The Colorado River and its tributaries on the Colorado Plateau are home to unique desert river ecosystems and changing environmental conditions. Within this region, the Glen Canyon National Recreation Area (GCNRA) is comprised of rugged, high desert terrain and is managed by the United States National Parks Service as both a recreational and conservation area. Despite the ecological and economic importance of GCNRA, significant components of the ecological communities therein remain poorly characterized, including lichens. Accurately characterizing lichen-forming fungal diversity is challenging due to poorly known taxonomic groups, underexplored regions/habitats, and varying interpretations of morphological differences, including the recognition of environmentally modified forms. To better understand lichen diversity in GCNRA, we used an integrative taxonomic approach, incorporating both traditional morphology-based identification and information from the standard fungal DNA barcoding marker, the ITS, to compile a thorough inventory of lichen-forming fungi in Fifty-Mile Canyon. Vouchered lichen specimens were collected in 2019, and from these the ITS marker was sequenced. Candidate species-level lineages were delimited from family-level multiple sequence alignments using the Assemble Species by Automatic Partitioning web server. Specimens comprising DNA-based candidate species were then evaluated using traditional taxonomically diagnostic characters to link these, where possible, to currently described species. For Fifty-Mile Canyon, we document 100 putative species in 15 families, each represented by vouchered specimens, ITS sequence data, and photographic documentation. For comparison, a survey of historic records from GCNRA revealed a total of 124 documented lichen-forming fungal species throughout the NRA and adjacent land. Approximately 50% of the species documented in Fifty-Mile Canyon had not previously been found in GCNRA, and similar proportions of species diversity have been documented in GCNRA but not observed in our survey. We report three species new to North America – Calogaya ferrugineoides (H. Magn.) Arup, Froden & Sochting, Endocarpon deserticola T. Zhang, X. L. Wei & J. C. Wei and Xanthocarpia ferrari (Bagl.) Frödén, Arup & Søchting – verified using ITS sequencing data. In addition, Circinaria squamulosa sp. nov. is formally described here, currently known only from sandstone slabs in Fifty-Mile Canyon. However, the taxonomic identity of many of the candidate species from Fifty-Mile Canyon remained ambiguous at the species level, and some collections likely represent undescribed species-level lineages. Our results revealed unexpected, high species-level diversity of lichen-forming fungi at local scales and that overall lichen diversity across the entire GCNRA is likely vastly undercounted. These data – including DNA barcodes for the vast majority of lichen-forming fungi occurring in this canyon – provide an important resource that can be integrated into subsequent lichen biodiversity research in the southwestern United States and other semi-arid climates.

Family-level multiple sequence alignments of the of the standard DNA barcoding marker – the internal transcribed spacer region (ITS).

Supplementary file S1. List of Herbarium records of lichens collected from the Glen Canyon National Recreation Area – search of the Consortium of North American Lichen Herbaria on 26 February 2021.

Supplementary file S2. Family-level multiple sequence alignments of the of the standard DNA barcoding marker – the internal transcribed spacer region (ITS).

Supplementary file S3. Photographic documentation of sampled lichens.

Supplementary file S4. Family-level topologies inferred from multiple sequence alignments of the standard DNA barcoding marker – the internal transcribed spacer region (ITS).

Supplementary file S5. Caliciaceae: A. Buellia elegans (Leavitt sl19085). Candelariaceae: B. Candelariella antennaria (Leavitt sl19083); C. Candelariella aurella (Leavitt sl19098); D. Candelariella citrina (Leavitt sl19124); E. Candelariella rosulans agg. 1 (Leavitt sl19038); F. Candelariella rosulans agg. 2 (Leavitt sl19044); G. Candelariella rosulans agg. 3 (Leavitt sl19128). H. Unknown cf. Candelariales (Leavitt sl19049). Catillariaceae: I. Thalloidima sedifolium (Leavitt sl19051). Collemataceae: J.Enchylium aff. polycarpon (Leavitt sl19118); K. Scytinium plicatile (Leavitt sl19045); L. Enchylium aff. polycarpon (Leavitt sl19127). Parmeliaceae: M. Montanelia saximontana (Leavitt sl19093); N. Xanthoparmelia aff. plittii (Leavitt sl19079); (O) Xanthoparmelia maricopensis (Leavitt sl19102. Lichenotheliaceae: P. Heppia lutosa (Leavitt sl19120). Physciaceae: Q.Phaeophyscia aff. hirsuta 1 (Leavitt sl19082); R. Phaeophyscia cf. hirsuta (Leavitt sl19090); S. Phaeophyscia aff. hirtella 1 (Leavitt sl19081); T. Phaeophyscia aff. hirtella 2 (Leavitt sl19132); U. Rinodina aff. luridata 3 (Leavitt sl19042); V. Rinodina cf. athallina 2 (Leavitt sl19067); W. Rinodina aff. luridata 1 (Leavitt sl19047); X. Rinodina aff. luridata 2 (Leavitt sl19060). Psoraceae: Y. Protoblastina aff. rupestris (Leavitt sl19099). Stereocaulaceae: Z. Lepararia aff. membranacea (Leavitt sl19101). i. Lepararia aff. vouauxii (Leavitt sl19116). Thelotremataceae: ii. Diploschistes scruposus (Leavitt sl19107). All specimens housed at BRY-C.