We conducted a floristic revision of calicioid lichens and allied fungi from Alberta, Canada, including the descrioption of 13 species new to science, seven of which were assigned to three newly described genera (Albocalicium candidum, Brevicalicium roseum, Paracalicium betulae, P. caraganae, P. chamaedaphnes, P. piceae, and P. recedens) and six to two existing genera (Chaenothecopsis abscondita, C. caelumsaltator, C. calicii-viridis, C. epifurfuracea, C. yukonensis, and Phaeocalicium alnophilum). Seventy-three new sequences from 57 specimens across 34 species were generated (43 ITS, 9 LSU, and 21 mtSSU), including sequences for all newly described species and the first published sequences for Chaenotheca selvae, Chaenothecopsis ochroleuca, C. penningtonensis, C. parasitaster, and Phaeocalicium flabelliforme. We provided phylogenies for the Caliciaceae, Coniocybaceae, and Sphinctrinaceae, using all available public sequences. Novel clades and traits in Chaenotheca phaeocephala and Stenocybe pullatula are explored. Here we deposit the individual alignments for each locus and family, as well as the concatenated matrices (minus excluded regions) used to generate our phylogenies.

Molecular methods: To test species hypotheses, we generated new sequences of three loci from material in Alberta, Northwest Territories, and Yukon Territory: the fungal genetic barcode, the internal transcribed spacer (ITS, nuclear ribosomal DNA consisting of the internal transcribed spacer regions 1 and 2, the embedded 5.8S region, and small sections of the flanking large and small ribosomal subunits, LSU and SSU), a longer segment of the nuclear ribosomal large subunit (LSU), and the mitochondrial small subunit (mtSSU). Sanger sequencing was carried out in the Canadian Museum of Nature’s Laboratory of Molecular Biodiversity (RDB), the Spribille Laboratory, and the Molecular Biology Service Unit at the University of Alberta (EP). We used previously unpublished sequences generated by the Canadian Centre for DNA Barcoding (BOLD, https://boldsystems.org/) as part of the Arctic Probe Project, as well as most NCBI sequences of calicioids from the three target families.

DNA extraction, PCR amplification, and sequencing: Canadian Museum of Nature: Genomic DNA was extracted from dried material following a silica column purification protocol similar to commercially available DNA extraction kits (modified from Alexander et al. 2007). DNA extraction success was assessed via gel electrophoresis on 1.25% agarose gels stained with ethidium bromide. After extractions, the three gene loci were amplified with the following primers: ITS with ITS1, ITS2, ITS3 and ITS4 (White et al. 1990); mtSSU with mrSSU1, mrSSU2, mrSSU2R and mrSSU3R (Zoller et al. 1999), and LSU with LIC24R (Miadlikowska & Lutzoni 2000), LR0R, LR3, LR3R, LR5, LR6 and LR7 (Vilgalys & Hester 1990).

DNA was amplified using polymerase chain reaction (PCR) in a 15 μL volume with 9.05 μL of DNA-grade H₂0, 3 μL of 5x reaction buffer, 0.3 μL of 10 mM dNTP, 0.75 μL of 10 μM each primer, 0.3 U of Q5 DNA Polymerase (New England BioLabs Inc.), and 1 μL of DNA template. Some LSU and mtSSU amplifications were carried out using DreamTaq DNA Polymerase (ThermoFisher Scientific) in a 15 μl volume with 11.3 μL of DNA-grade H₂0, 1.5 μL of 10x reaction buffer, 0.3 μL of 10 mM dNTP, 0.375 μL of 10 μM each primer, 0.75 U of polymerase, and 1 μL of DNA template. DNA template was doubled for difficult samples. For Q5 amplifications, an initial denaturation of 98°C for 30 sec was followed by 34 cycles of 98°C for 10 sec, 56°C for 30 sec, 72 °C for 30 sec and a final extension step of 72°C for 5 min. For DreamTaq amplifications, an initial denaturation of 95°C for 3 min was followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec, 72 °C for 90 sec and a final extension step of 72°C for 10 min. Amplification success was assessed via gel electrophoresis in 1.25% agarose gels stained with ethidium bromide. Sequencing reactions were performed in 10 μL reactions containing 6.2 μL of DNA-grade H₂0, 1.8 μL of 5x reaction buffer, 0.5 μL of primer, 0.5 μL of BigDye Terminator v3.1 Ready Reaction Mix (ThermoFisher Scientific), and 1 μL of diluted PCR products. An initial denaturation of 95°C for 3 min was followed by 30 cycles of 96°C for 30 sec and 50°C for 20 sec followed by a final step at 60°C for 4 min. Reaction products were purified via an EDTA-NaOH-ethanol precipitation protocol recommended by the sequencing kit manufacturer. Purified DNA pellets were resuspended in HIDI formamide, denatured at 95°C for 5 min, cooled for 2 min, and sequenced via automated capillary electrophoresis on an Applied Biosystems 3500xL Genetic Analyzer (ThermoFisher Scientific).

