Opuscula Philolichenum, 13: 155-176. 2014. *pdf effectively published online 12December2014 via (http://sweetgum.nybg.org/philolichenum/) Studies in lichens and lichenicolous fungi – No. 19: Further notes on species from the Coastal Plain of southeastern North America 1 2 JAMES C. LENDEMER & RICHARD C. HARRIS ABSTRACT. – Geographically disjunct and ecologically unusual populations of Cladonia apodocarpa from hardwood swamps are reported from southeastern North Carolina, and assignment to that species is confirmed with analyses of nrITS sequence data. The separation of Lecanora cinereofusca var. cinereofusca and L. cinereofusca var. appalachensis is discussed in the light of analyses of mtSSU and nrITS sequence data. Lecanora cinereofusca var. appalachensis is considered to merit recognition at the species level, for which the name L. saxigena Lendemer & R.C. Harris (nomen novum pro L. appalachensis (Brodo) non L. appalachensis Lendemer & R.C. Harris) is introduced. Phlyctis ludoviciensis is formally placed in synonymy with P. boliviensis. Phlyctis willeyi is shown to belong to the genus Leucodecton and the new combination L. willeyi (Tuck.) R.C. Harris is proposed. Piccolia nannaria is hypothesized to be a parasite on Pyrrhospora varians and is shown to be more widespread in the Coastal Plain than previously thought. Schismatomma rappii is revised, illustrated, and shown to be widespread in the Coastal Plain of southeastern North America. Tylophoron hibernicum is confirmed to be the correct name for all North American records of T. protrudens. KEYWORDS. – Sandhills, maritime forest, barrier island, Mid-Atlantic, taxonomy, floristics. INTRODUCTION In 2012 we initiated an inventory of lichen biodiversity in the Mid-Atlantic Coastal Plain (MACP) with support from the U.S. National Science Foundation. The purpose of this inventory was to document and describe lichen biodiversity, and its patterns, in a little-studied region of North America whose remaining natural habitats are imperiled by anthropogenic forces including climate change (Auch 2000, Griffith et al. 2003, Lendemer & Allen 2013, Ricketts et al. 1999). Our work has led to the discovery of many species new to science, as well as large numbers of disjuncts and previously overlooked rare species (Lendemer 2013; Lendemer & Harris in press; Lendemer & Harris 2014a, b). Concurrently it has fundamentally reshaped our understanding of biogeographic patterns in the Coastal Plain, with respect to both the northern and southern distributional limits of taxa (see examples in Lendemer & Harris 2014b). During the course of this work we have also encountered many cases where newly collected material has prompted us to reexamine our understanding of recognized species, their distributions, and ecologies. Here we present a series of notes that fall into the latter category. We hope that these notes will be of use for those working in the region, as well as towards maintaining the Checklist of North American Lichens (Esslinger 2014). 1 JAMES C. LENDEMER– Institute of Systematic Botany, The New York Botanical Garden, Bronx, NY, 10458-5126, U.S.A. – e-mail: [email protected] 2 RICHARD C. HARRIS – Institute of Systematic Botany, The New York Botanical Garden, Bronx, NY, 10458-5126, U.S.A. – e-mail: [email protected] 155 MATERIALS AND METHODS Fieldwork and herbarium vouchers This study is based largely upon the fieldwork conducted by the authors, together with their colleagues at The New York Botanical Garden, throughout the Mid-Atlantic Coastal Plain between 2012 and 2014. This includes >14,000 vouchers specimens deposited at The New York Botanical Garden (NY). These data were complemented by reference material already available at NY, and supplemented by a loan from H. Morphological and chemical study The morphology of specimens was examined following the techniques of microscopy outlined by Lendemer (2011a). Chemistry was studied with standard spot test reagents (K, C, P and UV) following Brodo et al. (2001) and with Thin Layer Chromatography using Solvents A or C and the Peanut Butter Jar method outlined by Lendemer (2011a). Micrographs were captured following the methods outlined by Lendemer (2011a). DNA extraction, amplification, and sequence generation Subsamples used for DNA extraction were those used in TLC analyses above such that the chemistry and identification of the vouchers had been confirmed. DNA extraction, PCR amplification, and sequence assembly followed the methods of Hodkinson and Lendemer (2012). Molecular dataset assembly and taxon sampling Three molecular datasets were assembled for this study: 1) a dataset of nrITS sequences of Cladonia to confirm the identity of newly discovered populations of C. apodocarpa, 2) a dataset of mtSSU sequences to examine the