DOI: 10.5061/dryad.2ngf1vj04
title: Data from: Host control by Acmispon strigosus constrains fitness gains of ineffective Bradyrhizobium symbionts in mixed infections

description: Plant hosts can gain significant growth benefits from symbiosis with
 microbes, but these benefits could be threatened by divergent fitness
 interests among partners. Here, we measured fitness outcomes in symbiosis,
 by varying the genotypes of both microbes and hosts, to examine scenarios
 that might favor uncooperative symbionts. We studied associations between
 Acmispon strigosus, an annual legume native to California, and its
 nitrogen fixing symbionts in the genus Bradyrhizobium. Bradyrhizobium
 symbionts form root nodules on compatible hosts, with strains varying from
 effective, fixing substantial nitrogen for the host, to ineffective
 strains that do not fix nitrogen and provide no benefit to host growth. We
 co-inoculated four A. strigosus plant lines with nine combinations of
 effective and ineffective Bradyrhizobium strains and measured the relative
 fitness of ineffective strains within individual nodules, as hosts must
 select against uncooperative symbionts to maintain benefits. In mixed
 infections, ineffective strains always had lower relative fitness in
 nodules compared to beneficial strains, consistent with efficient
 punishment of nonfixing rhizobia. However, ineffective strains exhibited
 genotypic variation in their fitness in nodules within individual nodules
 co-infected with a beneficial strain, suggesting a role for symbiont
 competitiveness in shaping this joint phenotype. Variation in symbiont
 fitness during co-inoculations did not measurably affect plant
 performance, suggesting that predicted conflict over the joint phenotype
 of rhizobia fitness has negligible effect on the host.
description: <em>Data analysis –</em> Generalized linear mixed
 models (GLMMs) were used to test hypotheses using JMP Pro 13.0.0 (SAS
 Institute Inc., Cary, NC, USA). Dependent variables were
 log<sub>10</sub>-transformed as needed to improve normality.
 Proportional data was logit-transformed after applying a linear
 transformation to account for zeros and ones in the dataset (i.e., 1% was
 added to all datapoints except ones, from which 1% was subtracted). All
 models included a random effect of harvest week; models using plant
 biomass data (and not nodule culturing data) also included a random effect
 of block nested within harvest week. For each GLMM, all possible
 interactions among main effects of interest were initially tested.
 Nonsignificant interactions were removed from the model if this reduced
 the corrected AIC (AICc) by at least 2 units, and results from trimmed
 models were reported. Significant differences among levels of main effects
 were assessed with pairwise t-tests (Tukey’s HSD) of least squares means.
 The Test Slices option was used to explore interaction effects when only
 specific contrasts were of interest. Mean values discussed below were
 backtransformed (if applicable) from raw means and presented alongside 95%
 confidence intervals. Rhizobia strains were categorized
 as effective or ineffective depending on whether the total dry plant
 biomass of inoculated plants was significantly greater than that of the
 uninoculated control plants in the single inoculation experiment. Two
 different fitness proxies were estimated for rhizobia strains, including
 rhizobia population size per nodule (in the single inoculation experiment)
 and relative strain frequency within a nodule (i.e., proportion of CFU
 from a nodule of a particular strain in the co-inoculation experiment).
 Rhizobia population size (i.e., CFU) per nodule was averaged between
 replicate nodule cultures with the plant as the unit of replication (n =
 4) and was tested for effects of strain genotype and host line. Nodule
 occupancy for a strain was quantified as its relative abundance (i.e.,
 proportion of CFU/nodule) on each replicate plant (n = 4), for which the
 null expectation was 50% (i.e., relative abundance in the inoculum), and
 was tested for effects of the ineffective strain genotype, effective
 strain genotype, and host line. Plant relative
 performance was examined in the co-inoculation experiment by dividing the
 total plant biomass of each co-inoculated plant by the biomass of plants
 singly inoculated with the effective strain. Relative performance less
 than one would indicate that plants performed worse during mixed
 inoculations than with the effective strain alone, suggesting a cost to
 encountering the ineffective strain. Relative performance of co-inoculated
 plants was tested for significant deviation from one based on whether the
 confidence interval overlapped with one. Plant performance was tested for
 effects of the effective strain genotype, ineffective strain genotype, and
 host line.
