DOI: 10.5061/dryad.cvdncjt5s
title: Pollinators adjust their behavior to presence of pollinator-transmitted pathogen in plant population

description: Interactions between pollinators and plants can be affected by the
 presence of plant pathogens that substitute their infectious propagules
 for pollen in flowers and rely on pollinators for transmission to new
 hosts. However, it is largely unknown how pollinators integrate cues from
 diseased plants such as altered floral rewards and floral traits, and how
 their behavior changes afterward. Understanding pollinator responses to
 diseased plants is crucial for predicting both pathogen transmission and
 pollen dispersal in diseased plant populations. In this study, we
 investigated pollinator responses to contact with plants of Dianthus
 carthusianorum diseased with anther smut (Microbotryum carthusianorum). We
 combined three approaches: 1) observation of individual pollinators
 foraging in experimental arrays of pre-grown potted plants; 2)
 measurements of floral rewards and floral traits of healthy and diseased
 plants; and 3) quantification of pollen/spore loads of pollinator
 functional groups. We found that pollinators showed only weak preferences
 for visiting healthy over diseased plants, but after landing on plants,
 they probed fewer flowers on the diseased ones. Since diseased flowers
 offered lower nectar and no pollen rewards, this behavior is consistent
 with the prediction of optimal foraging models that pollinators should
 spend less time exploring less rewarding patches or plants. Furthermore,
 pollen-foraging solitary bees and hoverflies responded to diseased plants
 more negatively than nectar-feeding butterflies did. Lastly, based on
 group-specific behavior and typical pollen/spore loads, we suggest that
 solitary bees and hoverflies contribute to both pollen and pathogen spore
 dispersal mainly over short distances, while butterfly visits are most
 important for long-distance dispersal.
description: For the complete description of materials and methods, see the
 paper <em>Pollinators adjust their behavior to presence of
 pollinator-transmitted pathogen in plant population</em> published
 in Behavioral Ecology.  The study system for the
 experiments was the herbaceous perennial
 plant <em>Dianthus carthusianorum</em> together with the
 plant's specialized anther smut pathogen <i>Microbotryum
 carthusianorum</i> and the plant's pollinators from three
 broadly defined functional groups (butterflies, hoverflies, solitary
 bees).<i> </i>The study site was a dry grassland on shallow
 soil in the Czech Republic (N 49°38'10.9", E
 14°13'35.7") with a natural population of the plant and
 long-term occurrence of the pathogen.
 <strong>Experiment 1 – Pollinator foraging
 behavior</strong> We observed pollinator foraging
 behavior in experimental arrays of potted pre-grown plants in order to
 examine pollinator response to diseased plants. We set up the arrays at
 the study site. Each array consisted of 36 plants divided into four
 clusters; one third of the plants was diseased with anther smut and two
 thirds were healthy. We set up four different types of arrays to reflect
 variable spatial aggregation of plants in natural populations
 (within-cluster spacing was dense or loose, between-cluster spacing was
 near or far, for details see the published paper). We
 observed the arrays in 15-min observation blocks from ca 9:00 a.m. to
 17:00 p.m. For each observed pollinator, we wrote down the sequence of
 visited plants and afterward, we constructed three variables that
 might reflect pollinator response to diseased plants. 1) We estimated each
 pollinator’s avoidance of diseased plants as the proportion of visited
 diseased plants out of the total visited by that pollinator. 2) We
 estimated the plant visit duration using the number of visited flowers on
 that plant. 3) We calculated the flight distance travelled between visited
 plants from the positions of the visited plants.
 <strong>Experiment 2 – Floral traits and nectar rewards of
 healthy and diseased plants</strong> We measured
 several floral traits of healthy plants and plants diseased with anther
 smut that could explain the pollinator behavior observed in Experiment 1.
 We used both potted pre-grown plants and plants naturally occurring at the
 study site. 1) We measured flower size as corolla
 diameter (the average of two measurements per flower). 2) We measured
 nectar volume in flowers using 0.5-µl microcapillary tubes (Hirschmann
 Laborgeräte, Eberstadt, Germany); we measured nectar standing crop in
 unmanipulated flowers from ca 8:00 a.m. to 17:00 p.m. and we also measured
 nectar production in flowers bagged for 12 hours prior to the
 measurements. 3) We measured reflectance of flowers between 300 and 700 nm
 on an AvaSpec-ULS2048CL-EVO spectrometer calibrated against a WS-2 diffuse
 reflectance standard (Avantes, Apeldoorn, Netherlands) using a BSD 50W
 lamp (Lucky Reptile, Waldkirch, Germany) as the light source.
 <strong>Experiment 3
 – Pollen and spore loads of the main pollinator functional
 groups</strong> We estimated pollen and spore
 loads carried on pollinator bodies in order to evaluate potential
 contribution of the main pollinator functional groups to pollination and
 pathogen transmission at the study site. We captured pollinators leaving
 diseased or healthy flowers using a hand net and we dabbed their bodies
 with a cube of fuchsin-stained gelatin. The gelatin cubes were then melted
 and mounted on microscope slides. We counted pollen grains and
 spores at 400× magnification. We used a 1×1 mm square grid and scored the
 numbers in every other grid cell on a semiquantitative scale (0, 1, 2-10,
 11-25, 26-50 pollen grains/spores). For further calculations, we used the
 mid values of the ranges. Next, the first 200 pollen grains on each slide
 were identified to species or to the lowest taxonomical level possible. We
 thus obtained the proportion of <i>D. carthusianorum</i>
 grains, which could be used to extrapolate the numbers to the whole sample
 (if the sample contained more than 200 pollen grains).
description: Each data file (numbered 1-8) contains data for one of the
 published analyses. The numbering of the data files correspond to the
 order in which analyses are presented in the published paper. The data
 files can also be divided into three groups according to the three
 separate experiments in the published paper: Experiment 1 (files 1-3),
 Experiment 2 (files 4-7), and Experiment 3 (file 8). There is also the
 additional file0 that contains primary data for Experiment 1 prior to
 constructing response variables for analyses. Handling
 of the data is explained in the "read_me" files, either
 "read_me.txt" or "read_me.r" with working examples of 
 R code.