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Home , Agaricomycetes, Arthuriomyces, Artomyces, Artomyces pyxidatus, Auriscalpium, Auriscalpium vulgare

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 vulgare

Gail J. Celio1 fungal , 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 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 (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 () 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 exclusion surrounded the septal pore apparatus and nuclear division, including . Metaphase I of and metaphase of pole body forms and cycles, among the first to be 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 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 (Agaricomyco- additional signal in phylogenetic analyses. tina), a that fruits in the laboratory. This Key words: , , cytology, is represented in the AFTOL Molecular Database informatics, phylogeny, (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 and structures nonmedian sections prevented data entry for many have been observed with the electron microscope septal characters. In the Russulales only since the late 1950s (Girbardt 1958, Shatkin and coralloides (Flegler et al 1976) and pyxidatus Tatum 1959) and have been helpful in developing (5 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 . 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 .—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 had a simple Digital micrographs were processed in Adobe PhotoshopH pore with swollen margins approximately 230 nm CS2 (Adobe Systems Inc., San Jose, ) 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 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. The either side of a septum (FIG.4,SUPPLEMENTARY FIGS. specimens were placed in plastic mesh baskets in a poly- 1–3). Some caps terminated at the base of the septal styrene lid and covered with an infiltration pore swelling but other caps spread out up to 360 nm solution made up of equal parts 100% Quetol 651 and past the septal pore swelling (FIGS. 2–4). Although hardeners (nonenyl sussinic anhydride and nadic methyl septa in clamp connections may be narrower than 646 MYCOLOGIA

those of main hyphae, pore caps at these septa were observed extending to the clamp hyphal wall (FIGS.2, 3). In some sections the area between the cap and septal pore appeared more electron-transparent than the abseptal area of the cap (FIG.4,SUPPLEMENTARY FIGS. 1–3). A zone of organelle exclusion 50–530 nm thick was observed on the abseptal side of the pore cap and had a textured appearance (FIG. 1).

Nuclear division.—Interphase nuclei observed with transmission electron microscopy (TEM) (n 5 6) in dikaryotic basidia of A. vulgare were spherical (1.8– 2.3 mm diam) and the two nuclei usually were oriented one above the other along the long axis of the cell (FIGS. 5, 11). Nucleoli were electron opaque with a granular texture. Small electron-opaque patches presumed to be condensed chromatin were visible in some sections (FIG. 11). The size of the fusion nucleus observed with TEM (n 5 4) increased to 2.6–3.8 mmdiam(FIG.6,SUPPLEMENTARY FIG.4) and synaptonemal complexes were present. SPB were not observed in interphase or prophase. In metaphase I (n 5 3) the spindle was positioned at an angle oblique to perpendicular to the long axis of the cell. Condensed chromatin surrounded the central spindle and often was distributed symmetri- cally (FIGS. 7, 12, 13, SUPPLEMENTARY FIG. 5), although it sometimes appeared in greater volume on one side. The one complete spindle observed was 1.7 mm long (not illustrated). Globose to ovoid SPB (n 5 4) were 140–240 nm h 3 210–350 nm w and had an electron- opaque narrow ovoid area (70–140 nm h 3 160– 240 nm w) in the center. This area usually was oriented with its long axis oblique to perpendicular to the spindle and was surrounded by a more electron-transparent layer (FIG. 12). Each SPB was set in a loose fenestra at opposite ends of the nucleus

r

both sides of the pore. A zone of organelle exclusion (arrowheads) is present on the abseptal side of the pore caps. 2. Transverse section of a septum from a . The pore cap extends along the lateral hyphal wall and has evenly spaced perforations in the central area. Perforations also occur throughout the pore cap adjacent to the septum wall. Arrows delimit the extent of the internal layering of the pore cap. 3. Nonmedian section of a septum and pore cap from a clamp connection. Arrow and rectangle delimit the extent of the internal layering of the pore cap. Inset: internal layering of the pore cap along the lateral wall. 4. Section of the septal pore cap demonstrating FIGS. 1–4. Transmission electron micrographs of septa the extension of the cap along the hyphal cross wall. Note from the hymenophore trama of Auriscalpium vulgare.1. the absence of a pore occlusion. Arrows delimit the extent Nonmedian section of a hyphal septum with bell-shaped of the internal layering of the pore cap. Bars: 1 5 0.5 mm, 2– perforated pore caps that extend along the septum wall on 4 5 0.25 mm. CELIO ET AL:ULTRASTRUCTURE OF AURISCALPIUM VULGARE 647

