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Morphological Analyses of Basidiomata
Extensive collecting was undertaken throughout the United States and parts of eastern and western Canada. The following states and provinces were sampled: British Columbia, California, Colorado, Florida, Georgia, Idaho, Illinois, Kentucky, Louisiana, Massachusetts, Michigan, Minnesota, Mississippi, New York, North Carolina, Nova Scotia, Ohio, Ontario, Oregon, South Carolina, Tennessee, Texas, Virginia, West Virginia, Washington, Wisconsin, and Wyoming. Field work was also undertaken in Sweden, Mexico, Costa Rica, and much of South America as part of an ongoing project to produce a world monograph of the genus and to obtain comparative material. Dried specimens on loan from numerous herbaria including most extant type specimens were also examined.
Collections were made and assembled using standard techniques (Smith, 1949; Largent, 1977). Descriptive terms were taken from Snell and Dick (1971) and Largent (1977). Unless otherwise noted, color names within parentheses and quotation marks are from Ridgway (1912), colors from Kornerup and Wanscher (1978) are listed by (page-column-row) while color names outside of parentheses are author-generated. Color names followed by M&P; were taken from Maerz and Paul (1930).
Basidiomata were preserved by warm-air drying and deposited in either F, TENN, UPS or WTU (Holmgren et al., 1981). Most examinations were made directly using either a Nikon Model S-Kt research microscope, a Zeiss Universal photomicroscope, or a Zeiss RA standard microscope equipped with both bright field and phase contrast optics. Recent data acquisition and remeasurements were made using JAVA 1.31 (Jandel Video Analysis Software, 1989) running on an AT&T; 6386 computer from images captured through an Olympus BH-2 microscope with Nomarski differential interference optics. Illustrations of micromorphological characters were made with the aid of a drawing tube. All measurements were taken on material mounted in 3% KOH. Iodine reactions were determined in Melzer's reagent and the cyanophilic reaction was determined in cotton blue (Kotlaba and Pouzar, 1964; Singer, 1972; Largent et al., 1977). Descriptive terminology was taken from Snell and Dick (1971) and Largent et al. (1977).
Micromorphological data used in taxon descriptions were based on a complete, detailed examination of at least five (when possible) representative collections per taxon. The number of collections examined was dependent upon the availability of material and the amount of variability encountered within the taxon. All specimens listed in Specimens Examined (Appendix A) were examined, and any deviations from the norm were noted. Measurements and observations were taken from several basidiomata per collection to check for uniformity. At least 10 randomly sampled cheilocystidia, pleurocystidia, and terminal cells of cuticular hyphae, and 15 randomly sampled basidia were measured per collection. Width and diameter measurements of these elements were taken at the widest point and rounded to the nearest 0.5 µm. Arrangement of hyphae comprising the pileipellis was observed both in radial and scalp sections.
All basidiospore measurements were taken from hymenial tissue and not from spore prints to treat all specimens equally. The number of basidiospores measured and the number of collections examined for calculating mean size (= ) and length/width ratio (= Q) were included in brackets with basidiospore size data to give some indication of reliability of these data (Bas, 1974). When available, data on basidiospores from additional collections were included in basidiospore size range data. Ranges of collection means (, ) for basidiospore data, rather than overall mean values for the taxon, are provided to give a better indication of intraspecific variation. Basidiospore size data are always given without ornamentation and hilar appendix, with the hilar appendix in profile. Means and other descriptive statistics were obtained using SAS for Personal Computers version 6.03 (SAS, 1985).
Scanning Electron Microscope Analyses
Lamellar fragments from air-dried collections were rehydrated in an acetone series, fixed in 2.5% glutaraldehyde in phosphate buffer for 3-4 hrs at 4°C, dehydrated in an acetone series and critical point dried in a Balzers CPD 030 apparatus with CO2 as the transition fluid (Cheeseman and Grund, 1985). Samples were then attached to aluminum mounts with double-stick tape and coated with gold in a Denton Vacuum Desk II sputter coater. Basidiospores were examined and micrographs were taken at 20 kV with an Amray 1810 scanning electron microscope.
