Fungal Genetics and Biology 20, 243–253 (1996) Article No. 0039 Phylogeny of the Genus Agaricus Inferred from Restriction Analysis of Enzymatically Amplified Ribosomal DNA Britt A. Bunyard,* Michael S. Nicholson,† and Daniel J. Royse‡ *USDA-ARS, Fort Detrick, Building 1301, Frederick, Maryland 21701; †Department of Biology, Grand Valley State University, Allendale, Michigan 49401; and ‡Department of Plant Pathology, Pennsylvania State University, 316 Buckhout Laboratory, University Park, Pennsylvania 16802 Accepted for publication November 15, 1996 Bunyard, B. A., Nicholson, M. S., and Royse, D. J. 1996. accounting for 37% of the total world production of Phylogeny of the genus Agaricus inferred from restriction cultivated mushrooms. Although much is known about A. analysis of enzymatically amplified ribosomal DNA. Fungal bisporus, several aspects remain unclear, especially those Genetics and Biology 20, 243–253. The 26S and 5S concerning its genetic life history (Kerrigan et al., 1993a; ribosomal RNA genes and the intergenic region between Royer and Horgen, 1991; Castle et al., 1988, 1987; Spear et the 26S and the 5S rRNA genes of the ribosomal DNA al., 1983; Royse and May, 1982a,b; Elliott, 1972; Raper et repeat of 21 species of Agaricus were amplified using PCR al., 1972; Jiri, 1967; Pelham, 1967; Evans, 1959; Kligman, and then digested with 10 restriction enzymes. Restriction 1943). Fundamental processes such as the segregation and fragment length polymorphisms were found among the 21 assortment of genes during meiosis remain poorly defined putative species of Agaricus investigated and used to (Kerrigan et al., 1993a; Summerbell et al., 1989; Royse and develop a phylogenetic tree of the evolutionary history of May, 1982a). In addition, little information exists about the A. bisporus. The 58 end of the 26S gene showed more evolution and relatedness of species within the genus variability than the 38 end. A. excellens, A. chionodermus, Agaricus (Kerrigan et al., 1993b). and A. caroli represented the species most distantly In the past, strains of A. bisporus having desirable or related to A. bisporus. We present here the first comprehen- improved traits were developed from single- or multispore sive attempt at systematically resolving the entire genus cultures (Kligman, 1943). Suitable parental lines were Agaricus using modern techniques for molecular genetic selected on the basis of growth habit on defined media analysis. Our data indicate that previous taxonomic (Fritsche, 1981). New strains were then developed follow- schemes, based on morphological characters, are in need ing anastomosis between the hyphae of different parental strains grown in the same petri plate (Fritsche, 1981). of revision. r 1996 Academic Press Through continuous selection, those strains with undesired Index Descriptors: mushrooms; phylogeny; RFLPs; ri- characteristics were eliminated. bosomal DNA. Recently, researchers employed allozyme analysis to identify genotypic classes and to follow the segregation of allozyme- encoding alleles during meiosis (Royse and May, 1990, 1982a; INTRODUCTION Kerrigan and Ross, 1989; Royse et al., 1983; Spear et al., 1983) in various wild and commercial mushrooms. Genetic studies The white button mushroom, Agaricus bisporus (Lange) with allozymes have been conducted on more than 30 crop Imbach (5A. brunnescens Peck) is the most important species (Abler et al., 1991). Spear et al. (1983) created the first commercially cultivated mushroom species in the world, map of linkage groups in A. bisporus using allozymes. How- 1087-1845/96 $18.00 Copyright r 1996 by Academic Press All rights of reproduction in any form reserved. 243 244 Bunyard, Nicholson, and Royse ever, the lack of suitable markers resulted in a very limited map. tandem array of transcribed and nontranscribed stretches of Kerrigan et al. (1991) have added to this map of linkage groups DNA (Cassidy et al., 1984; Petes and Botstein, 1977). The large using restriction fragment length polymorphisms (RFLPs)1 subunit ribosomal RNAs (LSU) vary in length from about 3500 and randomly amplified polymorphic DNA. It is important to to 5000 nucleotides in most eukaryotes and contain regions of note that gene linkage maps for A. bisporus, as yet, do not link rapidly and slowly evolving regions. RFLP analysis of genes or markers to any agronomically important characteristics. Other intergenic regions of rRNA may provide new information methods, relying on DNA analysis, used to explore the relation- about evolutionary relationships of many different fungal taxa. ships among and between taxa have been discussed (Royse et As the sequences of the 26S–28S genes are highly conserved, al., 1993). they are useful for comparisons between distantly related RFLPs have been