DOI: 10.5061/dryad.s7h44j1fd
title: Beyond cyanogenesis: Temperature gradients drive environmental adaptation in North American white clover (Trifolium repens L.)

description: Species that repeatedly evolve phenotypic clines across environmental
 gradients have been highlighted as ideal systems for characterizing the
 genomic basis of local environmental adaptation. However, few studies have
 assessed the importance of observed phenotypic clines for local
 adaptation: conspicuous traits that vary clinally may not necessarily be
 the most critical in determining local fitness. The present study was
 designed to fill this gap, using a plant species characterized by
 repeatedly-evolved adaptive phenotypic clines. White clover is naturally
 polymorphic for its chemical defense cyanogenesis (HCN release with tissue
 damage); climate-associated cyanogenesis clines have evolved throughout
 its native and introduced range worldwide. We performed landscape genomic
 analyses on 415 wild genotypes from 43 locations spanning much of the
 North American species range to assess the relative importance of
 cyanogenesis loci vs. other genomic factors in local climatic adaptation.
 We find clear evidence of local adaptation, with temperature-related
 climatic variables best describing genome-wide differentiation between
 sampling locations. The same climatic variables are also strongly
 correlated with cyanogenesis frequencies and gene copy number variations
 (CNVs) at cyanogenesis loci. However, landscape genomic analyses indicate
 no significant contribution of cyanogenesis loci to local adaptation.
 Instead, several genomic regions containing promising candidate genes for
 plant response to seasonal cues are identified — some of which are shared
 with previously-identified QTLs for locally-adaptive fitness traits in
 North American white clover. Our findings suggest that local adaptation in
 white clover is likely determined primarily by genes controlling the
 timing of growth and flowering in response to local seasonal cues. More
 generally, this work suggests that caution is warranted when considering
 the importance of conspicuous phenotypic clines as primary determinants of
 local adaptation.
description: <strong>Sample Collection, DNA Extraction, and GBS Library
 Preparation</strong> Using a nationwide network
 of K-12 science teachers, citizen scientists and colleagues, we obtained
 mature seeds or stolon cuttings for 419 wild white clover accessions
 across 43 locations in North America during the growing seasons of
 2014-2017. Each location was represented by 6 to 11 accessions (individual
 genotypes), and latitude and longitude were recorded for each sample.
 Seeds and stolon cuttings were cultivated in the greenhouse at Washington
 University in St. Louis under standard greenhouse conditions (see Wright
 <em>et al</em>. 2022). Genomic DNA was extracted from young
 leaves using a standard DNA extraction protocol (Whitlock<em> et
 al.</em>, 2008). Extraction quantity and quality were assessed using
 a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer, and Qubit™
 dsDNA HS Assay Kits. Genotyping-by-sequencing (GBS)
 libraries were prepared following Elshire<em> et al.</em>
 (2011) with <em>ApeK</em>I methylation sensitive restriction
 enzyme. Barcoding and protocol modifications are described in
 Olsen<em> et al.</em> (2021). Paired-end sequencing (150-bp
 reads) was performed using the Illumina Hi-Seq 2500 platform (Novogene
 Corp., Chula Vista, CA, USA). <strong>Read
 Mapping, SNP Calling, and SNP Filtering </strong>
 Raw GBS reads were demultiplexed with SABRE
 (https://github.com/najoshi/sabre) and adaptor-trimmed with CUTADAPT
 (Martin, 2011). The processed reads were mapped back to our white clover
 reference genome (Kuo<em> et al.</em>, 2024) using BWA (Li
 & Durbin, 2009) with default paired-end settings. SNP calling from
 the alignments followed GATK best practices, with omission of the
 duplicated-read removal step as recommended for GBS data (Poplin<em>
 et al.</em>, 2017). The output SNP dataset in vcf format underwent
 hard filtering (bcftools filter -e 'QD &lt; 2.0 || FS &gt;
 60.0 || MQ &lt; 40.0 || MQRankSum &lt; -12.5 || ReadPosRankSum
 &lt; -8.0 || INFO/DP &lt; 2500'). It was then filtered for
 sites with missing accessions &lt; 0.25, missing sites &lt; 0.35,
 and minor allele frequency &gt; 0.05. Missing genotypes were imputed
 with Beagle v5.4 (Browning<em> et al.</em>, 2018). A relaxed
 Hardy-Weinberg filter was applied to remove sites with skewed
 heterozygosity suggesting sequencing error (p &lt;
 1´10<sup>-50</sup>). This dataset was used for GWAS and
 genome-wide environmental association (GEA). For population structure
 analyses and genomic differentiation scans, three different levels of
 LD-pruning were performed using PLINK2 (--indep-pairwise 100kb 0.8;
 --indep-pairwise 200kb 0.5; --indep-pairwise 500kb 0.2) before analysis
 (Chang<em> et al.</em>, 2015).
description: # Beyond cyanogenesis: Temperature gradients drive environmental
 adaptation in North American white clover (*Trifolium repens* L.)
 [https://doi.org/10.5061/dryad.s7h44j1fd](https://doi.org/10.5061/dryad.s7h44j1fd) The dataset includes the vcf files using in this study, which are the documentations of the genetic polymorphism of the 415 genotypes from the 43 locations. ## Description of the data and file structure bwa_wild_DP0_diploid_hardfilter_miss0.25_maf0.05_GT.vcf.gz - A vcf file including all the polymorphic sites after filtering. See methods for detailed filtering conditions. bwa_wild_DP0_diploid_hardfilter_miss0.25_maf0.05_GT_imputed.vcf.gz - A vcf file with imputations for the missing data in bwa_wild_DP0_diploid_hardfilter_miss0.25_maf0.05_GT.vcf.gz. All the vcf files have not been subjected to LD pruning. LFMM_output_20230606.txt - LFMM result output.