description: # Host control by Acmispon strigosus constrains fitness gains of
 ineffective Bradyrhizobium symbionts in mixed infections
 [https://doi.org/10.5061/dryad.2ngf1vj04](https://doi.org/10.5061/dryad.2ngf1vj04) ## Description of the data and file structure *Data analysis –* Generalized linear mixed models (GLMMs) were used to test hypotheses using JMP Pro 13.0.0 (SAS Institute Inc., Cary, NC, USA). Dependent variables were log10-transformed as needed to improve normality. Proportional data was logit-transformed after applying a linear transformation to account for zeros and ones in the dataset (i.e., 1% was added to all datapoints except ones, from which 1% was subtracted). All models included a random effect of harvest week; models using plant biomass data (and not nodule culturing data) also included a random effect of block nested within harvest week. For each GLMM, all possible interactions among main effects of interest were initially tested. Nonsignificant interactions were removed from the model if this reduced the corrected AIC (AICc) by at least 2 units, and results from trimmed models were reported. Significant differences among levels of main effects were assessed with pairwise t-tests (Tukey’s HSD) of least squares means. The Test Slices option was used to explore interaction effects when only specific contrasts were of interest. Mean values discussed below were backtransformed (if applicable) from raw means and presented alongside 95% confidence intervals. Rhizobia strains were categorized as effective or ineffective depending on whether the total dry plant biomass of inoculated plants was significantly greater than that of the uninoculated control plants in the single inoculation experiment. Two different fitness proxies were estimated for rhizobia strains, including rhizobia population size per nodule (in the single inoculation experiment) and relative strain frequency within a nodule (i.e., proportion of CFU from a nodule of a particular strain in the co-inoculation experiment). Rhizobia population size (i.e., CFU) per nodule was averaged between replicate nodule cultures with the plant as the unit of replication (n = 4) and was tested for effects of strain genotype and host line. Nodule occupancy for a strain was quantified as its relative abundance (i.e., proportion of CFU/nodule) on each replicate plant (n = 4), for which the null expectation was 50% (i.e., relative abundance in the inoculum), and was tested for effects of the ineffective strain genotype, effective strain genotype, and host line. Plant relative performance was examined in the co-inoculation experiment by dividing the total plant biomass of each co-inoculated plant by the biomass of plants singly inoculated with the effective strain. Relative performance less than one would indicate that plants performed worse during mixed inoculations than with the effective strain alone, suggesting a cost to encountering the ineffective strain. Relative performance of co-inoculated plants was tested for significant deviation from one based on whether the confidence interval overlapped with one. Plant performance was tested for effects of the effective strain genotype, ineffective strain genotype, and host line. ### Files and variables #### PER-PLANT RAW DATA Plant_ID: Unique identifier for the focal plant (ranges 1-512 for Main experiment and 601-660 for Small experiment) Experiment: Experiment (Main versus Small) to which the focal plant was assigned; the Main experiment included 6 Bradyrhizobium strains in single-inoculations (6 treatments) and co-inoculations (9 treatments; each pair of beneficial and ineffective strains); the Small experiment only included Bradyrhizobium strains #2 and #18 in single-inoculations Wash_date: Date the plant was brought into the lab, removed from its pot, and washed free of sand; plants were wrapped in damp paper towels and stored at 4C until 'Harvest_date' Harvest_date: Date the plant was dissected into root, shoot, and nodule portions Wash_week: Weeks post-inoculation that the plant was removed from the greenhouse, depotted, and washed; if plants were not immediately