The Bryologist 106(1), pp. 61 79 Copyright q 2003 by the American Bryological and Lichenological Society, Inc. The Lichen Genus Sticta in the Great Smoky Mountains: A Phylogenetic Study of Morphological, Chemical, and Molecular Data TAMI MCDONALD* Department of Plant Biology, University of Minnesota, 1445 Gortner Ave., St. Paul, MN 55108, U.S.A. JOLANTA MIADLIKOWSKA Department of Biology, Duke University, Box 90338, Durham, NC 27708, U.S.A. and Department of Plant Tax- onomy and Nature Conservation, Gdansk University, Al. Legionow 9, 80-441 Gdansk, Poland; e-mail: jolantam@ duke.edu FRANCËOIS LUTZONI Department of Biology, Duke University, Box 90338, Durham, NC 27708, U.S.A. e-mail: ¯[email protected] Abstract. In this paper we segregate specimens from the genus Sticta in the Great Smoky Mountains National Park into phenotypic groups corresponding to putative species using tradi- tional taxonomic methods, paying particular attention to specimens from the S. weigelii s. l. group, then employ phylogenetic analyses and rigorous statistics to test the robustness of these species groups. In order to circumscribe putative species and to resolve the S. weigelii complex, mor- phological, chemical, and molecular characters from the nuclear ribosomal DNA sequences of the entire Internal Transcribed Spacer region are analyzed separately and simultaneously using maximum parsimony or maximum likelihood. In addition to the bootstrap method, Bayesian sta- tistics with the Markov Chain Monte Carlo algorithm are used to estimate branch robustness on the resulting reconstructed trees. Five out of six analyses recover the same ®ve monophyletic putative species from the genus Sticta, indicating the concordance of DNA-based and morphology- based species delimitation. The phylogenies show that lichens identi®ed as S. weigelii represented S. beauvoisii and the two new species described here ± S. carolinensis and S. fragilinata. Sticta weigelii s. s. does not occur in the park. Specimens from Oregon identi®ed as S. weigelii belong to another unnamed Sticta taxon. The remaining two monophyletic groups represent two species well known from the park±S. fuliginosa and S. limbata. Characteristics of secondary compounds detected by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) in S. fragilinata thalli are provided. Detailed descriptions, including morphology and chemistry, are provided for four Sticta species found in the Smoky Mountains: S. beauvoisii, S. carolinensis, S. fragilinata and S. fuliginosa. The genus Sticta (Schreber) Ach. includes ma- Ach., purportedly found in the western United crolichens that are found throughout the world States; and Sticta weigelii (Ach.) Vainio, a species (Galloway 1994, 1997, 1998; Joshi & Awasthi with coralloid marginal isidia (Hale 1979). How- 1982). Though the genus is primarily tropical in ever, according to Harris (1984), an additional Stic- distribution, its representatives are present as far ta species, S. beauvoisii Delise, which is geograph- north as Norway (Jùrgensen 1969) and as far south ically restricted mainly to the northeastern part of as the southern tip of South America (Galloway the U.S., is commonly misidenti®ed as S. weigelii. 1994). Historically, there have been thought to be Dey (1976) recognized a lobulate, chemically four frequently collected species of Sticta in the distinct variant of S. weigelii s. l. in the southern continental United States: Sticta fuliginosa Appalachians. A phyllidiate lichen lacking anthra- (Hoffm.) Ach., a substipitate lichen with a thin thal- quinones, also present in the region, is misclassi®ed lus and laminal isidia; Sticta limbata (Sm.) Ach., a as S. weigelii (Brodo et al. 2001). A third unnamed lichen with grayish marginal soralia; the large- species with pale, long, tufted tomentum is con- lobed, laminally isidiate Sticta sylvatica (Huds.) fused with S. weigelii in the southeastern United States (Harris 1984). In the northwest, a fuliginous *Current address: University of Wisconsin, Department lichen with anthraquinones in the medulla is con- of Plant Pathology, Russell Labs 885, 1630 Linden Drive, Madison, WI 53706, U.S.A. e-mail: [email protected]. fused with S. weigelii, as also noted by Brodo et edu al. (2001). Thus the distribution of S. weigelii s. s. 0007-2745/03/$2.05/0 62 THE BRYOLOGIST [VOL. 106 in temperate North America is not clear, nor is the baria oregana, and Pseudocyphellaria crocata, all from identity of temperate North American specimens the Paci®c Northwest, and tropical specimens of S. wei- gelii s. s. were included. identi®ed as S. weigelii. As many as six distinct Morphological, anatomical, and chemical studies were species may comprise S. weigelii s. l. in the United carried out on the Sticta collection from the Smoky Moun- States alone, and more in the Caribbean (Harris tains, including the specimens listed in Table 1, and on 1984). selected material from various herbaria (see Taxonomy of This paper focuses on the Sticta of the Great the genus Sticta section; Specimens examined). For