mycological research 110 (2006) 511– 520

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Ruth DEL PRADOa, Imke SCHMITTa, Stefanie KAUTZb, Zdenek PALICEc, Robert LU¨ CKINGa, H. Thorsten LUMBSCHa,* aDepartment of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605, U.S.A. bFachbereich Biologie und Geografie, Universita¨t Duisburg-Essen, Campus Essen, Universita¨tsstraße 5, D-45517 Essen, Germany cInstitute of Botany, Academy of Sciences of the Czech Republic, CZ-25243 Pruhonice, Czech Republic article info abstract

Article history: The phylogenetic position of Trypetheliaceae was studied using partial sequences of the Received 15 July 2005 mtSSU and nuLSU rDNA of 100 and 110 ascomycetes, respectively, including 48 newly ob- Accepted 31 August 2005 tained sequences. Our analysis confirms Trypetheliaceae as monophyletic and places the Published online 18 April 2006 family in Dothideomycetes. , which were previously classified with Trypethelia- Corresponding Editor: Martin Grube ceae in or Melanommatales, are supported as belonging to Chaetothyriomycetes. Monophyly of Pyrenulales, including Trypetheliaceae is rejected using three independent Keywords: test methods. Monophyly of Arthopyreniaceae plus Trypetheliaceae, the two families includ- ing -forming fungi in Dothideomycetes, is also rejected, as well as a placement of Try- petheliaceae in (incl. Melanommatales). Molecular phylogeny ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Pleosporales Pyrenulales

Introduction The latter has previously been excluded from Trypethe- liaceae by Veˇzda (1968) based on the thin-walled, muriform as- Trypetheliaceae is a medium-sized family of tropical and sub- cospores, and classified in a separate family Laureraceae (Poelt tropical crustose pyrenocarpous mainly lichenized fungi with 1974), but Eriksson (1981) showed that Laureraceae fits well about 200 species (Trevisan 1861; Malme 1924; Letrouit-Galinou into Trypetheliaceae. Subsequently, three additional genera 1957, 1958; Harris 1984, 1990, 1991, 1995, 1998; Makhija & Pat- were described in the family by Aptroot (1991). wardhan 1988, 1993; Aptroot 1991; Aptroot et al. 1997). Most Based on morphological characters, such as bitunicate asci species grow endophloedically on bark and occur in lowland and graphidean ascospores, Trypetheliaceae have always been to submontane tropical rainforests, gallery forests, and man- regarded as closely related to Pyrenulaceae (Barr 1981; Eriksson groves. The family is characterized by bitunicate asci, asco- 1981; Henssen & Jahns 1973; Poelt 1974; Aptroot 1991; Harris spores with angular-wall thickenings and diamond-shaped 1995), and the family is currently placed in Pyrenulales (Eriksson lumina (syngraphidean sensu Sherwood 1981), rather thin et al. 2004). Thus far, molecular data to test this view are scarce. and richly branched and anastomosing pseudoparaphyses, The only molecular study including Trypetheliaceae was published and if present, a photobiont. Most taxa have asco- by Lutzoni et al. (2004),whoincludedoneTrypethelium sp. se- mata that are concentrated in pseudostroma. Nine genera quence which fell into Dothideomycetes. However, the relation- were placed in this family by Harris (1984), including . ships lacked support and hence no conclusions were drawn.

* Corresponding author. E-mail address: tlumbsch@fieldmuseum.org 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2005.08.013 512 R. del Prado et al.

