*3.2. ITS-Based Phylogeny*

Our new ITS tree with 1325 sequences contained 1091 unique samples, collected from 29 different countries (Figures 5 and S1). By utilizing historical specimens, we were able to add records for an additional 11 countries for which the diversity of *Cora* and *Corella* species was previously unknown (Figure 2; a singular fresh collection from Sri Lanka not mapped, see [33]).

**Figure 5.** ITS fungal barcoding tree inferred by maximum likelihood for the genus *Cora*, including the *Acantholichen*-*Corella* clade as outgroup (1325 terminals), and two examples of the results of species delimitation approaches. *Cora hawksworthiana* (**a**) is currently considered a single species, but could potentially be further divided into two—one from Brazil and another for Chile, Colombia, and Costa Rica—based on the ad hoc, GMYC, DNADIST, and ABGD-231 approaches. In contrast, *Cora reticulifera* (**b**), an abundant common species from southeastern Brazil and Uruguay, with a uniform phenotype and ecology, was delimited as a single species by all methods, except in bPTP, which recovered each sample as a separated species. For the full-length tree as well as species delimitations for all specimens, see Figure S1 and Table S4.

Given this information, we were able to extend the distribution of six species in *Corella* and 28 in *Cora*; for example, *Cora davibogotana* previously only known from Colombia, is now also reported from Venezuela; and *Cora* spec-84, before known exclusively from Brazil, is now also known from Uruguay (Figure S1). In all cases, range extensions were to adjacent countries. Countries such as Ecuador, Jamaica, Mexico, Colombia, and Brazil, continue to be well represented by fresh specimens, while for other countries, the addition of new specimens from the herbarium was invaluable. For example, for Panama, we added 18 herbarium collections to compliment the one available fresh specimen, now corresponding to seven or eight species as opposed to just one previously known from the country. Overall, approximately 25–30 additional novel lineages were detected among the herbarium collections alone; however, since these require further studies to be formally described (as either species or at infraspecific level, following the approach proposed by Lücking et al. [34]), they will be treated in a separate publication.

### *3.3. Comparison between ITS and Astral Six-Marker Tree*

Our microfluidics PCR followed by Illumina sequences yielded 239 novel sequences belonging to *EF3*, mtLSU, nuLSU and nuSSU. ITS data were also highly successful with this approach; however, we already had those sequenced with other methods. Our ITS-based ML tree and the ASTRAL six-marker coalescent tree exhibited very similar topologies (Figure 6), with a normalized Robinson–Foulds distance of 0.0278, suggesting that an ITS-based phylogeny reliably recovers a multi-marker phylogeny in this particular genus.

**Figure 6.** Comparison between inferred ASTRAL six-marker species tree (**a**) and ITS maximum likelihood tree (**b**). The different colors simply denote the two major clades in *Cora*, with *Corella* in black as the outgroup.
