DOI: 10.5061/dryad.79cnp5hz5
title: Defensive mutualists affect outcross pollen transfer and male fitness in their host plant

description: Ant guards can increase plant fitness by deterring herbivores, but they
 may also reduce it by interfering with pollination. While ant impacts on
 herbivory have been well-studied, much less is known about their impacts
 on pollinators and associated consequences for plant pollination,
 particularly pollen transfer dynamics and outcrossing/selfing rates. We
 used field experiments to quantify the effect of ant guards on pollinator
 community composition, frequency and duration of flower visits, and
 cascading effects on outcrossing pollen transfer and pollen exports in
 Turnera velutina (Passifloraceae). Although ant patrolling did not affect
 pollinator community composition or visitation frequency, it decreased
 flower visit duration and the time pollinators spent foraging inside
 flowers. Such behavioural changes resulted in reduced pollen deposition on
 stigmas, decreased pollen exports (a proxy for male fitness) and
 significantly doubled outcross pollen transfer. This study contributes to
 our understanding of how nonpollinator mutualists can shape plant
 reproductive processes. We discuss the downstream effects that variation
 in biotic defences, such as rewards for guarding ants, can have on plant
 pollen transfer patterns and fitness. In conclusion, guarding ants
 influence pollen transfer patterns in Turnera velutina, increasing
 outcrossing in a self-compatible species at the cost of male fitness. We
 show how non-pollinators, such as defensive ant mutualists, can shape
 plant reproductive traits and discuss the consequences these interactions
 may have for plant mating systems.
description: <em><strong>Study site and
 system</strong></em> Field experiments were
 conducted in coastal sand scrub at Troncones, Guerrero, on the southern
 Pacific coastline of Mexico (17°47’ N, 101° 44’ W, elevation &lt; 50
 m). <em>Turnera velutina</em> (Passifloraceae) is a Mexican
 endemic shrub (Cuautle and Rico-Gray 2003, Arbo 2005) that establishes a
 facultative mutualism with ants (Zedillo-Avelleyra 2017), rewarding them
 with extrafloral nectar (Villamil et al. 2013). <em>Turnera
 velutina</em> is a self-compatible, herkogamous, polyploid species
 that requires insect pollinators for seed production (Sosenski et al.
 2016). Although it flowers year-round, flowering peaks during summer
 (Cuautle et al. 2005), and flowers last only one day, having a very short
 anthesis period (4-6 h) (Sosenski et al. 2016, Villamil et al. 2018).
 Pollinator rewards are pollen and floral nectar (Sosenski et al. 2016,
 Villamil et al. 2018). At Troncones, native butterflies are the dominant
 flower visitors of <em>T. velutina</em>, followed by the
 introduced honeybee (Apis mellifera); native bees, wasps, and occasionally
 flies also visit the flowers. All field experiments were conducted during
 June 2017. <em><strong>Ant exclusion
                                                       
 </strong></em> We identified large plants
 producing six or more flowers per day, surrounded by other <em>T.
 velutina</em> plants, but not within dense vegetation patches, and
 with few vegetation links to other plants, so that they could be isolated
 for the ant exclusion treatment. From within this selection, we
 haphazardly chose twelve plants (hereafter focal plants) forming six pairs
 of nearby plants with similar height and foliage abundance. In each plant
 pair, one focal plant was haphazardly designated as control, with natural
 levels of ant-guard activity. The second plant had ants excluded from all
 stems using a sticky barrier (Tanglefoot) (Fig. 1a). Both focal plants in
 each pair were isolated physically from other plants by trimming or tying
 back any surrounding vegetation. Exclusion treatments were checked daily
 and Tanglefoot was reapplied if required. We ensured similar ant
 patrolling dynamics between plant pairs. All control plants were patrolled
 by <em>Dorymyrmex bicolor</em> ants with similar instantaneous
 counts per branch: 10.86 ± 0.77 ants/plant (mean ± se). Plants within the
 pair were located 2-4 m apart, whilst plant pairs were separated from any
 other plant pair by at least 10 m (range: 10-150 m). Each focal plant pair
 was surrounded by 6-10 neighbouring adult plants of <em>T.