A placement of Trypetheliaceae in Dothideomycetes would be nu-LSU-1125-30 (¼LR6) (Vilgalys & Hester 1990), nu-LSU-0654- surprising, as the supposedly related Pyrenulaceae belong to 50 (¼LR3R), nu-LSU-0635-30 (¼LR3) and nu-LSU-0948-30 (¼LR5) Chaetothyriomycetes (Lumbsch et al. 2004; Lutzoni et al. 2001, (Rytas J. Vilgays, website; http//www.biology.duke.edu/fungi/ 2004; Schmitt et al. 2005). The families containing lichen- mycolab/primers.htm); and (2) for the mtSSU rDNA: mrSSU1, forming fungi in the Chaetothyriomycetes, Pyrenulaceae and mrSSU2, mrSSU2R, mrSSU3R (Zoller et al. 1999) and MSU 7 are characterized by ascohymenial ascoma de- (Zhou & Stanosz 2001). The 25 ml PCR reactions contained velopment (Doppelbaur 1960; Janex-Favre 1970a, b), while 2.5 ml buffer, 2.5 ml dNTP mix, 2 ml of each primer (20 mm), 5 ml Dothideomycetes include species with ascolocular ascoma BSA, 2 ml Taq, 2.5 ml genomic DNA extract and 6.5 ml distilled ontogeny (Janex-Favre 1970a, b; Nannfeldt 1932; Parguey- water. Thermal cycling parameters were: initial denaturation Leduc 1966, 1967). Loculoascomycetes sensu Luttrell (1955) in- for 3 min at 94 C, followed by 34 cycles of 45 s at 94 C, 1 min clude species that are currently placed in either Chaetothyrio- at 50 C (mtSSU primers) or 54 C (nu-LSU-0155-50/LR6), mycetes or Dothideomycetes (Barr 1981; Barr & Huhndorf 2000; 1.5 min at 72 C, and a final elongation for 10 min at 72 C. Eriksson et al. 2004), although ascoma development of non- Amplification products were viewed on 1 % agarose gels lichenized Chaetothyriomycetes is poorly understood. Most mo- stained with ethidium bromide and subsequently purified us- lecular studies confirmed that Loculascomycetes sensu Luttrell ing the QIAquick PCR Purification Kit (Qiagen) or Nucleo Spin (1955) are not monophyletic (Berbee 1996; Winka et al. 1998; DNA purification kit (Macherey-Nagel, Dueren, Germany). Silva-Hanlin & Hanlin 1999; Lumbsch et al. 2000; Lindemuth Fragments were sequenced using the Big Dye Terminator et al. 2001): Chaetothyriomycetes are closely related to Eurotiomy- reaction kit (ABI PRISM, Applied Biosystems, Forster City, cetes (Berbee 1996; Liu et al. 1999; Silva-Hanlin & Hanlin 1999; USA). Sequencing and PCR amplifications were performed us- Lumbsch et al. 2000), sometimes classified as a subclass Chae- ing the same sets of primers. Cycle sequencing was executed tothyriomycetidae of (Lutzoni et al. 2004), while with the following program: initial denaturation for 1 min at Dothideomycetes are related to Arthoniomycetes (Lumbsch et al. 96 C followed by 32 cycles of 96 C for 15 s, 50 C for 10 s, 2004; Lutzoni et al. 2004). The only recent study that recovered 60 C for 4 min. Sequenced products were precipitated with a monophyletic Loculoascomycetes, based on RPB2 sequences 10 ml sterile dH2O, 2 ml of 3 m NaOAc, and 50 ml of 95 % ethanol (Liu & Hall 2004), lacked significant support in the critical before they were loaded on an ABI 3100 or 3730 (Applied Bio- nodes. systems) automatic sequencer. Sequence fragments obtained The aim of the present study is to test the placement of Try- were assembled with SeqMan 4.03 (DNASTAR, Madison, USA) petheliaceae within Dothideomycetes by means of molecular and manually adjusted. data. For this purpose, we generated new sequences of several lichen-forming pyrenomycetes aiming at resolving the phylo- Sequence alignments and phylogenetic analysis genetic position of Trypetheliaceae. The mitochondrial data set contains sequence portions that are highly variable. Standard multiple alignment programs, Material and methods such as Clustal (Thompson et al. 1994) become less reliable when sequences show a high degree of divergence. Therefore Taxon sampling we used an alignment procedure that uses a linear Hidden Markov Model (HMM) as implemented in the software SAM New sequence data of the nuLSU rDNA and mtSSU rDNA were (Sequence Alignment and Modelling system) (Karplus et al. obtained from 28 pyrenocarpous lichen species. Two separate 1998) for the mitochondrial alignment. Regions that were analyses were performed: (1) A data matrix of 115 samples not aligned with statistical confidence were excluded from from 110 species was assembled using mitochondrial small the phylogenetic analysis. The nuLSU rDNA is much less vari- subunit rDNA sequences. Twenty-nine sequences were newly able and alignment was straightforward. This data set was obtained and 86 downloaded from GenBank; four taxa of Sor- aligned using Clustal X and all ambiguous regions were ex- dariomycetes were included as outgroup. (2) A combined data cluded from the alignments following an alignment done us- matrix of nuLSU sequences and mtSSU rDNA sequences of ing SAM (SAM deleted the last part of the alignment, as it 103 samples from 100 species was produced, including 19 was missing in some sequences, therefore the SAM alignment newly obtained nuLSU sequences. Specimens and sequences has not been used, but the modified longer alignment instead). used for the molecular analyses are compiled in Table 1. In the combined data sets only those species were included for which sequences of both gene portions were available. DNA extraction, PCR, and sequencing The alignments were analysed using minimum evolution (ME) and a Bayesian approach (B/MCMC) (Huelsenbeck et al. Total DNA was extracted from freshly collected material and 2001; Larget & Simon 1999). herbarium specimens, using the DNeasy Plant Mini Kit ME analyses were performed using the program PAUP* (Qiagen, Hilden, Germany) following the instructions of the (Swofford 2003). A heuristic search using the general time re- manufacturer. Dilutions (101 up to 103) or undiluted DNA versible nucleotide substitution model (Rodriguez et al. 1990) was used for PCR amplifications of the genes coding for the assuming a gamma shape parameter of 0.5 was conducted nuLSU rRNA and the mtSSU rRNA. Primers (nu-rDNA primer with TBR branch swapping and MulTrees option in effect. nomenclature follows Gargas and DePriest (1996) for amplifi- Bootstrapping (Felsenstein 1985) was performed based on cation were: (1) for the nu-LSU rDNA: nu-LSU-0155-50 (Do¨ ring 2000 replicates with the same settings as in the heuristic et al. 2000), nu-LSU-0042-50 (¼LR0R), nu-LSU-1432-30 (¼LR7), search. Phylogeny of Trypetheliaceae 513

Table 1 – Species and specimens used in the current studya Species name Order Collection nuLSU mtSSU (after Eriksson et al. 2004)

