species uniformly
distributed among sampling sites, specifically Tridax procumbens,
Wollastonia biflora , and Scaevola taccada .