Nelsen & al. • Phylogeny of Trypetheliaceae TAXON 63 (5) • October 2014: 974–992 S YS T E M AT I C S A N D PH Y LO G E N Y Elucidating phylogenetic relationships and genus-level classification within the fungal family Trypetheliaceae (Ascomycota: Dothideomycetes) Matthew P. Nelsen,1,2 Robert Lücking,2 André Aptroot,3 Carrie J. Andrew,2,4 Marcela Cáceres,5 Eimy Rivas Plata,2 Cécile Gueidan,6 Luciana da Silva Canêz,7 Allison Knight,8 Lars R. Ludwig,8 Joel A. Mercado-Díaz,9 Sittiporn Parnmen2 & H. Thorsten Lumbsch2 1 Committee on Evolutionary Biology, University of Chicago 1025 E. 57th Street, Chicago, Illinois 60637, U.S.A. 2 Science & Education, The Field Museum, 1400 S. Lake Shore Drive, Chicago, Illinois 60605, U.S.A. 3 ABL Herbarium, Gerrit van der Veenstraat 107, 3762 XK Soest, The Netherlands 4 Department of Biology, Northeastern Illinois University, 5500 North St. Louis Avenue, Chicago, Illinois 60625, U.S.A. 5 Departamento de Biociências, Universidade Federal de Sergipe, CEP: 49.500-000, Itabaiana, Sergipe, Brazil 6 Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, U.K. 7 Laboratório de Botânica Criptogâmica, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Brazil 8 Department of Botany, University of Otago, P.O. Box 56, Dunedin, 9054, New Zealand 9 Herbario, Jardín Botánico, Universidad de Puerto Rico, 1187 Calle Flamboyán, San Juan, Puerto Rico 00926-1177 Author for correspondence: Matthew Nelsen, mpnelsen@gmail.com DOI http://dx.doi.org/10.12705/635.9 Abstract While the phylogenetic position of Trypetheliaceae has been the subject of recent molecular studies, the relationships within this family have been little studied. Here we construct a detailed genus-level phylogeny of the family. We confirm previous morphology-based findings suggesting that a substantial proportion of genera are not monophyletic, and that an overemphasis has been placed on certain character state combinations which do not strictly reflect phylogenetic relationships. Specifically, patterns of ascospore septation, ostiole orientation and type of ascomatal aggregation are evolutionarily labile, and of limited utility for the delimitation of genera as currently circumscribed. We show that species from a number of genera including Astrothelium, Bathelium, Cryptothelium, Laurera and Trypethelium together form a strongly supported group, referred to here as the “Astrothelium” clade. Species from Aptrootia, Architrypethelium, Campylothelium, Marcelaria (L. purpurina and L. cumingii groups), Pseudopyrenula and species from the Trypethelium eluteriae group fall outside of the “Astrothelium” clade and each form monophyletic groups. In contrast, species from Arthopyrenia, Mycomicrothelia and Polymeridium fall outside of the “Astrothelium” clade, and do not form monophyletic groups. The data presented here validate earlier morphology-based findings suggesting generic delimitations are in need of revision, and provides a first step towards identifying the utility of individual characters and identifying which characters and character state combinations may be useful for future classification. Keywords crustose; lichen; ostiole; systematics; taxonomy; tropical Supplementary Material The Electronic Supplement (Figs. S1–S2) is available in the Supplementary Data section of the online version of this article at http://www.ingentaconnect.com/content/iapt/tax INTRODUCTION A central goal of systematics is to place taxonomy in a phylogenetic context, such that classification schemes reflect evolutionary relationships (Darwin, 1859). Among the Fungi, molecular sequence data have greatly illuminated relationships, thereby facilitating taxonomic revisions in an evolutionary framework, and forcing a re-examination and re-interpretation of characters that have appeared homoplasious in higher-level phylogenies and classification schemes (Hibbett & al., 2007; Aveskamp & al., 2010; Lumbsch & Huhndorf, 2010a, b). An excellent example is the fungal family Trypetheliaceae (Figs. 1–2), in which the classification of genera is based on suites of characters that have been suggested to conflict with evolutionary relationships, thereby resulting in a taxonomic scheme that is not strictly congruent with these relationships (Harris, 1989a, 1995; Del Prado & al., 2006; Aptroot & al., 2008; Nelsen & al., 2009, 2011b). Here we employ molecular sequence data to construct the first detailed phylogeny of Trypetheliaceae, including representatives of nearly all genera. We then use this framework to explore relationships among species as well as assess the validity of existing generic concepts. Most higher-level classification schemes have placed Trypetheliaceae in the order Pyrenulales in Eurotiomycetes Received: 5 Jan 2013 | returned for first revision: 5 Nov 2013 | last revision received: 13 Apr 2013 | accepted: 14 Apr 2014 | not published online ahead of inclusion in print and online issues || © International Association for Plant Taxonomy (IAPT) 2014 974 Version of Record (identical to print version). TAXON 63 (5) • October 2014: 974–992 Nelsen & al. • Phylogeny of Trypetheliaceae Fig. 1. Habit images of the thallus and ascomata from select Trypetheliaceae taxa. A, Arthopyrenia cinchonae (Costa Rica, Lücking 45); B, Mycomicrothelia hemisphaerica (Costa Rica, Lücking s.n.); C, Pseudopyrenula subnudata (Fiji, Lumbsch 19845g); D, Polymeridium catapastum (Bahamas, Britton 6650); E, Trypethelium tropicum (Colombia, Lücking 32544); F, Campylothelium puiggarii (Brazil, Osorio SM25); G, Marcelaria purpurina (Colombia, Moncada 3467); H, Trypethelium eluteriae (Colombia, Moncada 3399); I, Laurera sanguinaria (Brazil, Brako 7136); J, Aptrootia elatior (New Zealand, Walker s.n.); K, Architrypethelium seminudum (Costa Rica, Lücking 15212b); L, Astrothelium galbineum (Florida, Harris 41750); M, Trypethelium nitidiusculum (Costa Rica, Lücking 34530); N, Cryptothelium rhodotitthon (Brazil, Dumont 596); O, Laurera megasperma (Costa Rica, Lücking s.n.). — Scale bars = 1 mm. All images by R. Lücking. Version of Record (identical to print version). 975 Nelsen & al. • Phylogeny of Trypetheliaceae TAXON 63 (5) • October 2014: 974–992 Fig. 2. Microscopic images of ascospores (immature and mature) and an ascus, illustrating some of the diversity in ascospore types found in Trypetheliacae. A–C, ascospores from Astrothelium diplocarpoides (Florida, Lücking 26627): A, extremely immature; B, immature; C, mature. D–G, ascospores from Laurera gigantospora (Philippines, Rivas Plata 2128): D, extremely immature; E, immature; F, immature (more mature than E); G, mature. H, immature ascus from Laurera megasperma (Florida, Lücking 26710). I, mature ascospore from Bathelium tuberculosum (India, Lumbsch 19735). J, mature ascospore from Trypethelium subeluteriae (Costa Rica, Lücking 17611). K, immature ascospore from Architrypethelium seminudum (Costa Rica, Lücking 15212). L, mature ascospore from Architrypethelium nitens (Panama, Lücking 27038). M–N, ascospores from Aptrootia terricola (Costa Rica, Lücking 17211): M, immature; N, mature. O, post-mature ascospore from Aptrootia robusta (Tasmania, Lumbsch 20012n). — Scale bars: A, I–J = 20 µm; B–C, K–M = 50 µm; D–H, N–O = 100 µm. All images by R. Lücking. 976 Version of Record (identical to print version). TAXON 63 (5) • October 2014: 974–992 Nelsen & al. • Phylogeny of Trypetheliaceae (Kirk & al., 2001; Eriksson & al., 2004; Cannon & Kirk, 2007) or in the order Melanommatales (now Pleosporales) in Dothideomycetes (Barr, 1979, 1987; Harris, 1984, 1991). However, molecular data have since demonstrated its placement in Dothideomycetes (Lutzoni & al., 2004; Del Prado & al., 2006; Spatafora & al., 2006), but outside the order Pleosporales (Del Prado & al., 2006; Schoch & al., 2006, 2009; Spatafora & al., 2006; Nelsen & al., 2009, 2011b; Hyde & al., 2013). Dothideomycetes is the most speciose class of fungi within the phylum Ascomycota, including approximately 20,000 known species (Kirk & al., 2008). While this class is primarily composed of saprotrophs and plant pathogens, several recent studies have demonstrated the occurrence of lichen-forming or lichen-like (Lutzoni & al., 2004; Lumbsch & al., 2005; Del Prado & al., 2006; James & al., 2006; Muggia & al., 2008, 2013; Nelsen & al., 2009, 2011b; Schoch & al., 2009) and lichenicolous fungi (Lawrey & Diederich, 2003; Lawrey & al., 2011, 2012) within Dothideomycetes. The most diverse of these lichen-forming lineages in Dothideomycetes is the family Trypetheliaceae, placed in its own order, Trypetheliales (Chadefaud, 1960; Aptroot & al., 2008). Trypetheliaceae species are primarily lichen-forming, and have a predominantly tropical to subtropical distribution (Harris, 1984; Sipman & Harris, 1989; Aptroot, 1991b), with high numbers of species being reported from Central and northern South America (Harris, 1984; Aptroot & al., 2008), India (Makhija & Patwardhan, 1988, 1993; Awasthi, 2000; Singh & Sinha, 2010), Southeast Asia (Harris, 1984; Aptroot & Sipman, 1991; Aptroot & al., 1997, 2007; Vongshewarat & al., 1999; Wolseley & al., 2002; Aptroot, 2009b) and Australia (Aptroot, 2009a). Studies, especially from Costa Rica and Venezuela, have demonstrated that species occur primarily on tree trunks and branches