Absconditella sphagnorum AY300825 AY300873 Adelolecia pilati AY300826 AY300874 sp. AY300827 AY300875 Agonimia repleta Verrucariales Czech Republic DQ329014 DQ328985 (Veˇzda, Lich. Rar. Exs. 446,F) Agonimia tristicula Verrucariales AY300828 AY300876 salicis 1 Dothideomycetes et AY607730 AY607742 Chaetothyriomycetes incertae sedis A. salicis 2 Dothideomycetes et AY538339 AY538345 Chaetothyriomycetes incertae sedis Aspergillus flavus Eurotiales AF109342 AFU29214 Aspergillus nidulans Eurotiales AF109337 V00653 Bacidia rosella Lecanorales AY300829 AY300877 Bathelium degenerans Pyrenulales Costa Rica (Lu¨cking 17502 b, F) DQ329016 DQ328987 B. degenerans Pyrenulales Costa Rica (Lu¨cking 16657, F) DQ329017 DQ328988 Botryosphaeria ribis Dothideomycetes et AY004336 AF271148 Chaetothyriomycetes incertae sedis Cainia graminis Xylariales AF431949 AF431952 Calicium viride Lecanorales AF356670 AY143402 Caloplaca flavorubescens Lecanorales AY300831 AY143403 Campylothelium cf superbum Pyrenulales Costa Rica (Lu¨cking s.n.,F) d DQ328991 Capnodium citri Capnodiales AY004337 AF346421 mansonii AY004338 AF346422 Celothelium aciculiferum Pyrenulales Costa Rica (Lu¨cking 16591,F) DQ329019 DQ328992 C. chinchonarum Pyrenulales Costa Rica (Lu¨cking 17105 f,F) DQ329020 DQ328993 Cephalotheca sulfurea Sordariales AF431950 AF431953 Ceramothyrium carniolicum Chaetothyriales AY004339 AF346423 Chromatochlamys muscorum Family of uncertain position AY607731 AY607743 Cladonia rangiferina Lecanorales AY300832 AY300881 cucurbitula AF274092 AF329161 C. maritimum Pertusariales AF329164 AF329163 Conotrema populorum Ostropales AY300833 AY300882 Curvularia brachyspora Pleosporales AF279380 AY584701 biennense Verrucariales Turkey (John, Lich. Anat. Exs. 86,F) DQ329021 DQ328994 D. luridum Verrucariales AY607732 AY607744 D. miniatum Verrucariales AY607733 AY607745 cinereocaesius Ostropales AY300835 AY300885 Ostropales AY300836 AY300886 D. thunbergianus Ostropales AF274095 AF431955 Dothidea ribesia Dothideales AY016360 AY538346 pusillum Verrucariales Germany d DQ329012 (Northrhine-Westphalia, 2002, Lumbsch,F) Eupenicillium javanicum Eurotiales AF263348 L14501 Eurotium rubrum Eurotiales AY004346 AF346424 Farlowiella carmichaeliana Hysteriales AY541492 AY571387 Glyphium elatum Chaetothyriales AF346420 AF346425 Gyalecta ulmi AF465463 AY300888 Hysteropatella clavispora Hysteriales AY541493 AY571388 Laurera sp. Pyrenulales Costa Rica (Lu¨cking 17211,F) d DQ328995 Lecania cyrtella Lecanorales AY300840 AY300891 Lecanora intumescens Lecanorales AY300841 AY300892 Lecidella meiococca Le`canorales AY300842 AY300893 Myriangium duriaei Myriangiales AY016365 AY571389 Neobelonia sp. Ostropales AY300830 AY300879 peltigericola Verrucariales AY300845 AY300896 androgyna Pertusariales AY300846 AY300897 O. parella Pertusariales AF274097 AF320173 O. tartarea Pertusariales AY300848 AY300899 Orceolina kerguelensis Agyriales AY212830 AF381561 Paecilomyces tenuipes Hypocreales U47838 AB027358 (continued on next page) 514 R. del Prado et al.

Table 1 – (continued) Species name Order Collection nuLSU mtSSU (after Eriksson et al. 2004)

