DOI: 10.5061/dryad.2fqz612mg
title: A revision of Hypotrachyna subgenus Parmelinopsis (Parmeliaceae) in eastern North America

description: 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.
description: <i>Field and herbarium study</i>. – 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
 <i>Hypotrachyna</i> 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 <i>Hypotrachyna </i>subgenus
 <i>Parmelinopsis</i> 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. <i>Morphological and chemical
 study</i>. – 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. <i>DNA extractions, PCR,
 and sequencing</i>. – 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. <i>Taxon sampling
 and molecular dataset assembly</i>
 (<i>mtSSU</i>)<i>. –</i> To assess the
 relationships between clades within <i>Hypotrachyna</i>, 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).<i> </i>All sequences tagged with
 <i>Cetrariastrum, Everniastrum,</i>
 <i>Hypotrachyna</i> and<i> Parmelinopsis</i> were
 downloaded from GenBank (06 November 2016). Taxa belonging to the
 <i>Remototrachyna</i> clade as defined by Divakar et al.
 (2010) were pruned, as were taxa assigned to other genera (e.g.,
 <i>Bulbothrix</i>). A selection of
 <i>Myelochroa</i> 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 (<i>H.
 rockii</i>) was removed because it contained a large section of
 missing data. AY611131 (<i>H. immaculata</i>), AY611168
 (<i>P. minarum</i>), and DQ287793 (<i>C.
 ecuadorense</i>) 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. <i>Taxon sampling and dataset
 assembly</i> (<i>ITS</i>)<i>. </i>– To
 examine the relationships between members of <i>Parmelinopsis
 </i>as recovered by Divakar et al. (2010) we assembled a dataset of
 ITS sequences. All sequences of <i>Hypotrachyna,
 Parmelinopsis</i> and <i>Remototrachyna</i> 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
 (<i>H. minarum</i>) was removed because it was highly
 divergent from the other sequences and did not BLAST to other
 <i>Hypotrachyna</i>. 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 <i>Hypotrachyna
 </i>subgenus <i>Parmelinopsis</i>, selected members of
 <i>Hypotrachyna </i>subgenus <i>Hypotrachyna</i>,
 and selected members of <i>Hypotrachyna </i>subgenus
 <i>Everniastrum</i> 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.
 <i>Molecular phylogenetic analyses</i>. – The mtSSU
 dataset and the final, smaller <i>Hypotrachyna </i>subg.
 <i>Parmelinopsis</i> 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).
description: 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.