DOI: 10.5061/dryad.v9s4mw76h
title: Impact of mulches on floral visitors in day-neutral strawberries and contribution of syrphid flies to strawberry pollination

description: Animal-mediated pollination is essential for the production of many
 specialty crops with insects, primarily bees, serving as the predominant
 pollinators in many agroecosystems. However, increasing management
 problems with honey bees and declines in native bee populations due to
 various environmental stressors have highlighted the importance of
 promoting wild bees and other insect pollinators to ensure sustainable
 pollination services. This study explores the impact of various mulches on
 the abundance and richness of floral visitations by insects and the role
 of syrphid flies in a day-neutral strawberry system. Four different types
 of mulches were evaluated at the University of Wisconsin West Madison
 Agricultural Research Station in Wisconsin, USA, during the 2022 and 2023
 growing seasons, and included three polyethylene mulches of different
 color (black, white on black, and reflective metallic) and one brown
 soil-biodegradable paper mulch. Observations of pollinator floral
 visitations were conducted and insects were grouped into broad
 morphotypes. In addition, syrphid flies were sampled to assess their
 pollen loads. Mulch type did not significantly affect the overall
 abundance or richness of pollinators visiting flowers. Syrphid flies were
 the most abundant floral visitors, followed by small dark bees, green
 bees, Orius spp., and honey bees, while Bombus spp. were found in low
 numbers. Toxomerus marginatus, the margined calligrapher, was the most
 abundant syrphid species collected, representing 72% of all syrphid flies
 collected. Overall, 31% of syrphid flies captured carried strawberry
 pollen.  A few syrphid species carried significant amounts
 (&gt;200 grains) of strawberry pollen on their body. The findings
 suggest that while mulch type did not impact pollinator visitation,
 syrphid flies may be important for strawberry pollination as they were
 abundant, at least in 2022, and carry strawberry pollen. Our results
 underscore the need for further research to understand the complex role of
 non-bee pollinators in strawberry pollination, and to further investigate
 the role of syrphid flies as pollinators in different agroecosystems.
description: <strong>Floral Visitor
 Observations</strong> To assess pollinator
 visitation, observations were conducted between 9:30 am and 3:30 pm on
 days with no or partial cloud cover, and sustained wind speeds below 24
 kmph. Observations were done every other week from July 13 to October 5 in
 2022 and weekly from July 27 to October 5 in 2023. In
 each row, up to five flower clusters were observed for 10 min and any
 flower visitors were recorded, with the order of rows observed randomized
 on each sampling date. The total number of flowers observed was recorded
 as the number of open flowers varied across clusters. A flower visitor was
 defined as any insect which appeared to be interacting with flowers, or
 which landed on open flowers. Insects visiting were identified to species
 when possible, including all honey bees and bumble bees. If a species was
 not identifiable due to short visit duration, small size, or complex
 identification traits (such as syrphid flies), they were recorded into one
 of the following visually discernible groups (morphospecies): green bees,
 small dark bees, large dark bees, or the most specific possible
 classification such as family or order (McIntosh et al, 2023). The number
 of individuals visiting flowers per species or morphospecies per flower
 was recorded. <strong>Syrphid Collection and
 Pollen Assay</strong> Syrphid flies were
 collected in 2023, due to their high abundance in 2022, to assess their
 pollen load for the percentage of strawberry pollen. Flies were collected
 via sweep net, taking 20 sweeps over the tops of strawberry plants in each
 bed, weekly.  Targeted sweeps were made to capture flies observed foraging
 in rows where the 20 sweeps did not capture any individuals. Captured
 flies were euthanized in a potassium cyanide kill jar and stored in
 individual 2 ml microcentrifuge tubes at -20ºC until processing.
 Pollen load was measured by thawing the frozen flies for 30 min
 at 2ºC and submerging them in 1.25 mL of 50% ethanol. Flies were then
 vortexed for 30 sec before being rinsed with 0.5 ml of 50% ethanol and
 placed into a clean microcentrifuge tube with 70% ethanol for long term
 preservation. Rinsate containing pollen was collected and allowed to
 evaporate for 5 d at 65ºC, before being reconstituted with 40 µL of 95%
 ethanol and 10 µL of Calbera’s Stain (1 g acid fuchsin, 150 ml DI water,
 50 ml glycerol). This solution was then added to a 10 µL haemocytometer
 (Hausser Scientific model 3100 improved neubauer, Horsham, PA). The total
 number of all pollen and strawberry pollen grains were counted under a
 compound microscope (Olympus BX60, Center Valley, PA). Pollen was
 identified only as strawberry pollen or other species’ pollen, based on
 reference images of pollen sourced from the Pollen Atlas (Stebler, 2024),
 an open-source pollen imaging database; a stained sample of pollen from a
 flower collected in the field was used as a secondary comparison source.
 Pollen ID was aided in that most other pollen observed was likely from
 various weeds in the family <em>Polygonaceae</em>, and bared
 little resemblance to the smaller, smooth, and roughly triangular pollen
 of <em>Fragaria x ananassa.</em> Flies were
 identified to the furthest taxonomic level using the key provided by
 Miranda et al., 2023. The flies we were unable to identify based on our
 abilities were categorized as either large or small syrphids, with small
 syrphids being smaller and large syrphids being as large or larger than
 <em>Toxomerus marginatus</em>.