molecular phylogenetic analyses based on ITS Smoky Mountains National Park located in North nrDNA, 24 specimens of Sticta were selected (Table 1). Carolina and Tennessee, U.S.A. The area is at the Twenty-one of them, collected in the Smoky Mountains, convergence of the projected ranges for several of represent three recognized taxa (S. beauvoisii, S. fuligi- the S. weigelii s. l. taxa, and therefore is a starting nosa, and S. limbata) and two potentially new species ± point for unraveling the S. weigelii problem in S. carolinensis and S. fragilinata. One specimen of S. lim- bata and a single collection of Sticta representing a mor- North America. photype commonly identi®ed as S. weigelii (named here The main goals of this study are to: 1) recognize Sticta sp.) come from the Paci®c Northwest (Oregon). In and delimit putative Sticta species occurring in the addition, one tropical specimen of S. weigelii s. s. was Great Smoky Mountains National Park with special included in the molecular data set. Except for Sticta sp., emphasis on specimens and taxa from the S. wei- neotropical S. weigelii, and outgroup species, each Sticta taxon was represented by more than one collection from gelii complex, using phenotypic characters as different localities in both types of data sets: morphology grouping criteria; 2) test their monophyletic status 1 chemistry and ITS nrDNA. To produce a stable root based on separate and simultaneous phylogenetic for the ingroup, three ITS sequences from GenBank were analyses of morphology, chemistry, and molecular added, two from Lobaria (L. oregana and L. pulmonaria) data obtained from specimens representing the phe- and one from Pseudocyphellaria crocata, for a total of 27 specimens from 10 putative species. notypic variation within each putative species; 3) Morphological data. All 221 Sticta specimens were propose S. carolinensis and S. fragilinata as new separated into putative species groups based on phenotyp- species, if the results from the phylogenetic analy- ic characters derived from species descriptions and from ses are in agreement with our conclusion based on literature on S. weigelii s. l. Five groups were recognized: morphological and chemical evidence; and 4) re- S. beauvoisii groups I and II; S. fuliginosa; S. carolinensis; construct relationships among these two new Sticta and S. fragilinata. Individuals from S. beauvoisii group II were chosen to be as different as possible from the ®rst species and the existing taxa based on a combined group of this taxon to assess the stringency of the subse- data set, if no con¯ict is detected among data par- quent analyses of the morphological characters. First, titions. based on similarity to descriptions of the type for existing To reach these goals, we conducted a detailed species, one representative specimen was chosen to ex- assessment of morphological, anatomical, and emplify the group. Then a survey was made of the re- maining specimens in the group and the ®ve morpholog- chemical features, and sequenced the entire Internal ically most diverse lichens were chosen to represent the Transcribed Spacer region of the nuclear ribosomal variation in that group. In addition to these thirty lichens, DNA (ITS1, 5.8S and ITS2 nrDNA) for the ®ve ®ve tropical specimens of S. weigelii were included in the potential species of Sticta present in the Great morphological data matrix. Five specimens each of Lo- Smoky Mountains, including reference material for baria oregana, L. pulmonaria, and Pseudocyphellaria crocata were used as the outgroup representatives. All S. weigelii s. s. from South America, for Sticta morphological data subjected to phylogenetic analyses specimens recognized in the Paci®c Northwest area were obtained from this set of 50 lichens. Sticta sp. was of the U.S. as S. weigelii, and for three outgroup not included in the morphological data matrix due to the species from the Lobariaceae, closely related to the limited fresh material available. genus Sticta. This phylogenetic study of the genus A total of 26 morphological, anatomical, and ecological traits were scored for this 50-specimen set of lichens, in- provides a framework for future systematic work cluding various measurements of the tomentum on the on Sticta, as well as a stable foundation for detailed lower surface of the thallus, rhizines, cyphellae, vegetative study of the weigelii complex. propagules (isidia and soredia), and overall size, shape, and appearance of the thallus. All observations were made with a Heerbrugg Wild M20 compound microscope or MATERIALS AND METHODS Heerbrugg Wild M5-41053 dissecting microscope. Cal- Taxon sampling. Specimens were collected in the culations of the density of cyphellae were made using a summer of 1999 at 43 localities in the Great Smoky 0.5 by 0.5-cm grid. For anatomical studies, lichens were Mountains National Park. At each locality, GPS position sectioned by hand or with a freezing microtome. The sec- and habitat were recorded; substrate was recorded for each tions were mounted in distilled water. specimen.