DOI: 10.5061/dryad.hx3ffbgkj
title: Soil composition, phenotypic and genetic data to: Adaptive differentiation on serpentine soil in diploid versus autotetraploid populations of Biscutella laevigata (Brassicaceae)

description: Serpentine soils exhibit extreme properties (e.g. high magnesium content)
 influencing plant growth and survival and have been repeatedly documented
 to promote adaptive edaphic differentiation in plants. Individuals from
 four pairs of nearby diploid and autotetraploid populations of Biscutella
 laevigata sampled on serpentine vs non-serpentine soils in a factorial
 design are used to assess the genetic and phenotypic changes associated
 with edaphic origin and ploidy level. Individual samples from natural
 populations were subjected to soil elemental analysis and genotyping using
 restriction site-associated DNA sequences (RAD-seq) to link genetic
 variation with contrasting soils and ploidy levels. In diploids, genetic
 variation was consistent with demographic contraction and a pattern of
 isolation by environment with respect to the ratio of calcium / magnesium
 concentrations, whereas tetraploids presented evidence of expansion with
 limited edaphic differentiation. The genetic basis of tolerance and
 adaptation to serpentine was further assessed experimentally on seed-grown
 individuals from all populations subjected to high (serpentine-like) vs
 low (control) concentrations of magnesium in hydropony. Fitness-related
 phenotypic traits under experimental cultivation were consistent with
 adaptive differentiation among diploid ecotypes but not among the
 tetraploids that similarly grow in both habitats and consistently present
 higher investment in roots. Further work comparing experimentally
 resynthesized polyploids to natural diploids and polyploids has to tease
 the role of whole genome duplication apart from the impact of
 post-polyploidy evolution.
description: Field sampling of material was conducted in proximate pairs of
 <em>Biscutella laevigata</em> populations growing either on
 serpentine or non-serpentine soils. These covered four diploid populations
 (<em>B. laevigata</em> subsp. <em>kerneri</em>
 Mach.-Laur.) in the Wachau region of Austria and four tetraploid
 populations (<em>B. laevigata</em> subsp.
 <em>laevigata</em> L.) in the Swiss Alps around Davos and
 Arolla. At each site, eight randomly selected individuals growing at a
 minimum distance of 3 m apart were georeferenced using a GPS receiver and
 sampled for silica gel-dried leaves, open-pollinated seeds and &gt; 10
 g of soil next to the main root. Soil samples were
 dried at 65°C for 24h, sieved with a 1 mm-sized mesh, and 10 g of each
 sample was mixed with 30 mL of 1M ammonia acetate at pH 7. After 2h of
 shaking, the suspension was filtered and diluted (between 1:100 and
 1:1000) with distilled water prior to analyses. Cation exchange capacity
 was assessed quantitatively by using inductively coupled plasma–optical
 emission spectrometry (ICP-OES) at the Platform of Analytical Chemistry of
 the University of Neuchâtel, Switzerland. Elemental concentration of
 Ca<sup>2+</sup>, Mg<sup>2+</sup>,
 K<sup>+</sup>, Na<sup>+</sup>,
 Ni<sup>2+</sup>, Cu<sup>2+</sup>,
 Zn<sup>2+</sup>, Cr<sup>2+</sup>,
 Al<sup>3+</sup> was measured for each of the 64
 samples. We used a hydroponic experiment to mimic the
 specific chemistry of serpentine vs. non-serpentine soils and compare the
 corresponding phenotypic responses of plants originating from serpentine
 vs. non-serpentine sites across two different ploidy cytotypes. Each of
 the eight plants genotyped per population was represented in the
 hydroponic experiment by six randomly selected seeds. Half the plants were
 exposed to low magnesium concentrations (Mg-, 0.5 mM, control setting)
 whereas the other half were exposed to high magnesium concentrations (Mg+,
 5 mM, serpentine-mimicking setting). For details on the hydroponic system
 and used nutrient solutions see the original article. After eight weeks of
 hydroponic cultivation, plants were harvested and the following traits
 were measured: overall number of leaves, length and width of the largest
 leaf, chlorophyll content and specific leaf area (SLA). The harvested
 biomass (root systems and leaf rosettes separately) was dried at 80°C for
 4 days and weighed. Relative chlorophyll content was obtained with
 SPAD-502 meter (Konica Minolta Sensing Europe B.V.) by averaging three
 measurements on a fresh largest leaf. A 0.64 cm<sup>2</sup>
 square punched out of the largest leaf was dried at 80°C and used to
 estimate the SLA. Additionally, two derived phenotypic characters were
 estimated: an approximate largest leaf size (i.e., length × width of the
 largest leaf) and root / shoot ratio (i.e. belowground biomass /
 aboveground biomass).  ddRAD seq libraries including the eight
 individuals sampled per population were prepared following a protocol
 adapted from Peterson et al. (2012) using restriction enzymes EcoRI and
 MseI. Subsequent ligation of uniquely indexed EcoRI adapters and
 library-specific MseI adapters, including four degenerate bases to
 identify PCR duplicates, enabled each sample to be distinguished. Library
 pools were size-selected, targeting a mean length of 550 bp with AMPure XP
