Field sampling and isolation
We rediscovered A. vaillantii sensu lato from a Shimin no Mori. Additional five fresh samples were also collected from B. japonica in similar ecological environments with sunny but humid grasslands and a water source around the Kanto area of Japan, which were suspected to be A. vaillantii sensu lato. Seven samples were collected from the six study areas in total, and six single teliospore strains were established (Table 1).
Phylogenetic Analyses
ML and Bayesian phylogenetic analyses were conducted using an aligned sequence dataset composed of 870 nucleotides from ITS and 565 from LSU. The alignment contained a total of 38 taxa, which consisted of 38 taxa (100%) in ITS and 37 (97.3%) in LSU (Table 1, 2). Of the 1435 characters included in the alignment, 633 were variable while 672 were conserved. The ML tree with the highest log likelihood (–7326.7656) is shown in Fig. 1. The topology recovered by the Bayesian analysis did not show any topological conflicts with the significant support (≥ 70% ML BP). The six isolates of A. barnardiae formed a robust clade (99% ML BP/1.00 Bayesian PP) and were grouped with Antherospora spp. with strongly supported values (100% ML BP/1.00 Bayesian PP, Fig. 1).
Table 2
List of Antherospora spp. with teliospore size and wall thickness, color, host plant species, country and references.
Species | Teliospore size | Teliospore wall | Teliospore color | Host plant species (Subfamily) | Country | References |
A. albucae | 9.5–22.5 × 7–14.5 µm | ca. 0.5 µm | Yellowish brown | Albuca sp. (Ornithogaloideae) | Rwanda | Bauer et al. (2008) |
A. barnardiae | 7.9–12 × (5–)6–8.5 µm (av. 9.7 × 7.2 µm, n = 30) | 0.6–1.1 µm thick | Yellowish brown to olivaceous-brown | Barnardia japonica (Hyacintheae) | Japan | This study |
A. eucomis | 5.5–8(–9) × 7–12(–13.5) µm | ca. 0.5 µm | Yellowish brown | Eucomis punctata (Massonieae) | South Africa | Vánky (2009) |
A. hortensis | (5.5–)6–10.5(–13.0) × 5.0–8.5(–9.5) µm | ca. 0.5–0.7 µm | Olive-brown | Muscari armeniacum, M. schliemannii, M. cyaneoviolaceum (Hyacintheae) | Germany, UK | Piątek et al. (2013) |
A. muscari-botryoidis | (5–)6–10.5(–11.0) × (4.5–)5.0–8.5(–9.5) µm | ca. 0.5–0.8 µm | Olive-brown | Muscari botryoides (Hyacintheae) | Europe, New Zealand | Piątek et al. (2013) |
A. peglerae | 9–24(–27) × 7–12 µm | ca. 0.5 µm | Yellowish or pale oliveaceous brown | Ornithogalum lacteum (Ornithogaloideae) | South Africa | Bauer et al. (2008) |
A. scillae | 7–11.5(–13) × (6–)6.5–9.5(–10) µm | ca. 0.5 µm | Olive–brown | Scilla bifolia (Hyacintheae) | Germany | Bauer et al. (2008) |
A. sukhomlyniae | (6.5–)7–10 × 7.5–13(–14) µm | ca. 0.8 µm | Yellowish brown | Hyacinthella sp. (Hyacintheae) | Ukraine | Savchenko (2015) |
A. tourneuxii | 7–16 × 6–10 µm | No data | No data | Bellevalia trifoliate (Hyacintheae) | Egypt | Bauer et al. (2008) |
A. tractemae | 6.5–14.5(– 16.5) × (5.5–)6.0–10.5(–11.5) µm | ca. 0.5–1.3 µm | Olivaceous or yellowish-brown | Tractema verna (Hyacintheae) | UK | Piątek et al. (2011) |
A. urginea | 9.5–15(–17.5) × 7–12 µm | ca. 0.5–0.8 µm | Yellowish brown | Urginea maritima (Urgineoideae) | Morocco | Bauer et al. (2008) |
A. vaillantii | (5.5–)6–10.5(–13) × 5–8.5(–9) µm | ca. 0.5–0.7 µm | Olive-brown | Muscari comosum, M. tenuiflorum (Hyacintheae) | Europe, Africa, North America | Piątek et al. (2013) |
A. vindobonensis | (6–)7–11(–15) × (6–)7–9(–10) µm | ca. 0.5 µm | Olive-brown | Scilla vindobonensis, S. vindobonensis subsp. borhidiana (Hyacintheae) | Europe | Bauer et al. (2008) |
Morphological And Ontogenic Observations
Morphological characterization was conducted using a mirrorless interchangeable lens camera and stereo microscope for field and macro morphological observations, respectively (Fig. 2) and light microscopy for microscopic (Fig. 3) and time-lapse (Fig. 4) observations.