in shaded to exposed microhabitats of lowland to lower montane (0–1000 m) rainforests, and also in savannas with distinct dry seasons (Aptroot & Seaward, 1999; Komposch & Hafellner, 2000, 2003; Komposch, & al., 2002; Rivas Plata & al., 2008; Aptroot & al., 2008). Thus far, lichen-forming Trypetheliaceae taxa have been found to form associations with algae from the order Trentepohliales (Johnson, 1940; Lambright & Tucker, 1980; Harris, 1984; Matthews & al., 1989; Aptroot, 1991b; Tucker & al., 1991; Komposch & al., 2002; Aptroot & al., 2008; Nelsen & al., 2011a). About 23%–31% of all lichen-forming fungi associate with these algae (Ahmadjian, 1993; Nelsen & al., 2011a), which are also primarily restricted to the tropics (Thompson & Wujek, 1997; López-Bautista & al., 2002, 2007). Within the tropics and subtropics, relatively high proportions (38%–45%) of the lichen-forming fungi associate with Trentepohliaceae algae (Ahmadjian, 1967; Tucker & al., 1991), while this proportion drops to only about 9% in temperate regions (Santesson, 1952; Ahmadjian, 1967). Consequently, Trypetheliaceae fungi associate with one of the more common lichen photobionts found in tropical ecosystems. At a cellular level, the nature of the fungal-algal attachment appears to vary, with some fungal species attaching through intraparietal haustoria (Matthews & al., 1989; Tucker & al., 1991), while others attach by means of appressoria (Lambright & Tucker, 1980). Trypetheliaceae fungi are nearly exclusively corticolous, forming a crustose, often in part endoperidermal (barkinhabiting) thallus. Thalli often grow in intimate contact with the host, with host cells often being integrated into the thallus or even occurring in specific layers within compound fruiting bodies (pseudostromata). Many species cause the host to form galls (Aptroot, 1998; Aptroot & al., 2008), and the family thus represents the only known group of cecidiogenous lichens. Species of Trypetheliaceae produce closed ascomata solitarily or aggregated in pseudostromata, with either separate ostioles or with ascomatal chambers laterally fused to share a common ostiole (Johnson, 1940; Harris, 1984; Aptroot, 1991b). Ascomata typically contain branched and anastomosing paraphysoids forming a distinct network, along with bitunicate asci containing hyaline (or brown in some lineages), transversely septate to muriform ascospores often with diamond-shaped lumina and angular wall thickenings (Morgan-Jones, 1972; LetrouitGalinou, 1973; Eriksson, 1981; Harris, 1984; Aptroot, 1991b; Lücking & al., 2007; Nelsen & al., 2009, 2011b; Sweetwood & al., 2012). Ontogenetic studies of muriform-spored taxa revealed these spores initially form transverse septa with diamond-shaped lumina and subsequently develop muriform septation, thus suggesting a close evolutionary connection between species producing these different ascospore types (Sweetwood & al., 2012). Ascospores from several species appear to germinate readily in culture and produce mycelia (Johnson, 1940; Mathey & Hoder, 1978a; Mathey, 1979; Mathey & al., 1980; Crittenden & al., 1995; Sangvichien & al., 2011). Consequently, Trypetheliaceae may prove a useful model for studying lichen-forming fungi, due to the relative ease of symbiont isolation. Secondary metabolites produced in Trypetheliaceae are polyketide-derived aromatic compounds, which are formed through the acetyl-polymalonyl pathway (Elix & StockerWörgötter, 2008). These metabolites are primarily restricted to xanthones, anthraquinones and a small number of perylenequinones, often providing thalli and/or ascoma with brilliant colors ranging from yellow to red (Stensiö & Wachtmeister, 1969; Culberson & Culberson, 1970; Mathey & Hoder, 1978b; Harris, 1984; Mathey & al., 1987, 1994; Aptroot, 1991b; Mathey & Lukins, 2001; Manojlovic & al., 2010). The synthesis of these substance classes is not restricted to lichen-forming fungi (C.F. Culberson, 1969; Elix & Stocker-Wörgötter, 2008; Daub & Chung, 2009; Mulrooney & al., 2012); instead these classes of substances are found in non-lichen-forming fungi and plants, suggesting that their production is not a consequence of the lichen-forming state (C.F. Culberson, 1969; Daub & Chung, 2009). The synthesis of perylenequinones is of interest, as these are photoactivated compounds, which in non-lichenized fungi frequently elicit pathogenic responses from host plants (Daub & al., 2005; Daub & Chung, 2009; Mulrooney & al., 2012). Little is known about the polyketide synthase genes responsible for the production of secondary metabolites in Trypetheliaceae, but Amnuaykanjanasin & al. (2009) have sequenced five type I reducing polyketide synthases from an unidentified Trypethelium Spreng. species and demonstrated their placement in subclades I, II and IV of Kroken & al. (2003), Version of Record (identical to print version). 977 Nelsen & al. • Phylogeny of Trypetheliaceae TAXON 63 (5) • October 2014: 974–992 with one gene forming part of what may be a new subclade (D3) of genes. The use of secondary metabolites as taxonomic characters to discern species has a long history in lichenology (Brodo, 1986; W.L. Culberson, 1969; Hawksworth, 1976; Rogers, 1989; Culberson & Culberson, 1994; Lumbsch, 1998), with recent molecular studies both supporting (Lumbsch & al., 2008; Lücking & al., 2008) and refuting (Articus & al., 2002; Buschbom & Mueller, 2006; Wirtz & al., 2008; Nelsen & Gargas, 2009) their correlation with species limits. The relationship between species classification and the presence/ absence of the secondary metabolite lichexanthone has been questioned by Harris (1991, 1995, 1998), who concluded that this metabolite is of little use for species classification in the family, while Aptroot & al. (2013a) regarded the presence/ absence of this metabolite as sufficient to segragate species. Future molecular-based studies should focus on the utility of this metabolite for species delimitation in Trypetheliaceae. The family Trypetheliaceae has traditionally included about ten genera (Lumbsch & Huhndorf, 2010a) and approximately 200 species (Harris, 1984; Aptroot, 1991b; Del Prado & al., 2006). Using sequence data, Nelsen & al. (2009, 2011b) further demonstrated the inclusion of species from Arthopyrenia A.Massal., Julella Fabre and Mycomicrothelia Keissl. in this family. Since these species share important characteristics with Trypetheliaceae, Nelsen & al. (2009, 2011b) suggested that the species included in their studies be placed in Trypetheliaceae under their current generic assignment, but refrained from formal taxonomic changes until the phylogenetic position of the types of these genera and their synonyms has been determined. Further work is needed to clarify the position of other species in these genera, as well as the genus Naetrocymbe Bat. & Cif. (Harris, 1995; Dai & al., 2013). Combinations of ecological, morphological and anatomical characters have traditionally been employed to delimit genera within Trypetheliacaceae (Table 1), including: substrate (corticolous vs. terricolous/muscicolous), ascomatal arrangement (solitary vs. aggregated vs. fused), ostiole orientation (apical vs. eccentric), ascospore septation (transversely septate vs. muriform), ascospore color (hyaline vs. darkened) and ascospore size (Harris, 1990, 1995; Del Prado & al., 2006) (Table 1). While conidial and pycnidial characters have been utilized for taxonomic purposes in other lineages, the infrequent occurrence of pycnidia in Trypetheliaceae taxa has generally prevented their use for taxonomic purposes in this family (but see Harris, 1991). Harris (1989a, 1995) anticipated that the classification scheme employed for Trypetheliaceae was artificial, and would result in the polyphyly of some genera. His contention was predicated on two central arguments: (1) differences in ascospore septation (muriform vs. transversely septate) were minor and could not be used to delimit genera, a principle which he had applied to the Trypetheliaceae genera Bathelium Ach., Mycomicrothelia and Polymeridium (Müll.Arg) R.C.Harris (Harris, 1989b, 1991, 1995); (2) differences between compound ascomata (with a shared ostiole) and simple ascomata were minor, citing species of the Astrothelium variolosum (Ach.) Müll.Arg./ Trypethelium variolosum Ach., complex as a continuum with numerous intermediate morphologies; consequently, he argued against the use of these ascomatal differences for the segregation of genera (Harris, 1995). Though Harris (1989a, 1995) was aware of the problems pertaining to generic concepts in Trypetheliaceae, he refrained from formally making taxonomic changes until it was clear what character or character state combinations reflected phylogeny (Harris, 1989a, 1995), fearing that if changes were made prematurely, a large number of Table 1. Anatomical and morphological character state combinations used to distinguish genera within Trypetheliaceae (from Harris, 1984; Aptroot & al., 2008, 2013b). Aptrootia Ascospore color Ascospore septation Ascospore walls Ascospore size Ascomatal distribution Ostiole type Ostiole orientation Brown Muriform Thick Large Solitary Single Apical Architrypethelium Hyaline/rown Transverse Thick Large Solitary Single Apical/Lateral Astrothelium Hyaline Transverse Thick Small-Medium Aggregate Shared Lateral Bathelium Hyaline Transverse/Muriform Thick Small-Medium Aggregate Shared Apical Campylothelium Hyaline Muriform Thick Medium-Large Solitary Single Lateral Cryptothelium Hyaline Muriform Thick Medium-Large Aggregate Shared Lateral Laurera Hyaline Muriform Thick Medium-Large Solitary (to aggregate) Single Apical Marcelaria Hyaline Muriform Thin Medium-Large Solitary (to aggregate) Single Apical Polymeridium Hyaline Transverse/Muriform Thin Small-Medium Solitary Single Apical/Lateral Small Pseudopyrenula Hyaline Transverse Thick Trypethelium Hyaline Transverse Thin/Thick Small-Medium Solitary Single Apical Solitary to Aggregate Single Apical (Arthopyrenia) Hyaline Transverse Thin Small Solitary Single Apical (Julella) Hyaline Muriform Thin Small Solitary Single Apical Transverse Thin Small Solitary Single Apical (Mycomicrothelia) Brown Some species of Arthopyrenia, Julella and Mycomicrothelia have recently been shown to belong to Trypetheliaceae, and are included in parentheses as these genera have not traditionally been included in the family. 