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RAD-seq data from: Evidence of local adaptation despite strong drift in a Neotropical patchily distributed bromeliad

Citation

Simões Santos Leal, Bárbara et al. (2021), RAD-seq data from: Evidence of local adaptation despite strong drift in a Neotropical patchily distributed bromeliad, Dryad, Dataset, https://doi.org/10.5061/dryad.zpc866t84

Abstract

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.

Methods

ddRAD sequencing and de novo assembly 

Total genomic DNA was extracted from leaf samples of 115 individuals from 19 populations of Pitcairnia lanuginosa (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 <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).

Ecophysiological traits

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 P. lanuginosa 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 cm2 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 (CTmax), sensitivity to temperature change (z), maximum relative electron transport rate (ETRmax), initial slope of the curve of light-response (α), light saturation coefficient (Ik), and maximal photochemical efficiency (Fv/Fm). To calculate heat tolerance parameters CTmax and z, 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 Fv/Fm (D50) in each leaf sample. To draw the ‘thermal tolerance landscape’ of each ramet, we calculated D50 under four distinct temperatures (40, 45, 50, and 60°C) and performed a linear regression to measure CTmax and z parameters. Measures of Fv/Fm 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. 

Usage Notes

De novo assembled RAD data

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.

Ecophysiological traits

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. CTmax = Upper critical thermal limit, z = sensitivity to temperature change, ETRmax = maximum relative electron transport rate, α = initial slope of the curve of light-response, Ik = light saturation coefficient, and Fv/Fm = maximal photochemical efficiency. The decay of each of these traits under drought stress is 

Funding

Fundação de Amparo à Pesquisa do Estado de São Paulo, Award: 2014/15588-6

Fundação de Amparo à Pesquisa do Estado de São Paulo, Award: 2016/04396-4

Fundação de Amparo à Pesquisa do Estado de São Paulo, Award: 2014/08087-0

Fundação de Amparo à Pesquisa do Estado de São Paulo, Award: 2016/20273-0

Agence Nationale de la Recherche, Award: CEBA:ANR-10-LABX-25-01

Conselho Nacional de Desenvolvimento Científico e Tecnológico, Award: 300819/2016-1

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Award: 001