*Article* **DNA Barcoding of Fresh and Historical Collections of Lichen-Forming Basidiomycetes in the Genera** *Cora* **and** *Corella* **(Agaricales: Hygrophoraceae): A Success Story?**

**Manuela Dal Forno 1,2,\*, James D. Lawrey 3, Bibiana Moncada 4,5,6, Frank Bungartz 7,8,9, Martin Grube 10, Eric Schuettpelz 2 and Robert Lücking 4,5**


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> **Abstract:** Lichens collected worldwide for centuries have resulted in millions of specimens deposited in herbaria that offer the potential to assess species boundaries, phenotypic diversification, ecology, and distribution. The application of molecular approaches to historical collections has been limited due to DNA fragmentation, but high-throughput sequencing offers an opportunity to overcome this barrier. Here, we combined a large dataset of ITS sequences from recently collected material and historical collections, obtained through Sanger, 454, or Illumina Sequencing, to test the performance of ITS barcoding in two genera of lichenized Basidiomycota: *Cora* and *Corella*. We attempted to generate new sequence data for 62 fresh specimens (from 2016) and 274 historical collections (collected between 1888 and 1998), for a final dataset of 1325 sequences. We compared various quantitative approaches to delimit species (GMYC, bPTP, ASAP, ABGD) and tested the resolution and accuracy of the ITS fungal barcoding marker by comparison with a six-marker dataset. Finally, we quantitatively compared phylogenetic and phenotypic species delimitation for 87 selected *Cora* species that have been formally described. Our HTS approach successfully generated ITS sequences for 76% of the historical collections, and our results show that an integrative approach is the gold-standard for understanding diversity in this group.

> **Keywords:** ASTRAL; biological collections; cryptic species; diversification; fungal barcoding; fungarium; historical specimens; natural history collections; phenotype
