Genetic analysis
Genomic DNA was extracted from ~0.2g silica-dried leaf
tissue from all 192 genotypes 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
(Catchen et 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. All loci were used in the
measure of FST were found to be in Hardy-Weinberg
equilibrium. To calculate FST and a 95% confidence
interval around FST, we bootstrapped population-level
pairwise FST values 500 times using thedivPartCalc function in the R package diversity (Keenanet al. 2013).