DOI: 10.5061/dryad.xsj3tx9q2
title: Data from: Seasonal assembly of nectar microbial communities across angiosperm plant species: Assessing contributions of climate and plant traits

description: Plant-microbe associations are ubiquitous, but parsing contributions of
 dispersal, host filtering, competition, and temperature on microbial
 community composition is challenging. Floral nectar-inhabiting microbes,
 which can influence flowering plant health and pollination, offer a
 tractable system to disentangle community assembly processes. We
 inoculated a synthetic community of yeasts and bacteria into nectars of 31
 plant species while excluding pollinators. We monitored weather and, after
 24 hours, collected and cultured communities. We found a strong signature
 of plant species on resulting microbial abundance and community
 composition, in part explained by plant phylogeny and nectar peroxide
 content, but not floral morphology. Increasing temperature reduced
 microbial diversity, while higher minimum temperatures increased growth,
 suggesting complex ecological effects of temperature. Consistent nectar
 microbial communities within plant species could enable plant or
 pollinator adaptation. Our work supports the roles of host identity,
 traits, and temperature in microbial community assembly, and indicates
 diversity-productivity relationships within host-associated microbiomes.
description: <strong>Creating nectar microbiome
 inoculum</strong> We selected five microbe
 species (Table S1) that are common, widely distributed representatives of
 nectar microbiomes in various plant species, including those in Northern
 California (Vannette 2020; Vannette <em>et al.</em> 2021): the
 yeasts <em>Metschnikowia reukaufii</em> and
 <em>Aureobasidium pullulans</em>, and the bacteria
 <em>Neokomagataea thailandica</em>, <em>Acinetobacter
 pollinis</em>, and <em>Apilactobacillus micheneri</em>.
 We created our microbial inoculum (Fig. 1A; Supporting Information:
 Inoculum preparation) as described in Cecala & Vannette (2024).
 The inoculum contained ~10<sup>4</sup> cells µL<sup>-1
 </sup>of each species (5 x 10<sup>4</sup> total cells
 µL<sup>-1</sup>). <strong>Floral
 bagging and inoculation</strong> We conducted 11
 rounds of floral inoculation on the University of California, Davis campus
 (38.540 °N, 121.756 °W) (USA: California: Yolo County) from 22 March to 29
 June 2023. In the morning the day before inoculations, we bagged ~10
 unopened flower buds on each of 5 to 8 species of flowering plants (Fig.
 S1) to prevent the transfer of microbes by pollinators. We secured organza
 bags (7x8.5 cm, 10x13 cm, or 13x18 cm) around flowers, removing all open
 flowers prior to sealing the bag. Each time we handled flowers, we
 inspected for any breaches by ants or thrips. Flowers
 that opened within bags were inoculated between 0900 to 1100 h. To
 inoculate a flower, 1 µL inoculum, carried into the field on ice, was
 delivered onto the nectary using a micropipette and autoclaved tips (Fig.
 1B), then flowers were tagged with a unique identifier, and re-bagged.
 Each week, we inoculated roughly 5 to 8 flowers per plant species (~40
 flowers per week). Over the course of the study, we recorded temperature
 extrema (afternoon highs and overnight lows) for all inoculation days from
 a local weather station (Fig. 1C, Fig. S2).
