DOI: 10.5061/dryad.zpc866t84
title: RAD-seq data from: Evidence of local adaptation despite strong drift in a Neotropical patchily distributed bromeliad

description: Both genetic drift and divergent selection are predicted to be drivers of
 population differentiation across patchy habitats, but the extent to which
 these forces act on natural populations to shape traits is strongly
 affected by species' ecological features. In this study, we infer the
 genomic structure of Pitcairnia lanuginosa, a widespread herbaceous
 perennial plant with a patchy distribution. We sampled populations in the
 Brazilian Cerrado and the Central Andean Yungas and discovered and
 genotyped SNP markers using double-digest restriction-site associated DNA
 sequencing. In addition, we analyzed ecophysiological traits obtained from
 a common garden experiment and compared patterns of phenotypic and genetic
 divergence (PST-FST comparisons) in a subset of populations from the
 Cerrado. Our results from molecular analyses pointed to extremely low
 genetic diversity and a remarkable population differentiation, supporting
 a major role of genetic drift. Approximately 0.3% of genotyped SNPs were
 flagged as differentiation outliers by at least two distinct methods, and
 Bayesian generalized linear mixed models revealed a signature of isolation
 by environment in addition to isolation by distance for
 high-differentiation outlier SNPs among the Cerrado populations. PST-FST
 comparisons suggested divergent selection on two ecophysiological traits
 linked to drought tolerance. We showed that these traits vary among
 populations, although without any particular macro-spatial pattern,
 suggesting local adaptation to differences in micro-habitats. Our study
 shows that selection might be a relevant force, particularly for traits
 involved in drought stress, even for populations experiencing strong
 drift, which improves our knowledge on eco-evolutionary processes acting
 on non-continuously distributed species.
description: <strong>ddRAD
 sequencing and de novo assembly </strong> Total genomic DNA was extracted
 from leaf samples of 115 individuals from 19 populations of
 <em>Pitcairnia lanuginosa </em>(Bromeliaceae) using the Qiagen
 DNA Plant Mini kit (Qiagen, Finland). Sample quality and concentration
 were checked on 1% agarose gels and using a NanoDrop 2000
 spectrophotometer (Thermo Scientific) and normalized to 60 ng/ul as
 quantified by the Qubit dsDNA BR assay (Invitrogen). The ddRAD libraries
 were prepared and sequenced on the Ion Torrent Proton platform using a
 modified version of Peterson et al.’s (2012) protocol at the Genome
 Transcriptome Facility of Bordeaux (INRAE, Cestas, France). In brief, we
 digested 50ul (300 ng total) of genomic DNA using rare- and
 frequent-cutter restriction enzymes (PstI and MspI, New England
 Biosciences) at 37° C for two hours and inactivated them at 80°C for 20
 minutes. After bead-purification, a P1 adaptor (the same for all samples)
 and a unique barcode adaptor were added to each sample, and ligation was
 performed at 22°C for 2 hours followed by inactivation at 65°C for 20
 minutes. We then used a qPCR to quantify each sample before normalizing
 and pooling samples in a 48-plex. We employed an automated size-selection technology
 (Pippin, Sage
 Science) to select fragments with the expected size (270-290
 pb). Each library was then amplified by PCR, quantified at
 the Agilent 2100 Bioanalyzer, and sequenced on the Ion Torrent Proton P1v2
 chip. We analyzed
 raw read data using PYRAD v3.0.5 software (Eaton 2014). Following
 preliminary testing, we settled the parameters as follows: a maximum
 number of sites with quality score &lt;20 (NQual): 5, clustering
 threshold (Wclust): 0.85, minimum coverage for a cluster (Mindepth): 6,
 and maximum individuals with a shared heterozygous site (MaxSH): 3. All
 other parameters were kept at default values. PYRAD was run on the
 GenoToul bioinformatics
 facility (INRA, Toulouse,