978 Version of Record (identical to print version). TAXON 63 (5) • October 2014: 974–992 Nelsen & al. • Phylogeny of Trypetheliaceae species would be placed temporarily in a single, variable genus (Laurera Rchb.). Del Prado & al. (2006) and Aptroot & al. (2008) subsequently echoed Harris’s (1989a, 1995) contentions, further emphasizing the need for a revision of generic concepts within Trypetheliaceae. Utilizing molecular data, Del Prado & al. (2006) and Nelsen & al. (2009) began assessing Harris’s (1989a, 1995) assertions, and demonstrated the non-monophyly of Trypethelium and Astrothelium Eschw. With the exception of these studies, which focused primarily on establishing the relationship of Trypetheliaceae to other higher-level taxa, no molecular-based studies have explicitly studied relationships within Trypetheliaceae or appraised generic concepts. Here we expand taxon sampling to examine relationships among taxa and assess existing generic concepts, while evaluating the utility of morphological and anatomical character state combinations for systematic purposes in Trypetheliaceae. MATERIALS AND METHODS Taxon selection. — We included multiple species from all genera traditionally referred to Trypetheliaceae (including the Arthopyrenia, Julella and Mycomicrothelia species demonstrated to belong to Trypetheliales by Nelsen & al. (2009, 2011b), with the exception of the monospecific genera Exiliseptum R.C.Harris (Harris, 1984), which has now been placed in Polymeridium (Müll.Arg.) R.C.Harris (Aptroot & Cáceres, 2014), and Melanophloea P.James & Vězda (Aptroot & Schumm, 2012), for which we were unable to obtain fresh material. Additional genera and species traditionally placed in Arthopyreniaceae and Naetrocymbaceae were not included, and further work is needed to elucidate their placement. Additionally, Trypetheliopsis Asahina (= Musaespora Aptroot & Sipman) was not included, as T. kalbii (Lücking & Sérus) Aptroot has been shown to belong to another family, Monoblastiaceae (Nelsen & al., 2009, 2011b; Hyde & al., 2013). Several taxa from Botryosphaeriales, Capnodiales, Dothideales and Myriangiales were included as outgroups. Taxa included in the analyses, along with GenBank accession numbers and collection information for newly sequenced samples are located in Appendix 1. DNA extraction, amplification and sequencing. — The Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit (St. Louis, Missouri, U.S.A.) was used to isolate DNA, following the manufacturer’s instructions, except only 10–30 µl of extraction buffer and 10–30 µl dilution buffer were used, and a 20× DNA dilution was then used in subsequent PCR reactions. A portion of the fungal mitochondrial small subunit (mtSSU) was amplified and sequenced using combinations of the following primers: mrSSU1, mrSSU2, mrSSU2R, mrSSU3R (Zoller & al., 1999), MSU7 (Zhou & Stanosz, 2001), mrSSU-1/2-5′mpn and mrSSU-2/3-3′-mpn (Nelsen & al., 2011b). Additionally, a portion of the fungal nuclear large subunit (nuLSU) was amplified and sequenced using combinations of the primers f-nu-LSU-0116-5′/ITS4A-5′ (Nelsen & al., 2011b, 2012; reverse complement of D.L. Taylor’s ITS4A in Kroken & Taylor, 2001), AL2R (Mangold & al., 2008), f-nu-LSU-0287-5′-mpn (Nelsen & al., 2011b), LR3 (Vilgalys & Hester, 1990), LR3R (reverse complement of LR3 from Vilgalys & Hester, 1990), LR4 (http:// www.biology.duke.edu/fungi/mycolab/primers.htm), LR5 (Vilgalys & Hester, 1990) and LR6 (Vilgalys & Hester, 1990). The 10 µl PCR reactions consisted of 5 µM of each PCR primer, 3 mM of each dNTP, 2 µl of 10 mg/mL 100× BSA (New England BioLabs, Ipswich, Massachusetts, U.S.A.), 1.5 µl 10× PCR buffer (Roche Applied Science, Indianapolis, Indiana, U.S.A.), 0.5 µl Taq, approximately 2 µl diluted DNA, and 2 µl water or 2.5–5 µl REDExtract-n-Amp PCR Ready Mix (SigmaAldrich), 5 µM of each PCR primer, 2 µl diluted DNA and 2–4.5 µl water. The PCR cycling conditions were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, 53°C (mtSSU), 55°C (nuLSU: AL2R/LR3) or 60°C (nuLSU: f-nuLSU-0116-5′/ITS4A-5′ with LR3 or LR6) for 1 min, and 72°C for 1 min, followed by a single 72°C final extension for 7 min. Samples were visualized on a 1% ethidium bromide-stained agarose gel under UV light and bands were gel extracted, heated at 70°C for 5 min, cooled to 45°C for 10 min, treated with 1 µl GELase (Epicentre Biotechnologies, Madison, Wisconsin, U.S.A.) and incubated at 45°C for at least 24 hours. The 10 µl cycle sequencing reactions consisted of 1–1.5 µl of Big Dye v.3.1 (Applied Biosystems, Foster City, California, U.S.A.), 2.5–3 µl of Big Dye buffer, 1–6 µM primer (primers listed above), 0.75–2 µl GELase-treated PCR product and water. Cycle sequencing was performed using one of the following conditions: 96°C for 1 min, followed by 25 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 4 min or instead 96°C for 1 min, followed by 40 cycles of 96°C for 10 s, 45°C for 5 s and 60°C for 4 min. Samples were precipitated and sequenced in an Applied Biosystems 3730 DNA Analyzer, and sequences assembled in Sequencher v.4.9 (Gene Codes, Ann Arbor, Michigan, U.S.A.). Most DNA analyses were performed at the Pritzker Laboratory for Molecular Systematics and Evolution at the Field Museum. Phylogenetic analyses. — Sequences were aligned using a combination of automated (Muscle v.3.6: Edgar, 2004) and manual (Se-Al v.2.0a11: Rambaut, 1996; Mesquite v.2.73: Maddison & Maddison, 2010) alignment. Ambiguous regions were visually identified and excluded together with introns. The final alignment has been deposited in TreeBase (http:// treebase.org, ID 15181). A combined, partitioned (by locus) maximum likelihood (ML) analysis was performed in RAxML v.7.2.8 (Stamatakis, 2006), using the GTR-GAMMA model, and support was estimated by performing 10,000 fast bootstrap pseudoreplicates (Stamatakis & al., 2008). Additionally, a Bayesian analysis was performed using Markov chain Monte Carlo (MCMC) sampling (Larget & Simon, 1999) in MrBayes v.3.2.1 (Ronquist & al., 2012). We performed reversible-jump MCMC analysis (Huelsenbeck & al., 2004), partitioning the dataset by gene and employing the time-reversible class of substitution models with gammadistributed rate heterogeneity. This precluded the a priori selection of a single substitution model for each data partition, instead permitting models to be sampled in proportion to their posterior probability. Two parallel analyses were then run at a temperature of 0.05 in MrBayes for 30 million generations, Version of Record (identical to print version). 979 Nelsen & al. • Phylogeny of Trypetheliaceae TAXON 63 (5) • October 2014: 974–992 with four chains each, sampling every 1000 generations. The program AWTY (Wilgenbusch & al., 2004; Nylander & al., 2008) was used to assess convergence between parallel runs by creating a bivariate plot of bipartitions. Additionally, the average standard deviation of split frequencies (Lakner & al., 2008) was monitored to ensure it dropped below 0.01, and the potential scale reduction factor (Gelman & Rubin, 1992) for all parameters was examined and found to approach 1.0. Initial burn-in trees (first 25%) were discarded for each run and a majority-rule consensus tree constructed. The ML phylogeny and character states were plotted using the ape (Paradis & al., 2004) and phyloch (Heibl, 2012) packages in the R statistical language (R Core Team, 2012). Relationships were considered supported if they had ML bootstrap support (BS) values of 70 or greater and Bayesian posterior probabilities (PP) of 0.95 or greater. To assess potential conflict between loci, individual phylogenies for each locus were generated in RAxML v.7.2.8 (Stamatakis, 2006) using the methods described above. We then examined whether supported (bootstrap support values greater than or equal to 70) clades from single-locus phylogenies were in conflict, using the python program compat.py v.3.0 (Kauff & Lutzoni, 2002, 2003). Conflict between supported clades was taken as evidence for topological incongruence. RESULTS The final dataset consisted of 85 OTUs (79 ingroup) and 1016 unambiguously aligned characters (nuLSU: 406; mtSSU: 610). In the Bayesian analysis, several 4–6 parameter models (including the gamma distributed rate heterogeneity parameter) obtained the highest posterior probabilities (Table 2). No single model achieved an exceptionally high posterior probability; instead, several models, especially in the nuLSU dataset, contributed to the final sample. Conflict was detected among loci for a number of taxa (Aptrootia terricola (Aptroot) Lücking & al., Astrothelium cf. robustum Müll.Arg., Bathelium madreporiforme (Eschw.) Trevis., Campylothelium sp. 1, C. cartilagineum Table 2. The GTR + G substitution submodels with the three highest posterior probabilities for each partition from the Bayesian analysis. Locus Posterior Standard GTR submodela probability deviation Minimum Maximum probability probability nuLSU 121341 0.201 nuLSU 121131 0.096 0.003 0.094 0.098 nuLSU 121343 0.093 0.003 0.090 0.095 mtSSU 123121 0.227 0.000 0.227 0.227 mtSSU 123141 0.226 0.006 0.222 0.231 mtSSU 123451 0.057 0.002 0.056 0.059 a 0.204 0.004 0.207 Numbers indicate the rate parameter categories in each submodel for: A↔C, A↔G, A↔T, C↔G, C↔T and G↔T, respectively. Results highlight that no individual submodel obtained an exceptionally high posterior probability; instead, numerous submodels were sampled. 