EXPERIMENTAL MYCOL()(;Y 19,7-15 (1995) Phylogeny of Discomycetes and Early Radiations of the Apothecial Ascomycotina Inferred from -SSU rONA Sequence Data 1 ANDREA GARGAS",·2 AND JOHN W. T A YLORt ·Department of BOlany, NHB-I66, National Museum of Natural His/ory, Smithsonian Ins/itution, Washington, DC l056fJ; and tDeparlment of Plant Biology, I I J Koshland Hail, University of CaUfornia, Berkeley, California 94720 Accepted for publication October 16. 1994 G ARGAS, A" AND TAYLOR, J. W. 1995. Phylogeny of discomycetes and earl y radiati ons of the apothecial Ascomycotina inferred from SSU rDNA sequence data. Experimen/al Mycology 19, 7- 15. We used nucleotide sequences of the small subunit ribosomal genes (SSU rONA) to examine evolutionary relationsh ip s of apothecial ascomycetes (division Ascomycota; class Discomycetes sensu), commo nl y known as the cup fungi. The apothecial ascomycetes include both lichen­ fonning and free-living fungi. We sequenced the SSU rONA from representatives of 10 funga l genera from four orders: Pe zizale~ (Ascobolus Uneola/us, Morchel/a elata agg" Peziw badia); Leotiales (Leo/ia lubrica, Sclero/inia sclerotiorum); Caliciales (CaUdum tricolor, Myc()Calicium albonigrum, Sphaerophorus globosus); and Lecanorales (Lecanora dispersa, Porpidia crus/ula/a). Of these, C. Iricolor, S. globosus, L. dispersa, and P. cruslillata are lichen-forming fungi. Based on parsimony analyses of approximately 1750 aligned nucleotides of their SSU rO NA, we deter­ mined a most parsimonious tree (MPT). Thi s hypothesis suggests that the apothecial ascomycetes are a paraph yletic assemblage, basal to other groups of filamentous ascomycetes including repre­ sentati ves of the perithecial fungi and cleistothecial fungi. The most parsimonious tree produced using thi s dataset suppor1ed the monophyly of the orders Pezizales, Leotiales, and Lecanorales. However, there was no suppor1 for monophyly of the representative Caliciales: S. g/oboslls had affi nities with member5 of the Lecanorale~. This ph ylogenetic hypothesis recognizes Pezizales as basal and suppor1 S Nannfeldt's hypothesis (1932) of a primitive apothecial ascomata with su b ~e­ quent evolution of perithecial and cleistothecial forms. Thi~ MPT provide~ a foundation for un­ derstanding evolution of the ascomycetous fungi. c 1 99~ Acadcm;c I'r< ... II><, I NDEX DESC RIPTORS: fungi: Ascomycetes; discomycetes; lichen-forming; apothecia; 18S rONA; SSU rDNA; small subunit ribosomal DNA; molecular evolution; phylogeny. Fungi that produce cup-shaped ascomata mycetes, nor on their relationships to other (apothecial ascomycetes) are commonl y fungi . Most of the apothecial ascomycetes known as the cup fungi or Discomycetes. (around 6000 spp.) are lichen-forming fungi The placement of this group within the fil­ in symbiotic re lationships with green algae amentous ascomycetes is important since or cyanobacteria, The 13,000 species of li­ the apothecial fungi represent almost 9000 chen-forming ascomycetes, including mem­ of the 28 ,000 described ascomycete species bers of the apothecial ascomycetes as we ll (Hawksworth et al., 1983). There is no hi s­ as members of other classes, constitute torical consensus on the evolutionary rela­ nearly half of the descri bed species of as­ tionships within the apothecial asco- comycetes. These fungi have been treated separately from pathogenic and free-living 1 Sequence data from this article have been depos­ fungi, but must be considered to produce a ited with the GenBank Data Library under Accession Nos. U5732- US74 I, comprehensive phylogeny of the asco­ 2 To whom correspondence shou ld be addres~ed. mycetes. To address the relationships of Fax: (:ro2) 786-2563. E- mai l: garga~@onyx . si.edu. the apothecial ascomycetes. we sequenced 7 0147-5975195 $6.00 Copyright c 1 \l9~ by A ca.d~mk I'r< ... In<:. All righ,. 0( ~producti"" in any form ~.., .... ed. 8 GARG ..... S AND TAYLOR the small subunit rONA gene (SSU rO NA») (Berbee and Taylor, 1992) from asco­ from representatives of 10 species and llsed mycetes with closed ascomata (cleistothe­ these nucleotide sequences to produce a cia) and fl ask-shaped ascomata (perithecia) phylogenetic hypothesis. have suggested that these are useful phylo­ Classification schemes for apolhecial as­ genetic characters. and that the classes comycetes have been reviewed by Kim­ based on these characters, Plectomycetes brough (1970) and for all ascomycetes by and Pyrenomycetes, respectively, are sup­ Hawksworth (1985), Based on reproductive ported as monophyletic lineages. Landvik structures of open, simple cups in contrast et af. (1993) included members of the Leo­ to the closed structurcs of the cleistothecial tiales and Pezizales, but found no clear ascomycetes or the fl ask-shaped structures