 beads. DNA fragments containing both EcoRI and MseI adapters were
 amplified with 12 PCR cycles and the final libraries were obtained after
 cleaning of the PCR products. Libraries were sequenced as paired-end 2 × 250
 bp reads on two Illumina Novaseq 6000 lanes. Raw reads have been deposited
 in the European Nucleotide Archive (ENA) at EMBL-EBI under accession
 number PRJEB48869 (https://www.ebi.ac.uk/ena/browser/view/PRJEB48869). The correspondence between samples and runs included in this study is provided in the ‘ENA_samples_list.txt’. All tetraploid samples were included twice, as independent samples from the start of the procedure to account for the greater depth required to accurately genotype tetraploids. The process_radtag command implemented in Stacks (Catchen et al. 2013) was used to demultiplex raw fastq reads and remove bad quality bases. PCR duplicates were filtered out with clone_filter. Trimmomatic (Bolger et al. 2014) was used to trim the first four low-quality bases and keep only reads with a minimum length of 100 bp. The de novo reference catalog of RAD tags was generated based on reads from the 32 diploid samples using the dDocent pipeline (parameters: dDocent Cutoff1 = 5, dDocent Cutoff2 = 5, first clustering rate 80%, second clustering rate 80%; Puritz et al. 2014). Then reads from all 64 samples were mapped to the de novo reference catalog using BWA mem (Li 2013). SNPs were called with the Genome Analysis Toolkit ver. 4.1.0.0 (McKenna et al. 2010), without base recalibration. In a first step, samples were individually genotyped according to their ploidy by local re-assembly of haplotypes with the HaplotypeCaller tool. Single-sample GVCFs were then merged and jointly genotyped using GenomicsDBImport and GenotypeGVCFs tools. The resulting VCF file was first filtered to keep only biallelic SNPs covered in at least 50% of samples with sufficient quality according to GATK best practice (Supporting information). To avoid filters relying on expectations of the Hardy–Weinberg equilibrium that differ among ploidy, positions with an overall depth higher than two standard deviations above the mean were further removed as putative paralogous loci. Individual genotypes were filtered for a minimal depth of three and genotype quality of 20. Filtered genotypes were set to no-call and only SNPs with a maximum of 10% missing data were kept. The dataset was further pruned using PLINK2 (Purcell et al. 2007) based on linkage disequilibrium (–indep-pairwise 1000 100 0.2) and only positions with a minor allele count (MAC) above eight and a minor allele frequency (MAF) above 0.05 were finally kept. The resulting filtered VCF including 1146 biallelic SNPs and the derived input for STRUCTURE, which was obtained by randomly selecting one SNP per radtag, are provided here.
description: # Soil composition, phenotypic and genetic data to: Adaptive
 differentiation on serpentine soil in diploid versus autotetraploid
 populations of *Biscutella laevigata* (Brassicaceae)
 [https://doi.org/10.5061/dryad.hx3ffbgkj](https://doi.org/10.5061/dryad.hx3ffbgkj) The dataset contains elemental compositon of soil immediately surrounding *Biscutella laevigata* individuals growing on eight serpentine/non-serpentine sites in Austria and Switzerland and phenotypic traits measured on *B. laevigata* individuals in hydroponic cultivation mimicking the specific chemistry of serpentine vs. non-serpentine soils. We also provide the input data used in population genetic analyses and an overview of the raw sequencing reads deposited in the European Nucleotide Archive (ENA) for 63 *B. laevigata* individuals sampled on the eight sites and genotyped using ddRAD-seq. ## Description of the data and file structure The attached spreadsheet "Bürki_et_al_2023_primary_data.xlsx" consists of the following: **Sheet 1:** Elemental composition of soil surrounding diploid (2x) or tetraploid (4x) individuals of *Biscutella laevigata* sampled in field populations occurring on serpentine (S) or nonserpentine (NS) sites. Concentrations of particular elements are given in parts per million (ppm). At each locality, eight individual samples were included for genetic analyses and elemental concentrations. **Sheet 2:** Plant traits and fitness proxies scored on diploid (2x) and tetraploid (4x) individuals of *Biscutella laevigata* originating either from serpentine (S) or nonserpentine (NS) sites after eight weeks of hydroponic cultivation from seeds in magnesium enriched (Mg+) or control (Mg-) solutions, the former mimicking a specific chemistry of serpentine soils. The attached archive "Bürki_et_al_2023_genetic_data.zip" consists of the following: **ENA_samples_list.txt:** provides ENA project ID, run ID (i.e. raw fastq files), sample ID, and alias for each sample included in the study. **blaevserp_filtered_MD01_pruned02_MAC3MAF05rm.vcf.gz:** final filtered vcf file including 1146 biallelic SNPs for 63 samples **1snp_percontig.structure:** input dataset for STRUCTURE analysis, obtained by randomly selecting 1 SNP per RAD tag resulting in 944 SNPs ## Sharing/Access information Raw sequencing reads have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under the accession number PRJEB48869: * [https://www.ebi.ac.uk/ena/browser/view/PRJEB48869](https://www.ebi.ac.uk/ena/browser/view/PRJEB48869) ## Code/Software