Somatic Culture Mat Analyses
The following procedure was employed to obtain heterokaryotic tissue cultures of Laccaria. Small pieces of tramal tissue excised from the pileus-stipe interface were aseptically placed on modified Melin Norkrans Medium (=MMN) plus benomyl (10 mg/1) in disposable test tubes (Marx, 1969; Molina and Palmer, 1982; Mueller, 1984). The benomyl was added to reduce ascomycetous contamination. Six to ten replicates were taken for each collection utilized. Subculturing of each resulting isolate was undertaken until a pure culture was obtained. Stock isolates were then transferred to tubes containing modified MMN or N6:5 (Fries, 1983a) and stored in the dark at 2-4° C. Voucher herbarium material of all specimens used for tissue cultures were described and deposited in F, TENN, UPS and WTU. All isolates are housed in the mycological culture collection at Field Museum of Natural History.
Culture mat analyses were based on the classic work of Nobles (1948, 1965). Five millimeter round plugs of agar containing hyphal tips taken from the advancing zone of each of 2-week-old "stock" plates were transferred to the edge (mycelium side down) of a Petri plate containing 15-20 ml of either MMN, Malt Extract Agar (= MEA), or Potato Dextrose Agar (= PDA). Seven replicates of each medium for each isolate were inoculated and placed in a dark incubator at 24°C. Macromorphological descriptions were taken during the third and the sixth weeks. Photographs of representative isolates were taken during the fourth week, and micromorphological characters were examined during the sixth week.
Macromorphological characters noted included: (a) radius of the culture mat, (b) form and character of the advancing zone, (c) mat color and topography, and (d) the presence or absence of exudates. Terminology used was taken from Nobles (1948, 1958b, 1965).
Micromorphological characters were observed by mounting hyphae from the advancing zone, mat, and plug of each isolate in 3% KOH and examining the slide under phase contrast. Micromorphological characters examined included the presence or absence of: (a) modified hyphae, (b) chlamydospores or oidia, etc., and (c) basidiomata or hymenial structures such as basidia or cystidia along the mat surface. Descriptive terminology used was that of Nobles (1948, 1965).
Extracellular oxidase activity of each isolate was tested using both the Bavendamm (Davidson et al., 1938, 1942) and gum guaic (Nobles, 1958a, 1965) tests (Mueller, 1984).
Intra- and Intercollection Pairing Analyses
Homokaryotic (single basidiospore) and heterokaryotic isolates derived from multiple-germinated basidiospores were obtained following the techniques of Fries (1983a, 1983b) and Fries and Mueller (1984). Homokaryotic isolates originating from one basidioma were assigned a stock number. Within a stock, each isolate received a unique extension number. All isolates are stored at 2-4°C on N6:5 medium in the mycological culture collection at Field Museum of Natural History.
Intra- and interstock pairing analyses were carried out following the procedures outlined in Mueller and Gardes (1991). Isolates to be tested were placed * 10 mm apart on N6:5 plates and allowed to grow together (2-4 wks). After an additional one week or more, plugs of tissue were cut from the interface and placed on fresh N6:5 plates. Mycelium growing from these plates were checked for the presence or absence of clamp connections by examining them through the bottom of the inverted Petri plate at 200 x magnification. Pairings that resulted in hyphae that bore clamp connections were considered positive while those that did not yield clamped hyphae were scored as negative. Two or more testers for each stock, each containing different mating type alleles, were used, when possible. As in Mueller and Gardes (1991) and Mueller (1991c), the terms noncompatible and intracompatible are restricted to intrastock pairings. I used the terms intersterile for intercollection pairings that did not form clamp connections and intercompatible (rather than interfertile) for positive intercollection matings since data regarding fruiting and progeny analysis were not available.