FIGS. 5–13. Micrographs of longitudinal sections of basidia from the layer of Auriscalpium vulgare. 5–10. Light micrographs. 11–13. Transmission electron micrographs. 5. Paired prefusion nuclei with nucleoli (Nu). 6. Postfusion nucleus with nucleolus (Nu). 7. Metaphase I nucleus with a narrow area clear of chromatin presumed to be the spindle (S) traversing the nucleus and terminating at a probable spindle pole body (SPB). 8. Metaphase II nuclei. Condensed chromatin in each nucleus surrounds a clear core presumed to be the spindle, suggesting a chiastic second division. 9. Interphase II nuclei congregating subapically in the basidium. 10. Basidium with sterigmata and developing basidiospores just out of the plane of section. A nucleus (N) migrating into the sterigmata. 11. Two prefusion interphase nuclei, each with granular nucleolus. White arrows indicate patches of condensed chromatin. 12–13. Four and six of six serial sections of a metaphase I nucleus. The central spindle is perpendicular to the long axis of the cell and connects directly to the SPB. The globular SPB has an electron- opaque area (white arrowhead) surrounded by a more electron-transparent layer. Fragments of endoplasmic reticulum (ER) are visible near the pole. Arrows delimit loose polar fenestrae. Arrowheads indicate other gaps in the nuclear envelope. Note the astral microtubules (AM) extending into the cytoplasm and the nonkinetochore microtubules (NM) crossing the nucleus to emerge through the opposite polar fenestra. A kinetochore (K) is visible. NE 5 nuclear envelope. Bars: 5–10 5 2.5 mm; 115 1 mm; 12 5 0.5 mm, and same bar applies to 13. 648 MYCOLOGIA

FIGS. 14–19. Transmission electron micrographs of longitudinal sections of basidia and sections of a basidiospore of Auriscalpium vulgare. 14. Meta-anaphase I nucleus with spindle pole bodies not in plane of section. Spindle length has increased compared to metaphase I, and the nuclear envelope (NE) is more discontinuous. Arrows delimit margins of polar fenestrae. Arrowheads indicate other gaps in the nuclear envelope. 15–16. Skipped serial sections of telophase I nuclei with the CELIO ET AL:ULTRASTRUCTURE OF AURISCALPIUM VULGARE 649 and was attached to astral microtubules (MT) and daughter nuclei were observed congregated subapi- kinetochore and nonkinetochore spindle MT cally, each having distinct nucleoli (FIGS.9,17). (FIG. 13). The connections between kinetochores Nuclei subsequently separated and could be seen and MT were difficult to observe; they did not appear migrating into the sterigmata (FIG. 10). differentiated although they often were found at the Mitotic metaphase (n 5 2) observed in basidio- terminal end of MT that expanded slightly. Astral MT appeared similar to metaphase I. We also projected away from the dividing nucleus into the observed what was likely mitotic metaphase in one cytoplasm. Some nonkinetochore MT traversed the basidium with two nuclei that were dividing during condensed chromatin, emerging through the nuclear migration into the developing spores (SUPPLEMENTARY envelope at the opposite fenestra. Except at the poles FIGS. 6, 7). Globoid SPB (140–210 nm high 3 210– the nuclear envelope usually remained intact with 310 nm wide) (n 5 6) had a central ovoid electron- occasional gaps. However the nuclear envelope opaque area (70–120 nm high 3 90–190 nm wide). sometimes appeared absent adjacent to the cell wall SPB were located in polar fenestrae and the nuclear (SUPPLEMENTARY FIG. 5). Fragments of endoplasmic envelope had occasional gaps but was mostly contin- reticulum (ER) were visible near the SPB. A meta- uous (FIGS. 18, 19). Spindles (n 5 3) were 1.6–1.8 mm phase plate was absent. long and chromatin was distributed both symmetri- The transition from metaphase I to anaphase I was cally and asymmetrically around the central spindle. difficult to distinguish. We observed nuclei (n 5 2) As in metaphase I, kinetochore and nonkinetochore with increased spindle length (2.7 mm, n 5 1) spindle MT and astral MT were present. Fragments of compared to that of metaphase I but it was unclear ER were present near the poles of the nucleus but whether the chromatin had started to migrate toward a SPB cap was absent. Neither a metaphase plate nor the poles. The nuclear envelope displayed wider and the nucleolus was observed. more frequent gaps, and the amount of ER increased both at the poles and around the nucleus (FIG. 14). DISCUSSION Nucleoli were not observed during metaphase I and meta-anaphase I. Septum and septal pore characters.—Septa of Auriscal- A telophase I basidium had daughter nuclei on pium vulgare had curved perforated septal pore caps opposite sides of the cell (FIGS. 15, 16). Their nuclear similar to those reported for species in the Russulales, envelopes were reformed almost completely except , , and many species for the area around the SPB and a remnant of the in the and (see Hibbett degrading spindle. One ovoid SPB (190 nm high 3 and Thorn 2001). Except for 300 nm wide) with an internal electron-opaque area (Flegler et al 1976) and (M 120 nm high 3 210 nm wide appeared adjacent to Berbee unpubl) detailed comparisons with other taxa the cell wall and was estimated to be 3.6 mm from the in the Russulales is not possible because of the other SPB. incompleteness of the septal data. The diameter of Events from the second stage of nuclear division pore cap perforations in A. vulgare appears to be rarely were seen. One possible metaphase II basidium greater than those reported for select species from was observed with the light microscope (FIG. 8) and the Phallomycetidae, Polyporales, Agaricales and showed two areas of condensed chromatin, each with Boletales and closer in size to the perforations of a central chromatin-free area. These areas were fuciformis (5 fuciforme) (Patton interpreted to be spindles oriented parallel to each and Marchant 1978, Orlovich and Ashford 1994). other and perpendicular to the long axis of the cell However stercorea (5 stercorar- suggesting a chiastic division. At interphase II the four ius) and commune in the Agaricales