Parmelia saxatilis Lecanorales AY300849 AF351172 albescens Pertusariales AF329176 AF329175 P. amara Pertusariales AF274101 AY300900 P. erythrella Pertusariales AF274100 AF431958 P. flavicunda Pertusariales AF279299 AF381562 P. leioplaca Pertusariales AY300852 AY300903 P. pertusa Pertusariales AF279300 AF381565 P. rupicola var. coralloidea Pertusariales AY300853 AY300904 P. subventosa Pertusariales AY300854 AY300905 P. tejocotensis Pertusariales AF279301 AF381566 Phaeotrichum benjaminii Pleosporales AY004340 AY538349 Physcia aipolia Lecanorales AY300857 AY143406 Placopsis bicolor Agyriales AY212832 AY212857 Placopsis gelida Agyriales AY212836 AY212859 gelatinosa Verrucariales Sweden (Torne Lappmark, 2002, DQ329022 DQ328996 Palice, hb. Palice) Protothelenella corrosa Family of uncertain AY607734 AY607746 position Protothelenella sphinctrinoidella Family of uncertain position AY607735 AY607747 subnudata Pyrenulales Costa Rica (Lu¨cking 17619,F) d DQ328997 cf acutalis Pyrenulales Australia (Queensland, Lumbsch DQ329026 DQ329001 19092 b & Mangold,F) P. laevigata Pyrenulales AY607736 AY568029 P. cf leucostoma Pyrenulales Australia (Queensland, Lumbsch DQ329024 DQ328999 19082 a & Mangold,F) P. nitida 1 Pyrenulales AY607737 AY568030 P. nitida 2 Pyrenulales Czech Republic (Bohemia, Palice DQ329023 DQ328998 5929,F) P. subpraelucida Pyrenulales Costa Rica (Lu¨cking 17550 f,F) DQ329015 DQ328986 Pyrenula sp. 1 Pyrenulales Australia, (Queensland, Lumbsch DQ329025 DQ329000 19082 r & Mangold,F) Pyrenula sp. 2 Pyrenulales Australia (Queensland, Lumbsch DQ329027 DQ329002 19113 n & Mangold,F) Pyrrhospora quernea Lecanorales AY300858 AY300908 Raciborskiomyces longisetosum Dothideomycetes et AY016367 AY571386 Chaetothyriomycetes incertae sedis Scoliciosporum umbrinum Lecanorales AY300861 AY300911 fissa Verrucariales Sweden (Torne Lappmark, 2002, DQ329028 DQ329003 Palice, hb. Palice) Staurothele rufa Verrucariales Germany (Northrine-Westphalia, DQ329029 DQ329004 2002, Lumbsch, F) radiata Ostropales AY300864 AY300914 Stylodothis puccinioides Dothideales AY004342 AF346428 Tephromela atra Lecanorales AY300865 AY300915 Thelenella antarctica Family of uncertain position d AY607749 papulare Verrucariales Sweden (Torne Lappmark, 2002, DQ329030 DQ329005 Palice, hb. Palice) T. zwackhii Verrucariales Czech Republic (Bohemia, 2002, DQ329031 DQ329006 Palice, hb. Palice) lepadinum Ostropales AY300866 AY300916 T. suecicum Ostropales AY300867 AY300917 Thrombium epigaeum 1 Family of uncertain position d AY607750 T. epigaeum 2 Family of uncertain position d AY607751 Toninia sedifolia Lecanorales AY300868 AY300918 Trapelia coarctata Agyriales AF274117 AY212874 T. placodioides Agyriales AF274103 AF431962 Trapeliopsis flexuosa Agyriales AF274118 AY212875 T. granulosa Agyriales AF274119 AF381561 Trematosphaeria heterospora Pleosporales AY016369 AF346429 Trypethelium sp. Pyrenulales AY584652 AY584632 Trypethelium eluteriae 1 Pyrenulales Australia (Queensland, Lumbsch d DQ328990 19112g & Mangold,F) Phylogeny of Trypetheliaceae 515

Table 1 – (continued) Species name Order Collection nuLSU mtSSU (after Eriksson et al. 2004)

T. eluteriae 2 Pyrenulales Australia (Queensland, Lumbsch DQ329018 DQ328989 19113 k & Mangold,F) T. floridanum 1 Pyrenulales Costa Rica (Lu¨cking 17090 b,F) d DQ329007 T. floridanum 2 Pyrenulales Costa Rica (Lu¨cking 16306 a,F) d DQ329008 T. subeluteriae Pyrenulales Costa Rica (Lu¨cking 17611,F) d DQ329009 nigrescens Verrucariales Czech Republic (Bohemia, Palice DQ329032 DQ329010 4229, hb. Palice) V. phloeophila Verrucariales Ukraine (1997, Palice, hb. Palice) d DQ329013 V. viridigrana Verrucariales Slovakia (Palice 5769, hb. Palice) DQ329033 DQ329011 Westerdykella cylindrica Pleosporales AY004343 AF346430 Xanthoria parietina Lecanorales AF356687 AY143406 Xerotrema sp. Gyalectales AY300871 AY300921 Xylaria hypoxylon Xylariales AF132333 AF431964

a Newly obtained sequences are in bold face.