used to define genotypes of hetero- taxonomic groups. The 5S gene is much less conserved and, as karyotic and homokaryotic strains and to confirm crosses such, may change more rapidly over time. The regions between among several isolates (Summerbell et al., 1989; Castle et the genes, or intergenic regions (IGRs), are not transcribed al., 1987, 1988), as well as to investigate the postmeiotic and, therefore, show much lower conservation when compared distribution of nuclei in A. bisporus (Kerrigan et al., 1993a; to genic regions. The 5S gene and IGRs are useful for Summerbell et al., 1989). Conclusions about reproductive elucidating differences between closely related taxa, at the events are scarce due to the small number of known species or race level. Johansen et al. (1992) determined the genetic markers available for genetic analysis of the species time of divergence between the myxomycetes Didymium and (Kerrigan, 1990; Castle et al., 1987), as well as the lack of Physarum based on the amount of variation in the LSU of the an extensive genetic map of linkage groups. Also of interest two genera. Vilgalys and Gonzales (1990) recently used RFLP is the evolutionary history of A. bisporus which remains analysis of the rDNA subunits for the determination of uncertain (Singer, 1986). Although addressed in great genotypic classes for the fungal species Rhizoctonia solani detail by Kerrigan (1990), many questions still exist concern- Ku¨ hn. Using similar methods, other researchers have begun ing the evolutionary origin of A. bisporus, the relationship taxonomic work involving species of Phanerochaete and Sporot- of cultivars to wild populations of this species, and its richum (Raeder and Broda, 1984), Pythium (Martin, 1990), position within the Agaricales. and Sclerotinia (Kohn et al., 1988). Anderson and Stasovski Phylogenetic assessment of Basidiomycetes based on DNA (1992) determined the molecular phylogeny of species of offer many advantages over traditional morphological methods. Armillaria from the Northern Hemisphere using sequence Divergence times between species are directly related to analysis following polymerase chain reaction (PCR) amplifica- changes in the DNA sequence (see pp. 11–18 in Nei, 1987). tion of the intergenic region between the 26S and the 5S rRNA These changes can be determined through direct sequencing genes. Other researchers have used RFLP analysis of PCR- of the DNA or through analysis of RFLPs found by cutting the amplified rDNA to distinguish isolates of the Gaeumannomy- DNA with restriction endonucleases. Researchers conducting ces–Phialophora complex of fungi (Ward and Akrofi, 1994), phylogenetic studies have begun to utilize the variation found strains of Rhizobium (Laguerre et al., 1994), genotypes of in the sequences of the ribosomal RNA genes (rDNA) (for Pleurotus (Bunyard et al., 1996), and species of Morchella review, see Bruns et al., 1991). These variations are used to (Bunyard et al., 1995, 1994). determine relatedness between species, as well as among a The goals of this investigation were to explore novel single species. The use of RFLP analysis (Bunyard et al., 1996, 1994; Cubeta et al., 1991; Hibbett and Vilgalys, 1991; Kohn et methods of genotypic classification of A. bisporus and to al., 1988; Magee et al., 1987) and direct sequencing (White et elucidate the evolutionary history of A. bisporus using al., 1990; Medlin et al., 1988; Woese and Olsen, 1986) to molecular methods of genetic analysis. investigate the genes coding for the production of 16S–18S, 5.8S, 26S–28S, and 5S ribosomal RNA are well established methods for the assessment and comparison of phylogenetic relationships of organisms (Sogin, 1990) over a wide range of MATERIALS AND METHODS taxonomic levels. The rRNA genes of fungi are located on a single chromosome and are present as repeated subunits of a Isolates 1 Abbreviations used: RFLP, restriction fragment length polymor- Fifty-four isolates, comprising 21 putative species of phism; rDNA, ribosomal RNA gene; IGR, intergenic region; LSU, large Agaricus spp. collected from worldwide sources were used subunit ribosomal RNA. in this study. Cultures were grown in liquid potato dextrose Copyright r 1996 by Academic Press All rights of reproduction in any form reserved. Phylogeny of the Genus Agaricus 245 broth supplemented with yeast extract. Mycelia were Phylogeny Construction filtered and air dried prior to DNA extraction. Isolate numbers from the Pennsylvania State University Mush- Phylogenies were inferred from data collected by RFLP room Culture Collection, species, source, and geographic analysis of the 26S rDNA and the IGR-1/5S rDNA. site are listed in Table 1. PHYLIP, a phylogeny inference package written by J. Felsenstein (1993), was used to determine