DOI: 10.5061/dryad.z8w9ghxgr
title: Seed and pollen deposition on Salvia przewalskii

description: Generalized pollinators visit multiple co-flowering plant species and may
 transfer heterospecific pollen grains. Recent studies have indicated that
 the effect of heterospecific pollen (HP) on reproduction success is
 variable and depends on the identity of donor and recipient species.
 However, few studies have documented variation in HP receipt and evaluated
 the reproductive effects of HP receipt across geographic locations under
 natural conditions. We investigated the spatial variation of pollen
 deposition across eight sites and how the pollen receipt related to seed
 set of Salvia przewalskii, a subalpine perennial herb in Hengduan Mountain
 in southwest China. We found that stigmatic pollen loads substantially
 varied among sites for several metrics, including quantities of
 conspecific and heterospecific pollen, the proportion of HP, and species
 composition of HP donors. Five different plant families were the most
 common HP source at one or two sites, and the proportion of HP ranged from
 3.4–51.3% across sites. The association of conspecific pollen with seed
 set was positive and variable among sites, whereas the association of HP
 receipt and seed set was negative and not significantly different among
 sites. Our results demonstrate variation in the quantity and fitness
 effect of pollen receipt across sites, which is a precondition for
 evolution of local adaptation. Further study of variation in patterns and
 effects of HP receipt for the same recipient species across natural
 communities would allow for better understanding of the ecological and
 evolutionary consequences of HP receipt. 
description: Field collections occurred
 from July 25 to August 19, 2021, during peak flowering of
 <em>Salvia</em> <em>przewalskii</em>. We collected
 2.9 (median, 3) stigmas per plant, with a range from 1 to 10 stigmas due
 to variation in plant size. Because the wilted corolla and pistil usually
 abscise, we secured the corolla to the corona with a single horizontal pin
 at the corona’s midpoint to keep it from falling off. For each site, we
 performed a given procedure (labeling, stigma collection or seed counts)
 for all flowers on the same day. After three or four days,
 when the corolla wilted, we removed the stigma with clean forceps and
 stored it in a microcentrifuge tube containing 70% ethanol. In most cases,
 the style abscised naturally, which indicated that the fertilization had
 already occurred, and germinated pollen exines were still affixed to the
 stigma. Ten days after stigma collection, we counted the developed and
 undeveloped seeds of each flower, which were readily distinguishable based
 on size. This approach allowed us to link pollen receipt and seed number
 in the same flower. In some cases, stigmas were damaged or flowers
 (plants) were missing or showed evidence of herbivore damage. In total,
 878 stigmas from 335 individuals were retrieved. We then observed the
 stigmatic pollen grains in the laboratory at 400× magnification and
 captured a series of images using a digital camera linked to a microscope,
 with one image per species of HP pollen on each stigma, the pollen from
 each HP species being sufficiently aggregated to be captured in a single
 image. Pollen grains were identified to the species level on the basis of
 morphological features including size, shape, and exine ornamentation,
 with use of a pollen reference library of co-flowering species within
 sites. We excluded the observations for which there was no pollen
 deposition (10 flowers) or conspecific pollen quantities were smaller than
 the number of seeds (19 flowers). We assume that these observations were
 due to loss of stigmatic pollen in the field during fertilization and
 stigma collection.