DOI: 10.5061/dryad.6m905qfw4
title: Limited divergent adaptation despite a substantial environmental cline in wild pea

description: Isolation by environment (IBE) is a wide spread phenomenon in nature. It
 is commonly expected that the degree of differences among environments is
 proportional to the level of divergence between populations in these
 environments. Consequentially, it is assumed that species’ genetic
 diversity displays pattern of IBE in the presence of a strong
 environmental cline if geneflow does not mitigate isolation. We tested
 this common assumption by analyzing the genetic diversity and demographic
 history of Pisum fulvum. P. fulvum inhabits very contrasting habitats in
 the southern Levant and is expected to display only minor migration rates
 between populations what makes it an ideal test case. Ecogeographic and
 subpopulation structure was analyzed and compared. Correlation of genetic
 with environmental distances was calculated to test the effect of IBD and
 IBE and detect the main drivers of these effects. Historic effective
 population size was estimated using stairwayplots. Limited overlap of
 ecogeographic and genetic clustering was observed, and correlation of
 genetic with environmental distances was statistically significant yet
 small. We detected a sharp decline of effective population size during the
 last glacial period. The low degree of IBE may be the result of genetic
 drift due to the past bottleneck. Our findings contradict the expectation
 that strong environmental clines cause IBE in the absence of extensive
 geneflow.
description: <strong>Plant material and habitat
 characteristics</strong><br> Naim-Feil et al. (2017) collected
 135 P. fulvum accessions spanning its documented ecological amplitude in
 Israel and the Palestinian Territories. Our collection was comprised of
 126 of these accessions (Supplementary Table. S1). Within our study area,
 comprising Israel and the Palestinian Territories, it occupies a wide
 range of different habitats. In these habitats average temperature in the
 main growing season during winter ranges from 4.0°C to 14.3°C. The annual
 precipitation, which occurs almost entirely during the winter months,
 spans a range from 295 mm to 914 mm. The echo system of each sampling
 site, i.e., the climate conditions, soil content, and the vegetative
 vicinity is tabulated in Supplementary Table S1. For more detailed
 description of P. fulvum habitats see Abbo et al. (2013). We are unaware
 of published census size data for wild pea of which is rather difficult to
 assess. The number of individuals in a population strongly vary over
 years, and often in certain locations, no plants can be found in some
 years. Yet, P. fulvum, like most Mediterranean legumes, produces seeds
 exhibiting strong physical dormancy, and wild populations build
 considerable soil seed banks (Abbo et al., 2011; Berger, Shrestha,
 & Ludwig, 2017; Smýkal, Vernoud, Blair, Soukup, &
 Thompson, 2014). These seed banks allow repopulation after years with low
 germination rates. Flowers of P. fulvum are usually cleistogamous. Yet,
 flowers can open in later stages and they have been documented to
 potentially be prolific after opening (Bogdanova & Berdnikov,
 2000). Although Pisum sp. are mostly considered as selfing species, the
 degree of self-fertilization is debated (Polowick et al., 2002, Smýkal et
 al., 2018). Due to its selfing nature and its big seeds is can be expected
 that long range geneflow in P. fulvum is unlikely and populations are well
 diverged, similar to the situation in Pisum sativum ssp. elatius, the only
 other wild pea species (Smýkal et al., 2018).
 <br> <strong>Genotyping by
 sequencing</strong><br> DNA samples from individuals of the
 association panel were digested by MspI/PstI restriction enzymes.
 Genotyping-by-sequencing (GBS) libraries were prepared by ligating the
 digested DNA to unique nucleotide adapters (barcodes) using 48-plex,
 followed by PCR amplification. Libraries were placed in an Illumina
 NextSeq 500 instrument using 75-base single-end sequencing at the LGC
 Company. Preprocessing was performed by the LGC Company who generated the
 GBS sequence reads as follows. All library groups were demultiplexed using
 the Illumina bcl2fastq (v2.17.1.14) software. No mismatches or Ns were
 allowed in the inline barcodes, but Ns were allowed in the restriction
 site. Sequencing adapter remnants were clipped from all raw reads. Reads
 with final length &lt;20 bases were discarded and reads with 5’ ends
 not matching the restriction enzyme sites were filtered out. The high
 quality reads were aligned to the reference genome of Pisum sativum
 (Kreplak et al., 2019) using the Burrow-Wheelers Alignment tool (Version
 0.5.9; Li & Durbin, 2009) with default alignment parameters, the
 alignment data was processed with SAMtools (version 0.1.19; Li et al.,
 2009). Sequence variants were identified using freebayes (version
 1.1.0-50-g61527c5; Garrison & Marth, 2012). The combined read
 depth of 10 was used across samples for identifying an alternative allele
 as a variant, with the minimum mapping and base quality filters of 1 and
 3, respectively. Subsequent filtering steps included only biallelic SNPs
 with genotype calls across the population ≥ 50% and a minor allele
 frequency (MAF) ≥ 0.01. The QUAL estimate was used for estimating the
 phred-scaled probability. Sites with a QUAL value less than 20 were
 removed. The filters were applied using VCFtools (Danecek et al., 2011)
 and in-house Perl scripts. Afterwards, missing genotype calls were imputed
 with Beagle (version 5.0; Browning, Zhou, & Browning, 2018;
 Browning & Browning, 2007) using 30 iterations, 10 burn-in steps
 and an effective population size of 50,000 because P. fulvum presents
 rather small population sizes with high inbreeding rates, so the default
 value was not appropriate. We used individual’s heterozygosity rates to
 test for potential sample contamination or multilocus mapping. A deviation
 of more than three standard deviations from the mean heterozygosity rates
 were considered as too high. All accessions passed this criterion. Linkage
 disequilibrium (LD) pruning was conducted with PLINK (version 1.90b5.4;
 Purcell et al., 2007). LD was estimated by r2 in a sliding windows
 approach. 50 kb windows were shifted by 5 variants in each step and SNPs
 within one window with r2 ≥ 0.2 were dropped. After application of all
 filters, we obtained 5144 SNPs in total and 3951 LD pruned SNPs.