r nuclear envelope almost completely reformed except around the spindle pole body (SPB) and remnant spindle microtubules (MT). SPB contains electron-opaque area. 17. Interphase II nuclei congregated subapically in the basidium. 18–19. Three and five of eight serial sections of a mitotic metaphase nucleus. The globular SPB contains an electron-opaque area (white arrowhead) and is set within a loose polar fenestra (delimited by arrows). The nuclear envelope is mostly continuous with occasional gaps (black arrowheads). Cytoplasm contains glycogen (G) and lipid droplets (L). A kinetochore microtubule with an expanded end (K). AM 5 astral microtubules, ER 5 endoplasmic reticulum, N 5 nucleus, NM 5 nonkinetochore microtubule, S 5 spindle, SP 5 spindle pole. Bars: 14–16, 18, 19 5 0.5 mm; 17 5 1 mm. 650 MYCOLOGIA have pore cap perforations similar in size to those nuclear division in the Russulales (Berbee and Wells observed in this study (Ellis et al 1972, Mu¨ller et al 1989) was possible and desirable. Microtubules and 1998). Some of these studies used chemical fixation the nuclear envelope were well preserved. Reports (CF) while others employed FS. Tangential sections differ on improved nuclear envelope fixation with FS of pore caps preserved by both methods that display compared to CF (see O’Donnell 1994). SPB structure measurable perforation diameters are rarely pre- appeared somewhat diffuse. Chemically fixed SPB sented for comparison, so it is not clear whether the may display better internal definition than those diameter of pore cap perforations varies with fixation undergoing FS (Lu¨ and McLaughlin 1994, Frieders method. and McLaughlin 1996). Many septal pore caps contain internal layers and The basic shape of the SPB of A. vulgare was are continuous with the ER adjacent to the septal wall. globular, corresponding to those observed in the The transition between layered pore cap and un- Agaricomycotina (Celio et al 2006). The stages of layered ER occurs at the base of the septal swelling meiosis I reported here share similarities with A. (Bracker and Butler 1964, Ellis et al 1972, Mu¨ller et al pyxidatus (Russulales) (Berbee and Wells 1989). Both 1998). Pore caps of A. vulgare displayed internal taxa have globular SPB during meiosis I and in A. layering that extended along the cross wall identical vulgare a SPB often contained an electron-opaque to that seen but undescribed in chemically fixed A. area whose long axis was oblique to perpendicular to vulgare (Keller 1997). Limited observations in the the spindle. This area described as an inclusion in A. phylogenetically related Artomyces pyxidatus do not pyxidatus also was observed at angles ranging from show such layering along the cross wall (M Berbee perpendicular to parallel to the spindle but did not unpubl). appear in the monoglobular SPB of meiosis II and Extended internal layering in A. vulgare was more mitosis until late telophase. Berbee and Wells (1989) pronounced in pore caps in clamp connections than compare the inclusion to the electron-opaque codisk in main hyphae and more likely was to be found in found in Puccinia malvacearum () the hymenophore trama than in the subhymenium. (O’Donnell and McLaughlin 1981). The codisk in P. This variation may indicate tissue and/or cell - malvacearum is generated within the disk-shaped SPB specific pore cap differentiation. Keller (1997) pre- during meiosis I; at prometaphase II it is presumed to sents only one micrograph of the septal pore cap in A. fuse with the SPB, which subsequently splits into two vulgare and its cell type is not mentioned. Also septal SPB. Berbee and Wells (1989) suggest that the pore occlusions were absent from the hymenophore inclusion in A. pyxidatus could be the half middle hyphae we examined in A. vulgare. Flegler et al piece that joins the duplicated SPB during prophase (1976) observed differences in septal pore occlusions andthatitishomologoustothecodiskinP. between vegetative hyphae and sporocarp tissue in malvacearum. various Basidiomycota including Hericium coralloides The spindle pole body cap is a membranous (Russulales); occlusions were imperforate in vegeta- structure found on the cytoplasmic side of the SPB tive hyphae and perforate or absent in the hymenium in many Basidiomycota. Among members of the and lamellae. phylum the cap may be continuous with the nuclear A zone of organelle exclusion was observed on the envelope with or without perforations or distinct from abseptal side of pore caps in the trama of A. vulgare. the nuclear envelope. Membrane fragments also may Similar zones have been reported so far only for be present near the SPB that together do not form subhymenial and basal septa in basidia of the a distinct cap. SPB caps that are continuous with the Agaricales (Thielke 1972, Craig et al 1977, McLaugh- nuclear envelope have been reported for those lin 1974) and Boletales (McLaughlin 1982) and may in members of the Boletales (Yoon be a synapomorphy for the group containing these and McLaughlin 1987), Agaricales (Lerbs 1971, Raju clades. McLaughlin (1982) associates such outer and Lu 1973, Wells 1978), and Polyporales (Girbardt zones with basidiocarp tissue, although their presence 1968). In contrast species in the (Lu¨ may depend on developmental stage and type of cell and McLaughlin 1994), (Taylor 1985), within the basidiocarp (Gull 1976). (Setliff et al 1974) and Russulales (Berbee and Wells 1989) lack SPB caps. Mapping the Nuclear division.—The nuclear division and spindle SPB cap character states onto an abbreviated clado- pole body data presented here are not intended to be gram based on molecular data (Matheny et al 2007) a comprehensive study. Our original goal was to demonstrates the potential phylogenetic signal of this examine the septal pore apparatus and cystidia, but ultrastructural character (FIG. 20). James et al (2006) preservation of basidia by FS exceeded our expecta- and Matheny et al (2007) reported weak to moderate tions and a comparison with the single study of support for the branch leading to a group containing CELIO ET AL:ULTRASTRUCTURE OF AURISCALPIUM VULGARE 651

