DOI: 10.5061/dryad.k98sf7mfm
title: Biotic interactions promote local adaptation to soil in plants - Supplementary data

description: Although different ecological factors shape adaptative evolution in
 natural habitats, we know little about how their interactions impact local
 adaptation. Here we used eight generations of experimental evolution with
 outcrossing Brassica rapa plants as a model system, in eight treatment
 groups that varied in soil type, herbivory (with/without aphids), and
 pollination mode (hand- or bumblebee-pollination), to study how biotic
 interactions affect local adaptation to soil. First, we show that several
 plant traits evolved in response to biotic interactions in a soil-specific
 way. Second, using a reciprocal transplant experiment, we demonstrate that
 significant local adaptation to soil-type evolved in the “number of open
 flowers”, a trait used as a fitness proxy, but only in plants that evolved
 with herbivory and bee pollination. Whole genome re-sequencing of
 experimental lines revealed that biotic interactions caused a 10-fold
 increase in the number of SNPs across the genome with significant allele
 frequency change, and that alleles with opposite allele frequency change
 in different soil types (antagonistic pleiotropy) were most common in
 plants with an evolutionary history of herbivory and bee pollination. Our
 results demonstrate that the interaction with mutualists and antagonists
 can facilitate local adaptation to soil type through antagonistic
 pleiotropy. 
description: Plant, Brassica rapa, phenotype and genotype were measured.
 Samples result from a 8 generations experimental evolution where selective
 agents were different soils, herbivory and pollinators (Dorey and Schiestl
 2024). Sample size for trait measurements was 36 per replicate, this was
 chosen according to previous studies, and values published in the
 literature (Gervasi and Schiestl 2017, Ramos and Schiestl 2020, Dorey and
 Schiestl 2024). For trait measurements and DNA extraction, we used all
 individuals if available (some plants lacked sufficient leaf tissue for
 DNA extraction).
description: # Supplementary Data
 [https://doi.org/10.5061/dryad.k98sf7mfm](https://doi.org/10.5061/dryad.k98sf7mfm) ## Description of the data and file structure **File Name**: Supplementary Data 1. **Dataset.** **Description**: Traits values of plants growing in different soil (L = limestone, T= Tuff), and in different soil treatment (fertilizer: with use of fertilizer, nofertilizer: without use of fertilizer, standardized: grown in standardized soil, which provides optimal conditions for cultivation). Plantlabel: Identity of plants during the experiment PlantlabelDNA: Identity of plants for DNA collection (Labels used for DNA sequences, available on the platform NCBI). Replicate: Replicate of the experiment Generation: either plants from the initial population (generation one) or evolved plants (generation 10) Soil: soil in which plants grew during the experiment Soilline: Soil in which plants evolved during the experimental evolution Herbivoryline: herbivory treatment in which plant evolved during the experiment evolution (NH: non herbivory; H: herbivory) Pollinationline: pollination treatment in which plant evolved during the experiment evolution (B: bumblebee, H: hand pollination) Treatment: overal treatment in which plant evolved during the experiment evolution Native soil: Binary variable determining whether plants are growing in their native soil during the reciprical transplant experiment Seedssownout: refers to the number of seeds used for germination Germination: refers to the number of seeds that germinated Germination rate (%) Weightperseed: refers to the average weight per seed used for germination (mg) NbFlowers: number of open flower during choice tests Height: plant height during choice tests in cm Leaf_size: product of leaf length and leaf width, divided by 2; expressed in cm2 Lengthperbranche: average length per branche (in cm) Timetofirstflower: onset of flowering after germination (days) Flowerproduction: total flower production between flowering onset and choice tests.  Flowerdiameter: average flower diameter of three flowers (in mm) Petal_width: average petal width of three flowers (cm) Style:  average style length of three flowers (cm) Stamen:  average stamen length of three flowers (cm) Herkogamy: average difference between style and stamen length (cm) Visits: corresponds to how many bees visited the plants during the choice tests. MatriceNb: corresponds to identity of the different matrices of choice tests **File name**: Supplementary Data 2. **Dataset.