Peziza and Pezizaceae Inferred from Multiple Nuclear Genes: RPB2, -Tubulin, and LSU Rdna
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Molecular Phylogenetics and Evolution 36 (2005) 1–23 www.elsevier.com/locate/ympev Evolutionary relationships of the cup-fungus genus Peziza and Pezizaceae inferred from multiple nuclear genes: RPB2, -tubulin, and LSU rDNA Karen Hansen ¤, Katherine F. LoBuglio, Donald H. PWster Harvard University Herbaria, Cambridge, MA 02138, USA Received 5 May 2004; revised 17 December 2004 Available online 22 April 2005 Abstract To provide a robust phylogeny of Pezizaceae, partial sequences from two nuclear protein-coding genes, RPB2 (encoding the sec- ond largest subunit of RNA polymerase II) and -tubulin, were obtained from 69 and 72 specimens, respectively, to analyze with nuclear ribosomal large subunit RNA gene sequences (LSU). The three-gene data set includes 32 species of Peziza, and 27 species from nine additional epigeous and six hypogeous (truZe) pezizaceous genera. Analyses of the combined LSU, RPB2, and -tubulin data set using parsimony, maximum likelihood, and Bayesian approaches identify 14 Wne-scale lineages within Pezizaceae. Species of Peziza occur in eight of the lineages, spread among other genera of the family, conWrming the non-monophyly of the genus. Although parsimony analyses of the three-gene data set produced a nearly completely resolved strict consensus tree, with increased conWdence, relationships between the lineages are still resolved with mostly weak bootstrap support. Bayesian analyses of the three- gene data, however, show support for several more inclusive clades, mostly congruent with Bayesian analyses of RPB2. No strongly supported incongruence was found among phylogenies derived from the separate LSU, RPB2, and -tubulin data sets. The RPB2 region appeared to be the most informative single gene region based on resolution and clade support, and accounts for the greatest number of potentially parsimony informative characters within the combined data set, followed by the LSU and the -tubulin region. The results indicate that third codon positions in -tubulin are saturated, especially for sites that provide information about the deeper relationships. Nevertheless, almost all phylogenetic signal in -tubulin is due to third positions changes, with almost no signal in Wrst and second codons, and contribute phylogenetic information at the “Wne-scale” level within the Pezizaceae. The Peziza- ceae is supported as monophyletic in analyses of the three-gene data set, but its sister-group relationships is not resolved with sup- port. The results advocate the use of RPB2 as a marker for ascomycete phylogenetics at the inter-generic level, whereas the -tubulin gene appears less useful. 2005 Elsevier Inc. All rights reserved. Keywords: Fungi; Ascomycota; Pezizales; Protein coding genes; Third codon saturation; Combining data; Parsimony, Maximum likelihood and Bayesian inference 1. Introduction fruit bodies (ascomata) that range in size from a few mm to more than 10 cm in diameter. It includes epigeous, The cup-fungus family Pezizaceae (Pezizales) are Wla- semi-hypogeous to hypogeous (truZe) taxa. Ten out of mentous ascomycetes recognized macro-morphologi- 22 currently accepted genera in the family (Eriksson cally by the often Xeshy, soft, and brittle, cup-shaped et al., 2004) are exclusively truZe or truZe-like genera, but several truZe species have also been described in Peziza Fr.. A shared derived character, the operculate * Corresponding author. Fax: +1 617 495 9484. E-mail address: [email protected] (K. Hansen). ascus, characterizes Pezizales. Molecular phylogenetic 1055-7903/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.03.010 2 K. Hansen et al. / Molecular Phylogenetics and Evolution 36 (2005) 1–23 studies show the Pezizales (along with Orbiliaceae) to be ful for both species level and deep phylogenetic work, the most basal lineage(s) within the Euascomycetes (e.g., depending on the segment used. The region of RPB2 Liu et al., 1999; Lumbsch et al., 2000; Platt and Spata- with the highest evolutionary rate is spanning conserved fora, 2000), which makes work toward a robust phylog- regions 6–7 (Liu et al., 1999). It has provided phyloge- eny of this group of general importance in netic information in analyses of ascomycetes at the spe- understanding ascomycete phylogeny and character evo- cies level (within Leotia, Zhong and PWster, 2004) using lution. The Pezizaceae is chieXy characterized by amy- the nucleotides, and between families and orders (Dia- loid asci (asci that turn blue in iodine solutions) a feature porthales, Microascales, and Sordariales) using the only shared with the Ascobolaceae within the Pezizales. amino acids (Zhang and Blackwell, 2001). Here, we eval- The ascospores are uninucleate, usually thin-walled, glo- uate the usefulness of RPB2 to infer intra- and interge- bose, ellipsoid, or fusiform, hyaline or pale brownish, neric phylogenetic relationships within Pezizaceae, using smooth or with cyanophylic ornaments. The excipulum the exonic nucleotides spanning conserved regions 6–11. consists, at least