 <strong>Nectar extraction and
 plating</strong> Roughly 24 hours after
 inoculation, we excised flowers from plants, sealed them in containers and
 transported them to the laboratory. Inside a laminar flow hood, we used
 glass microcapillary tubes (VWR, Drummond) to extract and measure the
 volume of total nectar in each flower (Fig. 1D). We quantified microbes in
 nectar as in Cecala and Vannette (2024). Briefly, we diluted pure nectar
 in DPBS, plated aliquots on each of three agar media types, and incubated
 for 6 days, after which CFUs were identified and tallied (Supporting
 Information: Quantifying microbes in nectar). Microbial growth from four
 bagged flowers breached by crawling insects did not differ markedly from
 that of other flowers, and remained in analyses. For
 each flower, we calculated: (1) the density of CFUs per µL nectar, by
 dividing the number of CFUs per plate by the actual volume of pure nectar
 in the aliquot; and (2) the estimated total abundance of CFUs per flower,
 by multiplying our calculated density (1) by the total volume of nectar
 originally extracted from that flower. The above values (1 and 2) were
 calculated for each inoculated microbe individually and for all five
 species collectively. For comparison with real nectars
 and to test our inoculum in artificial solutions, we also added 1 µL
 inoculum to 10 µL of 30% m/m sucrose and an artificial nectar containing
 sugars and peptone (“experimental controls”; <em>n</em>=6
 replicates each; Supporting Information: Media recipes) in strip tubes.
 Tubes were sealed and incubated at 25 °C for 24 hours, then processed
 identically to actual nectar samples.
 <strong>Determination of floral
 traits</strong> We estimated concentrations of
 hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), a
 known antimicrobial reactive oxygen species found in some nectars (Carter
 & Thornburg 2004; Mueller <em>et al.</em> 2023), in
 the nectar of separate, non-inoculated flowers of most sampled plant
 species (Supporting Information: Peroxide quantification, Table S2).
 Peroxide values from non-inoculated nectar represent initial conditions
 which would be experienced by microbes arriving in flowers. To assess the
 contribution of  floral morphology, we scored floral phenotypes of all
 plant species on the basis of 28 binary traits used in past studies
 (Faegri & Pijl 1979; Ollerton <em>et al.</em> 2009) to
 represent pollination syndromes in multivariate space using Bray-Curtis
 dissimilarity. We determined trait states through a combination of
 observation and reference with the Jepson eFlora (ucjeps.berkeley.edu/eflora). We also encoded other traits of particular interest such as inflorescence density and corolla fusion. <strong>Scope of collected data</strong> We excluded from analysis five plant species for which we had few, low quality samples (Table S2). In total, we inoculated 398 flowers across 31 species of plants, 372 of which (93.5%) contained nectar after 24 hours (range: 7 to 16 flowers per species; mean=12 flowers per species). The absence of nectar in flowers did not coincide with any recorded variables. These species comprised 29 genera in 21 families. From the 372 nectar samples, we tallied 1,016,048 CFUs on agar media, of which 99.94% were our inoculated species: 72,242 <em>Metschnikowia</em>; 16,640 <em>Aureobasidium</em>; 20,121 <em>Neokomagataea</em>; 795,332 <em>Acinetobacter</em>; 111,149 <em>Apilactobacillus</em>. We classified 564 CFUs as non-inoculated bacteria or fungi (0.056% of all CFUs), likely originating from other plant tissues or the environment, and excluded these from analyses. <strong>Statistical analysis</strong> We conducted analyses in R (R Core Team 2024). Using package <em>lme4</em> (Bates <em>et al.