2.1.2. DNA Extractions

DNA was isolated from lichen fragments of approximately 1 cm<sup>2</sup> using the PowerSoil®- htp 96 Well Soil DNA Isolation Kit (MO BIO, USA, now the DNeasy® PowerSoil® HTP 96 kit, Qiagen, Germantown, MD, USA) following Marotz et al. [45]. Prior to DNA extraction, each individual lichen fragment was cleaned in 0.85% NaCl and subsequently in sterile water.

### 2.1.3. PCR, ITS Sanger Sequencing and Assembly

PCR amplification and Sanger sequencing followed the protocols established by Dal Forno et al. [28,40]. Newly generated sequences were organized and assembled in Geneious Prime 2 January 2020. We considered as an "accepted sequence" any piece longer than 100 bp (before any trimming) that matched the target genus.

### 2.1.4. PCR, ITS Illumina Sequencing and Bioinformatic Analyses

Foliose members of *Dictyonematinae* (*Cora* and *Corella*) were processed with protocols from the Earth Microbiome Project (EMP, https://earthmicrobiome.org/protocols-andstandards/ accessed on 5 July 2017). For the ITS Illumina Amplicon Protocol, primers ITS1F [46] and ITS2 [47] were ordered with Illumina constructs, so there is no need for additional PCR steps to attach Illumina barcodes, following Caporaso et al. [48]. Each sample was amplified with PCR in triplicate [49], with reactions combined after gel electrophoresis. DNA concentrations were measured with QubitTM dsDNA Broad Range Assay Kit (InvitrogenTM, Waltham, MA, USA) and 240 ng of each PCR product was pooled into a single tube for each plate (amplicon pool). Each pool contained 96 samples plus two negatives. The quantity of negative sample added to each amplicon pool was the average used volume per plate (ranging from 11 μL to 30 μL). The post-PCR DNA yields from fresh specimens ranged from 16.7 to 54.4 ng/μ<sup>L</sup> and from 1.9 to 50.48 ng/μ<sup>L</sup> for historical samples. Amplicon pools were then cleaned with UltraClean PCR Clean-Up Kit (MO BIO, Carlsbad, CA, USA) following the manufacturer's protocol, but followed by one or more additional pool clean ups with AMPure XP beads (Beckman Coulter, Brea, CA, USA; modification from EMP protocol needed, given that only utilizing the suggested clean up kit would not be sufficient to remove primer dimers, unincorporated DNTPs, etc. when visualized in a 1% gel). The two pools (one for each plate) were then combined and a qPCR with a KAPA Library Quantification Kit for Illumina® platforms was performed (Kapa Biosystems, Wilmington, MA, USA). A 2-nM dilution was made and finally sequenced in a MiSeq Kit v2 500 cycles (paired end 2 × 250 bp; Illumina, Inc., San Diego, CA, USA) in house at the Laboratories of Analytical Biology (LAB) of the SI-NMNH (Washington, DC, USA).

Samples were de-multiplexed and processed bioinformatically using QIIME2 [50]. Sequences were de-noised using DADA2 [51]. Taxonomy was assigned with the UNITE dynamic fungal classifier [52] with the "feature-classifier classify-sklearn" option in QI-IME2. Newly generated tables were compiled into a single file with Amplicon Sequence Variation (ASV) reads, number of reads per ASV and per sample, number of times that ASV was observed across samples, and taxonomy. All analyses were run on the Smithsonian Institution High Performance Computing Cluster [53] or locally.

### 2.1.5. Microfluidics PCR, Multi-Marker Illumina Sequencing and Bioinformatic Analyses

For a subset of samples, a microfluidics PCR followed by Illumina sequencing methodology was performed following Gostel et al. [54,55], but adapted to commonly used primer combinations in lichens. This approach and its application in lichenology will be fully treated in a separate publication; but, in summary, this is a PCR-based target enrichment procedure that allows a large number of samples and primer combinations (including genetic markers for both myco- and photobionts and associated microbiome) to be amplified in a single step due to a series of microtubes that intersect, forming a central matrix of thousands of reaction chambers [55]. It is here applied for the first time in lichens. For this current study, five fungi markers were selected (EF3, mitLSU, ITS, nuLSU, nuSSU), amplified with a Microfluidic PCR on a Fluidigm Access Array at the Center for Conservation Genomics (CCG) at the Smithsonian's National Zoo (Washington, DC, USA) and sequenced on an Illumina MiSeq Kit v3 (600 cycles) at the LAB (SI-NMNH). Samples were

de-multiplexed and de-noised using QIIME2 [50], and then assigned taxonomy with a custom database with reference sequences for each of the loci sequenced.