dissected, they were wrapped in damp paper towels and stored at 4C until the Harvest_date Block: Block in which the focal plant was grown; the Main experiment (8 blocks) and Small experiment (5 blocks) were arranged in different areas of the same greenhouse; each block contained one plant replicate of each inoculation/host treatment (64 plants per block in the Main experiment; 12 plants per block in the Small experiment) Sort_group: Size-matched group to which the focal plant belonged prior to inoculation; plants within the same sort group were randomly assigned to inoculum treatments; each sort group was randomly assigned to a block; sort group 1 = largest plants; sort group 8 = smallest plants; plants were assigned first to the Main experiment, and leftover plants (i.e., the smallest plants) were assigned to the Small experiment; within the Small experiment, sort group 1 = largest plants; sort group 5 = smallest plants Host_population: Population source of the focal plant (BMR, Gri, UCR, Yuc); BMR = Bodega Marine Reserve; Gri = Griffith Park; UCR = Riverside; Yuc = Burns-Pinyon Ridge Reserve near Yucca Valley Host_line: Inbred line identifier for the focal plant; for the Main experiment, BMR hosts were line AcS074_BMR_u01_g1_r04, Gri hosts were a mixture of lines AcS075_Gri_u01_g1_r13 and AcS075_Gri_u01_g1_r15, UCR hosts were line AcS027_UCR_u01_g1_r10, and Yuc hosts were line AcS052_Yuc_m01_g1_r02; for the Small experiment, BMR hosts were line AcS074_BMR_u01_g1_r04, Gri hosts were line AcS075_Gri_u01_g1_r15, UCR hosts were line AcS027_UCR_u01_g1_r09, and Yuc hosts were line AcS052_Yuc_m01_g1_r02 Inoculum_type: Category of inoculum to which the focal plant was assigned; Single = single-inoculation with strain specified by Inoculum; CoInoc = co-inoculation with strains specified by Inoculum; Uninoc = inoculation with sterile water Inoculum: Inoculum to which focal plant was assigned (beneficial strains = #18, #49, #138, CW09; ineffective strains = #2, #187, CW01); co-inoculation with a beneficial and ineffective strain is indicated with the names of the ineffective and beneficial strains, concatenated with an underscore Ben_inoc_prop: for co-inoculated plants, the proportional abundance of the beneficial strain in the inoculum; the inocula were prepared to ideally be 50% beneficial strain, but we used quantitative culturing of inocula to determine the actual proportional abundance in the inocula, which we used as the null expectation for the strain's abundance in nodules. A "." indicates that no beneficial strain was included. Strain_type: For singly-inoculated plants, indicates whether the inoculated strain was beneficial (#18, #49, #138, CW09) or ineffective (#2, #187, CW01). A "." indicates that there was more than one strain type. Ineffective_strain: For co-inoculated plants, indicates which strain in the co-inoculum was ineffective (#2, #187, CW01). A "." indicates that there was no ineffective strain included. Beneficial_strain: For co-inoculated plants, indicates which strain in the co-inoculum was beneficial (#49, #138, CW09). A "." indicates that there was no beneficial strain included. Nods_cultured: "Yes" = nodules from this plant were selected for culturing. A "." indicates that there was no culturing. Notes: notes may be combined for individual plant entries ·       "Lost_one_nod_before_weighing" = one nodule from this plant was lost before it could be weighed with the rest of the nodules; these difference are reflected in the columns 'Total_nodule_number_counted' and 'Total_nodule_number_weighed'; ·       "Non_nodulated" = this plant was inoculated with a strain(s) but failed to form nodules ·       "Flowering" = the plant bore flower buds or fully opened flowers; ·       "Plant_dead" = the plant was dead; ·       "Pods_(1,2,3)" = the plant bore pods in addition to flowers; the number of pods is indicated Initial_leaf_number: number of compound leaves (including cotyledons) counted on the focal plant 1 day before inoculation; all counted leaves were at least 1 mm in length; used to assess initial plant size and assign plant to a sort group Shoots_mg: Shoot dry