</em> 2015), we constructed linear mixed effect models with nectar volume, total and by-species CFU density, and CFU Shannon-Wiener diversity index as dependent variables. As independent variables, we included nectar volume and temperature extrema, and plant species as a random intercept effect. We obtained type III sums of squares, F- and P-values, and Kenward-Roger degrees of freedom using function ‘Anova’ in package <em>car </em>(Fox & Weisberg 2019). We inspected model residuals for normality and variance inflation factors to assess multicollinearity. We also created separate linear models with either plant species or nectar peroxide concentration as a fixed effect, as peroxide data was not collected for three species (Table S2). For linear models in which we included a quadratic predictor, we conducted a likelihood ratio test comparing the goodness of fit of the models with and without the quadratic term. To test if microbial community composition (as Bray–Curtis dissimilarity) differed by plant species and temperature extrema, we used function ‘adonis’ in package <em>vegan</em> (Oksanen <em>et al.</em> 2020) to perform a permutational multivariate analysis of variance. We used function ‘betadisper’ to examine multivariate homogeneity of dispersions across plant species. Community composition was visualized using non-metric multidimensional scaling (NMDS) ordination, and we tested for significant microbe species vectors using function ‘envfit’. As above, a separate analysis was conducted with peroxide concentration as a predictor variable. To test for co-occurrence between microbe species flowers, we generated Pearson correlation matrices on CFU densities, for both our entire dataset and for each plant species individually, and visualized matrices using package ‘corrplot’ (Wei & Simko 2021). To estimate plant phylogenetic relationships among sampled plant species, we used the function ‘phylo.maker’ in package <em>V.PhyloMaker2</em> (Jin & Qian 2022) using the reference plant phylogeny GBOTB.extended.TPL. Using this tree, we tested for a phylogenetic signal of nectar volume, CFU densities, and Shannon diversity using function ‘multiPhylosignal’ in package <em>picante</em> (Kembel <em>et al.</em> 2010) with 10,000 simulations. To test for relationships between plant phylogenetic relatedness and multivariate microbe community composition, we created a pairwise distance matrix of plant phylogenetic relatedness using function ‘cophenetic.phylo’ in package <em>ape</em> (Paradis & Schliep 2019). We compared this distance matrix to a Bray-Curtis dissimilarity matrix of the mean CFU densities of each microbe by plant species using a Mantel test via function ‘mantel’ in package <em>vegan</em>, calculating Spearman’s ρ with 10,000 permutations. We also created a Bray-Curtis dissimilarity matrix of plant species based on floral trait data and compared this to the two aforementioned matrices. We controlled for the effect of plant phylogenetic distance on pollination syndrome using a partial Mantel test via function ‘mantel.partial’. We generated correlograms for all Mantel tests using the function ‘mgram’ in package <em>ecodist</em> (Goslee & Urban 2007). Figures were created using package <em>ggplot2</em> (Wickham 2016) and tree plots using ggtree (Yu <em>et al.</em> 2017) and custom function ‘ggtreeplot’ (Hackl 2018).
description: # Seasonal assembly of microbial communities across angiosperm plant
 species: Assessing contributions of climate and plant traits
 [https://doi.org/10.5061/dryad.xsj3tx9q2](https://doi.org/10.5061/dryad.xsj3tx9q2) This README file was generated on 2024-06-11 by Jacob M. Cecala. # GENERAL INFORMATION 1. Title of Dataset: Seasonal assembly of microbial communities across angiosperm plant species: assessing contributions of climate and plant traits 2. Author Information A. Principal Investigator Contact Information Name: Jacob M. Cecala Institution: University of California, Davis Address: Davis, CA, USA Email: [jmcecala@gmail.com](mailto:jmcecala@gmail.com) B. Associate or Co-investigator Contact Information Name: Leta L. Landucci Institution: University of California, Davis Address: Davis, CA, USA Email: [llanducci@ucdavis.edu](mailto:llanducci@ucdavis.edu) ``` C. Associate or Co-investigator Contact Information Name: Rachel L. Vannette Institution: University of California, Davis Address: Davis, CA, USA Email: rlvannette@ucdavis.edu ``` 3\. Years of data collection: 2023-2024 1. Geographic location of data collection: University of California, Davis, CA, USA (38.540292, -121.755485) 2. Information about funding sources that supported the collection of the data: USDA NIFA Postdoctoral Fellowship # 2021-67034-35157 to J.M.C.; NSF DEB # 1846266 to R.L.V # SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data: CC0 1.0 Universal (CC0 1.0) Public Domain 2. Links to publications that cite or use the data: Cecala, J.M., Landucci, L.L., and Vannette, R.L. (2024). Seasonal assembly of microbial communities across angiosperm plant species: assessing contributions of climate and plant traits. Ecology Letters. 