Genetic analysis
Genomic DNA was extracted from ~0.2g silica-dried leaf
tissue from all 192 genotypes (12 genotypes per population, 16
populations) using the Thermo Scientific MagJET Genomic DNA Kit (Thermo
Scientific). Double-digest restriction-associated DNA (ddRAD) libraries
were prepared using 2-5ng of DNA per sample in 20μL reactions following
a modified Peterson et al. (2012) protocol. Briefly, restriction
and ligation was carried out simultaneously in 20μL reactions using
restriction enzymes MspI and EcoRI and universal adapter sequences for
indexing PCR. Ligation products were amplified using 25 cycles of PCR.
After indexing, products were checked on an agarose gel and purified.
Samples were then pooled and size selected for fragments between 200 and
350bp using a Pippin Prep (Sage Science, Inc., Beverly, MA). The
size-selected pool was quantified by qPCR and sequenced on an Illumina
MiSeq Desktop Sequencer (Illumina, Inc. San Diego, CA) in 2x75 mode.
Sequence reads were processed using a modified Stacks pipeline (Catchenet al. 2013; Andrews 2018). Potential chloroplast and
mitochondrial sequences were filtered from the dataset by comparing them
to other Populus sequences downloaded from GenBank. Specifically,
we removed sequences that matched chloroplast sequences from P.
fremontii and mitochondrial sequences from P. tremula x P.
alba. Parameter values for clustering were based on tests following
parameterization in Mastretta-Yanes et al. (2015). The minimum
stack depth for each individual was three and the minimum number of
individuals per locus cluster was three.
To ensure we measured FST for putatively neutral loci,
all SNP loci included in the measure of FST were tested
and found to be in Hardy-Weinberg equilibrium. Values of
QST were compared to the distribution range of
FST estimates and not the mean (Whitlock 2008; Leinonenet al. 2013). In order to obtain a 95% confidence interval
around FST, population-level pairwise
FST values were bootstrapped 500 times using thedivPartCalc function in the R package diversity (Keenanet al. 2013).