Note
Revisiting the isolation source after the first discovery: Myconymphaea yatsukahoi on excrements of Lithobiomorpha (Chilopoda)
2022 年 63 巻 4 号 p. 176-180
詳細
2022 年 63 巻 4 号 p. 176-180
Myconymphaea yatsukahoi is a fungus that has only been isolated once from a forest in the Sugadaira Research Station, Nagano, Japan. Over 20 y have passed since its first discovery but since then it has not been rediscovered. Here, we re-isolated M. yatsukahoi from the type locality and another location, Tambara Moor, Gunma, Japan. Sporophores of this species were detected by direct field observation in Sugadaira and by induction from soil from Tambara. We attempted to narrow down isolation sources of this species by investigating the excrements of Lithobiomorpha and Scolopendromorpha centipedes, which are frequently found in the two locations where the species is distributed. In both locations, we found M. yatsukahoi in the excrements of Lithobiomorpha but not Scolopendromorpha. Myconymphaea yatsukahoi appears to be a coprophilous fungus and the excrements of the predators living in soil may be promising isolation sources for understanding the hidden diversity of kickxellalean fungi.
Isolation sources are essential for understanding the life history of rarely encountered microfungi. Currently, twelve genera are recognized in the family Kickxellaceae and more than half of the genera are monotypic (Dipsacomyces, Kickxella, Martensiomyces, Mycoëmilia, Myconymphaea, Pinnaticoemansia, Spirodactylon) (Benjamin, 1959, 1961; Benny et al., 2016; Coemans, 1862; Kurihara & Degawa, 2006; Kurihara, Degawa, & Tokumasu, 2001, 2004; Meyer, 1957). These genera have been isolated from mammalian dung (Kickxella, Spirodactylon), a cadaver (Myconymphaea), or soil (the other four genera). Since most of these genera have not been detected since they were first discovered and described, their isolation sources remain unclear. Myconymphaea yatsukahoi is a fungus that was isolated from a cadaver of a soil arthropod, Metriocampa sp. (Diplura) (Kurihara et al., 2001). It has been over 20 y since the first discovery of this species but since then it has not been detected, including from its type locality. In the present study, we attempted to rediscover M. yatsukahoi from the type locality and another location, Tambara Moor, and to narrow down the isolation sources of this species by investigating the excrements of centipedes.
Isolation of M. yatsukahoi from unspecified isolation sourcesMyconymphaea yatsukahoi is a rarely encountered fungus in Kickxellales and is, so far, only known from a deciduous broad-leaved forest in Sugadaira Research Station, Ueda-shi, Nagano prefecture, Japan as the type locality (36°31’18”N, 138°21’00”E; Kurihara et al., 2001). Sporophores of this species have been rediscovered a few times in the type locality where the forest floor is dominated by Pinus densiflora (hereafter called “red-pine forest”), firstly on a frass inside a decaying log on 5 Jun 1996 by Y. Degawa [referred to in Kurihara et al. (2001)] and secondly on small excrements of unknown small animals under a decaying log on 4 Jul 2015 by Y. Takashima. The collection sites in the type locality, the deciduous broad-leaved forest (1st discovery) and the red-pine forest (2nd and 3rd rediscoveries), were adjacent and located within 600 m of each other (Iimura et al., 2010). During a survey of microfungi on 10 Oct 2018, a soil sample was collected from under a conifer tree, Thujopsis dolabrata, in Tambara Moor, Numata-shi, Gunma prefecture, Japan (36°47’14”N, 139°03’45”E). The straight-line distance between Tambara Moor and Sugadaira Research Station is about 70 km. To induce microfungi, a baited moist chamber (dried shrimps; Degawa & Tokumasu, 1997) was set on 14 Oct 2018 and incubated at room temperature (ca. 23 °C) under natural light (daytime) and room light conditions in the laboratory. On the soil in the moist chamber on 22 Oct 2018, there was a sporophore with unicellular sporocladia formed on apical vesicles, which is a characteristic of M. yatsukahoi. Two isolates were successfully established from sporangiospores of these sporophores that were encountered on 4 Jul 2015 and 22 Oct 2018, respectively. For the isolation, sporangiospores were inoculated onto fresh LCA medium [0.2 g yeast extract (Difco, Sparks, MD, USA), 1 g glucose (Wako Pure Chemical Industries, Osaka, Japan), 2 g NaNO3 (Wako), 1 g KH2PO4 (Wako), 0.2 g KCl (Wako), 0.2 g MgSO4·7H2O (Wako), 15 g Bacto agar (Difco) in 1 L distilled water] (Miura & Kudo, 1970) using a frame-sterilized fine needle. For the preparation of dried specimens, these isolates were incubated on LCA medium for 3 wk and then dried using an oven at 80 °C for 3 d and deposited in the Kanagawa Prefectural Museum of Natural History (KPM, Kanagawa Pref., Japan) under the specimen numbers, KPM-NC 28610 for YTK1 (Sugadaira isolate obtained from the sporophore on 4 Jul 2015) and KPM-NC 28611 for YTK2 (Tambara isolate obtained from the sporophore on 22 Oct 2018). Further detailed information of entire collection history of M. yatsukahoi from unspecified isolation sources was summarized in Supplementary Table S1.
