Identity of the fungal host
The representative isolate Sak4 obtained from litter collected from Haha-jima Island located in the Ogasawara Islands showed the unique morphology of sporangium having a long neck (Fig. 1), which is one of the characteristic morphologies of the genus Saksenaea in Mucorales (Saksena 1953). Six species (S. dorisiae, S. erythrospora, S. loutrophoriformis, S. oblongispora, S. trapezispora, and S. vasiformis) are currently recognized in this genus (Alvarez et al. 2010; Crous et al. 2016; Crous et al. 2017; Labuda et al. 2019; Saksena 1953). The genus Saksenaea is also known as one of the thermophilic fungi in Mucorales and the maximum growth temperature exceeds over 40°C in four species (Alvarez et al. 2010; Baijal 1965; Crous et al. 2017). The growth range of the representative isolate was determined as 13–37°C on CZA (Fig. 2). The growth at the threshold low and high temperatures (15 and 37°C, respectively) of the genus was ca. 6.0 mm/d (ca. 22 mm after 4 d on CZA) and ca. 3.7 mm/d (ca. 20 mm after 4 d on CZA), respectively. The lower maximum growth temperature (less than 40°C) was similar to that of S. dorisiae and S. trapezispora (Crous et al. 2016; Labuda et al. 2019). The minimum growth temperature of the representative isolate (13°C) was closer to that of S. dorisiae (12°C) than that of S. trapezispora (15°C). The growth of the representative isolate at the higher temperature (37°C) was 19–23 mm on CZA for 4 d, which was slightly slower than that of S. dorisiae (25–30 mm). Detailed morphological comparisons among the representative isolate and these physiologically related species showed that the isolate closely resembled these two species morphologically. However, the morphologies can be distinguished from those of S. trapezispora by the longer sporangium and neck (59.6–193.9 vs. 50–140 µm, respectively), and ellipsoidal to cylindrical sporangiospores (4.6–9.7 × 2.7–5.6 vs. 5.5–7.5 × 3.5–4 µm, respectively). The morphologies can be distinguished from those of S. dorisiae by having the longer neck (38.0–165.1 vs. 70–100 µm, respectively) and the slightly longer and wider sporangiospores (4.6–9.7 × 2.7–5.6 vs. 5.0–5.5 × 2.5–3.0 µm, respectively).
The Maximum likelihood (ML) phylogenetic trees showed that the isolates were phylogenetically identical to each other (Fig. 3, Additional file 2–4: Fig. S2–4). The isolates were located in a highly supported clade, “clade 3” defined by Alvarez et al. (2010), containing isolates of S. dorisiae, S. oblongispora, and S. trapezispora. This result suggests that the present isolates were phylogenetically close to these species. However, the present isolates were also distinct from these species. Therefore, the present isolates were morphologically, physiology, and phylogenetically distinguishable from known species and proposed as a new species, S. boninensis.
Saksenaea boninensis Y. Takash., K. Narisawa, sp. nov.
MycoBank no.: MB 843122
Figures 1, 2.
Diagnosis: The longer neck of sporangia, longer and wider sporangiospores, and slightly more sensitive growth at the threshold high temperature (37°C) are distinctive characters of this species compared with S. dorisiae.
Type: Japan, Tokyo, Ogasawara Islands, Haha-jima Island, Ogasawara-mura, Higashidai, near the north port, isolated from a culture plate (LCA medium) directly inoculated with litter by Y. Takashima on 1 Dec 2018; dried fungal materials on CZA (Holotype, KPM-NC0028612), ex-holotype strain (Sak4 = JCM 39173 = NBRC 114970 = CBS 147591).
Gene sequence ex-holotype: MK757863 (ITS), MK757859 (LSU), LC474957 (tef1).
Etymology: boninensis, referring to the Bonin Islands (Ogasawara Islands), the geographic origin of the type culture.