1. Links to other publicly accessible locations of the data: None 2. Links/relationships to ancillary data sets: None 3. Was data derived from another source? No A. If yes, list source(s): NA 4. Recommended citation for this dataset: Cecala, J.M., Landucci, L.L., and Vannette, R.L. (2024). Data from: Seasonal assembly of microbial communities across angiosperm plant species: assessing contributions of climate and plant traits. Dryad Digital Repository. [https://doi.org/10.5061/dryad.xsj3tx9q2](https://doi.org/10.5061/dryad.xsj3tx9q2) # DATA & FILE OVERVIEW 1. File # List: 1) floral inoculation datasheet_ANALYSIS.xlsx 2) florinoc_datC5.summary.xlsx 3) Floral nectar peroxide - metabolomics project.xlsx 4) Inoculation code JMC Dryad.R 1. Relationship between files, if important: None 2. Additional related data collected that was not included in the current data package: None 3. Are there multiple versions of the dataset? No A. If yes, name of file(s) that was updated: NA i. Why was the file updated? NA ii. When was the file updated? NA **GENERAL DESCRIPTION OF DATA AND FILE STRUCTURE** File #1: Contains data on inoculated flowers, including corresponding plant information and microbial growth / CFU count data from nectar plating. The last sheet in the file also contains temperature data for each experimental day. Also included on separate sheets within this file are the blank templates of the data sheets used for data collection in the field and laboratory. File #2: The first sheet contains data on plant taxonomic ranks and basic floral attributes, along with simple summary statistics of nectar volume which are derived from File #1. The second sheet in this file contains the matrix of binary (0=trait absent; 1=trait present) floral morphological traits used to encode multivariate pollination syndromes for plant species. For further reference on some floral morphological traits and how they were encoded, see Olesen et al. (2007). Olesen, J. M., Dupont, Y. L., Ehlers, B. K., & Hansen, D. M. (2007). The openness of a flower and its number of flower‐visitor species. Taxon, 56(3), 729-736. [https://doi.org/10.2307/25065856](https://doi.org/10.2307/25065856). File #3: Contains data on floral nectar samples used for hydrogen peroxide concentration analyses. File #4: The R script "Inoculation code JMC Dryad.R" analyzes data in the above three Excel files. In all four files, any cells which are empty/null are left intentionally so, either because the data is not applicable, not available or missing for that specific cell, not used in final analyses, etc. See data-specific information for files below for further clarification. **GENERAL ABBREVIATIONS USED IN FILES** * sp = species * individ = individual * inoc = inoculation * vol = volume * calib = calibration * CFU = colony-forming unit * NEG_CONTROL = negative control * wk = week * precip = precipitation * ppm = parts per million * conc = concentration * Aci, Acineto = *Acinetobacter pollinis* (SCC477) * Met, Metsch = *Metschnikowia reukaufii* (EC052) * Neo, Neoko = *Neokomagataea thailandica* (EC112) * Aur, Aureo = *Aureobasidium pullulans* (EC102) * Api, Apilacto = *Apilactobacillus micheneri* (HV60) **DATA-SPECIFIC INFORMATION FOR FILE #1: floral inoculation datasheet_ANALYSIS.xlsx** Sheets and variables: Sheet 1: field_data Empty cells in this Sheet were either not measured/available (e.g., if a flower contained no nectar, it lacks associated microbial growth data), or the columns were not used in final analyses. In all cases, empty cells are left blank intentionally. * plant sp.: the species of plant inoculated. * plant_individ_ID: arbitrary ID used to keep track of individual plants. * Flower_ID: unique identifier for a given flower. * inoc_date: date flower was inoculated. * mm nectar: total length of nectar