The B/MCMC analyses were conducted using the MrBayes 50 %-majority rule consensus tree of the MCMC sampling. For 3.0 program (Huelsenbeck & Ronquist 2001). Posterior proba- the hypothesis testing three different methods were employed: bilities were approximated by sampling trees using a MCMC (1) Bayesian hypothesis testing following Ihlen and Ekman method. The posterior probabilities of each branch were cal- (2002) and Lumbsch et al. (2004); (2) Shimodaira–Hasegawa culated by counting its occurrence in trees that were visited (SH) test (1999); and (3) expected likelihood weight (ELW) test during the course of the MCMC analysis. For all data sets the following Strimmer and Rambaut (2002). For the Bayesian hy- general time reversible model of nucleotide substitution pothesis testing a run of 1 M. generations was performed (Rodriguez et al. 1990) including estimation of invariant sites with the same settings as in the estimation of the phylogeny and assuming a discrete gamma distribution with six rate cat- using the combined data set. Five thousand trees at the equilib- egories (GTR þ IþG) was used. Each partition was allowed to rium state for the null hypothesis were used from this analysis. have its own model parameters as proposed by Nylander The probability of the null hypothesis being correct was calcu- et al. (2004). MrBAYES was run on each data set producing 2 lated by counting the presence of this topology in the MCMC M generations. Eight chains were run simultaneously. Trees sample (Lewis 2001). The frequency of trees in the MCMC sam- were sampled every 100 generations for a total of 20,000 trees. ple agreeing with the null hypothesis was calculated using the The first 200,000 generations (i.e. the first 2000 trees) were de- filter command in PAUP* with a certain constraint describing leted as the ‘burn in’ of the chain. We plotted the log-likeli- the null hypothesis. The SH and ELW tests were performed us- hood scores of sample points against generation time using ing Tree-PUZZLE 5.2 with the combined data set on a sample of TRACER 1.0 (http://evolve.zoo.ox.ac.uk/software.html?id¼ 200 unique trees, including the best trees agreeing with the null tracer) to ensure that stationarity was achieved after the first hypotheses, the unconstrained ML tree, and 197 trees with the 200,000 generations by checking whether the log-likelihood highest likelihood in the MCMC sample. values of the sample points reached a stable equilibrium value (Huelsenbeck & Ronquist 2001). Of the remaining 18,000 trees a majority rule consensus tree with average branch lengths Results was calculated using the sumt option of MrBayes. Posterior probabilities were obtained for each clade. Only clades that re- We generated 19 new nuLSU and 29 new mtSSU rDNA se- ceived bootstrap support equal or above 75 % under minimum quences for this study (Table 1). Two different analyses were evolution and posterior probabilities equal or above 0.95 were performed: (1) Single gene analysis of the mtSSU rDNA se- considered as strongly supported. Phylogenetic trees were quences, in which the newly obtained sequenced were visualized using the program Treeview (Page 1996). aligned with 86 sequences obtained from GenBank (Table 1). As the results of the phylogenetic analyses were incongru- The data matrix had 801 unambiguously aligned nucleotide ent with the current concept of Trypetheliaceae classification positions of which 606 were variable. (2) Combined analysis in suggesting that the family does not belong to Pyrenulales, of nuLSU and mtSSU rDNA sequences for which the newly we tested whether our data were sufficient to reject the follow- obtained sequences were aligned with 84 sequences down- ing three alternative topologies using the combined data set: loaded from GenBank to produce a matrix of 1158 unambigu- (1) monophyly of Pyrenulales including Trypetheliaceae; (2) place- ously aligned nucleotide positions in the nuLSU and 801 in the ment of Trypetheliaceae in Pleosporales as suggested (as ‘Mela- mtSSU rDNA partitions. We have decided to present the nommatales’) by Barr (1981); and (3) monophyly of the two mtSSU rDNA analysis in addition, as we failed in most cases families with lichen-forming species in Dothideomycetes: Artho- to obtain nuLSU rDNA sequences of Trypetehliaceae. Thus, pyreniaceae and Trypetheliaceae. Such topologies, which may not the mtSSU rDNA data set allows us to study a larger taxon be significantly worse than the obtained topology, might be sampling of the family. Five hundred and sixty-three charac- present in suboptimal trees not sampled or not present in the ters were variable in the nuLSU and 600 in the mtSSU data 516 R. del Prado et al.