Colonies fast growing, filling the 90-mm-diameter petri dish after 4 d of incubation (ca. 20 mm/d) on CZA at the optimum growth temperatures (28 and 30°C), hyaline, with scarce aerial mycelia. The minimum growth observed at 13°C (ca. 3.3 mm/d, ca. 15 mm after 4 d on CZA). No growth observed above 40°C. Mycelia 1.9–10.3 (Mean ± SD = 4.7 ± 2.0) µm wide. Sporulation abundant on CZA at 23°C. Sporangia generally single, rarely in twos, erect, developed at the end of a hyphal branch with a dichotomously branched rhizoidal structure below, rhizoids, hyaline, 2.9–5.8 (Mean ± SD = 4.3 ± 0.6) µm wide, stalk, brownish, 4.9–13.3 (Mean ± SD = 8.2 ± 2.2) × 44.9–89.4 (Mean ± SD = 68.0 ± 11.2) µm, flask-shaped with a brownish spherical venter, 18.8–41.3 (Mean ± SD = 28.9 ± 5.7) × 21.4–50.4 (Mean ± SD = 34.4 ± 6.8) µm, with a distinct dome-shaped columella; venter surmounted by a brownish long neck, 5.5–11.9 (Mean ± SD = 8.9 ± 1.3) × 38.0–165.1 (Mean ± SD = 93.2 ± 26.5) µm (venter + neck: 59.6–193.9 (Mean ± SD = 124.0 ± 31.2) µm long), apex of the neck slightly broader, 8.2–15.2 (Mean ± SD = 12.0 ± 1.8) µm in diameter, closed with a mucilaginous plug, which is gradually dissolved when mature. Surface of brownish parts of sporangia (stalk, venter, and neck) asperulate, ornamented with fine spines, dissolved in lactic acid within 12 h. Sporangiospores hyaline, ellipsoidal to cylindrical, 4.6–9.7 (Mean ± SD = 6.8 ± 1.1) × 2.7–5.6 (Mean ± SD = 3.5 ± 0.5) µm [Q (quotients of spore length and width) = 1.4–2.7, Qm (the mean Q value) = 1.9]. Zygospores unknown.
Note A Burkholderiaceae-related endobacterium has been associated with isolates of this species since the isolates were obtained. However, the morphological differences between isolates with/without the endofungal bacterium, such as sporulation of sporangiospores observed in R. microsporus (Lackner et al. 2011), were not examined in this study.
Discovery of new BRE lineage from isolates of Saksenaea
As the survey for the endofungal bacteria associated with Mucoromycota, all isolates obtained from different sporangia were subjected to PCR amplification of 16S rRNA using the DNA templates prepared from fungal mycelia. Since positive amplification was confirmed in all isolates, fluorescence microscopic observations were conducted to confirm the presence of endofungal bacteria. FISH observation of the representative isolate using the Cy3-labeled bacterial universal probe showed the presence of the endofungal bacterium within hypha (Fig. 4). The observation of the endofungal bacterium using the nucleic staining reagent also showed that the endofungal bacterium was present throughout the asexual stage, such as aerial mycelium, rhizoid, stalk, sporangium, sporangiospore, and geminated sporangiospore (Fig. 5). These observations strongly suggested that the amplification of 16S rRNA was derived from the endofungal bacterium, which was vertically transmitted through asexual sporogenesis of the fungal host. The 16S rRNA gene sequences determined from PCR amplicons with fungal mycelia of each isolate were identical to each other. ML phylogeny of the 16S rRNA gene clearly showed that the endofungal bacterium was phylogenetically related to the family Burkholderiaceae (Fig. 6, Additional file 1: Fig. S1). However, the phylogenetic position of the endofungal bacterium was not clustered with either “Glomeribacter-Mycoavidus clade” or Mycetohabitans spp. (Fig. 6). This result indicates that the endofungal bacterium, which thrived within isolates of S. boninensis was the new BRE lineage (hereafter, named “SakBRE”).