A taxonomic revision of the Hypotrachyna subgenus Parmelinopsis in eastern North America is presented based on molecular phylogenetic analyses of ITS and mtSSU data, extensive field observation and analyses of chemical and morphological data. Each species is described, illustrated with photographs, and the distribution in the region is mapped. An identification key is also presented. Eleven species are recognized: H. afrorevoluta, H. appalachensis, H. britannica, H. cryptochlora, H. horrescens, H. kauffmaniana, H. minarum, H. mcmulliniana, H. revoluta, H. showmanii and H. spumosa. Extensive discussion of prior studies is provided, particularly with respect to the delimitation of H. afrorevoluta and H. revoluta. Hypotrachyna kauffmaniana is described from the central and southern Appalachian Mountains and separated from H. afrorevoluta and H. revoluta by its ascending secondary lobes and pustulose soralia that are primarily confined to the secondary lobes. Hypotrachyna horrescens is shown to correspond to a taxon with narrow lobed, small thalli with ciliate isidia. Hypotrachyna mcmulliniana is described from material collected throughout southeastern North America that is chemically identical to H. horrescens but differs in having larger thalli and sparsely ciliate isidia. Hypotrachyna appalachensis is described to accommodate material previously referred to H. minarum but that differs in the production of 4,5-di-O-methylhiascic acid in high concentrations (vs. absent or present as a trace in H. minarum). Hypotrachyna britannica is reported for the first time from North America.

Field and herbarium study. – The genesis of this study derived from extensive fieldwork carried out by the authors in the southern Appalachian Mountains of eastern North America, a region long recognized as a center of diversity for the genus in North America north of Mexico (Dey 1978, Hale & Kurokawa 1964, Lendemer et al. 2017). Between 2010 and 2020 we visited numerous sites in the southern Appalachians in connection with multiple projects (e.g., Allen & Lendemer 2016, Lendemer & Allen 2015, Lendemer et al. 2017, Tripp et al. 2019). During this fieldwork, we collected many Hypotrachyna specimens and studied the species in the field. Additional species were studied and collected during field work in the Coastal Plain during this time (Lendemer et al. 2016). We made hundreds of Hypotrachyna subgenus Parmelinopsis collections, which we examined for this study together with the those already deposited in the herbarium of the New York Botanical Garden (NY) for a total of 2162 specimens. In addition to this material, we examined type specimens, sequenced vouchers, and selected general specimens from the following herbaria: KRAM, MAF, OSU, SP, US, WIS.

Morphological and chemical study. – Morphological studies were conducted in the field and the laboratory. Thalli were observed in the field to determine growth direction, lobe arrangement, and overall shape (e.g., rosette vs. lobes draping down, adnation to substrate), along with habitat and ecology. Herbarium specimens were then used to measure vegetative and reproductive structures with microscopy. Specimens were studied dry with an Olympus SZ-STB dissecting microscope. Microscopic morphology and anatomy were studied with an Olympus BX53 compound microscope and sections or preparations made by hand with a razor blade that were mounted in water. Chemistry was initially studied with standard spot tests (K, C, KC, P, UV) following Brodo et al. (2001). All specimens were also subjected to thin layer chromatography (TLC) using acetone extracts of thallus fragments run in solvent C following Culberson and Kristinsson (1970) as modified by Lendemer (2011) for use with a peanut butter jar and with the solvent ratio of 200:30 toluene:glacial acetic acid. The arrangement of substances in the species studied here, as detected with the above methods, is illustrated in Figure 1.

DNA extractions, PCR, and sequencing. – Molecular data generation followed Muscavitch et al. (2017) for sequencing of mtSSU and ITS. Thallus fragments were taken from selected specimens and secondary metabolites were first extracted with an acetone wash that was used for TLC as outlined above. DNA was extracted with methods and reagents modified from the Qiagen DNeasy Plant Mini Kit. ITS4/5 (White et al. 1990) and mtSSU1/3R (Zoller et al. 1999) primers were used for PCR amplification of target regions. Sanger sequencing of PCR products was conducted at Macrogen USA. Sequencher 5.2.4 (GeneCodes, Ann Arbor, Michigan) was used to assemble and edit the resulting sequences.

