Test of self-incompatibility of wiregrass (Aristida beyrichiana) in north central Florida

<h4>Study Species</h4> <p>Wiregrass (<em>Aristida beyrichiana</em>) is a perennial C4 bunchgrass that reproduces sexually following fire and vegetatively between fires through tiller production. It is an ecologically significant understory species in longleaf pine ecosystems and is commonly used in restoration projects. Wiregrass typically flowers in response to fires occurring early in the wet season, and pollination is primarily wind-mediated, although insects such as ants are occasionally observed visiting flowers.</p> <p> </p> <h4>Study Site</h4> <p>This study was conducted in a >840-hectare University research forest in northern Florida, USA (29.7524° N, 82.2184° W), which has a humid subtropical climate and averages 127 cm of annual rainfall, mostly during the summer wet season. The landscape is dominated by mesic pine savannas with longleaf pine (Pinus palustris), slash pine (P. elliottii), and turkey oak (Quercus laevis) in the overstory, and wiregrass (Aristida beyrichiana) with diverse forbs and grasses in the understory. Two mesic savanna sites differing in fire frequency were selected: an annual burn unit (3.6 ha) burned every year since 1979 and a triennial burn unit (5.3 ha) burned every three years since 2012. Both sites were burned during June 2020, shortly after the onset of the wet season, to stimulate flowering. The annual site had higher tree basal area (32.8 m²/ha) than the triennial site (21.1 m²/ha), less vegetative groundcover, and slightly higher elevation. Soils also differed: the annual site had deep, poorly drained Sparr fine sands, while the triennial site had Lochloosa fine sands. Shrubs such as saw palmetto (Serenoa repens), gallberry (Ilex glabra), and dwarf huckleberry (Gaylussacia dumosa) varied in abundance between sites.</p> <p> </p> <h4>Experimental Design</h4> <p>To assess pollen limitation and self-compatibility, 40 wiregrass individuals were selected at each site (80 plants total) during the early fall of 2020. Plants were at least 5 m apart to reduce the likelihood of sampling the same genet. On each plant, three inflorescences were randomly assigned to one of three pollination treatments:</p> <ol> <li> <p><strong>Open-pollinated:</strong> Inflorescences were left unmanipulated and exposed to natural wind and pollinator-mediated pollen transfer.</p> </li> <li> <p><strong>Outcross-supplemented:</strong> Inflorescences received hand-applied outcross pollen in addition to ambient pollen. Pollen was collected from ten wiregrass individuals located at least 10 m away from study plants. Pollen was applied using a paintbrush every 1–2 days during anthesis, for up to six applications over the flowering period (September 30 – October 19, 2020).</p> </li> <li> <p><strong>Autonomous self-fertilization:</strong> Inflorescences were enclosed in natural kraft pollination bags prior to stigma receptivity, excluding outcross pollen.</p> </li> </ol> <p>This design allowed direct comparison of seed production and germination across different pollination environments within the same plant, controlling for maternal effects.</p> <p> </p> <h4>Germination Trials</h4> <p>Germination was tested on seeds from undamaged inflorescences (annual: n = 19 plants; triennial: n = 18 plants). Seeds from each inflorescence, including attached lemmas and paleas, were placed in separate germination boxes on blotter paper. Each box received 4 mL of a 0.002% Plant Preservative Mixture solution to minimize fungal contamination. Germination boxes were maintained at room temperature (21–23°C), under ambient light conditions, for approximately 2.5 months.</p> <p>Seeds were inspected every three days, and germination was scored upon emergence of the epicotyl. Germinated seeds were removed from the boxes at each observation to prevent double counting.</p> <p> </p> <h4>Data and Variables</h4> <p>Two primary response variables were measured:</p> <ol> <li> <p><strong>Seed production</strong> – the number of seeds produced per inflorescence in each treatment.</p> </li> <li> <p><strong>Seed germination</strong> – the number of seeds per inflorescence that successfully germinated during trials.</p> </li> </ol> <p>These measures were used to estimate (a) pollen limitation and (b) degree of self-compatibility. The degree of self-compatibility was calculated as the ratio of autonomous self-fertilization relative to outcross-pollen supplementation. Pollen limitation was measured by comparing open-pollinated inflorescences with outcross-supplemented ones.</p>