980 Vain., Cryptothelium cecidiogenum Aptroot & Lücking, Laurera gigantospora (Müll.Arg.) Zahlbr., Pseudopyrenula subnudata Müll.Arg. 293, Trypethelium aeneum (Eschw.) Zahlbr., T. cinerorosellum Kremp., T. nitidiusculum (Nyl.) R.C.Harris, and the Julella fallaciosa (Stizenb. ex Arnold) R.C.Harris /Mycomicrothelia sp. nov. /M. oleosa Aptroot clade), with individual gene trees shown in Figs. S1–S2 (Electr. Suppl.). As supported differences in position were primarily confined to varying positions within terminal clades, we ignored these conflicts and retained all sequences. Several well-supported clades were recovered, although backbone relationships remained unresolved in some parts of the tree. However, the resulting topology (Fig. 3) illustrates that only five small genera traditionally or recently placed in Trypetheliaceae appear monophyletic (Architrypethelium Aptroot, Aptrootia Lücking & Sipman, Campylothelium Müll.Arg., Marcelaria Aptroot & al. and Pseudopyrenula Müll.Arg.), while the majority of genera, as currently circumscribed, are not monophyletic (Astrothelium, Bathelium, Cryptothelium A.Massal., Laurera, Polymeridium, Trypethelium). Two of the three included Arthopyrenia species formed a monophyletic group, while A. bifera Zahlbr. formed an unsupported relationship with Mycomicrothelia and Julella species. Clade composition. — Pseudopyrenula occupies an earlydiverging position among the genera traditionally placed in Trypetheliaceae. Pseudopyrenula is characterized by species forming solitary ascomata with hyaline, three-septate ascospores and an ecorticate thallus. The three Pseudopyrenula species we have studied, including the type, P. diluta (Fée) Müll.Arg., formed a strongly supported, monophyletic clade. Species in the Trypethelium eluteriae Sprengel group, i.e., Trypethelium s.str. (including T. eluteriae, T. platystomum Mont. and T. inamoenum Müll.Arg.) formed a strongly supported clade. Species in this group frequently produce ascomata in pseudostroma, which usually contain, and are often also covered with anthraquinone pigments (Fig. 1H). In addition, these species produce thin-walled ascospores with oval to rectangular lumina (Fig. 2J). Sister to the T. eluteriae group is a clade representing the recently segregated Marcelaria, composed of M. purpurina (Nyl.) Aptroot & al. (Fig. 1G) and M. cumingii (Mont.) Aptroot & al., which have traditionally been placed in Laurera. These species produce ascomata with a broad ostiole, as well as large muriform ascospores. Campylothelium species formed an unsupported, monophyletic group. These taxa produce ascomata with a lateral ostiole (Fig. 1F) and hyaline, muriform ascospores. Trypethelium virens Tuck. and an undescribed taxon, both of which produce hyaline, transversely septate ascospores, formed a strongly supported group together with Campylothelium species, even if clearly differing morphologically. Relationships within this clade remain obscure, due to low internal support; however, the results do suggest a relatively close relationship between these taxa producing either large, muriform or small, transversely septate ascospores. Two other small genera, Aptrootia and Architrypethelium, each formed strongly supported monophyletic groups, basal to a large, terminal “Astrothelium” clade. All described Aptrootia Version of Record (identical to print version). TAXON 63 (5) • October 2014: 974–992 Nelsen & al. • Phylogeny of Trypetheliaceae species were included in the present study; these taxa are primarily characterized by their unusual ecology (for Trypetheliaceae), occurring in temperate habitats (or montane, tropical regions) on moss, soil or rotting wood, and the production of one to two large, brown, muriform ascospores/ascus (Figs. 1J, 2M–O). Two of the three currently accepted Architrypethelium species were sampled in the present study, including the type, A. nitens (Fée) Aptroot. This genus is characterized primarily by the production of large ascospores with few (3–5) septa (Figs. 1K, 2K–L). Both sampled taxa differ in their ostiole orientation, with A. uberinum (Fée) Aptroot forming an apical ostiole, while A. nitens produces a lateral ostiole. Fig. 3. Phylogeny (mtSSU + nuLSU) of Trypetheliaceae obtained under ML. Bootstrap proportions ≥ 70 are placed above branches, and branches subtending nodes with Bayesian posterior probabilities ≥ 0.95 are bolded. Types for each genus (where sequenced) are followed by an asterisk. Several characters (and their states) are given on the righthand side of the figure: ascospore color (filled = brown, hollow = hyaline); ascospore septation (filled = muriform, hollow = transversely septate); ascomatal distribution (filled = aggregated, hollow = solitary); pseudostroma (filled = pseudostroma formed, hollow = absence of pseudostroma); ostiole type (filled = ostioles shared, hollow = ostioles not shared); ostiole orientation (filled = lateral, hollow = apical). Astrothelium Clade * * Architrypethelium * Aptrootia Bathelium s. str. * Trypethelium s. str. Marcelaria * Campylothelium * Polymeridium * * Pseudopyrenula Outgroup substitutions/site Version of Record (identical to print version). 981 Nelsen & al. • Phylogeny of Trypetheliaceae TAXON 63 (5) • October 2014: 974–992 Species currently placed in Bathelium were recovered in disparate locations, with B. lineare (C.W.Dodge) R.C.Harris, B. madreporiforme, B. tuberculosum (Makhija & Patw.) R.C.Harris, and an undescribed Bathelium species forming a strongly supported group corresponding to Bathelium s.str. In contrast, Bathelium degenerans (Vain.) R.C.Harris and B. endochryseum (Vain.) R.C.Harris formed a strongly supported group with species currently placed in Astrothelium and Trypethelium in the “Astrothelium” clade. Species in the Bathelium s.str. clade form large, sessile pseudostroma containing anthraquinones, and produce hyaline, muriform or rarely transversely septate ascospores (Fig. 2I), while those occurring outside this clade form small, semi-immersed pseudostromata and all produce hyaline, transversely septate ascospores. Finally, a large number of taxa grouped in the “Astrothelium” clade, which was composed of species currently placed in Astrothelium, Bathelium, Cryptothelium, Laurera and Trypethelium (Fig. 1L–O). Species from most of these genera do not form monophyletic groups within this clade but instead appear mixed. This clade is composed of taxa with variable ascomatal morphology and hyaline ascospores that are muriform or transversely septate (Figs. 1L–O, 2A–H). Support within this clade appears weak; however, the clade itself is strongly supported and further serves to illustrate the non-monophyly of genera such as Bathelium, Laurera, and Trypethelium. While at first glance there are no morphological characters supporting this phylogeny, upon closer inspection some patterns become apparent. Thus, species of Trypethelium within the “Astrothelium” clade have astrothelioid ascospores similar to those of Astrothelium, with diamond-shaped lumina, whereas those of Trypethelium s.str. have a different type of lumina. The species of Laurera falling outside this clade and in Marcelaria have a distinct ascoma anatomy. The two Bathelium species within the “Astrothelium” clade have pseudostromata that are morphologically different from those representing Bathelium s.str. Overall, the “Astrothelium” clade concentrates practically all species that have a well-developed, often greenish thallus with thick cortex and thick photobiont layer, combined with astrothelioid ascospores when transversely septate (or young stages of muriform ascospores), whereas the other clades either differ in thallus structure or in ascospore type. Character state distribution among clades. — Figure 3 also illustrates that taxa sharing particular character states or character state combinations used to define genera do not typically form a monophyletic clade. Species producing solitary ascomata are