Taxon sampling and molecular dataset assembly (mtSSU). – To assess the relationships between clades within Hypotrachyna, and the placement of North American taxa in those clades, we assembled an alignment of mtSSU sequences guided by the results of Crespo et al. (2010) and Divakar et al. (2010). All sequences tagged with Cetrariastrum, Everniastrum, Hypotrachyna and Parmelinopsis were downloaded from GenBank (06 November 2016). Taxa belonging to the Remototrachyna clade as defined by Divakar et al. (2010) were pruned, as were taxa assigned to other genera (e.g., Bulbothrix). A selection of Myelochroa mtSSU sequences were also downloaded for use as an outgroup following Crespo et al. (2010). One-hundred and eleven newly generated sequences were also included (see Table 1).

The sequences were assembled into a NEXUS file using Mesquite 3.04 (Maddison & Maddison 2015) which was then exported as a FASTA file and aligned using the MAFFT online interface. The resulting alignment from MAFFT was then downloaded and checked manually with adjustments made in Mesquite. During this initial alignment check, DQ287834 (H. rockii) was removed because it contained a large section of missing data. AY611131 (H. immaculata), AY611168 (P. minarum), and DQ287793 (C. ecuadorense) were removed because nearly one third of the sequences were lacking compared to the rest of the alignment. After pruning the above sequences the alignment was again subjected to auto-alignment in MAFFT with subsequent manual adjustment in Mesquite. Ambiguously aligned regions were then defined as part of an exclusion set in Mesquite.

Taxon sampling and dataset assembly (ITS). – To examine the relationships between members of Parmelinopsis as recovered by Divakar et al. (2010) we assembled a dataset of ITS sequences. All sequences of Hypotrachyna, Parmelinopsis and Remototrachyna were downloaded from GenBank (06 November 2016). The sequences were assembled into a NEXUS file, together with all newly generated sequences (see Table 1) and aligned in the same manner as was outlined for the mtSSU dataset above. After the initial stage of alignment and checking, KM250245 (H. minarum) was removed because it was highly divergent from the other sequences and did not BLAST to other Hypotrachyna. Following a subsequent cycle of alignment and checking, ambiguously aligned regions were excluded and the dataset was subjected to a rapid maximum likelihood (ML) analysis in RAxML 8.2.10 (Stamatakis 2006) with 500 bootstrap replicates and implementing the most complex nucleotide substation model available in the program (GTRGAMMA). The results were visualized in FigTree (Rambaut 2017). Using the results of the initial ML analysis as a guide, we pruned the large ITS dataset into a smaller dataset corresponding to Hypotrachyna subgenus Parmelinopsis, selected members of Hypotrachyna subgenus Hypotrachyna, and selected members of Hypotrachyna subgenus Everniastrum for use as an outgroup following Divakar et al. (2013). The alignment was then subjected to another round of auto-alignment and manual adjustment, and the ambiguously aligned regions were defined as part of an exclusion set.

Molecular phylogenetic analyses. – The mtSSU dataset and the final, smaller Hypotrachyna subg. Parmelinopsis ITS dataset were subjected to a rapid ML analysis in RAxML with 500 bootstrap replicates and implementing GTRGAMMA as the nucleotide substitution model. That model was selected because it is the most complex available in RAxML. The results were visualized in FigTree (Rambaut 2017).

The data for this study includes the following items:

1) The mtSSU molecular alignment in both NEXUS and PHYLIP format used to generate the phylogeny in Figure 1.

2) the ITS molecular alignment in both NEXUS and PHYLIP format used to generate the phylogeny in Figure 2.

3) The table from the publication that cross-references all of the sequences in the molecular alignments with their voucher data and NCBI GenBank accessions.

4) A PDF of a schematic TLC plate displaying the positions of substance discussed for each species and detected using Thin Layer Chromatography in Solvent C (modified, 200:30 toluene: glacial acetic acid).

5. The mtSSU phylogeny presented in the paper as Figure 1.

6) The ITS phylogeny presented in the paper as Figure 2.