mass (mg) of focal plant Roots_mg: Root dry mass (mg) of focal plant Totmass_ben_mg: for co-inoculated plants, the total plant mass (Shoots_mg + Roots_mg) of the size-matched plant (from the same block) singly-inoculated with the beneficial strain that was part of the focal plant's inoculum Rel_performance: for co-inoculated plants, calculated as (Shoots_mg + Roots_mg)/Totmass_ben_mg. Values greater than 1 indicate that co-inoculated plants had greater biomass than plants singly inoculated with the effective strain in the co-inoculum. Total_nodule_number_counted: total number of nodules counted on the focal plant Total_nodule_number_weighed: total number of nodules that were weighed; this is the value used to calculate mean individual nodule size; in most cases this column is the same as ‘Total_nodule_number_counted', but see 'Notes' for two exceptions Nodules_weighed_mg: total dry mass (mg) of all the nodules on the focal plant after some were removed for culturing Nodules_cultured_mg: total dry mass (mg) estimated from all the nodules removed from the focal plant for culturing; see PER-NODULE RAW DATA Cells_per_nodule: mean number of viable rhizobia per nodule for each single-inoculated plant from which nodules were cultured, calculated from the Per-nodule raw data (by averaging ‘Cells_per_nodule’ by each plant, after excluding co-inoculated plants); values are means of up to 2 nodules assayed per plant (Ineff/Ben)_ABCDE_col: For this co-inoculated plant, the total number of colonies subcultured from up to 4 nodules that were identified as the ineffective or beneficial strain, respectively (summed across all nodules cultured from the plant; see the PER-NODULE RAW DATA); uses colony counts from all plates (ABC, D, E). (Ineff_percent_abund: Ineffective strain percent abundance on the plant, calculated as (100*Ineff_ABCDE_col)/(Ineff_ABCDE_col + Ben_ABCDE_col) **PER-NODULE RAW DATA** Nodule_ID: Unique identifier for the focal nodule (composed of the Plant_ID number followed by the nodule position number in the harvest photograph) Plant_ID: See PER-PLANT RAW DATA, above Block: See PER-PLANT RAW DATA, above Sort_group: See PER-PLANT RAW DATA, above Host_population: See PER-PLANT RAW DATA, above Host_line: See PER-PLANT RAW DATA, above Inoculum_type: See PER-PLANT RAW DATA, above Inoculum: See PER-PLANT RAW DATA, above Strain_type: See PER-PLANT RAW DATA, above. Ineffective_strain: See PER-PLANT RAW DATA, above Beneficial_strain:  See PER-PLANT RAW DATA, above Purpose: Purpose of culturing focal nodule; nodules from singly-inoculated plants were cultured for 'Quantitative' purpose (estimating the number of viable rhizobia in the nodule) and nodules from co-inoculated plants were cultured to determine 'Nodule_occupancy' (which strain(s) from the inoculum colonized the nodule) Harvest_date: See PER-PLANT RAW DATA, above Wash_wpi: See PER-PLANT RAW DATA, above ABC_culture_date: the date when the nodule was surface-sterilized, crushed, and its contents cultured on plain MAG media (0-2 days after the Harvest_date); nodules for which Purpose = Quantitative were cultured onto four plates denoted B1, B2, C1, and C2; nodules for which Purpose = Nodule_occupancy were cultured onto three plates denoted A, B, C; crushed nodule slurries were stored at 4C until use on D_culture_date and/or E_culture_date D_culture_date: for nodules where Purpose = Nodule_occupancy, this is the date (4/17/17) when nodule slushes stored at 4C were re-cultured onto another plain MAG plate (denoted D) in an attempt to generate more colonies E_culture_date: for nodules where Purpose = Nodule_occupancy, this is the date (5/4/17 or 5/8/17) when nodule slushes stored at 4C were re-cultured onto another plain MAG plate (denoted E) in an attempt to generate more colonies Subculture_date: for nodules where Purpose = Nodule_occupancy, this is the date when colonies from this nodule were subcultured onto selective media from original A/B/C, D, or E plates ABX_conc: for nodules where Purpose = Nodule_occupancy, this is the antibiotic and concentration used for discriminating between the two strains that could occupy the