 velutina</em> 2-4 m away to ensure allogamous pollen supply (see
 details below, Fig. 1a). <em><strong>Pollen
 tracking </strong></em> To assess the
 effect of ant patrolling on pollen transfer and its consequences for rates
 of selfing, geitonogamy, and outcrossing, we dyed the anthers and pollen
 of control and ant-excluded plants using four contrasting dyes: red, blue,
 green or purple (Fig. 1b) using a focal-plant, focal-flower experimental
 design. Within a focal plant, a focal
 flower was selected and its anthers were dyed differently from the anthers
 in the rest of the flowers from that plant (hereafter referred to as
 satellite flowers), which were all dyed using a second colour
 (Fig. 1a). All satellite flowers from a given plant were dyed
 in the same colour. The focal flower of the other plant in the pair was
 dyed using a third colour, and all of its satellite flowers using a fourth
 colour. Colours were assigned independently of treatment to avoid
 confounding effects. Each pair of focal plants was surrounded by
 neighbouring <em>T. velutina</em> plants with 10-12 flowers
 (hereafter referred to as non-focal plants). Pollen from the neighbouring
 non-focal<em> T. velutina</em> plants was left undyed
 (naturally yellow-orange). The dyeing treatment was repeated in each of
 the six plant pairs for five days. Pollen grains of
 focal and satellite flowers were dyed once the corolla opened completely
 (~08:00-08:15 h), but before anther dehiscence. Anthers were individually
 embedded in a droplet of liquid dye until soaked, and flowers were bagged
 again until anthers dehisced and the released, dyed pollen grains were
 dry. The dyes used were methyl violet (purple; Drogueria Cosmopolita,
 Mexico), Green S (green; SigmaAldrich), safranin (red; SigmaAldrich), and
 methylene blue (blue; SigmaAldrich). Purple dye was prepared by diluting
 commercially available methyl violet with ethanol 70% [50:50]; the green
 dye used was commercially available Green S without dilution. Red
 (safranin) and blue (methylene blue) were prepared by diluting 1 g of
 either dye in 15.5 ml of distilled water, and then mixing this solution
 with ethanol 95% [50:50]. Previous studies showed that dyeing
 <em>Turnera velutina</em> anthers in these colours effectively
 dyed pollen grains and had no effect on pollinator visitation (Ochoa
 Sánchez 2016). Every morning before anthesis, six
 flower buds per plant (1 focal + 5 satellite buds) were bagged to exclude
 visitors. All additional pre-anthesis buds were removed to standardise
 floral display across focal plants. Once the corollas were fully open
 (~08:00-08:15 h), anthers were dyed and flowers were re-bagged until the
 dyed pollen dried (Fig. 1b). To ensure a minimum common supply of
 allogamous pollen in each experimental array, 10-12 undyed flowers from
 the neighbouring plants within the array were also bagged before anthesis.
 All flowers then remained bagged until visitation observations began at
 08:40 h. Towards the end of anthesis (11:30 h), pistils from the six focal
 flowers were collected in Eppendorf tubes and slide-mounted as a glycerine
 squash (Kearns and Inouye 1993, Ochoa Sánchez 2016) to allow counting of
 pollen grains received (Fig. 1c), and identification of their origins
 based on the dye-tracking.
 <em><strong>Pollinator visitation: community
 composition, visitation frequency, duration, and behaviour
 </strong></em> We recorded pollinator
 visitation to all six flowers on control and ant-excluded focal plants.
