There are not always the canonical four sterigmata per basidium observed in basidiomycete fungi but the frequency of variation from the 4-sterigmate basidium is rarely noted. While documenting the micromorphological characteristics of 25 species of Amanita, we surveyed basidia for the number of sterigmata each one had. The data summarize length and width measurements taken for 447 basidia from 39 mushrooms representing 25 species of Amanita. All basidia plus an additional 327 were also observed for sterigmata count. Sterigmata length was also measured for each of these basidia. We discovered deviations from the textbook number of four basidiospores are common. Only 10 mushrooms of 10 species housed entirely tetrasporic basidia, while the remaining mushrooms grew variable proportions of one to three-spored basidia. In 15 mushrooms of 12 species with at least 10 observed basidia each, noncanonical basidia made up more than 25% of each mushroom’s basidia.

Of 144 Amanita mushrooms collected from Wisconsin and Michigan’s Upper Peninsula, we chose 39 mushrooms representing 25 species for microscopy. Mushrooms were selected strategically to gather data from across the genus. All seven collected sections (sect. Roanokenses, sect. Amidella, sect. Validae, sect. Phalloideae, sect. Amanita, sect. Vaginatae, and sect. Caesareae) were represented by at least one mushroom. We did not find any mushrooms from sections Lepidella, Arenariae, Strobiliformes, or Amarrendiae. Small pieces of tissue (~3-8mm long) were broken off from the gills of each dried mushroom with fine-tipped tweezers, placed on a new Corning Incorporated (New York) glass microscope slide (2948-75x25), and rehydrated for one minute with a 3% potassium hydroxide (KOH) solution (three grams of KOH dissolved in 100mL of water). Once hydrated, lamellar tissues were cut longitudinally by hand into thin sections using a straight razor blade under a Meiji Techno® (Japan) dissecting microscope (model: EMZ-5). Razors were discarded after roughly 10 uses to ensure clean sectioning. Between three to six sections were subsequently transferred using two small sewing needles to a new drop of 3% KOH on a clean microscope slide. A coverslip was placed over the sections. To stain the sections, a drop of Congo Red solution (10mL of 1% sodium dodecyl sulphate solution, 0.5g congo red) (Emmett 2003) was placed on the edge of the coverslip and a KimTech Kimwipe® was dabbed on the opposite edge of the coverslip to pull the Congo Red stain through the sectioned material. Air bubbles were removed by gently pressing the coverslip with the eraser side of a pencil. 

Lamellar tissue sections were first observed at 100x magnification using a Zeiss® (Germany) Primostar3 light microscope to evaluate basidial density on each section. Afterwards, individual basidia were observed using a 100x Plan-ACHROMAT oil immersion objective with a numerical aperture of 1.25 and imaged with the microscope’s built-in digital camera. Images were taken with Zeiss® (Germany) Labscope microscope software (v4.6.1) on an Apple® (California) 10th generation iPad (model: A2696) with both the auto exposure and auto white-balance settings turned on. Sections of tissue were observed one-by-one from left to right to avoid recounting already recorded basidia. Only mature basidia with clearly visible sterigmata were examined. For each mushroom, we deemed it necessary to observe at least one basidia with spores connected to the sterigmata to confirm each sterigma grew one basidiospore (not more or fewer). If fewer than ten basidia were recorded for one slide, a new slide was prepared with new dried lamellar tissue from the original mushroom. If fewer than 5 basidia were observed from two slides, we excluded the mushroom from further study. Between five and 25 (mean: 19) basidia were recorded for each mushroom.

We took great care to image basidia thoroughly. The depth of field on our light microscope at 1000x magnification is roughly 0.7 µm, but the average width of our measured basidia was 12.5 µm. To account for this discrepancy, we focused through a single basidium using the fine focus knob of the microscope, moving from the part closest to the lens to the part furthest away. By doing so we were able to thoroughly view all aspects of the basidium and confirm no hidden sterigmata or spores. If a basidium presented any ambiguity (if we could not cleanly count the sterigmata for any reason), we did not include the basidium in our data and instead moved on to count another basidium. Ambiguities included ripped sterigmata, layers of cells from other parts of the tissue section found behind the sterigmata, or overlapping sterigmata that weren’t readily differentiated using light microscopy.