Mycologia, 98(6), 2006, pp. 896–905. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897 An overview of the higher level classification of Pucciniomycotina based on combined analyses of nuclear large and small subunit rDNA sequences M. Catherine Aime1 subphyla of Basidiomycota. More than 8000 species of USDA-ARS, Systematic Botany and Mycology Lab, Pucciniomycotina have been described including Beltsville, Maryland 20705 putative saprotrophs and parasites of plants, animals P. Brandon Matheny and fungi. The overwhelming majority of these Biology Department, Clark University, Worcester, (,90%) belong to a single order of obligate plant Massachusetts 01610 pathogens, the Pucciniales (5Uredinales), or rust fungi. We have assembled a dataset of previously Daniel A. Henk published and newly generated sequence data from USDA-ARS, Systematic Botany and Mycology Lab, Beltsville, Maryland 20705 two nuclear rDNA genes (large subunit and small subunit) including exemplars from all known major Elizabeth M. Frieders groups in order to test hypotheses about evolutionary Department of Biology, University of Wisconsin, relationships among the Pucciniomycotina. The Platteville, Wisconsin 53818 utility of combining nuc-lsu sequences spanning the R. Henrik Nilsson entire D1-D3 region with complete nuc-ssu sequences Go¨teborg University, Department of Plant and for resolution and support of nodes is discussed. Our Environmental Sciences, Go¨teborg, Sweden study confirms Pucciniomycotina as a monophyletic Meike Piepenbring group of Basidiomycota. In total our results support J.W. Goethe-Universita¨t Frankfurt, Department of eight major clades ranked as classes (Agaricostilbo- Mycology, Frankfurt, Germany mycetes, Atractiellomycetes, Classiculomycetes, Cryp- tomycocolacomycetes, Cystobasidiomycetes, Microbo- David J. McLaughlin tryomycetes, Mixiomycetes and Pucciniomycetes) and Department of Plant Biology, University of Minnesota, St Paul, Minnesota 55108 18 orders. Key words: basidiomycetes, molecular phylo- Les J. Szabo genetics, parasitic fungi, rDNA systematics, Uredi- USDA-ARS, Cereal Disease Lab, University of niomycetes Minnesota, St Paul, Minnesota 55108 Dominik Begerow INTRODUCTION Universita¨t Tu¨ bingen, Spezielle Botanik und Mykologie, Tu¨ bingen, Germany One-third of described Basidiomycota belong to Jose´ Paulo Sampaio subphylum Pucciniomycotina (5Urediniomycetes CREM, SABT, Faculdade de Cieˆncias e Tecnologia, sensu Swann and Taylor 1995). Roughly 7000 of Universidade Nova de Lisboa, 2829-516 Caparica, these, or ,90%, belong to a single order, Pucciniales Portugal (5Uredinales G. Winter) or the rust fungi (Kirk et al Robert Bauer 2001). The remaining fungi within the Pucciniomy- Michael Weiß cotina are remarkably diverse ecologically, biological- Franz Oberwinkler ly and physiologically; only with the relatively recent Universita¨t Tu¨ bingen, Spezielle Botanik und Mykologie, advent of ultrastructure, biochemical and molecular Tu¨ bingen, Germany systematic studies have the relatedness of the varied fungi now placed in the Pucciniomycotina become David Hibbett apparent. These include Microbotryales, phytopatho- Biology Department, Clark University, Worcester, Massachusetts 01610 gens once classified in the Ustilaginomycotina (true smuts and relatives, Ustilaginomycetes sensu Bauer et al 2001); numerous basidiomycetous yeasts formerly Abstract: In this study we provide a phylogenetically allied with Auriculariales and other groups of based introduction to the classes and orders of Agaricomycotina (mushroom-forming fungi and re- Pucciniomycotina (5Urediniomycetes), one of three lated taxa; Hymenomycetes sensu Swann and Taylor 1993); and at least one species, Mixia osmundae, Acepted for publication 7 September 2006. formerly classified within the Ascomycota (Nishida 1 Corresponding author. E-mail: [email protected] et al 1995). 896 AIME ET AL:PUCCINIOMYCOTINA OVERVIEW 897 Most species of Pucciniomycotina are parasitic and Pucciniomycotina (e.g. Fell et al 2001, Weiß et al the group includes phytopathogens (e.g. Pucciniales, 2004, Bauer et al 2006). However D1–D2 sequences Microbotryales), mycoparasites (e.g. Tuberculina, have provided only weak support for monophyly of Spiculogloea) and entomopathogens (e.g. Septobasi- Pucciniomycotina (Begerow et al 1997, Berres et al diales). A few are putatively saprotrophic or are of 1995), or have resolved the group as a paraphyletic unknown trophic habit. Pucciniomycotina species are assemblage from which the Ustilaginomycotina and present in most habitats including freshwater (Bauer Agaricomycotina have been derived (Weiß et al 2004). et al 2003) and marine environments (Swann et al Nor were analyses of a third gene, that coding for b- 2001). Dimorphism, cryptic