position and monophyly of Lecanora cinereofusca, and 3) a dataset of nrITS sequences to examine the differences between L. cinereofusca var. cinereofusca and L. cinereofusca var. appalachensis. For the first dataset (nrITS of Cladonia), all available nrITS sequences of C. apodocarpa, C. petrophila and C. stipitata were downloaded from GenBank. The taxon sampling was based on the MegaBlast similarity of the newly generated sequences to C. apodocarpa, and on the phylogeny of apodetiate Cladonia species published by Lendemer and Hodkinson (2009). The sequences were assembled into an alignment in Mesquite 2.0 (Maddison & Maddison 2009) and aligned manually. Ambiguously aligned and terminal regions were defined as part of an exclusion set. The second dataset (mtSSU of Lecanora) was constructed to examine the relationships and placement of Lecanora cinereofusca within the Lecanoraceae. Taxon sampling within Lecanoraceae and selection of Stereocaulon as an outgroup was based on Miadlikowska et al. (2006). For this dataset four new sequences of L. cinereofusca var. cinereofusca and three of L. cinereofusca var. appalachensis were generated (see table 1 for voucher data and GenBank accession numbers). The following sequences were downloaded from GenBank: 1) all mtSSU sequences tagged with “Lecanora” returned from an NCBI Nucleotide search on 22 April 2014, 2) all mtSSU sequences tagged with “Pyrrhospora” returned from an NCBI Nucleotide search on 22 April 2014, 3) all mtSSU sequences tagged with “Stereocaulon” returned from an NCBI Nucleotide search on 22 April 2014. The sequences were assembled into an alignment in Mesquite and the following were pruned: 1) all sequences identified as taxa not belonging to the genus Lecanora (i.e., taxa tagged with the identifier “Lecanora” but actually belonging to other taxonomic groups), 2) all sequences not belonging to Pyrrhospora s. str. (i.e., those belonging to Ramboldia following Kalb et al. (2008)), 3) all sequences identified only to genus, or with an indication of hesitation (i.e., “aff” or “cf”). The dataset was then subjected to a multiple alignment using the MAFFT online interface, and subsequently adjusted manually in Mesquite. During the process of manual alignment the AY464085 was pruned because more than half of the mtSSU region was missing. Terminal regions and ambiguously aligned regions were defined as part of an exclusion set. The third dataset (nrITS of Lecanora) comprised six newly generated sequences of Lecanora cinereofusca var. cinereofusca and L. cinereofusca var. appalachensis (three each). No additional sequences of this taxon were available in GenBank, and no sequences were downloaded for use as an outgroup because the L. cinereofusca was shown to be well-supported and monophyletic in analyses of the mtSSU dataset (see below) and because of overall poor resolution of relationships within the Lecanoraceae. The terminal ends of the alignment were defined as part of an exclusion set, but no ambiguously aligned regions were present. 156 GenBank Accesion Taxon mtSSU nrITS Isolate Voucher Herbarium Locality L. cinereofusca KP224462 NY1458 Lendemer 30827 NY-1598083 U.S.A., North Carolina, Gates Co. L. cinereofusca KP224463 NY1460 Lendemer 27805 NY-1222384 Canada, New Brunswick, Charlotte Co. L. cinereofusca KP224469 NY1457 Lendemer 30986 NY-1597992 U.S.A., North Carolina, Gates Co. L. cinereofusca KP224465 KP224470 NY1521 Lendemer 34415 NY-1772619 U.S.A., North Carolina, Dare Co. L. cinereofusca KP224464 KP224471 NY1520 Lendemer 35007 NY-1808085 U.S.A., North Carolina, Dare Co. L. saxigena KP224460 KP224467 NY1461 Lendemer 25832 NY-1217772 U.S.A., Arkansas, Madison Co. L. saxigena KP224459 KP224466 NY1445 Lendemer 32825 NY-1684429 U.S.A., North Carolina, Swain Co. L. saxigena KP224461 KP224468 NY1447 Lendemer 33186 NY-1684507 U.S.A., North Carolina, Swain Co. Table 1. GenBank accession numbers and associated voucher information for newly generates sequences of Lecanora cinereofusca used in this study. Molecular phylogenetic analyses The first two datasets (nrITS of Cladonia and mtSSU of Lecanora) were prepared for maximum likelihood (ML) analysis using Mesquite by manually deleting the excluded regions, transforming the gaps (-) to missing (?), and transforming uncertainties/polymorphisms to missing (?). They were then exported as PHYLLIP formatted files, and rapid ML topology searches and bootstrap analyses with 500 replicates were performed using the model GTRGAMMA implemented in RAxML 7.2.6 (Windows executable, Stamatakis 2006). The results of the ML analyses were visualized in FigTree 1.3.1 (Rambaut 2009). NEXUS formatted files of the same two datasets were then analyzed with Bayesian Inference (BI) using MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001). Before