FIG. 20. Cladograms of selected groups of Agaricomycetes. Cladograms based on current molecular phylogenetic hypotheses (Matheny et al 2007) illustrating variation of spindle pole organization at metaphase-anaphase. Taxa in bold have adjacent corresponding illustrations. Illustrations from top to bottom interpreted from araneosa (Cantharellales, Taylor 1985), auricula-judae (Auriculariales, Lu¨ and McLaughlin 1994), versicolor (Polyporales, Girbardt 1968), latemarginatus (Hymenochaetales, Setliff et al 1974), Auriscalpium vulgare (Russulales, this paper), (Agaricales, Lerbs 1971), rubinellus (Boletales, Yoon and McLaughlin 1987). Alternative topology on the right side beginning at branch with asterisk is suggested by spindle pole organization. Bar 5 0.5 mm. the Russulales, Agaricales, Atheliales and Boletales, metaphase I and mitotic metaphase. At these stages while other multigene analyses have resulted in the SPB was located within a loose fenestra of the slightly different topologies but also with weak nuclear envelope at the spindle pole while those support (Binder and Hibbett 2002, Lutzoni et al portions of the envelope surrounding the spindle 2004). The existing nuclear division data, represent- and condensed chromatin were mainly intact with ing a small portion of the Agaricomycotina, either minor discontinuities. This character state often suggest that the SPB cap is a highly homoplastic supports the Agaricomycetes (Celio et al 2006) and character or support an alternative, more parsimoni- is present in Russulales (Berbee and Wells 1989), ous topology. Specifically if the Russulales and Hymenochaetales (Setliff et al 1974), Polyporales Hymenochaetales share a most recent common (Girbardt 1968) and Cantharellales (Taylor 1985) as ancestor, then the most parsimonious explanation well as in the (Taylor and Wells would be a single gain of the SPB cap on the branch 1979). leading to the group containing the Polyporales Nucleoli could be seen in premeiotic fusion nuclei through Boletales and one reversal on the branch but were not observed in A. vulgare during meiotic leading to the Russulales and Hymenochaetales metaphase I and meta-anaphase I, or in mitotic (FIG. 20). However it is critical that the presence or metaphase. Berbee and Wells (1989) reported a nu- absence of a SPB cap in the Thelephorales be known cleolus during meiotic prophase I that was not before we can have confidence in this alternative evident during meta-anaphase I and II and that phylogenetic hypothesis. reappeared at telophase I and II; prophase II was not The nuclear envelope was easily discernable in A. observed. Whether the nucleolus is discarded wholly vulgare after FS, letting us determine its relative or dispersed in A. vulgare is uncertain. Data regarding position with respect to the SPB during meiotic nucleolus behavior within the Agaricomycotina is 652 MYCOLOGIA scarce, although some studies describe the disappear- However the success encountered with FS of A. ance of the nucleolus between prophase and meta- vulgare indicates that future acquisition of the phase (e.g. terrestris, Agaricales; Wells 1978) remaining data is feasible. or after metaphase (e.g. Chalciporus rubinellus, Ultrastructural characters can provide significant Boletales; Yoon and McLaughlin 1987). data for phylogenetic analyses at various taxonomic Spindle orientation in A. vulgare appears to be levels and have the potential to strengthen weakly chiastic during meiosis I and II. This pattern has been supported relationships determined with molecular observed in most Agaricomycotina studied, including data (FIG. 20; Swann et al 1999, Lutzoni et al 2004). A. pyxidatus (Berbee and Wells 1989), while both As taxon sampling widens the SB Database will adapt chiastic and stichic division are reported in many to new data and hypotheses to provide an up-to-date members of the Cantharellales, Auriculariales and and flexible resource. Continued analyses performed Dacrymycetales (see Hibbett and Thorn 2001). with both types of data will facilitate more compar- isons within and among related clades, resulting in Structural and Biochemical (SB) Database.—It pres- a better understanding of the evolutionary history of ently contains 18 characters for the septal pore the Fungi. apparatus in nonbasidial cells and nonascogenous /ascus (Celio et al 2006). The 16 nuclear division characters, four of which address the SPB, ACKNOWLEDGMENTS include many of the characters and states developed We thank D Hibbett and PB Matheny for reviewing the by Heath (1980, 1986) with modifications specific to manuscript and M Berbee for providing micrographs of the Fungi. Characters address both the general form Artomyces pyxidatus. This research was financed by the and detailed features of some structures because Assembling the Fungal Tree of Life project, NSF grant EF- different characters may be phylogenetically informa- 0228671 to DJ McLaughlin. tive at varying taxonomic levels. This range of specificity allows for greater customization of data LITERATURE CITED for phylogenetic analyses. Finalizing characters and states involved consideration of the homology of Abad AR, Cease KR, Blanchette RA. 1988. A rapid technique various structures, such as the numerous forms of using epoxy resin Quetol 651 to prepare woody plant tissues for ultrastructural study. Can J Bot 66:677–682. septal pore occlusions, and the developmental stage Beckett A. 1981. The ultrastructure of septal pores and and type of cell from which data were taken (e.g. the associated structures in the ascogenous hyphae and asci transition of the septal pore occlusion from immature of Sordaria humana. Protoplasma 107:127–147. to mature ascogenous hypha/ascus of Sordaria Berbee ML, Wells K. 1988. Ultrastructural studies of mitosis humana) (Beckett 1981). and the septal pore apparatus in globospora. The ultrastructure data collected from A. vulgare Mycologia 80:479–492. provided character states for many of the characters ———, ———. 1989. 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