The study area of this project is the Pacific Northwest bioregion of northwestern North America with an emphasis on Washington state. The target habitats covered for the project include temperate rainforests on the west slope of the Cascade Range in Washington and Oregon, temperate rainforests on the Olympic Peninsula in Washington state, and maritime forests in southwestern Alaska in the United States.
To assess the current distribution of P. rainierensis herbarium specimens dating from 1948 to 2012 were reviewed via the Consortium of North American Lichen Herbaria (CNALH). Revisits to 53 historic collection sites were conducted from June 2021 to September 2022 in the Gifford Pinchot, Olympic, and Mount Baker-Snoqualmie National Forests and Olympic, Mount Rainier, and North Cascades National Parks in Washington. During revisits presence/absence, abundance, and element occurrence data were collected.
To examine the population genetic structure of the species 96 thallus fragments and 1 whole thallus were collected from 11 populations located in the Gifford Pinchot, Olympic, and Mount-Baker Snoqualmie National Forests in Washington. Populations were defined as spatially discrete groups of individuals occupying a single forest stand (Devkota et al 2014). Whenever possible one thallus fragment (3 cm2) per carrier tree was randomly collected. The total number of thallus fragments collected was dependent on the local abundance of the species and no more than three thallus fragments were collected from a single carrier tree. Tissue samples were collected using nitrile gloves then kept in acid-free paper packets, air dried, and stored at 32oF (0oC).
DNA extractions were performed for genome sequencing and double digest restriction-site associated DNA sequencing (ddRAD-seq). 16 extractions from the whole thallus were completed to sequence the reference genome. One extraction was completed for each thallus fragment to reach a total of 96 extractions for ddRAD-seq. All extractions were conducted according to the manufacturer’s protocols using QIAGEN Plants Pro Kits.
Library preparation and whole genome sequencing was performed using the Oxford Nanopore MinIon Platform according to protocols outlined in Pfeffer et al. 2023 and the manufacturer’s protocols. Base calling was conducted with Guppy v5.0.7 in SUP (super high accuracy) mode. Reads were assembled using Flye v2.9.1 (Kolmogorov et al 2019). Contigs ascribable to the mycobiont nuclear genome were identified and metagenomic filtering was conducted using the modified blobology pipeline outlined in Pfeffer et al. 2023. Genome assembly completeness was assessed with BUSCO v5.4.4 and basic assembly metrics were calculated with Quast v5.2 (Manni et al 2021, Mikheenko et al 2019).
Library preparation for ddRAD-seq was performed at the University of Colorado Boulder (Boulder, Colorado USA) according to protocols described in Tripp et al. 2017. Sequencing was performed at the University of California, Riverside Genomics Core Facility (Riverside, California USA) using an Illumina NexSeq 500. A total of 259,603,356 raw reads were obtained. Quality control, demultiplexing, mapping, and single-nucleotide polymorphism (SNP) calling were conducted using protocols outlined in Alonso‐García et al. 2022. The process-radtags pipeline of Stacks v2.3 was used to demultiplex, filter, and trim reads obtained from ddRAD-seq (Alonso–Garcia et al 2022, Flynn et al. 2020). Reads were filtered based on a quality value of 24 using Phred+33 encoding. All filtered reads were trimmed to a minimum length of 40 base pairs. Up to 68% of the reference genome is comprised of interspersed repeats. RepeatModeler2 and RepeatMasker v4.1.4 were used to screen and mask repeats in the genome prior to mapping and SNP calling (Flynn et al. 2020, Smit et al. 2020). Processed reads were mapped to the masked reference genome using the alignment tool bwa v0.7 and the gstacks and populations pipelines in Stacks v2.3 to generate output loci (Alonso-Garcia et al 2022, Li & Durbin et al. 2009). Only loci present in 50% of the individuals sampled (R – 0.05) with a minimum minor allele frequency (MAF) of 0.02 were retained. The resulting output was comprised of 828 SNPs representing genomic data for 61 individuals.