Performance of Four Ribosomal DNA Regions to Infer Higher-Level Phylogenetic Relationships of Inoperculate Euascomycetes (Leotiomyceta)
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Molecular Phylogenetics and Evolution 34 (2005) 512–524 www.elsevier.com/locate/ympev Performance of four ribosomal DNA regions to infer higher-level phylogenetic relationships of inoperculate euascomycetes (Leotiomyceta) H. Thorsten Lumbscha,¤, Imke Schmitta, Ralf Lindemuthb, Andrew Millerc, Armin Mangolda,b, Fernando Fernandeza, Sabine Huhndorfa a Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA b Universität Duisburg-Essen, Campus Essen, 45117 Essen, Germany c Center for Biodiversity, Illinois Natural History Survey, 607 E. Peabody Drive, Champaign, IL 61820, USA Received 9 June 2004; revised 14 October 2004 Available online 1 January 2005 Abstract The inoperculate euascomycetes are Wlamentous fungi that form saprobic, parasitic, and symbiotic associations with a wide vari- ety of animals, plants, cyanobacteria, and other fungi. The higher-level relationships of this economically important group have been unsettled for over 100 years. A data set of 55 species was assembled including sequence data from nuclear and mitochondrial small and large subunit rDNAs for each taxon; 83 new sequences were obtained for this study. Parsimony and Bayesian analyses were per- formed using the four-region data set and all 14 possible subpartitions of the data. The mitochondrial LSU rDNA was used for the Wrst time in a higher-level phylogenetic study of ascomycetes and its use in concatenated analyses is supported. The classes that were recognized in Leotiomyceta ( D inoperculate euascomycetes) in a classiWcation by Eriksson and Winka [Myconet 1 (1997) 1] are strongly supported as monophyletic. The following classes formed strongly supported sister-groups: Arthoniomycetes and Doth- ideomycetes, Chaetothyriomycetes and Eurotiomycetes, and Leotiomycetes and Sordariomycetes. Nevertheless, the backbone of the euascomycete phylogeny remains poorly resolved. Bayesian posterior probabilities were always higher than maximum parsimony bootstrap values, but converged with an increase in gene partitions analyzed in concatenated analyses. Comparison of Wve recent higher-level phylogenetic studies in ascomycetes demonstrates a high degree of uncertainty in the relationships between classes. 2004 Elsevier Inc. All rights reserved. Keywords: Ascomycota; Evolution; Combining data; Ribosomal DNA; Bayesian analysis; Maximum parsimony 1. Introduction is a group of Wlamentous fungi in which the asci are usu- ally concentrated in a fruiting body of deWnite morphol- The Ascomycota is the largest group of fungi (Kirk ogy, the ascoma. The euascomycetes include species that et al., 2001) and is characterized by the endogenous for- form saprobic, parasitic, and symbiotic associations with mation of spores in a sac-like meiosporangium, the so- animals, plants (including algae, bryophytes, and phan- called ascus. These fungi colonize a large variety of habi- erogams), other fungi, or cyanobacteria (Alexopoulos tats and utilize a broad range of nutrient sources. Within et al., 1996). This study deals with the euascomycetes that the Ascomycota, the euascomycetes ( D Pezizomycotina) have inoperculate asci, which are classiWed in the super- class Leotiomyceta (Eriksson and Winka, 1997). They * Corresponding author. Fax: +1 312 665 7158. are distinguished from the basal Pezizomyceta, which E-mail address: [email protected] (H.T. Lumbsch). include the apotheciate Pezizales with operculate asci. 1055-7903/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.11.007 H.T. Lumbsch et al. / Molecular Phylogenetics and Evolution 34 (2005) 512–524 513 The higher-level classiWcation of inoperculate euasco- muth et al., 2001; Lumbsch et al., 2002). The analysis of mycetes has been unstable for almost 100 years. Most the two additional ribosomal data sets basically con- classiWcations employed only single characters to distin- Wrmed the classiWcation based on nuclear SSU rDNA. guish major groups within euascomycetes and these were In concatenated analyses some poorly supported clas- subsequently found to have limited phylogenetic infor- ses, such as Dothideomycetes and Lecanoromycetes, mation. In the classical period of the 19th and early 20th gained strong support, and some relationships among century, groups of euascomycetes were distinguished on classes, such as the sister-group relationship of Chaeto- the basis of morphology of the ascomata, resulting in a thyriomycetes and Eurotiomycetes, were strongly sup- schematic distinction of classes, such as Discomycetes ported. However, most relationships between classes including species with apothecia, Plectomycetes with cle- remained poorly supported. The analysis of -tubulin istothecia, and