** **Description**: Phenotypic, attractiveness and reproductive traits among first and seven generation plants and those having evolved with or without aphid-herbivory, with or without bee-pollination and either in limestone or tuff soil. LHB: limestone line plants (L) growing with aphid-herbivory (H) and bee-pollination (B). LHH: limestone line plants (L) growing with aphid-herbivory (H) and hand-pollination (H). LNHB: limestone line plants (L) growing without herbivory (NH) and bee-pollination (B). LNHH: limestone line plants (L) growing without herbivory (NH) and hand-pollination (H). THB: tuff line plants (T) growing with aphid-herbivory (H) and bee-pollination (B). THH: tuff line plants (T) growing with aphid-herbivory (H) and hand-pollination (H). TNHB: tuff line plants (T) growing without herbivory (NH) and bee-pollination (B). TNHH: tuff line plants (T) growing without herbivory (NH) and hand-pollination (H). ID: Identity of plants during the experiment Generation: Corresponds the generation to which plants belong (1:10) Replicate: Replicate of the experiment Soilline: Soil in which plants evolved during the experimental evolution Pollinationline: pollination treatment in which plant evolved during the experiment evolution (B: bumblebee, H: hand pollination) Herbivoryline: herbivory treatment in which plant evolved during the experiment evolution (NH: non herbivory; H: herbivory) Treatment: overal treatment in which plant evolved during the experiment evolution Nb_silliques: number of silliques produced by plants Nb_seeds: number of seeds produced by plants during the experimental evolution Nb_seeds_per_sillique: average number of seeds contained in each sillique Relativeseedset: relative seed set of the plant within the population. This value is proportionnal to the number of seeds produced by other plants of the same population (see paper for more details on formula).  Visits: corresponds to how many bees visited the plants during pollination Timetofirstflower: onset of flowering after germination (days) Height: plant height during pollination in cm NbFlowers: number of open flower during pollination logNectar: corresponds to nectar production of plants per flower (expressed in uL and then log(x+1) transformed) Flowerdiameter: average flower diameter of three flowers (in mm) Petal_area: average petal area of three flowers (cm2) Petal_length: average petal length of three flowers (cm) Petal_width: average petal width of three flowers (cm) Style:  average style length of three flowers (cm) Stamen:  average stamen length of three flowers (cm) Herkogamy: average difference between style and stamen length (cm) LNBenzaldehyde: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1` (LN(x+1) transformed) LN_1_Butene_4_isothiocyanate: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1) transformed) LN_Methyl_benzoate: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1) transformed) LN_Phenylethyl_Alcohol: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1) transformed) LN_2_Amino_benzaldehyde: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1) transformed) LN_p_Anisaldehyde: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1) transformed) LN_Methyl_anthranilate: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1)transformed) LN_Z_3_Hexen_1_ol_acetate: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1)transformed) LN_Phenylacetaldehyde: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1)transformed) LN_Benzyl_nitrile: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1)transformed) LN_Methyl_salicylate: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1)transformed) LN_Indole: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1) transformed) LN_E.E_a_Farnesene: floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1) transformed) LN_Totalvolatiles: total floral volatile emission expressed in pg.l`-1`.h`-1`.flower`-1`(LN(x+1) transformed) ## **Software uploaded to Zenodo** **File name**: Supplementary Data 3. **R Script.** **Description**: Script to produce the allele frequency change matrix. Fq.gz files are available on NCBI under the project PRJNA1105729. Both generation 1 and generation 10 of our experimental evolution were sequenced when growing in either local or foreign soil. This script requires the loading of the following packages: library('learnPopGen'), library ('tidyverse'), library ('vroom'), library(data.table), library (poolSeq), library (harmonicmeanp), library (remotes), library(ACER), library(plyr) **File name**: Supplementary Data 4. **R** **Script.** **Description:** Script for calculating the breeding values for markers, and merge them with the AF change matrix; check for conditional neutrality and antagonistic pleiotropy pattern in AF change. Fq.gz files are available on NCBI under the project PRJNA1105729. Both generation 1 and generation 10 of our experimental evolution were sequenced when growing in either local or foreign soil. This script requires the loading of the following packages: library(data.table), library(R.utils), library(rrBLUP), library(tidyverse), library (ggplot2), library(wesanderson),l ibrary("RColorBrewer"), library(plyr) **File name**: Supplementary Data 5. **R** **Script**. **Description:** Script to produce the list of annotate genes from the marker matrix. Fq.gz files are available on NCBI under the project PRJNA1105729. Both generation 1 and generation 10 of our experimental evolution were sequenced when growing in either local or foreign soil.  This script requires the loading of the following packages: library(dplyr), library(GenomicRanges), library(data.table), library(tidyverse)