Fungal Diversity Arizona gasteroid fungi I: Lycoperdaceae (Agaricales, Basidiomycota) Bates, S.T.1*, Roberson, R.W.1 and Desjardin, D.E.2 1School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA 2Department of Biology, San Francisco State University, 1600 Holloway Ave., San Francisco, California 94132, USA Bates, S.T., Roberson, R.W. and Desjardin, D.E. (2009). Arizona gasteroid fungi I: Lycoperdaceae (Agaricales, Basidiomycota). Fungal Diversity 37: 153-207. Twenty-eight species in the family Lycoperdaceae, commonly called ‘puffballs’, are reported from Arizona, USA. In addition to widely distributed species, understudied species (e.g., Calvatia cf. leiospora and Holocotylon brandegeeanum) are treated. Taxonomic descriptions and illustrations, which include microscopic characters, are given for each species, and a dichotomous key is presented to facilitate identification. Basidiospore morphology was also examined ultrastructurally using scanning electron microscopy, and phylogenetic analyses were carried out on nrRNA gene sequences (ITS1, ITS2, and 5.8S) from 42 species within (or closely allied to) the Lycoperdaceae. Key words: Agaricales, euagarics, fungal taxonomy, gasteroid fungi, gasteromycete, Lycoperdaceae, puffballs. Article Information Received 22 August 2008 Accepted 25 November 2008 Published online 1 August 2009 *Corresponding author: Scott T. Bates; e-mail: [email protected] Introduction Agaricales, Boletales, and Russulales. Accordingly, a vigorous debate concerning the Lycoperdaceae Chevall. has traditionally path of evolution within these series ensued been placed within the Gasteromycetes; a class (e.g., see Singer, 1962; Heim, 1971; Thiers, that workers have long suggested did not 1984). represent a natural assemblage of fungi (Heim, With the close of the twentieth century, 1971). By the early twentieth century, molecular phylogenetic analyses using systematic mycologists began to produce sequence data from the nuclear genes encoding alternative taxonomies, based in evolutionary ribosomal RNA presented convincing evidence theory, which included members of the that not only were the Gasteromycetes a Gasteromycetes among basidiomycetous agari- polyphyletic grouping, but that gasteroid forms coid (mushroom-like) fungi. For example, the had multiple independent origins from within classification of Heim and colleagues (1934) the Agaricomycotina (Hibbett et al., 1997). did not recognize the Gasteromycetes, but Taxa that were formerly placed within the rather placed both the Gastérales, which Gasteromycetes are now typically referred to as included Lycoperdaceae, and the Agaricales, ‘gasteroid’ fungi (e.g., Pegler et al., 1995; which exclusively contained agaricoid species, Binder and Bresinsky, 2002) or ‘gastero- in the Homobasidiés (Basidiomycetes). mycetes’ (e.g., Reijnders, 2000). Molecular Throughout the middle of that century, several phylogenetic analyses continue to support the evolutionary ‘series’ were proposed that linked position that Lycoperdaceae is a gasteroid taxa within the Gasteromycetes, secotioid lineage which has arisen from within the species (mushroom-like fungi having an lepiotoid mushrooms of the Agaricales (Hibbett unexpanded pileus that encloses malformed et al., 1997; Krüger et al., 2001; Moncalvo et lamellae), and agaricoid members of the al., 2002; Vellinga, 2004). It is also interesting 153 to note that as early as the mid-1960s Kreisel in Arizona. These biomes include xeric alpine (1967a) suggested this relationship and pointed tundra at extremely high elevations (above out similarities between Lepiota lycoperdoides 3500 m); mesic subalpine and montane conifer Kreisel [=Cystolepiota fumosifolia (Murrill) forests at high elevations (ca. 2000–3500 m); Vellinga] and Lycoperdon species. Recent arid adapted chaparral in the mid elevations (ca. studies (Krüger et al., 2001; Krüger and Gargas, 1050–2000 m); and xeric desertscrub in the 2008; Krüger and Kreisel, 2003; Larsson and lower elevations (below 1050 m). It is also Jeppson, 2008) based on nrDNA sequence data notable that boundaries of four major desert indicate that the type genus Lycoperdon is ecoregions (Chihuahuan, Great Basin, Mojave, polyphyletic and question the stability of and Sonoran) come in contact with some boundaries between the genera Bovista Pers., portion of the state. Calvatia Fr., Handkea Kreisel, Lycoperdon Members of the Lycoperdaceae have a Pers., Morganella Zeller, and Vascellum F. widespread distribution and are commonly Šmarda. found in temperate, arid, and tropical climates Prior to the twenty-first century, mention (Pegler et al., 1995). A large part of southern of puffballs from the state of Arizona in Arizona is occupied by the Sonoran Desert mycological literature is