 France). <b>Ecophysiological traits</b> To detect differences in plant responses to light, heat, and water stresses among populations, we conducted ecophysiological trait measurements in a total of 24 individuals (herein genets) from eight out of the 19 <i>P. lanuginosa</i> populations sampled (three genets/population); these eight populations represent the extent of the geographic distribution of the species in the Cerrado. Adult plants were sampled in the field and acclimated to the greenhouse for at least one year (from May 2014 to May 2015). We separated each genet into two or more ramets to grow in distinct pots with the same substrate composition for an additional acclimation period of three months prior to the experiments. We then selected 48 ramets of similar size lacking inflorescences and submitted them to the following treatments (24 ramets/treatment): (A) control, consisting in watering pots every two days (about 80 mL of water, which corresponded to field capacity); and (B) water shortage, consisting in no watering for 30 days. We measured ecophysiological traits based on photosynthetic responses of leaves in August 2016 (during the dry season). We selected an area of ca. 2 cm<sup>2</sup> on a fully developed leaf on each ramet from the control treatment to measure the photosynthetic response to light (i.e. rapid light curve) and to heat stress using a modulate fluorometer (MINI-PAM-II, Walz). We calculated the following parameters: upper critical thermal limit (CT<sub>max</sub>), sensitivity to temperature change (<i>z</i>), maximum relative electron transport rate (ETR<sub>max</sub>), initial slope of the curve of light-response (α), light saturation coefficient (Ik), and maximal photochemical efficiency (<i>F</i><sub>v</sub>/<i>F</i><sub>m</sub>). To calculate heat tolerance parameters CT<sub>max</sub> and <i>z</i>, we followed the framework described by Rezende et al. (2014), which considers the intensity and duration of thermal stress to drive the individual 'thermal tolerance landscape', or individual capacity to withstand heat at any given temperature. We measured the critical time duration under a static stressful temperature that promotes a 50% decay of the initial <i>F</i><sub>v</sub>/<i>F</i><sub>m</sub> (D<sub>50</sub>) in each leaf sample. To draw the ‘thermal tolerance landscape’ of each ramet, we calculated D<sub>50</sub> under four distinct temperatures (40, 45, 50, and 60°C) and performed a linear regression to measure CT<sub>max</sub> and <i>z</i> parameters. Measures of <i>F</i><sub>v</sub>/<i>F</i><sub>m</sub> at each temperature were simultaneously carried out using a thermal-gradient block. Finally, we used the decay of each heat and light parameter (i.e., values for ramets under controlled conditions minus the values under water shortage, divided by values under controlled conditions) as proxies of drought adaptation. 
description: <strong>De novo assembled RAD data</strong>
 The compressed folder pyrad_allpops.rar contains the parameters
 file and the result files from the de novo assembly of 115 individuals
 from 19 populations of Pitcairnia lanuginosa sampled in the Brazilian
 Cerrado and the Andean Yungas using the software PyRAD v3.0.5.
 The compressed folder pyrad_cerrado.rar contains the parameters
 file and the result files from the de novo assembly of 95 individuals from
 16 populations of Pitcairnia lanuginosa sampled in the Brazilian Cerrado
 using the software PyRAD v3.0.5.
 <strong>Ecophysiological traits</strong>
 The file pitcairnia_traits.xlsx contains information on 11
 physiological traits related to heat, light, and drought stresses measured
 in 24 individuals from a subset of eight populations of Pitcairnia
 lanuginosa sampled in the Brazilian Cerrado. CT<sub>max</sub>
 = Upper critical thermal limit, <i>z</i> = sensitivity to
 temperature change, ETR<sub>max</sub> = maximum relative
 electron transport rate, α = initial slope of the curve of light-response,
 Ik = light saturation coefficient, and
 <i>F</i><sub>v</sub>/<i>F</i><sub>m</sub> = maximal photochemical efficiency. The decay of each of these traits under drought stress is