 Every focal plant was observed for a total of 40 minutes in two 20-minute
 periods – one immediately after bag removal (08:40 h) when flowers had a
 full pollen and nectar load and the second 90 minutes later. We recorded
 floral visitor identity and behaviour, and the frequency and duration of
 visits, as detailed below, for 40 h of observations of 360 flowers on 12
 plants over five days (N=60 focal flowers), with all experimental plant
 pairs observed in each of the five days. Because <em>T.
 velutina</em> has one-day flowers, we considered daily assays as
 independent samples, whilst accounting for plant identity, pair and day in
 our models to avoid pseudo-replication. Visitor taxa were identified as
 potential pollinators if they were observed contacting male and female
 plant sexual organs. Most statistical models in this section shared a
 common fixed and random effects structure, even though their error
 distribution varied depending on the response variable. In general, the
 models included exclusion treatment, pollinator type and their interaction
 as fixed effects; plant pair identity and day were included as random
 effects to account for the non-independence of flowers sampled on the same
 day or on the same plant across several days. The error distribution of
 the models varied depending on the response variable and variations to
 this model structure are specified and detailed in Table 1.
 a)     Pollinator community composition Flower
 visitors regarded as potential pollinators (hereafter pollinators) were
 identified to one of five taxonomic categories: <em>Apis
 mellifera</em>, native bees, butterflies, flies, and wasps. To
 estimate the relative abundance of each pollinator category, we calculated
 the percentage of all visitors from each group summed over control and
 ant-excluded plants. For each taxonomic group, differences in the total
 number of visitors between control and ant-excluded plants were assessed
 using a Pearson Chi-squared test. Because <em>Apis
 mellifera</em> and butterflies jointly accounted for 94% of all
 visitors (Table S1), only these taxonomic groups were included in all
 further analyses. b)     Pollinator visitation
 frequency and duration Flower visits were scored each
 time a pollinator hovered over, landed, and contacted the reproductive
 organs of a flower. Visit duration was recorded until the pollinator
 departed. Re-visitation by the same insect was scored as a separate visit.
 Visitor abundance was estimated as the number of individual visitors per
 taxon landing on flowers of a particular plant. A visitor that landed,
 hovered, and landed again in another flower was registered as two visits
 from one visitor. Ant presence effects on visitation frequency were tested
 using a Poisson mixed model, which also included an observation-level
 random effect (OLRE) to cope with overdispersion (Table 1, M1).
 c)     Pollinator behaviour Visit duration was
 differentiated as inspection and contact behaviours, to test whether the
 time pollinators spent in these activities differed between ant-patrolled
 and ant- excluded plants. All pollinator visits were allocated to one of
 two behavioural categories following Villamil et al. (2018): inspection
 (approaching a flower  and hovering above it without landing) or contact
 (landing on the flower). The effect of ant patrolling on the likelihood of
 inspection behaviour was tested with a binomial mixed model, with
 presence/absence of inspection behaviour as the response variable; this
 model included pair identity as a random effect (Table 1, M2). We also
 used a binomial mixed model to test whether ant patrolling affected
 pollinator avoidance behaviours, defined as the pollinator failing to land
 on a flower following inspection behaviour; this model included pair
 identity as a random effect (Table 1, M3). For every pollinator that
 displayed an inspection behaviour, we recorded the presence (scored as 1)
 or absence (scored as 0) of subsequent contact behaviours and fitted this
 as a binomial response variable.  We used a separate Poisson mixed model
 to test for an effect of ant presence on the duration of inspection and
 contact behaviours (visit duration was measured in integers accounting for
 the number of seconds).  In addition to the exclusion treatment and
 pollinator type, this model included pollinator behaviour as a fixed
 effect, and pair identity and an OLRE as additional random effects (Table
 1, M4).  We decided a priori to include the full combination of two-way
 interactions, and deliberately avoided fitting a three-way interaction
 because their outcomes are usually hard to interpret and
 visualise. <em><strong>Pollen transfer
 dynamics (outcrossing, selfing and
 geitonogamy)</strong></em> The effect of
 ant patrolling on pollen transfer rates was assessed by counting
 differentially dyed pollen grains on focal flower stigma squash slides
 under a light microscope. The effect of ant patrolling on stigma pollen
 load, defined as the total number of pollen grains received per stigma,