nodule; Strep100 = 100 ug/mL streptomycin; Chlor140 = 140 ug/mL chloramphenicol; Chlor150 = 150 ug/mL chloramphenicol; Gent190 = 190 ug/mL gentamycin; Gent225 = 225 ug/mL gentamycin ABX_date: for nodules where Purpose = Nodule_occupancy, this is the date the antibiotic plates were prepared; if subcultured colonies from the same nodule were split between two different batches of plates, the older date is given Notes: combinations may be used for individual entries ·       Multiple_morphs = more than one colony morphology on the MAG plate, both suspected to be Bradyrhizobia (usually clean edges versus 'halos'); ·       Multiple_colors = more than one colony color on the plate (usually cream and tan) and not suspected to be contamination; ·       Contamination = distinctly different colors of colonies (or mold) on plates that were used for subculturing, although contaminated areas were avoided for colony-picking Nodule_area_mm2_(KA, PC): area of the focal nodule in mm2; measured from images of nodules on graph paper at time of harvest; measured by KA or PC Mean_nodule_area_mm2: Area for focal nodule in mm2 averaged across measurements by KA and PC Estimated_nod_mass_mg: Estimated nodule mass (in mg) of the nodule based off Mean_nodule_area_mm2 and the empirically-derived equation Nodule dry mass (mg) = (Nodule area (mm2) - 0.9097853) / 5.5258444. Estimated nodule mass values less than zero were set to zero. Colony_count_(B1, B2, C1, C2): Number of colonies counted on replicate plate 1 or 2 of dilution 'B' or 'C' of focal nodule slurry (B plates contained 2,000x dilution of total nodule population, and C plates contained 200,000x dilution of total nodule population); a '0' indicates zero colonies, a '.' indicates too many colonies to count Cells_per_nod_(B1, B2, C1, C2): Number of rhizobial cells per nodule estimated from the appropriate 'Colony_count_(B1, B2, C1, C2)' column and its dilution factor (2,000 for B plates; 200,000 for C plates); 'bad_range' is displayed if the colony count is outside the range 3-800 Cells_per_nodule: For quantitatively-cultured nodules, number of rhizobial cells per nodule averaged across the four preceding columns; '.' is displayed if the average could not be generated from at least two plates with colony counts in the acceptable 3-800 range Col_streaked \_(ABC, D, E): For nodules of co-inoculated plants, the number of rhizobial colonies streaked from each combination of subculture plate types; 'ABC' plates were plates generated from fresh nodule slurries the day nodules were crushed (0-2 days post plant harvest, median of 1 day); 'D' plates were generated from refrigerated nodule slurries (6-15 days post plant harvest, median of 11 days); 'E' plates were also generated from refrigerated nodule slurries (6-36 days post plant harvest, median of 19 days) Col_streaked_total: the total number of colonies streaked from this nodule (Ineff, Ben)_ABCDE_col: For nodules of co-inoculated plants, the number of rhizobial colonies identified as the ineffective (or beneficial) strain in the focal nodule based on sub-culturing of nodule isolates onto selective media; this sums up all scorable colonies from all plates (ABC, D, E) (Amb, Contam, NoGro)_ABCDE_col: For nodules of co-inoculated plants, the number of rhizobial colonies that were identified as ambiguous (not displaying an antibiotic resistance phenotype that was easy to score), contaminated (by having a different color or texture than expected, or not growing after subculturing. CW01_restreaked: The number of colonies originally identified as strain CW01 (i.e., resistant to Gent190) that were restreaked on selective media to check the antibiotic resistance phenotype CW01_consistent: Of the CW01_restreaked, the number of colonies that produced the same antibiotic resistance phenotype as before Ben_restreaked: The number of colonies originally identified as a beneficial strain (i.e., sensitive to Gent190) that were re-streaked on selective media to check the antibiotic resistance phenotype Ben_consistent: Of the Ben_restreaked, the number of colonies that produced the same antibiotic resistance phenotype as before