DNA extraction, PCR amplification and sequencing: University of Alberta, Genomic DNA was extracted from dried material using the QIAamp DNA Investigator kit (Qiagen, Hilden, Germany) following the manufacturer’s protocols for isolation of total DNA from tissue, except samples were first lysed with a mechanical bead beater for 30 sec and fragments were incubated in lysis buffer for 8 hours at 56℃. After extractions, samples were quantitated with a Nanodrop spectrophotometer (Implen NP80), and the three gene loci were amplified with the following primer sets: ITS with ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990), mtSSU with mrSSU1 and mrSSU3R (Zoller et al. 1999), and LSU with LR7 and LR0R (Vilgalys & Hester 1990).

DNA was amplified using PCR in 22 μL reactions for each gene locus of interest. Amplification of each gene region was performed as follows: ITS amplification used an initial denaturation at 95℃ for 5 min, and then 35 cycles of 95℃ for 30 sec, annealing at 57℃ for 30 sec and then extension at 72℃ for 30 sec, followed by a final extension at 72℃ for 7 min and holding at 4℃. LSU amplification used an initial denaturation at 95℃ for 5 min and then 35 cycles of 95℃ for 1 min, annealing at 56℃ for 1 min, 72℃ for 1 min 30 sec and then a final extension at 72℃ for 7 min and holding at 4℃. Finally, for mtSSU amplification an initial denaturation at 95℃ for 5 min and then 35 cycles of denaturation at 95℃ for 1 min, annealing at 54℃ for 1 min, extension at 72℃ for 1 min and then a final extension at 72℃ for 7 min and holding at 4℃. Once visualized using gel electrophoresis using 1% agarose to ensure PCR success and cleaned using a 4 μl ExoSAP reaction to remove unused primers and nucleotides, amplicons were sent for Sanger sequencing (ABI 3730, Thermo Fisher) at the University of Alberta Molecular Biology Service Unit.

Sequence screening and selection: We initially included almost all unique published sequences for the three families in NCBI. We screened both new and published sequences with ‘megaBLAST’ searches against the NCBI nucleotide database to identify sequences that may represent non-target organisms and to identify the closest published relatives (NCBI Resource Coordinators 2018). Because of gene tree conflict and difficulty with de novo sequencing, all newly generated and published sequences in Sphinctrinaceae were analyzed in T-BAS, a tree-based alignment selector toolkit (https://tbas.cifr.ncsu.edu/tbas2_3/pages/tbas.php, Carbone et al. 2017, 2019; Miller et al. 2015). We checked the placement of both individual loci and concatenated sequences within Pezizomycotina using the ‘place unknowns’ and environmental phylogenetic placement analyses. While we treat the name Sphinctrinaceae as synonymous with Mycocaliciaceae following Jaklitsch et al. (2016) and Ertz et al. (2023), the T-BAS reference tree retains Mycocaliciaceae as distinct from Sphinctrinaceae (Carbone et al. 2017). Subsequently, we excluded some published sequences because they i) blasted to non-calicioid sequences, ii) had low percent identities with any other sequences in GenBank (<88%), iii) T-BAS placed them in a family outside of the class Eurotiomycetes, and/or iv) they did not cluster with any of our putative new species using a local blast. For ITS, we excluded: Chaenothecopsis orientalis accession AY795863, C. rubescens OQ717807, C pusilla OQ717806, AF243132, AY795866 (placement variable, remote from Sphinctrinaceae); Mycocalicium victoriae AF243135, AJ312123, AY128702, AY128701, and M. sp. MN206996, MT558584, AJ972853 (97–98% percent identity to M. victoriae, placed in class Dothideomycetes, family Teratosphaeriaceae with high likelihood); Sphinctrina intermedia KJ865747 (placed in class Lecanoromycetes, clustered with Circinaria and Aspicilia, and the text in Tibell et al. (2014) states this sequence was an attempted sequencing of Phaeocalicium triseptatum; Tibell confirmed our interpretation, pers. com. Nov. 2024); Chaenotheca stemonea AF408683 and C. brunneola OQ843252. For LSU we excluded Chaenothecopsis hunanensis JX122784, C. proliferatus JX122783, and Phaeocalicium praecedens KC590486 (placed in Dothideomycetes). Finally, we concluded that accession KJ871615 (labeled in NCBI as Phaeocalicium triseptatum) represents Sphinctrina intermedia following the text in Tibell et al. 2014 and supported by our phylogenetic analyses (Tibell confirmed our interpretation, pers. com. Nov. 2024). We also excluded nine de novo sequences for similar reasons (5 LSU, 3 ITS, 1 mtSSU).