 was tested using a Poisson mixed model which included the exclusion
 treatment as a fixed effect, and plant identity, pair identity, day and
 OLRE as random effects (Table 1, M5). Outcrossing,
 selfing, and geitonogamous pollen transfer rates were characterised in
 terms of the proportions of pollen grains on focal stigmas identified by
 colour as transferred from the same flower (selfing), another flower
 within the same plant (geitonogamy), or another plant (whether the other
 focal plant in the same array or un-dyed plants surrounding the array)
 (Fig. 1a). The proportion of pollen grains from each source (selfing,
 geitonogamy, or outcrossing) was calculated for each focal stigma by
 dividing the number of pollen grains from each source by the total number
 of pollen grains on that stigma (pollen load). Proportional data were
 transformed to normality using the logit transformation, with infinite
 numbers resulting from impossible quotients replaced by zeros. The effect
 of ant patrolling on pollen source was determined using a linear mixed
 model, fitting the proportion of pollen grains from each source (selfing,
 outcrossing, or geitonogamy) as the response variable; ant exclusion
 treatment, mating system (pollen source) and their interaction as fixed
 effects, and plant identity, pair identity, day and an OLRE as random
 effects (Table 1, M6). Pollen transfer dynamics were
 analysed by further subdividing pollen flow into five categories depending
 on pollen transfer patterns and pollen source (with pollen-source,
 pollen-origin categories hereafter abbreviated to PSPO; Fig. 1a). Pollen
 source refers to whether the pollen comes from selfing, outcrossing or
 geitonogamy; whilst pollen origin refers to the provider of the pollen and
 it helps differentiate whether the pollen arrived from a short distance
 (dyed pollen from the other plant in the pair) or undyed pollen from the
 undyed, surrounding plants which comes from a greater distance. These
 categories summarise pollen grains received from, and donated to, every
 possible pollen source identifiable by colour in this experiment (Fig.
 1a), as follows: (i) received/donated by the same flower (selfing), (ii)
 received from another flower on the same plant (geitonogamy received),
 (iii) received from the reciprocal focal plant (outcrossing pair
 received), (iv) donated to the reciprocal focal plant (outcrossing pair
 donated), or (v) received from another plant of the same species
 (outcrossing unknown received). The effect of ant presence on pollen flow
 dynamics was tested using a Poisson mixed model, fitting the number of
 pollen grains in each of the five PSPO categories as the response
 variable. This model included the ant exclusion treatment, pollen origin
 category and their interaction as fixed effects; plant identity, pair
 identity, day and an OLRE were included as random effects (Table 1, M7). 
   <em><strong>Male fitness
 component</strong></em> The number of
 pollen grains donated per flower was used as an estimate of plant male
 fitness, quantified as the average number of pollen grains donated to
 focal stigmas. The total number of pollen grains from satellite flowers on
 the same plant was divided by five, to obtain the mean number of pollen
 grains donated per satellite flower. Similarly, the total number of pollen
 grains from the other plant in the same array was divided by six (1 focal
 flower + 5 satellite flowers). The effect of ants on male fitness was
 estimated using a Poisson mixed model fitting the number of pollen grains
 donated per flower as the response variable, the ant exclusion treatment
 as a fixed effect, and plant identity, pair identity, day and an OLRE as
 random effects (Table 1, M8). The effect of ant
 patrolling on the destination of the pollen grains donated per flower was
 tested using a Poisson mixed model fitting number of pollen grains as the
 response variable, the ant exclusion treatment as a fixed effect, and
 plant identity, pair identity, day and an OLRE as random effects (Table 1,
 M9). We contrasted the number of pollen grains donated by focal or
 satellite flowers in each array, but (in contrast to our pollen transfer
 pattern analysis, M6) ignored pollen from unknown, undyed plants for which
 the number of contributing flowers could not be determined.
 <em><strong>Statistical software and
 details</strong></em> Statistical analyses
 were conducted in R version 3.5 (R Core Team 2016).  All mixed effects
 models were fitted using the ‘lme4’ R package (Bates et al. 2016),
 residuals and model assumptions were checked using  the ‘DHARMa’ package
 (Hartig and Hartig 2017), and post-hoc Tukey comparisons were tested using
 the ‘multcomp’ R package  (Hothorn et al. 2008). Full details on the fixed
 and random effect structure of each model are provided in Table 1. To
 account for overdispersion, when needed, we included an
 observation-level-random effect in the models (Hinde 1982, Harrison
 2014).
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