Pyrenomycetes with perithecia. This clas- sequences in ascomycetes is complicated due to the siWcation was recognized as too coarse (e.g., von Höhnel, presence of paralogues (Landvik et al., 2001) and thus 1907) and did not allow proper placement of taxa with this data set was not further explored in higher-level intermediate ascoma-types. Consequently, other charac- phylogenetic studies in euascomycetes. The results of ters were used for the circumscription of major clades of the RPB-2 analyses (Liu et al., 1999; Liu and Hall, 2004) eusacomyetes, such as the ascoma development (Nann- supported the monophyly of most classes distinguished feldt, 1932) and ascus-type (Luttrell, 1955). However, by Eriksson and Winka (1997), but the relationships these classiWcations were also based on single characters between these classes diVered from analyses based on and became unstable with growing knowledge of the ribosomal genes in that loculoascomycetes formed a morphological diversity of these organisms. ConXicting monophyletic group. In a study including three large- classiWcations have been proposed for supraordinal cate- scale analyses of combined data sets of (a) nuclear small gories in euascomycetes and subsequently Eriksson and and large subunit ribosomal DNA, and in addition (b) Hawksworth (1993) avoided all supraordinal ranks in RPB-2, and (c) RPB-2 and mitochondrial small subunit their classiWcation of ascomycetes. ribosomal DNA sequences, Lutzoni et al. (2004) found Molecular data have been employed for more than a most of the classes distinguished by Eriksson and decade in mycology to test morphology-based classiWca- Winka (1997) as monophyletic except the Dothideomy- tions that were shown to produce unnatural groupings cetes and Leotiomycetes that were para- or polyphyletic when used as a single criterion. Phylogenetic studies in some analyses. Several changes to the classiWcation of employing nuclear SSU rDNA sequences provided sup- Eriksson and Winka (1997) were accepted by Lutzoni et port for a modiWed classiWcation. It was shown that a al. (2004). An additional class, the Lichinomycetes was combination of ascoma-types, ascoma development, accepted, which was described previously (Reeb et al., and ascus-type was useful to circumscribe monophyletic 2004). However, species in this class were nested within clades, even though the individual characters were Dothideomycetes or a paraphyletic Leotiomycetes in homoplasious (Berbee and Taylor, 1992, 1995; Gargas the diVerent analyses. No representative of the Lichino- and Taylor, 1995; Lumbsch, 2000; Spatafora, 1995; mycetes sensu Reeb et al. (2004) is included in our study, Winka, 2000). These Wndings resulted in a new supraor- since we were unable to obtain mt LSU rDNA dinal classiWcation including the distinction of several sequences from any species in this group. In the study classes of closely related orders that was based on the by Lutzoni et al. (2004) the Arthoniomycetes and Doth- combination of morphological characters and SSU ideomycetes were classiWed as subclasses of Sordario- rDNA molecular characters (Eriksson and Winka, mycetes, although they were paraphyletic in the analysis 1997). However, some of the classes proposed by Eriks- of the nuclear ribosomal DNA and only formed a son and Winka (1997), such as Dothideomycetes or Lec- monophyletic clade in the three and four gene analyses anoromycetes, did not receive support in nuclear SSU that included only one or two representatives of these rDNA phylogenies and the relationships among the two classes. The Chaetothyriomycetes and Eurotiomy- classes remained unclear. Further, Tehler et al. (2000, cetes were found as sister-groups in all three analyses 2003) conducted large-scale analyses of all nuclear SSU and hence classiWed as subclasses within one class Euro- rDNA data then available from fungi, and showed that tiomycetes sensu lato. this data set alone is insuYcient to resolve higher-level Given the uncertainty of the backbone of the euasc- phylogeny of euascomycetes with conWdence. Four omycete phylogeny, we have targeted the mitochondrial additional molecular data sets have so far been added to LSU rDNA as an additional data set to elucidate the the toolbox for the elucidation of the phylogeny of higher-level phylogeny within inoperculate euascomyce- higher-level euascomycetes: the protein-coding genes - tes. This gene has rarely been used for phylogenetic stud- tubulin (Landvik et al., 2001) and RPB-2 (Liu et al., ies in ascomycetes and mainly at intrageneric or 1999; Liu and Hall, 2004), the nuclear LSU rDNA intrafamiliar rank (e.g., Peever et al., 2004; Schmitt and (Bhattacharya et al., 2000; Lumbsch et al., 2000; Lutzoni Lumbsch, 2004). However, it has been employed at et al., 2001), and the mitochondrial SSU rDNA (Linde- higher-level phylogenetic studies