We report similarity metrics with accessioned sequences for newly generated loci when material is concordant morphologically with described species. We constructed de novo phylogenetic trees for Caliciaceae, Coniocybaceae and Sphinctrinaceae to confirm phylogenetic relationships in both described and putative new species. We used two species of Heterodermia as outgroups for Caliciaceae, following Prieto & Wedin (2017). For the Coniocybaceae phylogeny we adopted outgroups from Suija et al. (2023) and Tibell et al. (2019). For Sphinctrinaceae, we adopted outgroups from orders outside of Mycocaliciales following Ertz et al. (2023; adopted from Beimforde et al. 2023, 2017; Prieto et al. 2013; Thiyagaraja et al. 2022; Tibell & Vinuesa 2005; Tuovila et al. 2013; Vinuesa et al. 2001). Supplementary Table S2 provides information on the final 475 sequences used here. Across the three analyses, we included published sequences from: Aguirre-Hudson et al. 2007; Ariyawansa et al. 2015; Beimforde et al. 2023; Crous et al. 2013; De Leo et al. 2003; Ertz et al. 2023; Etayo et al. 2023; Hanani et al. 2022; Hofmeister et al. 2022; Houbraken et al. 2011; James et al. 2006; Kauff et al. 2018; Li et al. 2023; Lutzoni et al. 2001; Malíček 2022; Marthinsen et al. 2019; Mark et al. 2016; Masumoto et al. 2019; McMullin et al. 2024; Messuti et al. 2012; Gaya et al. 2012, 2014; Ohmura et al. 2022; Pavlov et al. 2023; Povilaitienė et al. 2022; Prieto & Wedin 2017; Prieto et al. 2013, 2021; Prieto 2020; Pykälä et al. 2019; Réblová et al. 2017; Rikkinen et al. 2014; Samson et al. 2009; Schoch et al. 2014; Schmull et al. 2011; Wiklund & Wedin 2003; Selva et al. 2023b; Sert et al. 2007; Spatafora et al. 2006; Spribille et al. 2020; Sterflinger & Prillinger 2001; Suija et al. 2016, 2023; Telfer et al. 2015; Temu et al. 2019, 2024; Thiyagaraja et al. 2022; Tibell 2001a, 2001b, 2002, 2003, 2006, 2007; Tibell & Beck 2001; Tibell & Knutsson 2016; Tibell & Koffman 2002; Tibell & Vinuesa 2005; Tibell et al. 2014, 2019; Tuovila et al. 2011, 2013, 2014; Vinuesa et al. 2001; Vondrák et al. 2022, 2023; Vu et al. 2019; Wang et al. 2005; Wedin et al. 2002, 2005; Weerakoon et al. 2012; Williams & Tibell 2008; Yahr 2015; Yang et al. 2022.

Phylogenetic analyses: For each family and locus, sequences were aligned in MAFFT (version 7.49, Katoh et al. 2019, 2002; Katoh & Standley 2013) via Mesquite (version 3.7, Maddison & Maddison 1997–2021) using the G-INS-i method. Alignments were vetted visually and adjusted manually using the Mesquite ‘Highlight Apparently Slightly Misaligned’ option. We used ITSx 1.1 (Bengtsson-Palme et al. 2013) on the DeCifr platform to split sequences into ITS, small subunit, and large subunit files to aid in sequence and alignment vetting. Ambiguously-aligned regions were identified using GBLOCKS (Castresana 2000) within Mesquite, using less stringent criteria (minimum length of a block was set to 2, all characters were allowed gaps, the minimum number of sequences for a flank position was set to 51% of taxa with non-gaps at that position) and excluded from further analyses. Terminal ends and introns were also excluded. Loci were concatenated in Mesquite without the excluded regions. Original (with excluded regions) and final alignments are deposited in Dryad, and sequence voucher data are provided in Supplementary Table S2.