Spore germination resulted in long-ellipsoidal, broadly fusiform to cylindrical, 1–4-celled phragmobasidia, usually on a narrow pedicel (Fig. 4A–P). Basidiospores were produced on sterigmata both laterally, apically, and basally on the basidia (Figs. 3O–R, 4Q–S). Young basidia appeared to be easily misidentified as holobasidia (Fig. 4D–G), but the growth observations for the basidia confirmed that they were phragmobasidia (Fig. 4H–K). Mature basidiospores were 1–3 celled, long ellipsoidal or broadly fusiform, giving rise to secondary yeast-like cells (Figs. 3W, 4T–Y). Overmature basidia after forming basidiospores blastically formed yeast-like cells (Fig. 3X). For the matured basidiospore and overmature basidia, yeast-like cells were similar to the basidiospore but could be distinguished from the latter by their budding scar, smaller spore size, and central septum position when compared with the eccentric septum position in the basidiospore. Yeast-like cells appeared to produce daughter cells by polar budding (Fig. 4S–W) or hyphae (Fig. 4I–K) in one day.
Taxonomy
Antherospora barnardiae S. Shibata & A. Hashim., sp. nov. Figures 2–5
MycoBank MB@@@@@
Etymology: Named after the host plant genus.
Typus: Japan: Kanagawa, Chigasaki, Tsutsumi, Shimin no Mori (24m), 23 Sept 2022, Collector Akira Hashimoto (SahoUro2 = KPM @ holotypus designated here, TPM @ isotypus)
ITS/LSU nucleotide sequences GenBank accession no. [DNA extracted from culture SahoUro2s (= JCM @) that isolated from SahoUro2]
Sori in flowers of an inflorescence, in the inner floral organs filled with a blackish-brown, powdery mass of spore.
Peridium absent. Teliospore circular or elliptic, in side view elliptic, 7.9–12 × (5–)6–8.5 µm (av. 9.7 × 7.2 µm, n = 30), l/w 1.0–1.9(–2.4) (av. 1.4, n = 30), in planeview elliptic, sometimes subpolygonally, concave in oil mount, yellowish brown to olivaceous-brown; wall 0.6–1.1 µm thick (av. 0.8 µm, n = 5), nailheaded, finely, densely, uniformly verruculose. Teliospore germination results in phragmobasidia. Phragmobasidia broadly fusiform, (1–)3-septated, 24.5–76 × 3.5–7.5(–9) µm, hyaline, smooth, usually on a narrow pedicel composed of 1.8–3.5 × (1.7–)2–2.8 µm. Matured basidia produce basidiospores from laterally or apically developed sterigmata and then produce yeast-like cells. Basidiospores 1-septated, eccentric, ellipsoidal or broadly fusiform, 19.5–26.5 × 3.5–4.5 µm (av. 23.7 × 3.9 µm, n = 20), hyaline, smooth. The basidiospores bud and eventually form hyphae or yeast-like cells from both ends. Yeast-like cells aseptate to 1-septated, ellipsoid, 11–21.5 × 2–3.5 µm (av. 15.5 × 3.2 µm, n = 20), hyaline, smooth, thin-walled (on WA). Matured yeast-like cells inflated, aseptate to 1-septated, central, budding to produce daughter cells and also developing hyphae (on WA).