monophyly for either group. Saenz el af. of the perithecial ascomycetes, mycologists (1994) showed the apothecial ascomycetes have variously proposed that the cup fungi to be a basal assemblage which included the constitute a monophyletic group of recent powdery mildew Blumeria graminis (Erysi­ origin, an ancestral group. or several inde­ phales). The fi rst analysis to include SSU pe ndent groups. Karf and Eriksson have rONA from lichen-forming fungi (Gargas, diffe red as to whether or not the Disco­ 1992) also found the Calic iales, Lecano­ mycetes are monophyletic. Korf (1973) rales, Leotiales, and Pezizales to be a basal placed the apothecial ascomycetes in the assemblage within the filamentous asco­ single class .. Discomycetes." Eriksson mycetes, although monophyly of each of (1981), in an outline of the ascomycetes, these groups was not rejected by maximum classified the apothecial ascomycetes into li kelihood analysis of the SSU rDNA data­ groups of one to several fami lies. Eriksson set. concluded that without a comprehensive in­ In the present study we have used SSU vestigation of their morphology and ontog­ rONA sequences to test the monophyly of eny, the ascomycetes could not be arranged the class Discomycctes, those fungi which into classes. Even when morphology and possess apothecia. We selected taxa of ontogeny were well known, interpretation apothecial ascomycetes that would address was difficult. Eriksson (1981) foc used atten­ the fo llowing questions: (I) with in all asco­ tion on the orders of ascomycetes and dis­ mycetes, are the fila mentous forms sup­ couraged the use of supraordinal taxa; the ported as a single lineage? (2) which type of Dictionary of Fungi (Hawksworth et 01., ascoma should be considered ancestral 1983) and Outline of Ascomycetes (Eri ks­ within the ascomycetes? (3) within the non­ son and Hawksworth, 1993) present the as­ yeast ascomycetes. arc the apothecial asco­ comycete fungi without class distinctions. mycetes supported as monophyletic and To examine monoph yly of the morpho­ separate from the c1cistothecial and peri­ logically defined class di scomycetes, as thccial ascomycetes? and (4) within the well as orders within this group, we have apothecial ascomycetes. are re presenta­ sought a source of characters independent tives of the fou r orders Pezizales. Cali­ from their morphology. An independent dales. Leoliales, and Lecanorales each test of phylogenetic hypotheses based on supported as monophyletic? morphological characters can be provided by molecular data. For example, recent MATERIALS AND METHODS analyses using S$U rONA sequence data Isolares ) Abbreviati ons used: PC R, polymerase chai n reac­ tion: MPT, most pa rsimonious tree: SSU. small sub­ We sequenced the SSU rONA from 10 unit. fungal species representing four orders of PHYLOGENY OF ASCOMYCETES FROM SSU rONA 9 ascomycete apothecial fungi . One species man) to sequence both the coding and the from a represe ntative genus for each order noncod ing strands. was sequenced, as we ll as one or two other To confirm the identity of the fungal species from ge nera considered to be within DNA being sequenced, sequence data for a the order. The species sequenced from diagnostic region was obtained from at least each order were: Pezizales-Ascobolus Ii­ one related species, or the fungal sequence neoimus Brumm. , Morcheffa elaw agg., was compared to that obtained by another Peziza badia Pers.; Leoli ale s-Leotia 111- researcher (5. Landvik, personal communi­ brica Pers.:Fr., Sclerotinia scJerotiorum cation). The S5U rONA sequences from (lib . DeBary); Ca li ciales-Calicillm tricolor the 10 fungi have been archived in Gen­ F . Wilson, Mycocalicium a/bonigrllm Bank. (Ny!.) TibelJ , Sphaerophorus globosus (Huds.) Vain. ; and Lecanorales-Lecanora Sequence Afignment and Construction 0/ dispersa (Pers.) Sommcrf., Porpidia crus­ Phylogenetic Trees tulata (Ach.) Hertel and Knoph (syn: Lecidea crustulata). The sequences were aligned with the fol­ lowi ng fungi (with GenBank accession DNA Extraction and Amplification Nos.): Saccharomyces cerevisiae Meyen ex Hansen (101353, M27607) (Rubtsov et We used standard fungal miniprep proto­ al., 1980); Neurospora crassa Shear and cols (Lee and Taylor, 1990; White et al., Dodge (X04971) (Chambers et al., 1986); 1990) to extract the tolal DNA from fungal Ascosphoeru apis (Maasen ~x Claussen) cultures, fre shly collected material, or her­ 1 Olive ex Spiltoir (M83264) , Byssochlamys barium specimens. From dilutions (10 - , 2 niveo Westling (M83256), Chaetomium ela­ 10 - ) of this total DNA we specificall y am­ tum Kunze (M83257), Eremascus albus Ei­ plified the fungal nuclear SSU rO NA using dam (M83258), Leucostoma persoonii polymerase chain reaction (PCR) primers Hoh n (M83259) ,