**3. Results**

### *3.1. Comparison of Sequence Performance between Fresh and Historical Collections*

The two Illumina MiSeq runs yielded the following raw data:

**Run 01** (Plates 1 and 2): 331.6 MB (I1 or Indexes), 3.35 GB (R1 or forward reads) and 3.94 GB (R2 or reverse reads) (samples: NSF-001–106 and US-001–086); 8417853 total sequences; **Run 02** (Plates 3 and 4): 289 MB (I1 or Indexes), 3.06 GB (R1 or forward reads) and 3.55 GB (R2 or reverse reads) (samples: US-087–278); 11250308 total sequences.

With amplicon sequencing in the Illumina system, we were able to successfully sequence 76.3% or 209 of the 274 historical specimens and 93.6% or 58 of the 62 fresh specimens. With Sanger sequencing, these numbers were substantially lower: 19% of the historical and 58.1% of the fresh specimens (Figure 3a). The difference was, thus, particularly marked for the historical specimens, with a 75% decrease in success between methods, compared to a 38% decrease for the fresh specimens. The pattern was largely consistent among the substrata where these lichens were growing (Figure 3b), although, for the rock substrate, Sanger fresh and historical success rates were more similar.

**Figure 3.** Overall ITS Success Rate in Illumina versus Sanger sequencing with historical and fresh samples (**a**), including a comparison by substrate (**b**). Color pallet proposed by Wong [77].

We did not find a linear pattern in sequencing success dependent on time of historical collections for the Illumina or Sanger sequencing, but overall there was an increase in success for the more recent specimens (Figure 4a,b). There was also no difference in sequencing success when analyzed by habitat in which the specimen was originally collected (Figure 4c), although this information was only available for 17% of the historical samples. Given the high rate of success even for older specimens in the two studied genera, sequencing success may also depend on how the material was collected, dried, and preserved, thus affecting potential initial DNA degradation independent of how long the material

has been stored. Unfortunately, data on collection and preservation methods were not available, but we used visual inspection of the condition of the material as a proxy: most successfully sequenced specimens appeared in good condition, as if preserved recently, their colors mostly grey to white-ish, whereas unsuccessfully sequenced specimens often showed discolorations, typically turning yellowish brown. Such discolorations, likely associated with oxidation and DNA degradation, are usually caused when material is collected in the hydrated stage and pressed before fully air-dried, or when a heat source has been used to dry the material, sometimes also in combination with placing the material in alcohol prior to heating, a commonly applied technique in the past.

**Figure 4.** Percentage of success in sequencing historical samples between the two tested sequencing platforms (Illumina, Sanger) by year (**a**), by period of time (**b**), and vegetation type (**c**).

Beyond the target mycobiont sequences, most Illumina-sequenced samples also produced ASVs for the most commonly found contaminants, such as *Penicillium*, *Cladosporium*, *Fusarium,* and *Aspergillus* (all Ascomycota). These fungi may already be present in the living lichen specimen but more commonly originate during the drying process or subsequent preservation. In addition, fungi typically occurring in the substrate where lichens were collected (e.g., soil), as well as human-related contaminants were found, including *Trichobolus* (Ascomycota), *Mortierella* (Mucoromycota), and *Wallemia* (Basidiomycota), despite the washes performed prior to DNA extraction. Beyond that, many mushroom species were detected, most likely by the presence of spores in the samples, as well as several basidiomycetous yeasts in the class Tremellomycetes.
