DOI: 10.5061/dryad.63xsj3v40
title: Experimental tests of pollinator dependence in Avicennia germinans, the black mangrove

description: 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.
description: Study organism <em>Avicennia
 germinans</em> 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, <em>A.
 germinans</em> seeds essentially germinate within the fruit wall,
 resulting in a reproductive structure called a "propagule".
 Each <em>A. germinans </em>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 <em>A. germinans</em> 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 <em>A. germinans</em>
 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 <em>A. germinans</em> 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<sup>®</sup> 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. <strong>Experiment 2: Pollinator exclusion
 only</strong>
 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 <em>A.
 germinans</em> 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 <em>n</em> = 45 and
 <em>n</em> = 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.
description: See ReadMe file.