set. The topology of the 95 % majority rule consensus tree of included species of Pleosporales form strongly supported the two Bayesian single-partition analyses was congruent; monophyletic groups. Dothideomycetes is monophyletic, but i.e. no clade that differed between the single-gene analyses this lacks support. was supported by posterior probabilities equal or above 0.95 The two Celothelium and the seven Pyrenula species each in both single-gene analyses (data not shown), and hence form monophyletic sister groups. However, this sister group a combined analysis was performed. The alignments of the relationship (i.e. Pyrenulaceae) is not supported. Pyrenulaceae two analyses are available in TreeBASE (http://www.treebase. cluster within the strongly supported Chaetothyriomycetes org/treebase/). that also include Chaetothyriales and Verrucariales. In the single-gene analysis (analysis 1), the likelihood param- The genus Dermatocarpon is monophyletic and well-sup- eters in the sample had the following mean values (Variance): ported, but clusters with Chaetothyriales and not with Verrucar- LnL ¼22300 (0.55), base frequencies p(A) ¼ 0.366 (0.0005), iales. However, this relationship lacks support. Within p(C) ¼ 0.108 (0.0004), p(G) ¼ 0.196 (0.0004), p(T) ¼ 0.331 (0.0004), Verrucariales, Agonimia, Thelidium and Verrucaria are not mono- rate matrix r(AC) ¼ 1.483 (0.012), r(AG) ¼ 3.169 (0.013), phyletic, while the two Staurothele species form a strongly r(AT) ¼ 1.561 (0.008), r(CG) ¼ 0.939 (0.007), r(CT) ¼ 5.224 (0.040), supported monophyletic group. Chaetothyriomycetes and Euro- r(GT) ¼ 1.0 (0), the gamma shape parameter alpha ¼ 0.757 tiomycetes have a strongly supported sister-group relationship. (0.0008) and the proportion of invariable site p(invar) ¼ 0.151 is monophyletic and none of the taxa currently (0.0002). placed in Pyrenulales cluster with this class. In the two-gene analysis (analysis 2), the likelihood param- The topology of the analysis of the combined data set basi- eters in the sample had the following mean values (Variance): cally agrees with analysis 1 with a few exceptions. The class LnL ¼38490 (0.52), base frequencies p(A) ¼ 0.289 (0.0004), Dothideomycetes is strongly supported, although the topology p(C) ¼ 0.179 (0.0004), p(G) ¼ 0.262 (0.0005), p(T) ¼ 0.27 (0.0003), of main clades within this class somewhat differs from rate matrix r(AC) ¼ 1.03 (0.069), r(AG) ¼ 3.079 (0.015), the single-gene analysis, such as the strongly supported r(AT) ¼ 2.089 (0.012), r(CG) ¼ 1.07 (0.006), r(CT) ¼ 5.209 (0.029), sister-group relationship of Capnodium citri and Raciborskiomyces r(GT) ¼ 1.0 (0), the gamma shape parameter alpha ¼ 0.705 longisetosum. Conversely, the sister-group relationship of Chae- (0.001) and the proportion of invariable site p(invar) ¼ 0.246 tothyriomycetes and Eurotiomycetes is not strongly supported in (0.0004). the combined analysis. Dermatocarpon now clusters with Verru- The topology of the trees obtained employing ME and cariales and hence this order is monophyletic and well Bayesian methods was very similar and hence only the phylo- supported in the combined analysis. In this combined analysis genetic trees of the Bayesian analyses are shown. Figs 1 and 2 Trypetheliaceae is also strongly supported. show the 50 % majority-rule consensus trees of the Bayesian Both Bayesian hypothesis testing and the SH and ELW tests analyses of the mtSSU rDNA and combined data sets, employed to test the probability of alternative topologies sig- respectively. nificantly rejected alternative topologies (P < 0.001 in all three In the single-gene analysis (Fig 1), the 11 Trypetheliaceae in- tests for the three topologies) that included: (1) monophyletic cluded in the study form a strongly supported monophyletic Pyrenulales including Trypetheliaceae; (2) monophyly of Artho- group within Dothideomycetes. The five included Trypethelium pyreniaceaeþTrypetheliaceae; and (3) placement of Trypethelia- species do not form a monophyletic group, but are scattered ceae within Pleosporales. in the Trypetheliaceae clade, confirming that the generic concept in this family awaits revision. Trypethelium floridanum, which is characterized by solitary to partly aggregated, immersed peri- Discussion thecia with thalline cover (similar to those of Laurera mega- sperma but much smaller), clusters with a Campylothelium, Our results confirm the placement of at least two families that which has the same perithecial gross morphology but much include lichen-forming fungi, Arthopyreniaceae and Trypethelia- larger perithecia and excentric ostioles. The two Trypethelium ceae, within Dothideomycetes. The placement of Arthopyreniaceae floridanum samples do not cluster together but are paraphy- within Dothideomycetes was expected, as its species have been letic. The taxa with perithecia aggregate in pseudostromata, demonstrated to have ascolocular ascoma development viz. Trypethelium eluteriae/subeluteriae versus Bathelium degener- (Janex-Favre 1970a), and was also demonstrated by Lumbsch ans, fall into two separate lineages, confirming their separation et al. (2005). The placement of Trypetheliaceae in this class, al- at generic level. We have not seen the original material of Try- though already suggested by Lutzoni et al. (2004), was unex- pethelium sp. and thus cannot make any statement about its pected, as this family is usually regarded as part of Pyrenulales, identity. The Laurera sp. is possibly an undescribed species, which is placed in Chaetothyriomycetes by molecular data (e.g. with fully immersed perithecia, muscicolous thallus, and Lutzoni et al. 2004). Previous placement of Trypetheliaceae in Pyr- rather large ascospores. Pseudopyrenula subnudata, with solitary enulales was mainly based on similar gross morphology of the perithecia and sparsely developed thallus, is sister to all mature ascomata, including bitunicate asci and ‘graphidean’ as- remaining Trypetheliaceae. However, the topology within the cospores. However, bitunicate asci are possibly a shared plesio- family lacks support, with the exception of the strongly sup- morphy between Chaetothyriomycetes and Dothideomycetes,and ported relationship of Trypethelium eluteriae and T. subeluteriae. ascospores with wall thickenings also occur in other groups of The sister group of Trypetheliaceae is not clear in the single- lichen-forming fungi, such as Ostropales (Sherwood 1977)and gene analysis, as the phylogeny of major clades within Dothi- Teloschistaceae (Letrouitia), suggesting that this character state deomycetes are only partially resolved and mostly lack support. has evolved independently in several groups. Other characters, Only the two included Dothideales taxa and four of the five such as theanastomosing pseudoparaphyses versus unbranched Phylogeny of Trypetheliaceae 517

