Mycologia, 99(5), 2007, pp. 644–654. # 2007 by The Mycological Society of America, Lawrence, KS 66044-8897 Septal pore apparatus and nuclear division of Auriscalpium vulgare Gail J. Celio1 fungal systematics, although their usefulness varies Mahajabeen Padamsee among taxonomic levels and clades (Bracker 1967, Bryn T.M. Dentinger Kimbrough 1994, McLaughlin et al 1995b, Lutzoni et Department of Plant Biology, University of Minnesota, al 2004). Improvements in chemical fixation methods Saint Paul, Minnesota 55108 for fungal ultrastructure resulted in better visualiza- Kelly A. Josephsen tion of nuclear division and the spindle pole body (SPB) (see Girbardt 1978). Freeze substitution (FS) Imaging Center, College of Biological Sciences, University of Minnesota, Saint Paul, Minnesota 55108 protocols were particularly helpful for observing the condition of the nuclear envelope during stages of Thomas S. Jenkinson division (Heath and Rethoret 1982; Kanbe and Esther G. McLaughlin Tanaka 1985; Tanaka and Kanbe 1986; Berbee and David J. McLaughlin Wells 1988; Berbee et al 1991; Swann and Mims 1991; Department of Plant Biology, University of Minnesota, O’Donnell 1992, 1994; Lu¨ and McLaughlin 1994; Saint Paul, Minnesota 55108 Frieders and McLaughlin 1996; Swann et al 1999). Such characters have supplemented or been used as the basis of phylogenetic analyses (Heath 1986, Abstract: Ultrastructure of the septal pore apparatus McLaughlin et al 1995a, Swann et al 1999). and nuclear division of Auriscalpium vulgare (Russu- The Structural and Biochemical (SB) Database for lales) was examined with freeze substitution and is the Fungi (http://aftol.umn.edu) (Celio et al 2006) presented for inclusion in the AFTOL Structural and associated with the Assembling the Fungal Tree of Biochemical Database (http://aftol.umn.edu). Pre- Life (AFTOL) project is a searchable database viously unreported septal characters for the Russu- containing data primarily from previous ultrastruc- lales (Agaricomycotina) were observed: Septa of the tural studies that have been coded for phylogenetic hymenophore had bell-shaped perforated septal pore analyses. Many traits were selected for inclusion in the caps that may extend along the septum and a zone of SB Database, with data on septum/septal pore organelle exclusion surrounded the septal pore apparatus and nuclear division, including spindle apparatus. Metaphase I of meiosis and metaphase of pole body forms and cycles, among the first to be mitosis were similar. Globular spindle pole bodies entered. The distribution of ultrastructural studies is with electron-opaque inclusions were set within polar uneven across taxonomic groups, hindering investiga- fenestrae of the nuclear envelope. The nuclear tions of evolutionary relationships and character envelope was mostly intact with occasional gaps. evolution. To fill in gaps we investigated taxa and Fragments of endoplasmic reticulum were present collected data from clades and cell types that are near the spindle pole bodies but did not form a polar poorly represented within the SB Database. This study cap. Structural characters may distinguish one or presents septal pore and nuclear division data from more clades of the Agaricomycotina and provide Auriscalpium vulgare in the Russulales (Agaricomyco- additional signal in phylogenetic analyses. tina), a species that fruits in the laboratory. This taxon Key words: basidiocarp, basidium, cytology, is represented in the AFTOL Molecular Database informatics, phylogeny, sporocarp (http://www.aftol.org) by sequence data for the nuclear internal transcribed spacer regions and the INTRODUCTION large and small subunits of the ribosomal DNA. Ultrastructural images of septa of some species in the Many ultrastructural characters have been useful in Russulales have been published (Besson and Froment systematic studies of the Fungi, including septal and 1968, Flegler et al 1976, Patrignani and Pellgrini 1986, nucleus-associated characters. Septa and the organi- Keller 1997); however low magnification and/or zation of the surrounding cytoplasm and structures nonmedian sections prevented data entry for many have been observed with the electron microscope septal characters. In the Russulales only Hericium since the late 1950s (Girbardt 1958, Shatkin and coralloides (Flegler et al 1976) and Artomyces pyxidatus Tatum 1959) and have been helpful in developing (5 Clavicorona pyxidata) (M Berbee unpubl) have character states entered in the database for more than Accepted for publication 29 July 2007. 