DOI: 10.25338/b8403v
title: Dispersal of nectar microbes in California flowering communities

description: Variation in dispersal ability among taxa affects community assembly and
 biodiversity maintenance within metacommunities. Although fungi and
 bacteria frequently coexist, their relative dispersal abilities are poorly
 understood. Nectar-inhabiting microbial communities affect plant
 reproduction and pollinator behavior, and are excellent models for
 studying dispersal of bacteria and fungi in a metacommunity framework.
 Here, we assay dispersal ability of common nectar bacteria and fungi in an
 insect-based dispersal experiment. We then compare these results to the
 incidence and abundance of culturable flower-inhabiting bacteria and fungi
 within naturally occurring flowers across two coflowering communities in
 California across two flowering seasons. Our microbial dispersal
 experiment demonstrates that bacteria disperse among habitat patches more
 readily than fungi via thrips. Across all flowers, bacterial and fungal
 incidence and abundance were positively correlated but bacteria were much
 more widespread, suggesting shared dispersal routes or habitat
 requirements but differences in dispersal and colonization frequency. The
 finding that bacteria are more common among flowers sampled here, in part
 due to superior insect-mediated dispersal, may have broad relevance for
 microbial life-history, community assembly of microbes and
 plant-pollinator interactions. 
description: During the peak flowering seasons of 2016 and 2017, standing crop
 floral nectar was sampled from two sites in northern California: Stebbins
 Cold Canyon Reserve (Stebbins) in Winters, CA and at flowering plots
 maintained at the Laidlaw Honey Bee Facility (Bee Biology) in Davis, CA.
 The sites are approximately 18 miles apart, so are unlikely to be linked
 by pollinator dispersal, and differ in pollinator species composition and
 anthropogenic influence, but share a subset of plant species.
 Between late February and early July, flowers were sampled every
 two-four weeks from available plant species, with approximately 10 flowers
 of each plant collected per week (collections were limited by floral
 availability and reserve collection restrictions to protect plant
 populations). When possible, flowers were sampled from multiple
 individuals and sub-populations or plots (Supplementary Table S1). Care
 was taken to sample flowers that had been open at least one day if
 possible, to allow the opportunity for floral visitation and microbial
 immigration to flowers. Individual inflorescences were collected, placed
 upright in humidified boxes and kept cool until extraction and plating, no
 more than 5 hours later. In the lab, flowers were destructively sampled.
 Nectar was collected using 10 µl microcapillary tubes, and volume
 quantified. Nectar was diluted in 30 µl of sterile water (D0), then
 diluted 10 and 100 fold (D1 and D2 respectively) in sterile
 phosphate-buffered saline. To assess fungal and bacterial abundance, 50 µl
 of the 10 fold dilution (containing 5 µl of D0) and 50 µl of the 100 fold
 dilution (containing 0.5 µl of D0) were plated on yeast media agar (YMA)
 containing chloramphenicol and Reasoner’s agar containing cycloheximide
 (R2A Oxoid formula with 20% sucrose), respectively. All samples were
 plated the day of collection. For convenience, we refer to the total
 number of colonies on YMA as “fungi” and colonies on R2A as “bacteria”
 throughout the manuscript although some colonies on each media type may be
 comprised by microbes resistant to the antimicrobial compounds used here
 (e.g. bacteria resistant to chloramphenicol (Dhami et al. 2018)). The
 threshold for detection was approximately 8 live cells for YMA media and
 60 cells for R2A media in the original nectar sample. Negative controls
 were included and plated to detect potential contamination and samples
 discarded if contamination was detected (detected on 1 date; these samples
 were removed from the analysis). Agar plates were incubated at
 28<sup>o</sup>C and colony-forming units (CFUs) counted after
 48-72 hours. The total number of CFUs and CFU density for each nectar
 sample was calculated based on dilutions and original sample volume. Over
 the course of the study, 1 825 nectar samples were collected and plated on
 two media types. In 2016, representative colonies were picked haphazardly
 from plates collected over the entire season including all sites and plant
 species and frozen in glycerol. A subset of microbial
 strains from glycerol stocks were identified using MALDI-TOF and spectra
 were compared to Bruker Bacteria and Eukaryote libraries and a custom
 in-house database curated from previously identified microbial isolates
 from nectar (Supplementary Methods 1).
 <i>Insect-based microbial dispersal
 assay</i> To empirically test whether nectar
 microbes differ in their dispersal ability, we developed an assay of
 microbial dispersal resulting from the feeding activity of western flower
 thrips (<i>Frankliniella</i> <i>occidentalis</i>).
 The assay consisted of 5 sterilized adult female thrips freely foraging
 within a 96-well plate in which 1/3 of wells contained a microbial
 suspension (200uL 10,000 cells/uL) in sweetened tryptic soy broth (TSB,
 adding 15% sucrose and 15% fructose) and the remaining 2/3 of wells were
 filled with sterile TSB (200uL). Thrips were sourced from a colony held by
 Diane Ullman (see supplementary methods S4 for assay methods details).
 Microbial isolates from field sampling in this study (see below), and
 other nectar-associated microbe collections were used (6 bacteria, 5
 fungi). After thrips foraged for 24 hours at 30°C under 16L:8D light,
 thrips were killed by adding 320uL of ethyl acetate to the plate bottom
 and the media was incubated for 5 additional days. Control replicates with
 thrips and no added microbes were included in all trials to ensure
 adequate sterilization and trial consistency. Following incubation, we
 assessed microbial occupancy of initially sterile wells by calculating the
 deviation of optical density (OD 600nm) from a blank control plate (no
 thrips, no microbes) with an occupancy threshold equal to the mean OD of
 control wells +6 standard deviations. PacBio sequences
 are available at NCBI SRA under Bioproject #727253 http://www.ncbi.nlm.nih.gov/bioproject/727353
description: Thrips analyses are contained in: thripsPlatesBulked.csv ,
 thrips_plate_reader_fileGat... Field microbial
 incidence and abundance data are analyzed in: DispersalMsCode_4.29.21.R
 which uses data in FieldData5.19.20.csv PacBio
 (full-length 16S) data are analyzed using :
 Stebbins_PacBio_DADA2_16S_marshall.R and
 Stebbins_PacBio_DADA2_ITS_marshall.R Output files from
 raw analysis are contained in: ITS_DADA2output.RDS and 16S_DADA2output.RDS
 and analyzed using: Stebbins_PacBio_Analysis.R which also incorporates
 metadata found in sampleSubmit_RECORD_2.