fructifications, heteroe- tubulin, able to recover a monophyletic Pucciniomy- cism and multiple spore types all have evolved in at cotina (Begerow et al 2004). Because of the published least some lineages of Pucciniomycotina, hampering discrepancies in tree topologies and support values, the collection of complete biological and life cycle monophyly for the group has yet to be established data for many taxa. Yet species of Pucciniomycotina conclusively by molecular analyses. are causal agents of some of the most devastating Current major classifications of Pucciniomycotina diseases of crops, may cause opportunistic disease in include those of Kirk et al (2001), Swann et al (2001), humans and have shown potential as biological Weiß et al (2004) and Bauer et al (2006). The latter control organisms of invasive plants and of other three classifications have many similarities (SUPPLE- pathogenic fungi (e.g. Evans 1993, Swann et al 2001). MENTARY TABLE I). However there are also differences A few Pucciniomycotina species produce basidio- between these systems in both resolution and carps, these typically being simple stipitate-capitate composition of taxa. Kirk et al (2001) divide the (stilboid) or resupinate (FIG. 1b) structures. Some Pucciniomycotina (as Urediniomycetes) into five Pucciniomycotina species form hyphae (filamentous) orders and do not include subclasses; thus their or yeasts or both (dimorphic) (FIG. 1d). Members of classification is quite different from the others. the Pucciniales produce spore-filled fruiting struc- The goal of the present study is to reassess the tures termed sori (FIG. 1f ). Basidia of teleomorphic higher level systematics of the Pucciniomycotina with species may take the form of teliospores, holobasidia combined analyses of nuc-lsu rDNA and nuc-ssu or phragmobasidia, and basidiospores may be sessile, rDNA sequences. Two other studies have included gasteroid or forcibly discharged. The Pucciniomyco- a simultaneous analysis of nuc-lsu and nuc-ssu rDNA tina is distinguished from the other two major clades sequences of Pucciniomycotina (Lutzoni et al 2004, of Basidiomycota by the possession of simple septal Bauer et al 2006). However the former was focused on pores that lack membrane-bound caps (Swann et al relationships across the Fungi and it included only 14 2001, Weiß et al 2004 and references therein). In species of Pucciniomycotina, of which 10 were addition the cell wall sugar composition of the members of the Pucciniales and the combined Pucciniomycotina differs from that in the Ustilagino- analysis in the latter employed only 25 species of mycotina and Agaricomycotina (Prillinger et al Pucciniomycotina and did not cover all major 2002). lineages. In the present study we compile and analyze Diverse approaches have been used to delimit taxa datasets containing new and previously published within the Pucciniomycotina, including studies of sequences that represent 174 species from all major ultrastructural, physiological, biochemical and eco- groups of Pucciniomycotina. We assess the ability of logical characters. Molecular characters have had different rDNA genes and gene regions to recover a major impact on our understanding of the and support each clade, and we provide an overview phylogenetic relationships of Pucciniomycotina. As of the classes and orders of Pucciniomycotina. early as 1985 Gottschalk and Blanz showed that the secondary structure of 5S ribosomal RNA in some MATERIALS AND METHODS species of Pucciniomycotina differs from that of some other lineages of Basidiomycota. The pioneering This study uses both newly generated sequences and studies of Swann and colleagues (Swann and Taylor sequences that were retrieved from GenBank from more 1993, 1995; Swann et al 1999) used nuclear small than 80 studies (SUPPLEMENTARY TABLE II). Some Pucci- niales sequences were generated with the methods de- subunit (nuc-ssu) rDNA sequences to support the scribed in Aime (2006); sequences of Jola spp. were monophyly of the Pucciniomycotina and a number of generated as in Frieders (1997). Descriptions of laboratory major clades. Other studies have employed a region methods pertaining to the majority of new sequences of about 500–650 bp at the 59 end of the nuclear large produced at Clark University can be found online (SUPPLE- subunit (nuc-lsu) rDNA, containing the highly vari- MENTARY APPENDIX 1). able divergent domains D1 and D2 (Hassouna et al The entire data matrix contains 208 operational taxo- 1984) providing resolution for many clades within the nomic units (OTUs) including six Ascomycota that were 898 MYCOLOGIA FIG. 1. Representatives of the Pucciniomycotina. a. Jola cf. javensis (Platygloeales) fruiting on Sematophyllum swartzii.b. Septobasidium burtii (Septobasidiales) fungal