Field Sampling and Sequencing
Field collections of plant material were made throughout the coastal and
inland PNW temperate rainforest for western redcedar, Thuja
plicata, and western hemlock, Tsuga heterophylla, between April
and June of 2016 and 2018. Fresh tissue for specimens was dried and
stored in silica gel. Voucher specimens of collections were preserved in
the Stillinger herbarium and can be located on the Consortium of Pacific
Northwest Herbaria data portal (http://pnwherbaria.org). Leaf
tissue from 137 Thuja plicata individuals (Fig. 1, Table S1) and
48 Tsuga heterophylla (Fig. 1, Table S2) individuals were
extracted using a modified CTAB protocol (Doyle & Doyle 1987), purified
using Sera-Mag SpeedBeads (Thermo Fisher Scientific; Rohland & Reich
2012), and their DNA quantified using a Qubit 2.0 Fluorometer (Life
Technologies).
Three double-digest restriction site associated DNA sequencing
(ddRADseq) libraries (Peterson et al. 2012) were prepared. Two of
these were for Thuja plicata , assigning half of the samples to
one or the other, and one for Tsuga heterophylla . For theThuja plicata libraries, the restriction enzymes used wereEcoRI and SbfI , while they were SbfI andMspI (New England Biolabs, USA) for Tsugaheterophylla . All libraries were size selected using a size
window of 200-500 bp using a BluePippin (Sage Science). All digestion,
ligation and PCR products were purified using Agencourt AMPure XP
purification system (Beckman Coulter). For Thuja plicata ,
sequences were generated as 50 bp single end reads using an Illumina
HiSeq 2500 at the Berkeley sequencing facility. For Tsuga
heterophylla , sequences were generated as 150 bp paired-end reads using
an Illumina HiSeq 4000 at The Ohio State University Wexner Medical
Center. Raw sequences were processed using Ipyrad (Eaton 2014, Eaton &
Overcast 2020) with a minimum coverage of 10 and clustering threshold of
0.80. Ipyrad includes Vsearch (Rognes et al. 2016) and Muscle
(Edgar 2004) for sequence clustering. Though we had overlapping reads
for Tsuga heterophylla , we opted to not merge them and only used
single-end reads. Complete assembly procedures were performed and
documented in Jupyter notebooks
(https://github.com/ruffleymr/ThujaTsugaAnalyses)