column in microcapillary tube after extraction from flower. * tube vol: corresponding microcapillary size (volume) used to extract nectar from flower. * tube calib length_mm: corresponding length of microcapillary tube. * nectar vol: calculated volume of nectar extracted in µL. * vol in dilution: volume of pure nectar added to DPBS solution prior to plating, either 5 µL (if total volume of nectar extracted was &gt;5 µL) or all (if &lt;5 µL extracted). * dilution check: internal check for appropriate dilution volume based on total volume of nectar extracted from flower. * thrips?: if thrips (Thysanoptera) were present in flower at time of nectar extraction. * corolla width: width of corolla at its widest point in mm. * corolla length: length of corolla, from base of petals to tip, in mm. * PBS dilution %v/v nectar: dilution factor of nectar in DPBS. * Brix pure: dissolved solids (°Brix) content of pure nectar samples. * Brix PBS: dissolved solids (°Brix) content of DPBS-diluted nectar samples. * Estimated Brix: estimated dissolved solids (°Brix) content of pure nectar based on the DPBS-diluted solution. * Metsch CFUs: number of CFUs of Metschnikowia reukaufii on yeast media (YM) agar plate. * Aureo CFUs: number of CFUs of Aureobasidium pullulans on yeast media (YM) agar plate. * Neoko CFUs: number of CFUs of Neokomagataea thailandica on tryptic soy (TS) agar plate. * Acineto CFUs: number of CFUs of Acinetobacter pollinis on tryptic soy (TS) agar plate. * Apilacto CFUs: number of CFUs of Apilactobacillus micheneri on MRS agar plate. * any other CFUs: number of CFUs of non-inoculated microbe colony morphotypes * total CFUs: total number of CFUs on all plates. * notes: miscellaneous notes. * Species inoculated: list of microbe species inoculated into flower. See abbreviations used. Sheet 2: field_datasheet Data sheet used for floral inoculation in the field. Sheet content not used in data analyses. Sheet 3: notes Miscellaneous notes. Sheet content not used in data analyses. Sheet 4: Plant_traits Various traits of inoculated plant species. Sheet content not used in data analyses. Sheet 5: plate_datasheet Data sheet used for counting microbial colonies in the laboratory. Sheet content not used in data analyses. Sheet 6: weather * wk number: number of week corresponding to experimental inoculation day, starting at 0. * inoc_date: date of inoculations for that week. * flower_IDs: range of flower identification numbers (see Sheet 1: field_data) inoculated in that week. * afternoon_high_F: maximum temperature in degrees Fahrenheit (°F) on afternoon of inoculation day, recorded by weather station. * overnight_low_F: minimum temperature in degrees Fahrenheit (°F) during the night after inoculation, recorded by weather station. * precip_in: precipitation in inches (") on day of inoculation. **DATA-SPECIFIC INFORMATION FOR FILE #2: florinoc_datC5.summary.xlsx** Sheet 1: Sheet1 Empty columns in this Sheet were not used in final analyses and are left blank intentionally. * species: plant species name. * genus: plant genus. * family: plant family. * plant sp.: plant species name (alternate formatting used in some files). * California.native: if the plant species is native to California or not (exotic). * NAmerica.native: if the plant species is native to North America or not (exotic). * flower.sym: flower symmetry, bilateral or radial. * flower.arch: flower architecture, single or born in an inflorescence. * petals: petals free or fused. * petal.color: predominant color of petals. * pollination.syndrome: * N.inoculated: number of flowers inoculated. * N.0nectar: number of flowers inoculated that had no retrievable nectar in them after 24 hours. * N.wnectar: number of flowers inoculated that had retrievable nectar in them after 24 hours. * mean: mean nectar volume. * median: median nectar volume. * SD: standard deviation of nectar volume. * SE: standard error of nectar volume. * CV: coefficient of variation of nectar volume. Sheet 2: Sheet2 Empty columns in this Sheet were not used in final analyses and are left blank intentionally. * species: plant species name. * trap: flower possesses a trap-like mechanism for insects or other animals. * open: flower is open and relatively