scattered across the phylogeny, while those producing pseudostromata are primarily restricted to the T. eluteriae, B. madreporiforme/tuberculosum, “Astrothelium” and Campylothelium clades. Taxa producing apical and lateral ostioles are scattered across the phylogeny, while taxa producing fused ostioles are restricted to the “Astrothelium” clade, although many taxa in this clade do not produce fused ostioles. The majority of Trypetheliaceae taxa produce hyaline ascospores, with brown ascospores being restricted to some species of Architrypethelium and all species of Aptrootia and Mycomycrothelia. The two types of ascospore septation 982 (transversely septate and muriform) appear scattered across the phylogeny, with the “Astrothelium” and Campylothelium clades containing both types of septation, while most other clades appear restricted primarily to a single type of septation. All taxa in the “Astrothelium” clade and its closest relatives produce thick-walled ascospores. This trait, however, is not confined to this group, as it is also found in Pseudopyrenula, T. tropicum (Ach.) Müll.Arg., the Marcelaria clade and part of the T. eluteriae group. Finally, lichexanthone is known from the “Astrothelium” clade as well as Marcelaria, Pseudopyrenula and Polymeridium, while anthraquinones are absent from earlydiverging lineages but especially common in the Marcelaria, Trypethelium eluteriae and “Astrothelium” clades. DISCUSSION The resulting molecular-based phylogeny of Trypetheliaceae strongly conflicts with traditional genus-level classification schemes within the family, as was anticipated by previous morphological (Harris, 1989a, 1995; Aptroot & al., 2008) and molecular (Del Prado & al., 2006; Nelsen & al., 2009) studies. Formal changes to the existing classification, in particular combination of most species of the family into the genus Astrothelium, will be put forward in a separate monograph of the family (Aptroot & al., in prep.), but we discuss the individual clades in detail below. The “Astrothelium” clade. — The overwhelming majority of Trypetheliaceae species are concentrated in the genera traditionally recognized as Astrothelium, Cryptothelium, Laurera and Trypethelium. Harris (1995) argued that the delimitation of these genera was artificial, and predicted that many species from these genera would eventually be placed in a single genus (Laurera). Our findings are largely consistent with Harris’s (1995) prediction for Trypetheliaceae, as species from these genera form non-monophyletic groups in the “Astrothelium” clade. However, the name Astrothelium (Eschweiler, 1824) has priority over Laurera (Reichenbach, 1841), so we instead use the name Astrothelium for this clade. This clade includes Cryptothelium sepultum (Mont.) A.Massal., the type of Cryptothelium. Although we did not have sequences from the type of Astrothelium (A. conicum Eschw.) and Laurera (L. varia (Fée) Zahlbr.), we anticipate Astrothelium conicum to be placed in this clade because of its close similarity with other sequenced species. Laurera varia, on the other hand, is expected to cluster either within the main “Astrothelium” clade or within the small clade immediately at the base, sister to the main clade. The “Astrothelium” clade is pantropical and composed of corticolous taxa, producing corticate thalli with ascomata embedded in pseudostromata or thalline warts/galls (Fig. 1L–O). A large amount of variability exists in the ascomatal arrangement (solitary vs. aggregated vs. fused), ostiole orientation (apical vs. eccentric), ascospore septation (transversely septate vs. muriform) and size. All taxa produce hyaline ascospores; transversely septate ascospores are of the astrothelioid-type, which are characterized by their thick-walled distosepta and diamond-shaped lumina (Fig. 2A–C). Even muriform-spored Version of Record (identical to print version). TAXON 63 (5) • October 2014: 974–992 Nelsen & al. • Phylogeny of Trypetheliaceae species, such as Laurera gigantospora (Fig. 2D–G), pass through an astrothelioid stage during ascospore ontogeny (Sweetwood & al., 2012). A few species of Bathelium, especially those with small, transversely septate ascospores, are placed in the “Astrothelium” clade. This genus was resurrected by Harris (1995) to include several taxa previously placed in Trypethelium and Laurera. These taxa were presumably united by pseudostromatal characteristics, such as aggregated ascomata produced in brown pseudostromata containing anthraquinones, as well as by the production of an outer pseudostromatal layer of jigsaw puzzle-shaped cells (Harris, 1995). Our results suggest that muriform-spored species producing this pseudostromatal type (Bathelium s.str.) form a monophyletic group (B. lineare, B. madreporiforme, B. tuberculosum) together with an undescribed species with transversely septate ascospores. In the nuLSU analysis, these three Bathelium species formed a supported monophyletic group with the undescribed species sister to it, while in the mtSSU analysis, the undescribed species formed a supported group with B. madreporiforme (Electr. Suppl.: Figs. S1–S2). Although we did not have sequence data from B. mastoideum Afzel. ex Ach., the type of Bathelium, we expect it to group with the other muriform-spored Bathelium species sequenced here in the clade referred to as Bathelium s.str., because of its close similarity with B. madreporiforme. This Bathelium clade falls outside the “Astrothelium” clade in the combined analysis (Fig. 3), while B. degenerans, B. endochryseum and B. feei (C.F.W.Meissn.) Aptroot, all species forming transversely septate ascospores, are placed within or near the “Astrothelium” clade. These findings partially confirm the early concept of Bathelium as recognized by Trevisan (1853) and Massalongo (1860), who did not include taxa with transversely septate ascospores in Bathelium. In the present case, it appears that pseudostroma type, rather than ascospore type, predicts phylogenetic placement, as the undescribed species with transversely septate ascospores clustering with Bathelium s.str. in Fig. 3 forms pseudostromata of the same type as the muriform-spored species, while the species clustering within Astrothelium produce smaller pseudostromata with a different morphology. Trypethelium s.str. — The Trypethelium eluteriae group (Fig. 3) appears quite distinct from the remaining Trypethelium species, which are primarily restricted to the “Astrothelium” clade. While Aptroot & al. (2008) suggested T. eluteriae may be related to Bathelium, our data do not confirm this hypothesis, and instead suggest the T. eluteriae group is closely related to Marcelaria (Laurera purpurina and L. cumingii groups). As T. eluteriae is the type of the genus, this clade represents Trypethelium s.str. This group is comprised of several corticolous species that produce ascomata aggregated in large pseudostroma that usually contain brightly colored anthraquinones (Fig. 1H). In addition, this group produces transversely septate ascospores with a reduced endospore forming rounded to oval lumina (Fig. 2J), which are different from the diamond-shaped lumina produced by most other species in the family (Fig. 2C). However, ascospores in T. eluteriae pass through an astrothelioid stage early in their ontogeny (Sweetwood & al., 2012). Makhija & Patwardhan (1993) extended and applied an ascomatal classification scheme to Trypethelium, which previously had been used to delimit infrageneric groups in Laurera (Letrouit-Galinou, 1957, 1958; Upreti & Singh, 1987a). While only six to seven groups were found in Laurera (LetrouitGalinou, 1957, 1958; Upreti & Singh, 1987a), twelve pseudostromal types were found in Trypethelium species from India alone, highlighting the variability in pseudostromal types within Trypethelium (Makhija & Patwardhan, 1993). When concentrating on the Trypethelium s.str. clade, species producing the T. eluteriae- and T. subeluteriae Makhija & Patw.