limited. Most which is arid with considerable tropical significant is Demoulin’s doctoral thesis (1972), influences, and the mid-elevations of the state which includes records of four Lycoperdon are covered by large tracts of temperate forest species occurring in the state. Other publica- (Brown, 1994). Considering these factors, it is tions (Morgan, 1890; Smith, 1974; Demoulin, not surprising that a biologically diverse and 1993) include only single records. In addition species rich assortment of these gasteroid fungi to these publications, numerous specimens of are present within the state. Arizona puffballs have been collected and This monograph documents 28 species of deposited in herbaria by the few field puffballs from nine of the biotic communities mycologists who have worked in the state, present in Arizona. Each of these gasteroid namely W.H. Long, R.L. Gilbertson, and J.S. species in the Lycoperdaceae is described as States. To supplement that material, specimens well as illustrated, and SEM micrographs have also been collected by one author (STB) typifying spore morphology are presented. over the course of four seasons in the field. Of Ribosomal RNA gene sequences were also the 28 species presented here, 22 were obtained from several of the specimens considered to be new records for the state examined as part of this study. These data were (Bates, 2004) and were subsequently published supplemented with rDNA sequences obtained as such in a checklist of Arizona macrofungi from GenBank, and were used in subsequent (Bates, 2006). In that publication, Calvatia phylogenetic analyses. In addition to this bicolor (Lév.) Kreisel was reported for the first publication, the reader may refer to Bates (2004) time from North America and Disciseda for further discussion of morphology and verrucosa G. Cunn. was reported for the first taxonomy of the Lycoperdaceae. time from the United States. In addition to those records, this publication also documents Materials and methods rare and/or understudied species, such as Calvatia cf. leiospora Morgan and Holocotylon Monography brandegeeanum Lloyd. Macro-morphological data are based on Arizona possesses a wide range of habitat. original descriptions of herbarium material or Brown (1994) outlined 27 major biotic collections made in the field. Micro- communities (biomes) that occur in the morphological data were gathered using an Southwestern United States and Northwestern Olympus BH2 conventional bright field Mexico. Of these, 14 biomes with 2 microscope. Illustrations of spores, capillitial subdivisions of Sonoran desertscrub are found threads, endoperidial hyphae, and exoperidial 154 Fungal Diversity cells were drawn using an Olympus U-DA Herbarium acronyms followed the Index drawing attachment. For microscopic examina- Herbariorum (Holmgren and Holmgren, 1998). tion, all material was first infiltrated with 95% aqueous ethanol before being mounted on glass Basidiospore ultrastructure slides. Observations of characters, such as Observations of spore ultrastructure were spore ornamentation and capillitial structure, made on a XL30 (FEI, Hillsboro, OR, USA) were made from glebal material mounted environmental scanning electron microscope separately in H2O (with a small amount of (ESEM) operated under high vacuum in detergent added as a surfactant), 3% aqueous scanning mode, at an accelerating voltage of 3– KOH, and/or Lactophenol Cotton Blue. In 5 kV. Spores were fixed in 2% aqueous addition, a small portion of the exoperidium glutaraldehyde after re-hydration from 24–48 was mounted in Melzer’s reagent to observe hours in deionized H2O. After fixation, spores structure of exoperidial hyphae and/or were washed in a phosphate buffer solution and sphaerocysts. Measurements were made from then subjected to a dehydration series from material mounted in KOH at 1000× using an 70% to 100% ethanol. After dehydration, ocular reticle with units calibrated to an optical spores were critical-point-dried using a CPD micrometer. All spore measurements include 020 (Balzer Union, Liechtenstein). Once ornamentation, and spore statistics include: X¯, removed from the critical-point-dryer, the the arithmetic mean of the spore length by spores were affixed to carbon coated aluminum spore width (± SD) for n spores measured; Q, specimen holders with poly-l-lysine and sputter the quotient of spore length and spore width in coated with palladium-gold for 5 min at 10 any one spore, indicated as the range of milliamps in a Technics Hummer II (Anatech variation in n spores measured; Qm the mean of LTD., Hayward, CA, USA). Q values. Standard classical taxonomic and DNA extraction descriptive methodologies for gasteroid fungi A small portion (15–20 mg) of glebal were used (see Calonge, 1998;