50% of the characters that apply to nonbasidial cells 1 Corresponding author. E-mail: [email protected] in taxa of the Basidiomycota. Also A. pyxidatus is the 644 CELIO ET AL:ULTRASTRUCTURE OF AURISCALPIUM VULGARE 645 only russuloid species with data on nuclear division anhydride) with an accelerator (2,4,6–tri[dimethylami- (Berbee and Wells 1989). The results presented here noethyl]phenol) equal to 1% of the total solution weight illustrate the extent of data needed to document (Abad et al 1988). The samples then were microwave septal structure and nuclear division. Even within embedded in a vacuum under 20–25 mm mercury pressure presumably well studied groups such as the Agarico- at 42 C for 2 min. The resin was replaced with fresh infiltration solution, and this process was repeated twice. mycotina we report a novel septal character state. The Single spines were separated from pileal tissue under organization of the spindle pole region during a dissecting scope and placed on glass slides coated with nuclear division might provide a morphological Crown dry film lubricant (Crown Industrial Products Co., feature characteristic of the Russulales. Hebron, Illinois). Flat embedding was achieved as described in Kleven and McLaughlin (1989) with glass slides instead of cover slips. The resin-infiltrated specimens were poly- MATERIALS AND METHODS merized in a 74 C oven for 48 h. Cells were selected with Light microscopy.—Sporocarps of Auriscalpium vulgare Gray a Zeiss Axioscope at 12503 with an Optivar and a Zeiss were collected on 16 Sep 1978 at Sand Dunes State Forest, diamond scribe in the ocular position, cut from the slides Zimmerman, Minnesota (voucher collection RJ Meyer 211, and mounted on resin blocks for sectioning. Ultrathin Herbarium, University of Minnesota) and were incubated in sections were cut with a Reichart-Jung Ultracut E ultrami- a moist chamber for 3 d. Specimens then were fixed in 3% crotome equipped with a diamond knife and collected on glutaraldehyde in 0.1 M phosphate buffer (pH 7) at room slot grids using the procedure of Rowley and Moran (1975). temperature approximately 1 h then stored at 4 C until Sections were poststained in 3% uranyl acetate followed by embedded. Material was rinsed in 0.1 M sodium cacodylate Sato’s triple-lead stain (Sato 1968) and examined with buffer (pH 7.2), dehydrated in an ethanol series and a Philips CM 12 transmission electron microscope operating embedded in plastic with a JB-4 embedding kit (Poly- at 60 kV. Detailed protocols for specimen fixation and sciences Inc., Warrington, Pennsylvania). embedding are at http://aftol.umn.edu/. Two micrometer-thick sections were cut with a glass knife on a JB-4 microtome, collected on glass slides and stained in RESULTS 0.05% toluidine blue O in borate buffer (pH 9) either at room temperature for 10 min or while heated by passing Septum and septal pore characters.—Plunge freezing the slide 6–83 over an alcohol flame, then rinsed with tap followed by FS of Auriscalpium vulgare resulted in water. Sections were air dried and cover slips mounted in good fixation, and organelle and plasma membranes Eukitt (Calibrated Instruments Inc., Ardsley, New York). appeared intact. Septa from the trama had a simple Digital micrographs were processed in Adobe PhotoshopH pore with swollen margins approximately 230 nm CS2 (Adobe Systems Inc., San Jose, California) with the Smart Sharpen filter at the default setting. high and the plasma membrane was continuous through the pore (FIGS. 1, 4). Pores were approxi- Electron microscopy.—Sporocarps of Auriscalpium vulgare, mately 130 nm wide and appeared unoccluded. No DJM 225 (Mycological Culture Collection and Herbarium, large organelles were observed in the pore or on the University of Minnesota) were produced axenically on Difco adseptal side of the septal pore cap, although cornmeal agar and Pseudotsuga menziesii cones under cool ribosomes sometimes were present (FIG. 4). Bell- white fluorescent light with a 12 h day at 20 C. Cultures shaped septal pore caps were present on both sides were grown in Pyrex deep storage dishes (100 mm diam, of the septum (FIGS. 1, 4) and each had circular 80 mm high) containing 50 mL of half-strength cornmeal agar (8.5 g/L) and a P. menziesii cone separately autoclaved perforations approximately 70 nm diam uniformly distributed throughout the rounded portion of the in distilled H2O. The dish was tilted at approximately 15u while the agar solidified to provide a reservoir for water cap (100–120 nm between the centers of adjacent addition during sporocarp formation. pores, FIG. 2). Perforations were present in the area Two to three spines attached to pileal tissue were excised of the cap adjacent to the septal wall but their size and from mature sporocarps from colonies approximately 60 d distribution were not determined. The pore caps old and plunged into 2178 to 2185 C liquid propane contained a central electron-opaque layer about 8 nm (Hoch 1986). Specimens were transferred to substitution thick between two less electron-opaque layers. This fluid consisting of 2% osmium tetroxide and 0.1% uranyl layering was present in the portion of the cap that acetate in 100% anhydrous acetone at 280 C, and stored at extended along the cross wall on both sides of the 280 C at least 48 h. Samples gradually were brought to pore (FIGS. 1–4). The length of the cap that extended room temperature (220 C, 2 h; 0 C, 2 h; room tempera- along the cross wall varied between septa as well as on ture, 1 h), then rinsed three times with 100% HPLC-grade acetone with 1% acidified 2, 2–dimethoxypropane.