**5. Conclusions**

We conclude that DNA barcoding in the foliose genera *Cora* and *Corella* is indeed a success story, considering that our knowledge of this group of basidiolichens has increased from a single perceived species to one of the largest genera of lichen-forming fungi, detected largely through rigorous application of the fungal ITS barcoding marker within our integrative approach, combining: (1) visual inspection of the molecular data (alignments + phylogeny); (2) phenotypic and anatomical data; and (3) ecology, including habitat and substrate preferences. Almost all species of *Cora* and *Corella* have been documented molecularly from the onset, resulting in a broad, near-complete, and steadily-growing phylogenetic framework where additional sequence data can be quickly added, particularly those obtained from further historical specimens, including types.

Our highly successful HTS results indicate that ITS barcoding of collections collected from a couple decades to over a century ago can be used to extend our knowledge of species-level diversity. In particular, the addition of archival samples not only lead to the discovery of novel lineages but made the phylogenies more robust, helped to better assess species distributions, and thus remarkably improved our understanding of levels of inferred endemism.

The high number of species now recognized in *Cora* is not surprising if compared to large lichenized genera in Ascomycota. What is extraordinary is that the numerous species are not actually cryptic. We emphasize that our broad phylogenetic sampling was only partially responsible for this change, with assessments of the genus in the field being equally important in this case. The key element to uncover this previously unrecognized diversity was our integrative approach, which allowed us to test the reliability of novel taxonomic characters and question classic concepts about environmental variation, in which the addition of historical specimens was invaluable. Traditional concepts largely ignored phenotypically distinct characters, dismissing them as variability. However, it is important to acknowledge that we are at a privileged position in research, where we can employ molecular data to test these phenotypic-based hypotheses. At first glance, phenotypical characters seem less clear in basidiolichens, including its variation and the lack of features usually applicable to lichens in general. In ascolichens, traditional concepts have often focused on both morphological adaptations resulting from the lichen symbiosis as a whole and on characteristic features of the mycobiont. We postulate that the patterns of diversification seen in *Cora*, namely that phenotypic variation previously regarded as environmentally triggered is actually diagnostic of species-level lineages, will also hold true for many ascolichen genera (or for some lineages within) that have not ye<sup>t</sup> been broadly assessed with molecular methods.

To assess evolutionary patterns related to so-called cryptic speciation, we, therefore, found it useful in this study to introduce more precise definitions of our concept of "crypticity", taking into account that species may often appear similar but are nevertheless not actually identical (kapo-(phylo-)cryptic vs. eu-(phylo-)cryptic), that phenotypically similar species might nevertheless not be closely related (pseudo-(phylo-)cryptic), and that species distributions patterns (allo- vs. sympatric) must be taken into account when looking at this phenomenon (allo-(phylo-)cryptic). These definitions might prove useful when evaluating cases of cryptic speciation in other lichens, or even more broadly across fungi, even though, in the case of *Cora*, the majority of the species should no longer be considered cryptic, but instead an example where phenotypic variation had not been ye<sup>t</sup> linked with genetic divergence.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/d14040284/s1, Figure S1: ITS full-length ML tree; Table S1: List of material used in this study; Table S2: Species utilized on the phenotypic assessment and their GenBank number; Table S3: Scores for the phenotype matrix (20 characters) for the 89 species utilized; Table S4: Full species delimitation results for all specimens; and 11 supplemental files containing data analyzed (File S1: ITS sequence alignment utilized as input in the ML analyses; File S2: Sixmarker trees utilized as input in ASTRAL-III, the map file to delimit species in ASTRAL and all six alignments utilized to generate the respective trees; File S3: Only the separated corresponding ITS alignment with the same terminals as ASTRAL six-marker data; File S4: Subset of the ITS sequence data limited to the genus *Cora*, retaining only terminals with less than 30% gaps; File S5: Subset of 758 terminals with (near) complete ITS sequences, similar to the *Cora* subset File S4, but also including *Acantholichen* and *Corella*; File S6: BLAST performance of dataset 1—with the ITS1 region only; File S7: BLAST performance of dataset 2—entire ITS region of all original terminals with less than 10% gaps, including Illumina ASVs; File S8: BLAST performance of dataset 3—with the ITS2 region only; File S9: BLAST performance of dataset 4—with a short terminal region of the ITS2; File S10: Alignment with 89 target sequences for the phenotypic assessment; File S11: Distance matrix utilized for the barcoding gap analyses).

**Author Contributions:** Conceptualization, M.D.F., J.D.L., M.G. and R.L.; methodology and analyses, M.D.F., R.L.; lab work, M.D.F., B.M.; funding acquisition, M.D.F., J.D.L., F.B., E.S. and R.L.; first manuscript versions, M.D.F., J.D.L. and R.L.; writing, reviewing and editing, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Science Foundation (DEB 0841405 and PRFB 1609022), the Smithsonian Institution (SI) and the National Museum of Natural History (Peter Buck Postdoctoral Fellowship), the Global Genome Initiative and the SI DNA Barcode Network.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in Supplementary Materials and deposited at GenBank.

**Acknowledgments:** M.D.F., J.D.L. and R.L. would like to thank the National Science Foundation for financial support and all collaborators who have helped in field expeditions, as well as those who sent material throughout the years. M.D.F. also wishes to thank the Smithsonian Institution and the National Museum of Natural History for a Peter Buck Postdoctoral Fellowship, a gran<sup>t</sup> from the SI DNA Barcode Network (FY18 Award Cycle, PI MDF) and an award from the Global Genome Initiative (GGI-Exp\_Sci-2016-069, PIs ES and MDF). M.D.F. would also like to thank Katherine (Katie) Murphy, Niamh Redmond, José Zuniga, Jeffrey Hunt, Jonathan (Jon) Coddington, Sergei Drovetski, Kenan Matterson, Gabriel (Gabe) Johnson, and Julia Steier, for their help in the LAB and project support at the SI-NMNH. Nancy McInerney is thanked for her help and permission to utilize equipment at the CCG. Morgan Gostel and Jerrod Stone from the Botanical Research Institute of Texas are acknowledged for their help with the microfluidics PCR and assessment of herbarium Sanger sequences, respectively. Galapagos specimens examined as part of this study were collected and examined from 2009 to 2013 as part of an NSF sponsored project (DEB 0841405), a project included in the Annual Operative Plan as part of the Galapagos Lichen Inventory by the Charles Darwin Foundation (CDF), approved at the time by the Directorate of the Galapagos National Park (DPNG). We are particularly grateful to successive scientific directors of the research station and technical directors of the Galapagos National Park for their continued support. The Galapagos Lichen Inventory is now part of the national biodiversity assessment "Biodiversidad Genética del Ecuador", led by the Instituto Nacional de Biodiversidad del Ecuador (INABIO). For research and specimen permits we are particularly indebted to Danny Rueda, Daniel Lara Solís, Galo Quedaza and Victor Carrión from DPNG, and Diego Inclán, Francisco Prieto and Rosa Batallas (INABIO). This publication is contribution number 2447 of the Charles Darwin Foundation for the Galapagos Islands. Lastly, we thank the reviewers for their contributions to the improvement of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