partly, of large isodiametric cells. The -Tubulin is well characterized in Ascomycota, but trophic status of the species is little known, and although has not been used in phylogenetics of cup-fungi. It is a the majority is considered to be saprotrophs, many more 445–449 amino acid protein, encoded in an approxi- are likely to be proven mycorrhizal. Ecologically it cov- mately 1.8 kb gene with 4–8 introns (May et al., 1987). At ers an extremely broad range of niches, fruiting on all the amino acid level partial -tubulin sequences have types of soil, dung, and wood. The family is most diverse recently been used by Landvik et al. (2001) to infer in temperate zones and arctic-alpine areas, but a few higher-level phylogenetic relationships in the ascomyce- strictly tropical taxa are known. The family includes ca. tes, but their results suggest it is less useful than other 200 known species, with Peziza as the largest genus, esti- genes at this level. At the nucleotide level, -tubulin has mated to comprise 84 species (Kirk et al., 2001). No been informative at low taxonomic levels within the intergeneric classiWcation has ever been proposed for the ascomycetes (e.g., Jong et al., 2001; O’Donnell et al., Pezizaceae, and furthermore, Peziza has never been 1998; Schoch et al., 2001). -Tubulin has been deter- monographed and infrageneric relationships are poorly mined to be a single copy gene in some genera of the understood. In addition, species delimitations within Ascomycota (e.g., Byrd et al., 1990; NeV et al., 1983; Peziza continue to be problematic, although many keys Orbach et al., 1986), but two highly divergent paralogs (e.g., Dissing, 2000; HäVner, 1995; Hohmeyer, 1986) and have been reported in others (e.g., May et al., 1987; Pan- descriptions (e.g., Donadini, 1978) of European Peziza accione and Hanau, 1990). Within Pezizaceae we are species exist. Molecular phylogenetic studies (Hansen using the nucleotide sequences from two large exons (6– et al., 2001; Norman and Egger, 1996, 1999) have shown 7 in Aspergillus nidulans benA, May et al., 1987). that Peziza is not monophyletic. Our previous results The goals of the present study were: (i) to reconstruct (Hansen et al., 2001), based on phylogenetic analyses of the evolutionary history of Peziza and the Pezizaceae, partial nuclear ribosomal large subunit RNA gene (ii) to explore and compare the utility, at the generic level sequences (LSU), suggest that Peziza is composed of at in ascomycete phylogeny, of partial sequences from the least six distinct lineages, most of which include other three nuclear genes, LSU rDNA, RPB2, and -tubulin, genera of the Pezizaceae. Higher-level relationships (iii) to explore, for the protein coding genes, if separate within the Pezizaceae were however, not resolved or only partitions of Wrst and second vs. third codon positions with low support, using LSU rDNA sequences. To contain the same phylogenetic information, and if diVer- improve phylogenetic inference within Pezizaceae addi- ent methods of phylogenetic inference aVect the relation- tional characters from alternative loci are needed. In this ships reconstructed for the Pezizaceae. study, we investigate the utility of two potential phyloge- netic markers, -tubulin and RPB2 (the gene encoding the second largest subunit of RNA polymerase II) for 2. Materials and methods intergeneric-level systematics within cup-forming fungi (Ascomycota). 2.1. Specimens RNA polymerase II is the key enzyme that tran- scribes pre-mRNA. The use of RPB2 was recently initi- A data matrix containing 69 unique species of Peziz- ated as a suitable alternative to the commonly used ales and Neolecta vitellina was constructed with nuclear ribosomal small subunit (SSU rDNA) region in sequences from LSU rDNA, RPB2, and -tubulin genes ascomycete molecular systematics (Liu et al., 1999). (Table 1). For some species more than one collection was RPB2 is a ca. 1000 amino acid protein, encoded in an sequenced to verify the sequences of a particular gene approximately 3.0 kb gene. A favorable attribute of and explore the intra-speciWc variation. Only a single RPB2 is that only a single copy of the gene has (so far) accession was included in the phylogenetic analyses been found in fungi. DiVerent regions of RPB2 have when all intra-speciWc sequences from diVerent acces- diVerent rates of evolutionary change, and are thus, use- sions were identical across all loci. Sixty-nine RPB2 and K. Hansen et al. / Molecular Phylogenetics and Evolution 36 (2005) 1–23 3 Table 1 Species examined, sequenced, and used in the molecular phylogenetic study Species Collection number (Herbarium)a LSU -Tubulin RPB2 Geographic origin Amylascus tasmanicus (Rodway) Trappe Trappe 18084 (C, dubl. OSC). Australia AF335113 AY513297b AY500465b Ascobolus carbonarius P. Karst. KH 00.008 (C). Denmark AY500526b AY513298b AY500459b Ascobolus crenulatus P. Karst. KH.02.005 (C). USA AY500527b AY513299b AY500462b Ascobolus denudatus Fr. KS-94-146 (C). Denmark AY500528b AY513300b AY500460b Boudiera dennisii Dissing & Sivertsen Rana 81.113 (C). Norway AY500529b — AY500508b,c Boudiera tracheia (Gamundí) Dissing & Rana 79.049 (C).