Nuclear staining

Nuclei of basidiospores, hyphae, and conidia were stained directly with DAPI (4′,6-diamidino-2-phenylindole in an antifade solution, Vector Laboratories, California, USA). Basidiospores were collected from the fruiting body T0984. Hyphae and conidia were obtained from culturing mycelia of FB, BSs, and paired BSs (Table 1) in 250 mL Erlenmeyer flasks, each of which contained a 100 mL liquid medium consisting of 3 g/L Bacto beef extract, 5 g/L Bacto peptone, and 15 g/L d-glucose, and incubated without shaking at 25 °C for 3 week. The stained fungal components were examined by fluorescence microscopy with a Zeiss LSM510 Meta confocal microscope equipped with C-Apochromat 63 ×/1.2 W corr objective. Two channels were used: DAPI channel (specifications: Two Photo Laser Excitation: 740 nm; Transmission 1.5%; Main Beam Splitter: KP650; Beam Splitter 2: mirror; BP 390–415IR; Detector Gain: 650; Amplifier Offset: −0.1) and DIC channel (specifications: Two Photo Laser Excitation: 740 nm; Detector Gain: 130; Amplifier Offset: −0.1).

Hyphae and conidia were also subjected to HCl-Giemsa stains (Giemsa’s azur eosin methylene blue solution, Merck KGaA, Darmstadt, Germany) in accordance with published protocols (Knox-Davies and Dickson 1960; Ward and Ciurysek 1962) with slight modification and is summarized as follows. A harvested mycelium was washed with MQ water, fixed in a freshly prepared Farmer’s fluid (95% ethanol: glacial acetic acid = 3:1) for 1 h, and successively washed in 95 and 70% ethanol, and immersed in MQ water for 10 min. It was then placed in 1 N HCl at room temperature for 10 min, hydrolyzed in 1 N HCl at 60 °C for 8 min, washed in MQ water with 5 changes, suspended in phosphate buffer (0.2% KH₂PO₄, 0.4% Na₂HPO₄·7H₂O, pH 7.2) for 5 min, and stained in HCl-Giemsa for 15 min or longer if required. The stained mycelium was washed thoroughly with MQ water and phosphate buffer in series, and was mounted in MQ water. The stained fungal components were examined by light microscopy with a Leica DMRB microscope equipped with a PL Fluotar 100 ×/1.30 oil objective.

Slide culture technique was also used with small blocks (ca. 0.5 × 0.5 cm) of Difco oatmeal agar (OA) (BD, Sparks, Maryland, USA), each of which was inoculated with a culture, sandwiched between a cover slip and a microscope slide, and grown for 2 week. The hyphal mats and conidia that attached on the cover slip were then stained with DAPI and HCl-Giemsa as described above.