2.2.1. ITS-Based Phylogeny

A total of 1325 ITS sequences were assembled to infer relationships within the *Acantholichen*-*Corella*-*Cora* clade (Table S1). *Acantholichen* was included as it represents the sister clade of *Corella*, with *Acantholichen* and *Corella* together sister to *Cora* [28]. Of the 1325 sequences, 28 represented *Acantholichen*, 54 *Corella*, and the remaining 1243 the genus *Cora*. We used a previously published alignment focusing on the genus *Cora* [33] as template and added further and newly generated sequences with MAFFT 7 "–add" [56] using the online server [https://mafft.cbrc.jp/alignment/server/add\_sequences.html]. The resulting alignment was manually inspected and, after running an initial phylogenetic analysis (see below for settings), terminals were sorted in a phylogenetic order and the alignment reinspected. This approach was repeated several times, until no further alignment inconsistencies were detected. Ambiguously aligned regions were found to be short and were detected only in a broader context between distantly related taxa, whereas closely related taxa within supported subclades did not show notable alignment ambiguities, as assessed through the Guidance2 Server [57] (http://guidance.tau.ac.il/) for smaller subsets of the data. Therefore, ambiguously aligned regions were not removed. The final alignment contained 1325 sequences and was 916 bases long after terminal trimming (File S1). Of these 1325 sequences, 965 were generated with Sanger, 330 with Illumina, and 30 with 454-pyrosequencing approaches. Sequence length (after trimming) varied between 86 and 724 bases (File S1). Short sequences generally corresponded to the ITS1 region obtained through Illumina and 454 pyrosequencing, but some short Sanger sequences covered only the ITS2 region.

We employed RAxML 8 [58] on the CIPRES Science Gateway [59] to reconstruct a maximum likelihood phylogeny based on the ITS data, using the universal GTRGAMMA model and bootstrap pseudo-replicates automatically determined through the RAxML Black Box on the CIPRES server. The resulting tree was visualized in FigTree 1.44 (http: //tree.bio.ed.ac.uk/software/figtree/). Using this tree, species-level clades (i.e., species hypotheses) were delimited ad hoc using a combination of stem branch length, support, ecology (substrate, habitat) and distribution of the corresponding terminals, without taking into consideration their morphology.