Culture characteristic: Colonies on PDA attaining 28–34 mm diam within 30 d at 20°C in the dark, velvety, plane, pale pink (Fig. 5D, E); asexual morph form. Hyphae septate, 2.5–3.5 µm wide, hyaline, smooth, thin-walled, sometimes sympodually or holoblastically produce yeast-like cells at the tip. Yeast-like cells aseptate to 1-septated, ellipsoid, 11.5–14.5 × 2.5–4 µm (av. 12.6 × 3.2 µm, n = 16), hyaline, smooth, thin-walled. Matured yeast-like cells inflated, aseptate to 1-septated, 16–23 × 3–4.5 µm (av. 18.4 × 4 µm, n = 18), budding to produce daughter cells and also developing hyphae.
Additional specimens examined (paratypes): JAPAN, Ibaraki, Tsukuba, Tennodai, Tsukuba University (27m), 17 Sept 2022, on Barnardia japonica, Collector S. Shibata, Y. Takashima, A. Hashimoto, SahoUro1 (= KPM @); Ibaraki, Tsukuba, Matsushiro, Kajihata Children's Park (25m), 25 Sept 2022, on B. japonica, Collector A. Hashimoto, SahoUro3 (= KPM @); Ibaraki, Tsukuba, Koyadai, around RIKEN BioResource Research Center (23m), Oct 3 2022, on B. japonica, Collector S. Shibata, Y. Takashima, A. Hashimoto, SahoUro4 (= KPM @); Chiba, Nagareyama, Nagasaki, around Saka river (18m), 3 Oct 2022, on B. japonica, Collector A. Hashimoto, SahoUro5 (= KPM @); Chiba, Nagareyama, Nishifukairi, around Tone Canal (21m), 3 Oct 2022, on B. japonica, Collector A. Hashimoto, SahoUro6 (= KPM @); Kanagawa, Chigasaki, Tsutsumi, Shimin no Mori (24m), 13 Sep 2007, Collector T. Yasui, KPM-NC 0015106.
Note: The abovementioned seven specimens were identified as A. barnardiae and were collected from September to October. They were all obtained from broadly similar habitats with sunny but humid grasslands and a water source, such as a pond or brook. The spore sizes were almost identical amongst the samples. We hypothesized that the specimen previously reported as “Ustilago vaillantii” that occurred on B. japonica from Shikoku Island Japan is the same species as was identified in this investigation, because the teliospore size, host, and seasonal characteristics were identical (vs. 7–12 × 7–9 µm, on B. japonica, at October 1921; Kakishima 1982; Ito 1936).
The phylogenetic analysis showed that the six identified strains of this species completely matched each other and were phylogenetically different from the six known species. Antherospora barnardiae is similar to A. scillae (Cif.) R. Bauer, M. Lutz, Begerow, Piątek & Vánky in teliospore size (7–11.5(–13) × (6–)6.5–9.5(–10) µm; Bauer et al. 2008) but can be distinguished from the latter species by its yellowish brown to olivaceous-brown spores and wider spore wall when compared with the previously reported olive–brown colour and approximately 0.5-µm-thick spore in Bauer et al. (2008). ITS nucleotide sequences between these two species differed in 21 positions with 31 gaps. Although A. barnardiae is also morphologically similar to Antherospora vaillantii and its segregates by Piątek et al. (2013), i.e. muscari-botryoidis (Cif.) Piątek & M. Lutz, A. hortensis Piątek & M. Lutz and A. vaillantii, these species can be separated from A. barnardiae by 20–25 bp differences in the ITS region and their tendency for somewhat smaller spores and host plant genera (Table 2). The five species without DNA sequence data could be distinguished using their spore size and the different plant genera (Table 2).