-types of pseudostroma were recovered. The T. eluteriae-type pseudostroma is separated from the thallus by the absence of cortical, algal and medullary layers; additionally, a cortical layer is produced beneath the pseudostroma, and ascomata are surrounded by a single layer either composed of a hyaline layer or a layer filled with yellow to orange crystals (Makhija & Patwardhan, 1993). In contrast, the T. subeluteriae-type pseudostroma contains a cortical layer both beneath the pseudostroma, as well as above it (Makhija & Patwardhan, 1992, 1993). Monophyletic genera. — The only genera recovered here as monophyletic (Aptrootia, Architrypethelium, Campylothelium, Marcelaria, Pseudopyrenula), are species-poor; consequently, their monophyly is not entirely surprising, given their low species richness and narrow generic delimitation, but also reflects their unique morphology and ecology. Architrypethelium as currently circumscribed contains three species, two of which were included in this study, including the type, A. nitens (Aptroot & al., 2008). These two species are strongly supported as a monophyletic group and occupy a position that is near to the “Astrothelium” clade. Architrypethelium was first described by Aptroot (1991b) for species with large, brown, 3–5-septate ascospores (Fig. 2L). These ascospores pass through an initial astrothelioid stage before producing their characteristic architrypethelioid ascospores (Sweetwood & al., 2012). All species are corticolous and are known only from the Neotropics. The collection of Architrypethelium uberinum, reported here, is of note as this taxon has only rarely (Aptroot, 2002; Aptroot & al., 2008) been collected since the 1820s–1890s (Aptroot, 1991b). All three species of Aptrootia form a strongly supported monophyletic group sister to the Architrypethelium + “Astrothelium” clade. Aptrootia species produce thin, cartilaginous and bullate or verrucose thalli (Fig. 1J), and form one to two, extremely large, dark brown, muriform ascospores per ascus (Fig. 2M–O), which pass through an initial astrothelioid stage during their ontogeny (Sweetwood & al., 2012). These taxa typically grow at high altitudes on soil, although A. elatior (Stirt.) Aptroot has also been found on bark; consequently, Aptrootia differs in its ecology and distribution from most other Trypetheliaceae taxa (which are tropical and corticolous). Aptrootia was described for Thelenella terricola Aptroot (Aptroot, 1999) from Papua New Guinea and Costa Rica (Lücking & al., 2007) and shown to be part of Trypetheliaceae by Del Prado & al. (2006). Subsequently, two additional species have been placed in Aptrootia (Aptroot, 2009a): Aptrootia robusta (P.M.McCarthy & Kantvilas) Aptroot, an alpine lichen from Tasmania which grows over soil and plants (McCarthy & Kantvilas, 1993), and A. elatior, a Version of Record (identical to print version). 983 Nelsen & al. • Phylogeny of Trypetheliaceae TAXON 63 (5) • October 2014: 974–992 corticolous or hepaticolous taxon, known from temperate habitats in New Zealand, as well as Victoria, Australia (Galloway, 1983, 2007; Upreti & Singh, 1987b; Aptroot, 2009a). Aptrootia elatior is quite unusual as it produces bi-layered ascospores with brown warts on the surface and has consequently been discussed as possibly representing its own genus (Aptroot, 2009a); however, our data suggest a placement within Aptrootia. Campylothelium is a small, pantropical, corticolous genus producing solitary ascomata with lateral ostioles and muriform ascospores (Fig. 1F). This genus differs from Laurera primarily in its production of lateral ostioles. Harris (1995) and Aptroot & al. (2008) argued that many of the genera found in the “Astrothelium” clade would contain species of the genus Campylothelium. In the present study, the Campylothelium species included were placed outside the “Astrothelium” clade and in a weakly supported monophyletic group. However, only two previously described species were included in our study, and one of these, Campylothelium puiggarii Müll.Arg. (Fig. 1F), which is the type of Campylothelium, has previously been regarded as an unusual species in the genus, with Harris (1995) suggesting it would be placed outside the large “Astrothelium” clade. Sampling of additional Campylothelium species may reveal their placement in the “Astrothelium” clade. Trypethelium virens and an undescribed species, both with transversely septate ascospores, formed a supported clade with Campylothelium species, although their morphology would not necessarily suggest such a placement. The description of a new genus or genera for these Trypethelium species may be necessary. Surprisingly, Marcelaria purpurina (Fig. 1G) and M. cumingii were sister to the T. eluteriae group. This genus was recently described to accommodate Laurera purpurina (Nyl.) Zahlbr., L. cumingii (Mont.) Zahlbr., and L. benguelensis (Müll. Arg.) Zahlbr. (Aptroot & al., 2013b). Marcelaria purpurina, the type species of the genus, produces large ascomata that are bright red. The chemistry of this taxon has been attributed to a diverse combination of five anthraquinones (Stensiö & Wachtmeister, 1969; Aptroot & al., 2013b). Marcelaria purpurina appears somewhat similar morphologically to the exclusively paleotropical M. cumingii and M. benguelensis (Müll.Arg.) Aptroot & al., but the pseudostroma of the latter are covered with a yellow pigment. Letrouit-Galinou (1957) divided Laurera into several groups according to their pseudostromatal type. Based on this grouping, M. purpurina was placed in the L. megasperma (Mont.) Riddle group; however, our results suggest these two species are only distantly related. Marcelaria cumingii and M. benguelensis (Upreti & Singh, 1987a) were part of the Laurera cumingii group (Letrouit-Galinou, 1957), but ascoma morphology is very similar in the two groups (Aptroot & al., 2013b). These species all share a similar ascomatal morphology, which includes the production of individual ascomata with a broad ostiole, the presence of a split separating the excipulum from the thallus, and the absence of thalline tissue covering the ascomata. Despite their supported sister relationship, these species differ, however, from the T. eluteriae group in their production of solitary to loosely aggregated ascomata, their broad ostioles, muriform ascospores and the separation of the excipulum from the thallus by a split. 984 A further genus recovered as monophyletic, including the type, P. diluta, was the pantropical and corticolous Pseudopyrenula (Fig. 1C). Pseudopyrenula forms an ecorticate thallus with solitary ascomata with hyaline, three-septate ascospores. Aptroot (1991a) suggested Pseudopyrenula occupied a basal position in Trypetheliaceae, and somewhat similarly, Harris (1998) suggested Pseudopyrenula represented an extreme in the family; however, it was unclear to Harris which extreme (derived or ancestral) this genus occupied. The present study includes three species, which together form a relatively earlydiverging monophyletic group within the historic sense of the family (which does not include Arthopyrenia, Julella and Mycomicrothelia), thereby supporting Aptroot’s (1991a) prediction. The number of species recognized in Pseudopyrenula depends largely on which characters are recognized as being of taxonomic utility. Harris (1998) found no taxonomic value at the species level for segregating species based on their thallus UV reaction, hamathecium color or hamathecium inspersion. If these characters are, however, found to correlate with species delimitations, the number of species in the genus will be much larger. Here we chose to recognize differences in these characters at the species level and have used names that reflect their inclusion. The three species included here do not form monophyletic groups, even when employing a narrow delimitation, as is shown in Fig. 3; ultimately, denser taxon sampling at a lower phylogenetic scale will be needed to resolve species delimitation in Pseudopyrenula. Additional genera. — Several early-diverging lineages within the family form thalli that lack pigmentation, produce black ascomata and form more or less ecorticate, and often weakly or non-lichenized thalli, although there is weak support along this part of the topology. These species are classified in Arthopyrenia, Julella, Mycomicrothelia, Polymeridium and the aforementioned Pseudopyrenula. Polymeridium was initially described as a section for a number of Arthopyrenia species (Müller, 1883). Harris (1975) later identified similarities between Arthopyrenia sect. Polymeridium and Trypetheliaceae, specifically noting their microconidia, hymenial type, photobiont association and lack of mesospore thickening. This section was later raised to the genus level (Harris in Tucker & Harris, 1980) and has been thoroughly treated in Harris (1991) and Aptroot & Cáceres (2014); an additional, moleculer study verified its inclusion in Trypetheliaceae (Nelsen & al., 2011b). Polymeridium species (Fig. 1D) are tropical, drought tolerant, high-light requiring, and relatively fast-growing taxa that occur mostly in dry forests such as the Brazilian Caatinga (Harris, 1991; Cáceres, 2007). Most species are bark-inhabiting, although a bambusicolous taxon, Polymeridium bambusicola Aptroot & Ferraro, is known (Aptroot & Ferraro, 2000). In the present study, our data suggest species of Polymeridium are not monophyletic, though there is weak support in this portion of the tree. Our results suggest that P. proponens (Nyl.) R.C.Harris does not group with P. catapastum (Nyl.) R.C.Harris and P. albocinereum (Kremp.) R.C.Harris, a result somewhat anticipated by Harris (Tucker & Harris, 1980; Harris, 1991). Harris initially placed P. proponens in Campylothelium (Tucker & Harris, 1980), but later argued for a placement in Version of Record (identical to print version). TAXON 63 (5) • October 2014: 974–992 Nelsen & al. • Phylogeny of Trypetheliaceae Polymeridium (Harris, 1991). At present it is unclear where the type (P. contendens (Nyl.) R.C.Harris) groups relative to the sequenced species; however, it is expected to be near P. catapastum based on morphological similarities. If this is confirmed, Harris’s initial intuition to not include P. proponens in Polymeridium (Tucker & Harris, 1980) would be correct, and a new genus may have to be established for P. proponens and related species (Aptroot & al., 2013a). Tucker & Harris (1980) suggested a close relationship between Polymeridium and Pseudopyrenula, with these