Avicennia germinans (L.) L. (Black Mangrove) is a common, sometimes dominant member of intertidal vegetation in tropical and subtropical regions of the Americas and West Africa. Its flowers are known to be attractive to animals – there are records of a variety of floral visitors – and it is widely believed to be zoophilous, but the degree to which it relies on pollinators for full fruit set is not entirely clear. Molecular methods have revealed that A. germinans is self-compatible; however, it has not been definitively shown whether this self-pollination occurs via spontaneous autogamy, via pollinator-mediated fertilization of flowers with either their own pollen or pollen from neighboring flowers on the same plant (geitonogamy), or via both mechanisms. This study's objective was to assess the ability of A. germinans to spontaneously self-pollinate, and to determine if A. germinans is capable of setting fruit in the absence of pollinators.

Study organism

Avicennia germinans flowers are syncarpous, two-carpellate, with each carpel containing two ovules. Following anthesis and fertilization, three of the four ovules typically abort, leaving a single fertilized ovule per flower. Like other species in the genus, A. germinans seeds essentially germinate within the fruit wall, resulting in a reproductive structure called a "propagule". Each A. germinans flower produces, at most, one propagule.

Study sites

Pollinator exclusion experiments were carried out during the summers of 2014 and 2015 at six sites in Florida, USA: Anastasia State Park (29.87°, -81.27°), the Guana-Tolomato-Matanzas NERR (29.74°, -81.25°), Merritt Island NWR (28.71°, -80.74°), Pine Island Conservation Area (28.49°, -80.73°), Avalon State Park (27.55°, -80.33°), and Saint Lucie Inlet Preserve State Park (27.15°, -80.16°).

Experiment 1: Pollinator exclusion, anther removal, and supplemental pollen

To assess 1) the ability of A. germinans to self without pollinators, 2) the contribution of within-flower selfing to fruit set, and 3) inter-site variation in its breeding system, we conducted an experiment to test reproductive strategy across all six sites in 2014. At each site, we haphazardly selected 12–21 A. germinans individuals, with most trees at least 10m apart. In June–July (depending on site), each tree received all of the following treatments: anther removal, pollinator exclusion, pollinator exclusion plus hand-pollination with self-pollen, and unmanipulated natural pollination. Up to 20 nearly-opened flowers, each on a different inflorescence, were selected within each tree. Each flower was haphazardly assigned to one of the treatment levels, such that all treatments were replicated up to five times within a tree. In order to identify each replicate at the end of the experiment, the calyx of each focal flower was marked with white correction fluid, and the pedicel of its inflorescence was tagged with flagging tape.

The first manipulative treatment – anther removal – effectively left flowers unable to self-pollinate. Therefore, this treatment’s fruit set, when compared to that of the unmanipulated natural-pollination treatment, would reveal the degree to which within-flower selfing boosts A. germinans fruit set. This treatment was applied by using dissecting scissors to cut out all four anthers from focal flowers, prior to pollen release; in total, 449 flowers received this treatment across all six sites.

For the pollinator exclusion treatment, inflorescences containing the focal flowers were covered with a 7.5 x 10cm fine-mesh (0.05mm) bag prior to anthesis, to ensure the flowers had not been pollinated prior to the treatment. The bags remained in place for 5–8 days, by which point the anthers and corollas had abscised. In total, 462 flowers received this treatment across all six sites.

To partly account for the confounding effect that increased temperatures inside the pollinator exclusion bags had on fruit set (confirmed using Onset HOBO^® temperature loggers), we included a hand-pollination treatment, in which replicates were bagged as in the pollinator-exclusion treatment, but also received supplemental pollen (applied with a brush from another open flower on the same tree). This allowed us to directly compare the fruit set of bagged flowers – for which spontaneous autogamy was the only means of pollination – to the maximal fruit set of bagged flowers that received abundant pollen. This treatment also gave some indication of the extent of fruit set decline due to heat/humidity within the pollinator exclusion bags: if there were no such negative effects, then fruit set of this treatment would be expected to be as high or higher than fruit set in the unmanipulated natural-pollination treatment. Across all six sites, 309 flowers received this treatment.

One week after applying the experimental treatments, we recorded whether or not each focal flower's ovary remained attached to the parent plant. Four to six weeks later, we once again recorded the presence/absence of a developing focal ovary/developing propagule, and covered those remaining with large-mesh (~1.0 cm) bags to catch them as they matured and dropped (which occurred 4–5 months later). In the fall, we recorded the presence/absence of a fully developed propagule from each focal flower, and weighed most remaining propagules.

Data from this experiment are presented here raw and unprocessed.

Experiment 2: Pollinator exclusion only

The second pollinator exclusion experiment, in 2015, was conducted at a single site (the Guana-Tolomato-Matanzas NERR) and was designed to better account for treatment artefacts that were revealed in the first experiment, by using pollinator exclusion bags with wider mesh. In early June, before flowers had opened, 20 A. germinans were selected, with all trees at least 10m from one another. We selected six inflorescences on each tree, randomly assigning three to a pollinator exclusion treatment and three as unmanipulated natural pollination (ultimately n = 45 and n = 54, respectively, after some replicates were lost over the course of the experiment). The pollination exclusion treatment here was the same as in the first experiment in 2014, except for the size of the mesh in the exclusion bags. Inflorescences assigned to the pollinator exclusion treatment were covered with large-mesh (~1.0cm) plastic bags, which were meant to exclude most large insect pollinators (however, we did observe one large wasp leaving one of the bags partway through the experiment, indicating that the bags were not impenetrable to large insects). Inflorescences assigned to the natural pollination treatment were left unbagged and were marked with flagging. In late August, once flowering had finished, bags were placed over the control inflorescences, as well, to catch propagules as they dropped. We collected all bags in early November, 2015, and counted both the number of propagules inside and the number of initial floral buds on the inflorescence.

Data from this experiment are presented with replicates that produced propagules but lack initial floral bud counts removed.

See ReadMe file.