Diploschistes cinereocaesius Diploschistes muscorum Diploschistes thunbergianus Thelotrema lepadinum Gyalecta ulmi Thelotrema suecicum Xerotrema sp. Conotrema populorum Stictis radiata sphagnorum Neobelonia sp. Chromatochlamys muscorum Thelenella antarctica Protothelenella corrosa Protothelenella sphinctrinoidella Thrombium epigaeum 1 Thrombium epigaeum 2 Placopsis bicolor Placopsis gelida Orceolina kerguelensis Trapelia coarctata Trapelia placodioides Trapeliopsis flexuosa Lecanoromycetes Trapeliopsis granulosa Pertusaria albescens Pertusaria amara Pertusaria erythrella Pertusaria subventosa Ochrolechia androgyna Ochrolechia tartarea Ochrolechia parella Coccotrema cucurbitula Coccotrema maritimum Lecanora intumescens Lecidella meiococca Adelolecia pilatii Pyrrhospora quernea saxatilis Cladonia rangiferina Tephromela atra Bacidia rosella Toninia sedifolia Lecania cyrtella Scoliciosporum umbrinum Caloplaca flavorubescens Xanthoria parietina Calicium viride Physcia aipolia Pertusaria rupicola var. coralloidea Pertusaria flavicunda Pertusaria tejocotensis Pertusaria leioplaca Agonimia sp. Norrlinia peltigericola Agonimia tristicula Agonimia repleta Verrucaria viridigrana Chaetothyriomycetes Staurothele rufa Verrucaria phloeophila Endocarpon pusillum Thelidium papulare Thelidium zwackhii Polyblastia gelatinosa Dermatocarpon biennense Capronia mansonii Ceramothyrium carniolicum Glyphium elatum Pyrenula cf. leucostoma Pyrenula sp. 1 Pyrenula cf. acutalis 1 Pyrenula nitida 2 Pyrenula sp. 2 Pyrenula laevigata Pyrenula subpraelucida Celothelium aciculiferum Celothelium chinchonarum Pyrenulaceae Aspergillus flavus Eurotium rubrum Eupenicillium javanicum Eurotiomycetes Aspergillus nidulans Campylothelium cf. superbum Trypethelium floridanum 1 Trypethelium floridanum 2 Bathelium degenerans 1 Bathelium degenerans 2 Trypetheliaceae Trypethelium sp. Dothideomycetes Laurera sp. Trypethelium subeluteriae Trypethelium eluteriae 1 Trypethelium eluteriae 2 Pseudopyrenula subnudata Stylodothis puccinioides Dothidea ribesia Capnodium citri Myriangium duriaei Farlowiella carmichaeliana Raciborskiomyces longisetosum Hysteropatella clavispora Botryosphaeria ribis Phaeotrichum benjaminii Arthopyrenia salicis 1 Arthopyrenia salicis 2 Westerdykella cylindrica Trematosphaeria heterospora Curvularia brachyospora Paecilomyces tenuipes Cephalotheca sulfurea Xylaria hypoxylon Cainia graminis 0.1

Fig 1 – Fifty percent majority rule consensus tree based on 18,000 trees from a B/MCMC tree sampling procedure of mtSSU rDNA sequences. ME-bootstrap values above 74 % and Bayesian posterior probabilities equal or above 0.95 are indicated as bold branches.

paraphyses, separate Trypetheliaceae from Pyrenulaceae (Barr currently placed and confirmed within Dothideomycetes differ 1981). The molecular data indicate that the similarities between from Trypetheliaceae in several aspects, including lifestyle, Pyrenulaceae and Trypetheliaceae have been overestimated. ascoma morphology and anatomy, hamathecium and As Trypetheliaceae were always placed within Pyrenulales type, and ascospore type (Eriksson 1981). Additional molecular (or Melanommatales s.lat.) but cannot longer be retained in data from a variety of Dothideomycetes is necessary to infer the that order, the ordinal assignment of the family within sister group of Trypetheliaceae and its ordinal placement. For Dothideomycetes is uncertain. Our data reject placement within the time being we propose to classify Trypetheliaceae among the clade including Arthopyreniaceae (Pleosporales), which is the families of uncertain position within Dothideomycetes. group that comes closest to Trypetheliaceae in terms of ascoma The low number of Trypetheliaceae taxa included in this morphology and anatomy (Eriksson 1981). Other major groups study does not allow any detailed conclusions regarding the 518 R. del Prado et al.

Diploschistes cinereocaesius Diploschistes muscorum Diploschistes thunbergianus Thelotrema lepadinum Thelotremas uecicum Gyalecta ulmi Xerotrema sp. Conotrema populorum Stictis radiata Absconditella sphagnorum Neobelonia sp. Chromatochlamys muscorum Placopsis bicolor Placopsis gelida Orceolina kerguelensis Trapelia coarctata Trapelia placodioides Trapeliopsis flexuosa Lecanoromycetes Trapeliopsis granulosa Protothelenella corrosa Protothelenella sphinctrinoidella Pertusaria pertusa Pertusaria rupicola var. coralloidea Pertusaria flavicunda Pertusaria tejocotensis Pertusaria leioplaca Pertusaria albescens Pertusaria amara Pertusaria erythrella Pertusaria subventosa Ochrolechia androgyna Ochrolechia tartarea Ochrolechia parella Coccotrema cucurbitula Coccotrema maritimum Lecanora intumescens Pyrrhospora quernea Adelolecia pilatii Lecidella meiococca Parmelia saxatilis Cladonia rangiferina Bacidia rosella Toninia sedifolia Lecania cyrtella Scoliciosporum umbrinum Tephromela atra Calicium viride Physcia aipolia Caloplaca flavorubescens Xanthoria parietina Agonimia sp. Norrlinia peltigericola Agonimia tristicula Verrucaria viridigrana Agonimia repleta Staurothele fissa Staurothele rufa Chaetothyriomycetes Thelidium zwackhii Verrucaria nigrescens Thelidium papulare Polyblastia gelatinosa Dermatocarpon biennense Dermatocarpon miniatum Dermatocarpon luridum Capronia mansonii Ceramothyrium carniolicum Glyphium elatum Pyrenula cf. leucostoma Pyrenula sp. 1 Pyrenula cf. acutalis Pyrenula nitida 1 Pyrenula nitida 2 Pyrenula sp. 2 Pyrenula subpraelucida