flat. * bell: flower is bell-shaped. * brush: flower possesses brush-like anthers and reduced petals. * gullet: flower possesses a deep, throat-like chamber at base. * flag: flower possesses a broad, banner-like petal. * tube: flower is narrow and tube-shaped. * zygomorphic: flower is zygomorphic (bilaterally symmetrical); 0 indicates a radially symmetrical flower. * medium_small: flower is average sized or below; 0 indicates large flowers (relative to sampled species). * mechanically_strong: [trait not used.] * pendulous: flower hangs downwards (stem above flower). * upright: flower is oriented upright (stem below flower). * horizontal: flower is oriented horizontally. * stiff_anthers: anthers are stiff and relatively inflexible. * exposed_anthers: anthers are exserted beyond rim of corolla / tips of petals. * narrow_tube: tube-shaped flower that is relatively narrow. * wide_tube: tube-shaped flower that is relatively wide. * short_tube: tube-shaped flower that is relatively short (from base to rim of corolla). * long_tube: tube-shaped flower that is relatively long (from base to rim of corolla). * day_anthesis * strong_scent: [trait not used.] * no_scent: [trait not used.] * sweet: [trait not used.] * fruity: [trait not used.] * fresh: [trait not used.] * musky: [trait not used.] * sour: [trait not used.] * decaying: [trait not used.] * drab: corolla color is dull. * vivid: corolla color is bright. * brown: corolla contains any amount of this color. * white: corolla contains any amount of this color. * red: corolla contains any amount of this color. * green: corolla contains any amount of this color. * yellow: corolla contains any amount of this color. * blue: corolla contains any amount of this color. * purple: corolla contains any amount of this color. * nectar_guides: [trait not used.] * nectar_absent: no nectar present in flower. * abundant_nectar: above average volumes of nectar present in flower (relative to sampled species). * moderate_nectar: average or low volumes of nectar present in flower (relative to sampled species). * dense_infloresence: flowers of inflorescence are densely packed, with less space between them than a floral width. * petals_fused: petals are fused for more than half their length to form a tube. **DATA-SPECIFIC INFORMATION FOR FILE #3: Floral nectar peroxide - metabolomics project.xlsx** Sheet 1: Sheet1 Empty cells in this Sheet were either not measured/available or not used in final analyses and are left blank intentionally. * tube_ID: unique identifier for nectar sample. * date: date of nectar sample collection. * plant_genus: genus of plant from which nectar as collected. * plant_species: species of plant from which nectar as collected. * peroxide_strip_ppm: concentration of peroxide in parts per million (ppm) based on peroxide strip tests. * peroxide_conc_uM: concentration of peroxide (µM) * sample_n: number of different flowers from which nectar was pooled for sample. * assay_type: type of assay used to measure peroxide concentration. * brix: dissolved solids (°Brix) content of pure nectar samples. * pH: pH of nectar sample. * notes: miscellaneous notes. * pooled: whether or not a sample was pooled. * proteomics_sample: whether sample was used in separate proteomics analyses. * storage location: location where sample is stored in -80° freezer. ## Sharing/Access information The data contained here are original and not derived from any other source. They are only accessible here. ## Code/Software The R script "Inoculation code JMC Dryad.R" analyzes data in the Excel files and was created in R version 4.1.1 (Kick Things) using RStudio version 2022.12.0+353. Operating system was Mac OS version 10.15.5. R Packages used in running the scripts (also listed in the scripts themselves) include, but are not limited to, the following: readxl, dplyr, ggplot2, car, lme4, AICcmodavg, MuMIn, emmeans, magrittr, multcomp, scales, plyr, visreg, tidyr, ggpubr, summarytools, fitdistrplus, vegan, arm, glmmTMB, ggfortify, goeveg, corrplot, Hmisc, stringr, ggrepel, data.table, writexl, patchwork, cooccur, ecodist, lmtest, purrr, factoextra, cluster, devtools, V.PhyloMaker2, BiocManager, ggtree, ape, picante, phytools, Rphylopars, V.PhyloMaker, phyloseq.