two genera being separated primarily by the reduced endospore in Polymeridium ascospores (Harris, 1984, 1995; Aptroot & al., 2008). A close relationship between the two groups cannot be confirmed here except that both diverge relatively early within the tree, although support is weak in critical parts of the tree bearing on this hypothesis. Two species of Polymeridium, P. albocinereum and P. catapastum, formed a supported relationship with Trypethelium tropicum. Harris (1984) had suggested that Trypethelium tropicum (Fig. 1E) produced a thallus similar to other Trypethelium species, but its ascomata were similar to those of Pseudopyrenula. The ascomata of T. tropicum are rather unusual among Trypetheliaceae taxa, producing a barrel-shaped fruiting body with a flat top. Our data confirm that T. tropicum is neither closely related to Trypethelium, nor to Pseudopyrenula; instead, it forms a supported relationship with P. albocinereum and P. catapastum, a finding which might be taken as support for the inclusion of T. tropicum in Polymeridium. Although T. tropicum produces astrothelioid type ascospores, which differ from those of Polymeridium, and its corticate, olive-green to olive-brown thallus is very different from Polymeridium species, P. sulphurescens s.l. produces a morphology that combines the flat-topped, barrel-shaped ascomata of T. tropicum and the unthickened, Polymeridiumtype ascospore. The inclusion of Arthopyrenia, Julella and Mycomicrothelia species in Trypetheliaceae has only recently been realized (Nelsen & al., 2009, 2011b). These lineages differ from the traditional circumscription of Trypetheliaceae by the production of cellular pseudoparaphyses (Eriksson, 1981; Hawksworth, 1985; Aguirre-Hudson, 1991), while core Trypetheliaceae produce trabeculate pseudoparaphyses, also known as paraphysoids (Eriksson, 1981; Harris, 1984; Barr, 1987; Aptroot, 1991). Additionally, Arthopyreniaceae taxa form broadly clavate asci with a broad, inamyloid ocular chamber, differing from the obclavate-cylindrical asci of Trypetheliaceae, which contain a refractive ring, and an exceptionally wide, inamyloid ocular chamber (Lücking & Nelsen, 2013; Lücking & al., 2013). Barr (1979) and Hawksworth (1985) both suggested a close relationship between Mycomicrothelia and Arthopyrenia s.str., with Hawksworth (1985) noting similarities between the two genera, such as the production of partially exposed ascomata and pycnidia containing bacillariform conidia (which are present in many Mycomicrothelia species). Hawksworth (1985) noted that Mycomicrothelia (Fig. 1B) species differ from Arthopyrenia (Fig. 1A) in the verruculose ornamentation of their ascospores, and also in that ascospores of Mycomicrothelia species turn brown while inside the asci. Aptroot (1995) suggested a probable placement in Arthopyreniaceae. Therefore, after finding Mycomicrothelia species belong to Trypetheliaceae (Nelsen & al., 2009), the inclusion of Arthopyrenia species in Trypetheliaceae, demonstrated by Nelsen & al. (2011b), may have been expected based on morphological similarities between these two genera. Based on these findings, the inclusion of Julella species in Trypetheliaceae (Nelsen & al., 2011b) is not surprising as Julella is considered the muriform-spored equivalent of Arthopyrenia s.l. (Harris, 1995; Aptroot & al., 2008). Additionally, Aptroot (2012) recently noted that the genus name Bogoriella Zahlbr. might precede that of Mycomicrothelia and may require the conservation of the name Mycomicrothelia, or the transfer of Mycomicrothelia species to Bogoriella. Our revision of the type material suggests that the type of Bogoriella, B. subpersicina Zahlbr., is conspecific with or at least related to what is currently known as Mycomicrothelia decipiens (Müll.Arg.) R.C.Harris or Ornatopyrenis muriformis Aptroot, a small group of non-lichenized species within Mycomicrothelia s.l. that are not related to the species sequenced here. Further studies are needed to determine whether Ornatopyrenis Aptroot s.str., based on O. queenslandica (Müll.Arg.) Aptroot (Aptroot, 1991b) is part of Mycomicrothelia, as has been suggested (Harris, 1995; Sipman & Aptroot, 2005; Aptroot, 2012). Mycomicrothelia s.l. suggested to be a highly polyphyletic taxon that requires more sequence data, but based on anatomical features, it appears that most species, including the type, M. macularis (Hampe ex A.Massal.) Keissl., are not related to Trypetheliaceae. Similarly, we expect that only a smaller part of all Arthopyrenia s.l. and Julella s.l. species are part of Trypetheliaceae. Several studies have demonstrated the placement of some Arthopyrenia and Julella taxa (or their segregates) outside of Trypetheliaceae and within Pleosporales (Lumbsch & al., 2005; Mugambi & Huhndorf, 2009; Nelsen & al., 2009, 2011b; Suetrong & al., 2009); therefore, these genera themselves are not monophyletic. Lücking & Nelsen (2013) have attempted to clarify where Arthopyrenia species are expected to group, and suggested that the type, A. cerasi (Schrad.) A.Massal., is unrelated to the tropical Arthopyrenia species included in the present study; however, sequence data are required before this can be formalized in describing a new genus for the tropical, lichenized species currently placed in Arthopyrenia. Work is also needed to clarify whether Julella species should be broadly delimited, as proposed by Aptroot & Van den Boom (1995) or narrowly delimited as suggested by Harris (1995), as well as to clarify the position of individual species. Sequences from the types of these genera will help illuminate and clarify the situation that is currently faced with these genera. Within Trypetheliaceae, the exact nature of the relationships between Arthopyrenia, Julella and Mycomicrothelia species is unknown, and there is weak support in this part of the tree; however, these groups do appear to form early divergent lineages within Trypetheliaceae. Broader context. — The results of this study agree with those from other fungal lineages, which suggest ascospore septation and ascomatal distribution are evolutionarily labile and not strongly conserved. For instance, the Dothideomycetes genus Lophiostoma Ces & De Not. has been shown to comprise Version of Record (identical to print version). 985 Nelsen & al. • Phylogeny of Trypetheliaceae TAXON 63 (5) • October 2014: 974–992 a monophyletic group of a relatively small number of species producing muriform or transversely septate ascospores that are hyaline or brown in color (Holm & Holm, 1988; Tanaka & Hosoya, 2008; Mugambi & Huhndorf, 2009). Somewhat similarly, the genus Misturatosphaeria Mugambi & Huhndorf is composed of species producing brown or hyaline ascospores that are muriform or transversely septate, and vary in their ascomatal immersion (erumpent vs. superficial) and distribution (solitary vs. aggregated) (Mugambi & Huhndorf, 2009). Differences in hamathecial tissue have previously been used to segregate the orders Pleosporales and Melanommatales (Barr, 1983). However, the significance of these differences has subsequently found to be overstated (Liew & al., 2000; Lumbsch & Lindemuth, 2001), and it is now accepted that families such as Massarinaceae (Zhang & al., 2009) and Tetraplosphaeriaceae (Tanaka & al., 2009), and even genera such as Lophiotrema Sacc. (Zhang & al., 2009), are not uniform in their hamathecial tissue. Consequently, the results of the present study do not conflict with observations in other fungal lineages. A classification scheme similar to that employed in Trypetheliaceae has also been employed in the family Pyrenulaceae. Again, Harris (1989b) has discussed the artificiality of this classification, citing the use of ostiole orientation and fusion (among others) as characters that have evolved multiple times within Pyrenulaceae. These characters are quite labile in Trypetheliaceae, as are ascospore-based characters, and characters based on ascomatal distribution, all of which are characters commonly used for genus-level delimitation in Pyrenulaceae (Harris, 1989b; Aptroot, 2012). It is unclear if the findings in the present study will also be observed in future studies of Pyrenulaceae, but initial molecular-based studies on this family have begun to reveal the non-monophyly of the genus Pyrenula Ach. (Gueidan & al., 2008; Weerakoon & al., 2012). Towards a new generic classification in Trypetheliaceae. — The non-monophyly of numerous genera currently recog- nized in Trypetheliaceae suggests that combinations of character states historically used to delimit genera are not strictly indicative of natural relationships. Refining classification in Trypetheliceae must rely on the use of presently employed character states in different combinations and/or the use of additional characters not presently employed. Specifically, luminal shape within ascospores appears as an important character for delimiting the T. eluteriae group. The findings of the present study illustrate the need for the recircumscription of genera, and future work will help clarify morphological synapomorphies of individual clades such that classification will reflect phylogeny. The findings of the present study also highlight the evolutionary lability of individual character states, ultimately suggesting they are of little use on their own for generic delimitations. This finding is especially true for the type of ascospore septation (septate vs. muriform), ostiole orientation (apical vs. eccentric), and ascomatal arrangement (solitary vs. fused or aggregated). Further study of the evolution of these and other characters may provide insight into their adaptive significance, by establishing whether these repeatedly evolved character states are correlated with environmental variables. 