Pyrenula laevigata Eurotiomycetes Celothelium aciculiferum Pyrenulaceae Celothelium chinchonarum Aspergillus flavus Eurotium rubrum Eupenicillium javanicum Aspergillusn idulans Trematosphaeria heterospora Westerdykella cylindrica Curvularia brachyspora Arthopyrenia salicis 1 Arthopyrenia salicis 2 Dothideomycetes Farlowiella carmichaeliana Botryosphaeria ribis Capnodium citri Raciborskiomyces longisetosum Myriangium duriaei Hysteropatella clavispora Phaeotrichum benjaminii Trypetheliaceae Bathelium degenerans 1 Bathelium degenerans 2 Trypethelium sp. Trypethelium eluteriae 2 Dothidea ribesia Stylodothis puccinioides Cephalotheca sulfurea Paecilomyces tenuipes Xylaria hypoxylon Cainia graminis 0.1

Fig 2 – Fifty percent majority rule consensus tree based on 18,000 trees from a B/MCMC tree sampling procedure of a com- bined data set of nuLSU and mtSSU rDNA. ME-bootstrap values above 74 % and Bayesian posterior probabilities equal or above 0.95 are indicated as bold branches.

generic delimitations in the family, except that our data con- in other recently revised groups, such as (Staiger firm that the present circumscriptions, mainly based on peri- 2002). thecial arrangement (solitary, aggregated, fused) and Our results showed the order Pyrenulales excluding Trype- ascospores (transverse, muriform) appear artificial. Harris theliaceae as a monophyletic group in both analyses, including (1995) already discussed possibilities towards a more natural the genus Celothelium, which is currently classified as Pyrenu- classification, and the available data suggest that more natu- lales incertae sedis (Eriksson et al. 2004). Pyrenulales forms ral generic delimitations in this family may be based on peri- a well-supported sister-group relationship to the Verrucar- thecial morphology and anatomy, as well as thallus iales/Chaetothyriales clade in Chaetothyriomycetes, as in previous development. This would be comparable with the situation studies (Lutzoni et al. 2004; Schmitt et al. 2005). Phylogeny of Trypetheliaceae 519

As for the groups traditionally considered ascolocular Doppelbaur AW, 1960. Studien zur Anatomie und Entwicklungs- (Dothideales, Chaeothyriales, Pleosporales), our results are in geschichte einiger endolithischen pyrenocarpen Flechten. agreement with others that show polyphyly of Loculoascomy- Planta 53: 246–292. Do¨ring H, Clerc P, Grube M, Wedin M, 2000. Mycobiont-specific cetes (Berbee 1996; Winka et al. 1998; Silva-Hanlin & Hanlin PCR primers for the amplification of nuclear ITS and LSU rDNA et al. et al. 1999; Lindemuth 2001; Lumbsch 2000; Lutzoni from lichenized ascomycetes. Lichenologist 32: 200–204. et al. 2001, 2004), and are in contradiction with a study by Liu Eriksson OE, 1981. The families of bitunicate Ascomycetes. Opera and Hall (2004) who found the Loculoascomycetes to be mono- Botanica 60: 1–209. phyletic. 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The family Trypetheliaceae (Loculoascomycetes: cetes are known to have an ascohymenial ascoma develop- lichenized Melanommatales) in Amazonian Brazil. Suplementum Acta Amazonica 14: 55–80. ment (Doppelbaur 1960; Janex-Favre 1970b) and thus raise Harris RC, 1990.Some Florida Lichens. Publ. by the Author, Bronx, N.Y. the question of the type of ascoma development present in Harris RC, 1991. A revision of Polymeridium (Muell. Arg.) R.C. Harris Chaetothyriales. A re-examination of ascoma development in (Trypetheliaceae). Boletim do Museu Paraense Emilio Goeldi se´rie several groups of pyrenomycetes is necessary to evaluate Botaˆnica 7: 619–644. the phylogenetic pattern of this character set for higher-level Harris RC, 1995. More Florida Lichens. Including the 10c¸Tourofthe classification of euascomycetes. Pyrenolichens. Publ. by the Author, Bronx, N.Y. Harris RC, 1998. A preliminary revision of Pseudopyrenula Mu¨ ll. Arg. (lichenized Ascomycetes, Trypetheliaceae) with a redispo- sition of the names previously assigned to the genus. In: Glenn MG, Harris RC, Dirig R, Cole MS (eds), Lichenographia Acknowledgments Thomsoniana: North American Lichenology in Honor of John W. Thomson. Mycotaxon, Ithaca, New York, pp. 133–148. Henssen A, Jahns HM, 1973. [‘1974’] Lichenes. Georg Thieme We thank Nora Wirtz for her assistance in the laboratory and Verlag, Stuttgart. Armin Mangold (both Chicago) for collecting material in the Huelsenbeck JP, Ronquist F, 2001. MrBAYES: Bayesian inference of field with H.T.L. Most of the material of Trypetheliaceae was col- phylogenetic trees. Bioinformatics 17: 754–755. lected in the frame of the TICOLICHEN project, and the Na- Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP, 2001. Bayesian tional Science Foundation is greatfully acknowledged for inference of phylogeny and its impact on evolutionary biology. Science 294: 2310–2314. financial support (DEB 0206125). 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