 <strong>Statistical Analysis</strong>
 Statistical analysis was conducted in R Studio Team version
 2023.12.1+402 (RStudio: Integrated Development for R. RStudio, PBC,
 Boston, MA) using R Studio Team packages.  Observational data were
 analyzed using a negative binomial general linear model. Models were
 evaluated for accurate fit of data using transformed QQ plot and residuals
 vs. fitted plot. Further fit analysis for zero-inflation and dispersion
 were conducted using the package DHARMA. A type III Wald chi-squared
 analysis of deviance test was conducted on all models to check for
 significance of specific terms. For all models, following significant
 fixed effects, Tukey post-hoc tests were performed to evaluate pairwise
 comparisons among the mulch treatments and interacting effects, and no
 reduced models were utilized.  A model of this type was
 used to analyze pollinator abundance of floral visitation as well as for
 the analysis of each of the seven individual bee and syrphid morphospecies
 and was fitted using the GLMTTB package. Total observed visits over the 10
 min observation period were used as the response variable; block (levels =
 A, B, C, D), year (levels = 2022 and 2023), treatment (levels = black,
 metallic, paper, and white), and morphospecies (see Table 1.1 for levels
 used) as well as year: treatment, year :morphospecies, and
 morphospecies:treatment were included as fixed-effect variables. An offset
 term of the natural log of the number of flowers included in each
 observation was included. No random-effects variables were used. Richness
 and abundance of floral visitation were calculated using the package VEGAN
 and were analyzed using a Poisson generalized linear model with log link.
 Block, treatment, year, and a treatment-year interaction were included as
 fixed-effects variables, with flower count as an offset term and no
 random-effect variables included.
description: # Impact of Mulches on Floral Visitors in Day-Neutral Strawberries and
 Contribution of Syrphid Flies to Strawberry Pollination
 [https://doi.org/10.5061/dryad.v9s4mw76h](https://doi.org/10.5061/dryad.v9s4mw76h) ## Description of the data and file structure The data is presented with two files. A file on syrphid observations and pollen collection from syrphid flies sampled and the number of pollen grains that belong to the target crop, strawberry. Another file reports on the floral observations made on strawberry flowers of all floral visitors. ### Files and variables #### File: Syrphid\_pollen.xlsx **Description:**  ##### Variables * Common names: syrphid species/morphogroups used in the manuscript * Letter: Letter is an indicator group for date of collection, * Date: date of observation in mm/dd/year format * Block (1-4) indexes the replication level of the observation, with a total of four blocks. * Row (M, P, W, B) represents the mulch treatments where the level represents the color of the mulch. B: black (polyethylene), M: metallicized (polyethylene), P: paper (cellulose), and W: white-on-black (polyethylene). * Sample ID: sample ID is the subsampling index * Total pollen: amount of pollen of any species identified in 1µL on an improved Nuebauer Haemocytometer out of a total of 50 µl of solution. Species were identified before pollen collection. * Fxa pollen: amount of strawberry pollen identified in 1µL on an improved Nuebauer Haemocytometer out of a total of 50 µl of solution. Species were identified before pollen collection. #### File: Flower\_Observations.csv **Description:**  ##### Variables * untitled column is an indexing column for the dataset. * Date: represents the date of observation in mm/dd/year format * Treatment: indexes each observation by mulch treatment, where the level represents the color of the mulch: Black (polyethylene), Metallicized (polyethylene), paper (cellulose), and White (on-black, polyethylene). * Block: indexes the replication level of the observation, with a total of four blocks. * Flower_count: total numbers of flowers watched during the observation * Time: length of the observation in minutes * Bombus_impatiens: Common Eastern Bumblebee, *Bombus impatiens* * Honey_bee: European honeybee, *Apis mellifera* * Syrphidae: syrphid flies * Small_Dark: small dark bees * Large_Dark: large dark bees * Green: Green haliticid bees * Wasp: Social wasps, family Vespidae, order Hymenoptera * Diptera: flies, exluding those in family *Syrphidae* * Orius: insects from genus Orius, family Anthocoridae, order Hemiptera * Totalpol: Total number of pollinators recoded for that observation across specied * Richness: Species richness index as calculated via the package “vegan” for R studio. * Bees: Total number of observed pollinators excluding wasps, Dipterans, and *Orius.* ## Code/software Statistical analysis was conducted in R Studio Team version 2023.12.1+402 (RStudio: Integrated Development for R. RStudio, PBC, Boston, MA) using R Studio Team packages. Observational data were analyzed using a negative binomial general linear model. Models were evaluated for accurate fit of data using transformed QQ plot and residuals vs. fitted plot. Further fit analysis for zero-inflation and dispersion were conducted using the package DHARMA. A type III Wald chi-squared analysis of deviance test was conducted on all models to check for significance of specific terms. For all models, following significant fixed effects, Tukey post-hoc tests were performed to evaluate pairwise comparisons among the mulch treatments and interacting effects, and no reduced models were utilized.  A model of this type was used to analyze pollinator abundance of floral visitation as well as for the analysis of each of the seven individual bee and syrphid morphospecies and was fitted using the GLMTTB package. Total observed visits over the 10 min observation period were used as the response variable; block (levels = A, B, C, D), year (levels = 2022 and 2023), treatment (levels = black, metallic, paper, and white), and morphospecies (see Table 1.1 for levels used) as well as year:treatment, year:morphospecies, and morphospecies:treatment were included as fixed-effect variables. An offset term of the natural log of the number of flowers included in each observation was included. No random-effects variables were used. Richness and abundance of floral visitation were calculated using the package VEGAN and were analyzed using a Poisson generalized linear model with log link. Block, treatment, year, and a treatment-year interaction were included as fixed-effects variables, with flower count as an offset term and no random-effect variables included.