986 ACKNOWLEDGEMENTS We are grateful to a number of organizations for funding including: NSF-DEB 0715660 “Neotropical Epiphytic Microlichens—An Innovative Inventory of a Highly Diverse yet Little Known Group of Symbiotic Organisms” to The Field Museum (PI Robert Lücking), a grant from the Committee on Evolutionary Biology (University of Chicago) to Matthew Nelsen, and the Caterpillar® company provided funds to study lichens from Panama. The American Society of Plant Taxonomists is also acknowledged for a Graduate Student Research Grant awarded to Matthew Nelsen. Additionally, Matthew Nelsen was supported by the Brown Family Fellowship (Field Museum). 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G.A.de Vries: DQ678057, FJ190628; Hortaea werneckii (Horta) Nishim. & Miyaji: GU301818, GU561844; Mycosphaerella punctiformis (Pers.) Starbäck: DQ470968, FJ190611; Dothiora cannabinae Froid.: DQ470984, FJ190636; Myriangium duriaei Mont. & Berk.: DQ678059, AY571389. — INGROUP TAXA: Aptrootia elatior (Stirt.) Aptroot: New Zealand, Knight O61815 (OTA) [MPN560B], KM453754, KM453821; Aptrootia robusta (P.M.McCarthy & Kantvilas) Aptroot: Australia, Lumbsch 20012 (F) [MPN235B], KM453755, KM453822; Aptrootia terricola (Aptroot) Lücking, Umaña & Chaves: Costa Rica, Lücking 17211 (F) [HTL1501], KM453756, DQ328995; Architrypethelium nitens (Fée) Aptroot: Panama, Lücking 27038 (F) [MPN257], KM453757, KM453823; Architrypethelium uberinum (Fée) Aptroot: Brazil, Nelsen s.n. (F) [MPN489], KM453758, –; Arthopyrenia bifera Zahlbr.: Thailand, Nelsen s.n. (F) [MPN574], –, KM453824; Arthopyrenia cinchonae (Ach.) Müll.Arg.: Brazil, Lücking 29583 (F) [MPN333], JN872351, JN872349; Arthopyrenia cinchonae (Ach.) Müll.Arg.: Brazil, Lücking s.n. (F) [MPN417], KM453759, KM453825; Arthopyrenia planorbis (Ach.) Müll.Arg.: Brazil, Lücking 29584 (F) [MPN334], JN872352, JN872350; Astrothelium cf. robustum Müll.Arg.: Costa Rica, Mercado-Díaz 586 (F) [MPN754], KM453760, KM453826; Astrothelium cinnamomeum (Eschw.) Müll.Arg.: Costa Rica, Lücking 15322b (DUKE) [AFTOL110], AY584652, AY584632; Astrothelium confusum Müll.Arg.: Peru, Nelsen s.n. (F) [MPN98], GU327710, GU327685; Astrothelium crassum (Fée) Aptroot: Brazil, Cáceres 6011 (F) [MPN335], KM453761, KM453827; Astrothelium eustomum (Mont.) Müll.Arg.: Panama, Lücking 27059 (F) [MPN258], KM453762, KM453828; Astrothelium galbineum Kremp.: Panama, Lücking 27077 (F) [MPN260], KM453763, KM453829; Astrothelium leucoconicum Nyl.: Peru, Nelsen 4000c (F) [MPN42], KM453764, KM453830; Astrothelium scorioides Nyl.: Fiji, Lumbsch 20556h (F) [MPN770], KM453766, KM453831; Astrothelium aff. scorioides Nyl.: Brazil, Cáceres & Aptroot 11137 (F) [MPN703], KM453765, –; Astrothelium sp. 1.: Brazil, Lücking 31242 (F) [MPN422], KM453767, KM453832; Astrothelium variolosum (Ach.) Müll.Arg.: Peru, Nelsen s.n. (F) [MPN43], KM453768, KM453833; Astrothelium versicolor Müll.Arg.: Panama, Lücking 27045 (F) [MPN259], KM453769, KM453834; Bathelium degenerans (Vain.) R.C.Harris: Brazil, Lücking s.n. (F) [MPN442], KM453771, KM453836; Bathelium endochryseum (Vain.) R.C.Harris: Brazil, Lücking 31088 (F) [MPN436], KM453772, KM453837; Bathelium feei (C.F.W.Meissn.) Aptroot: Ecuador, Rivas Plata 4065 (F) [MPN397], KM453773, KM453838; Bathelium aff. feei (C.F.W.Meissn.) Aptroot: Panama, Lücking 27109 (F) [MPN267], KM453770, KM453835; Bathelium lineare (C.W.Dodge) R.C.Harris: Vietnam, Gueidan 2078 (F) [MPN741], KM453774, KM453839; Bathelium madreporiforme (Eschw.) Trevis.: Brazil, Lücking 23290 (F) [MPN354], KM453775, KM453840; Bathelium tuberculosum (Makhija & Patw.) R.C.Harris: India, Lumbsch 19739z (F) [MPN81], KM453777, KM453842; Bathelium sp. 1: Vietnam, Gueidan 3040 (F) [MPN743], KM453776, KM453841; Campylothelium puiggarii Müll.Arg.: Venezuela, Lücking 32241 (F) [MPN399], KM453779, KM453844; Campylothelium sp. 1: Panama, Nelsen s.n. (F) [MPN646], KM453780, KM453845; Campylothelium cartilagineum Vain.: Panama, Lücking 27125 (F) [MPN268], KM453778, KM453843; Cryptothelium cecidiogenum Aptroot & Lücking: Nicaragua, Lücking 28529 (F) [MPN210], KM453781, KM453846; Cryptothelium purpurascens (Müll.Arg.) Zahlbr.: Peru, Nelsen s.n. (F) [MPN53C], KM453782, KM453847; Cryptothelium sepultum (Mont.) A.Massal.: Peru, Nelsen 4000d (F) [MPN52C], KM453783, KM453848; Cryptothelium sp. 1: Peru, Nelsen 4001a (F) [MPN63C], GU327714, GU327690; Cryptothelium sp. 2: Brazil, Lücking 31004 (F) [MPN438], KM453784, KM453849; Julella fallaciosa (Stizenb. ex Arnold) R.C.Harris: U.S.A., Nelsen s.n. (F) [MPN547], JN887400, JN887412; Laurera gigantospora (Müll.Arg.) Zahlbr.: Panama, Lücking 33037 (F) [MPN590], KM453786, KM453851; Laurera aff. megasperma (Mont.) Riddle: Philippines, Rivas Plata 2108 (F) [MPN189], KM453785, KM453850; Laurera megasperma (Mont.) Riddle: Philippines, Rivas Plata 2093 (F) [MPN190], KM453787, KM453852; Laurera sanguinaria Malme: Brazil, Canêz 3133 [MPN765], KM453788, KM453853; Marcelaria cumingii (Mont.) Aptroot, Nelsen & Parnmen: Thailand, Parnmen s.n. (F) [MPN552], KM453789, KM453854; Marcelaria purpurina (Nyl.) Aptroot, Nelsen & Parnmen: Brazil, Cáceres 2009 [MPN323A], KM453790, KM453855; Mycomicrothelia hemisphaerica (Müll.Arg.) D.Hawksw.: Nicaragua, Lücking 28641 (F) [MPN102], GU327719, GU327695; Mycomicrothelia miculiformis (Nyl. ex Müll.Arg.) D.Hawksw.: Nicaragua, Lücking 28637 (F) [MPN101B], GU327720, GU327696; Mycomicrothelia minutula (Zahlbr.) D.Hawksw.: Thailand, Nelsen s.n. (F) [MPN567], –, KM453856; Mycomicrothelia oleosa Aptroot: Peru, Nelsen 4007a (F) [MPN95], GU327721, GU327697; Mycomicrothelia megaspora Aptroot & M.Cáceres: Brazil, Cáceres & Aptroot 11821 (F) [MPN700], KM453794, KM453857; Polymeridium albocinereum (Kremp.) R.C.Harris: Brazil, Lücking s.n. (F) [MPN439], KM453795, KM453858; Polymeridium catapastum (Nyl.) R.C.Harris: Venezuela, Lücking 26052 (F) [MPN358], JN887402, KM453859; Polymeridium proponens (Nyl.) R.C.Harris: Venezuela, Lücking 26103 (F) [MPN359], JN887403, KM453860; Pseudopyrenula diluta (Fée) Müll.Arg.: Venezuela, Lücking 26062 (F) [MPN362], KM453797, KM453861; Pseudopyrenula diluta (Fée) Müll.Arg.: Brazil, Lücking 31068 (F) [MPN697], KM453798, KM453862; Pseudopyrenula subgregaria Müll.Arg.: Thailand, Lücking 24079 (F) [MPN106], GU327724, GU327699; Pseudopyrenula subgregaria Müll.Arg.: Version of Record (identical to print version). 991 Nelsen & al. • Phylogeny of Trypetheliaceae TAXON 63 (5) • October 2014: 974–992 Appendix 1. Continued. U.S.A., Nelsen 4082b (F) [MPN391], KM453799, KM453863; Pseudopyrenula subnudata Müll.Arg.: Panama, Lücking 27014r1 (F) [MPN293], KM453801, KM453865; Pseudopyrenula subnudata Müll.Arg.: Panama, Lücking 27053 (F) [MPN292], KM453800, KM453864; Trypethelium aeneum (Eschw.) Zahlbr.: Peru, Nelsen s.n. (F) [MPN62], KM453802, KM453866; Trypethelium cinereorosellum Kremp.: Philippines, Rivas Plata 2110 (F) [MPN191], KM453809, KM453873; Trypethelium eluteriae Spreng.: India, Lumbsch 19701a (F) [MPN111], GU327726, KM453874; Trypethelium aff. eluteriae Spreng.: U.S.A., Nelsen 4169 (F) [MPN382], KM453803, KM453867; Trypethelium inamoenum Müll.Arg.: Thailand, Lücking 24125 (F) [MPN228], KM453810, KM453875; Trypethelium marcidum (Fée) Müll.Arg.: Panama, Lücking 27131a (F) [MPN304], KM453811, KM453876; Trypethelium neogalbineum R.C.Harris: Brazil, Cáceres & Aptroot 11100 (F) [MPN711], KM453812, KM453877; Trypethelium nitidiusculum (Nyl.) R.C.Harris: Nicaragua, Lücking 28640 (F) [MPN217], KM453813, KM453878; Trypethelium ochroleucum (Eschw.) Nyl.: Panama, Lücking 27046 (F) [MPN313], KM453814, KM453879; Trypethelium aff. ochroleucum (Eschw.) Nyl.: Brazil, Cáceres & Aptroot 11297 (F) [MPN704], KM453804, KM453868; Trypethelium aff. ochroleucum (Eschw.) Nyl.: Brazil, Cáceres & Aptroot 11201 (F) [MPN713], KM453805, KM453869; Trypethelium papulosum (Nyl.) Makhija & Patw.: Peru, Nelsen 4009a (F) [MPN97], GU327729, GU327707; Trypethelium aff. platyleucostomum Makhija & Patw.: Argentina, Lücking 30512 (F) [MPN349], KM453806, KM453870; Trypethelium aff. platystomum Mont.: Peru, Nelsen s.n. (F) [MPN54], KM453807, KM453871; Trypethelium aff. scorioides Leight.: Brazil, Lücking 29814 (F) [MPN336], KM453808, KM453872; Trypethelium pupula (Ach.) R.C.Harris: Colombia, Lücking 26305 (F) [MPN224], KM453815, KM453880; Trypethelium sp. 1: Fiji, Lumbsch 20551a (F) [MPN764], KM453817, –; Trypethelium sp. 2: Argentina, Lücking 30515 (F) [MPN351], KM453816, KM453881; Trypethelium subeluteriae Makhija & Patw.: Peru, Nelsen s.n. (F) [MPN49C], KM453818, KM453882; Trypethelium tropicum (Ach.) Müll.Arg.: U.S.A., Nelsen s.n. (F) [MPN130], KM453819, KM453883; Trypethelium virens Tuck.: U.S.A., Nelsen s.n. (F) [MPN497], KM453820, KM453884. 992 Version of Record (identical to print version).