Isolation of M. yatsukahoi from a specified isolation sourceThe origin of the small excrements found on 4 Jul 2015 at the red-pine forest are unknown; however, we frequently found centipedes under the decaying logs. To narrow down the isolation sources of Myconymphaea, we considered centipedes as a candidate for the origin of the excrements. It was also suspected from the fact that the excrements often included the cysts of gregarines (Gregarinea, Apicomplexa) which is common gut inhabitants of centipedes. We collected centipedes belonging to the orders Lithobiomorpha and Scolopendromorpha from forests in the type locality (8 Jul 2019 to 23 Oct 2019) and from forests around Tambara Moor (7 Oct 2019) (Fig. 1A, B). Each centipede was kept separately and brought to the laboratory where each individual was placed onto wet wiping paper (Kimwipes, Kimberly-clark, USA) in its own clean plastic cup. The centipedes were morphologically identified to at least genus level following Aoki (2015). One or two d (mostly 1 d) after capture, if present, 1-9 excrements of each centipede were collected from the wet Kimwipes using a frame-sterilized fine needle (hereafter called “first collection”) (Fig. 1C, Supplementary Table S2). Then, 6-12 d (8 d on average) after capture, if present, 1-8 excrements of each individual were collected in the same way as above (hereafter called “second collection”) (Supplementary Table S2). The collected excrements were placed on LCA medium supplemented with antibiotics [100 μg mL-1 chloramphenicol (Wako), 50 μg mL-1 ciprofloxacin hydrochloride (Wako), and 100 μg mL-1 streptomycin sulfate (Wako) in final volume] and incubated for 5-13 d at room temperature (ca. 23 °C) under natural light (daytime) and room light conditions in the laboratory. The occurrence of sporophores of M. yatsukahoi on each excrement was observed under a stereomicroscope (SZ61, Olympus Corp., Tokyo, Japan) and isolates were established from each excrement, as much as possible, by inoculating sporangiospores onto fresh LCA medium using a frame-sterilized fine needle.
Fifty-eight Lithobiomorpha individuals and 27 Scolopendromorpha individuals were collected from Sugadaira Research Station (Table 1). Among them, 3 individuals of Lithobiomorpha were collected from a path leading to the Daimyojin waterfall located in the deciduous broad-leaved forest, and the others were collected from the red-pine forest (Supplementary Table S2). Most Lithobiomorpha individuals (54 of 58) were identified as belonging to the genus Bothropolys (Lithobiidae) based on the number of teeth on the tooth plate (more than 5 teeth). One Lithobiomorpha individual was identified as the genus Monotarsobius (Lithobiidae) based on the tooth plate having two teeth and the number of segments on legs. All Scolopendromorpha individuals were identified as Scolopocryptops quadristriatus (Scolopocryptopidae) based on the dorsal plates having four stripes. Three Lithobiomorpha individuals and two Scolopendromorpha individuals could not be identified because the body started to decay before identification. In total, 425 Lithobiomorpha excrements and 243 Scolopendromorpha excrements were investigated for the occurrence of M. yatsukahoi (Table 2). One to seventeen (8 on average) excrements were collected from each individual at least once. Myconymphaea yatsukahoi was found in Lithobiomorpha [18 out of 58 individuals (31%), 35 out of 425 excrements (8%)] but not in Scolopendromorpha (Fig. 1D, E, Tables 1, 2). Sporophores of M. yatsukahoi were found on excrements from 14 individuals during the first collection and from 9 individuals during the second collection. The excrements of 5 individuals had sporophores during both collections (Supplementary Table S2). The occurrence rate of M. yatsukahoi from Lithobiomorpha individuals over the sampling period varied between 20-47%, suggesting a constant moderate level of occurrence (Table 1).
| Location | Month | Lithobiomorpha | Scolopendromorpha | ||||
| No. of individuals | No. of individuals Myconymphaea detected | Occurrence rate | No. of individuals | No. of individuals Myconymphaea detected | Occurrence rate | ||
| Sugadaira | Jul | 23a | 6 | 26% | 4 | 0 | 0% |
| Aug | 10 | 2 | 20% | 8 | 0 | 0% | |
| Sep | 10 | 3 | 30% | 6 | 0 | 0% | |
| Oct | 15 | 7 | 47% | 9 | 0 | 0% | |
| Sub total | 58 | 18 | 31% | 27 | 0 | 0% | |
| Tambara | Oct | 15a | 3 | 20% | 12 | 0 | 0% |
| Total | 73 | 21 | 28% | 39 | 0 | 0% | |
a Unidentified Coemansia-like fungus was detected in one individual.
| Location | Month | Lithobiomorpha | Scolopendromorpha | ||||
| No. of excrements | No. of excrements Myconymphaea detected | Occurrence rate | No. of excrements | No. of excrements Myconymphaea detected | Occurrence rate | ||
| Sugadaira | Jul | 161a | 9 | 5% | 31 | 0 | 0% |
| Aug | 55 | 3 | 5% | 71 | 0 | 0% | |
| Sep | 61 | 6 | 9% | 46 | 0 | 0% | |
| Oct | 148 | 17 | 11% | 95 | 0 | 0% | |
| Sub total | 425 | 35 | 8% | 243 | 0 | 0% | |
| Tambara | Oct | 143b | 5 | 3% | 105 | 0 | 0% |
| Total | 568 | 40 | 7% | 348 | 0 | 0% | |
a Unidentified Coemansia-like fungus was simultaneously detected in two excrements from one individual in which M. yatsukahoi was detected.
b Unidentified Coemansia-like fungus was detected in four excrements from one individual.
Fifteen Lithobiomorpha individuals and 12 Scolopendromorpha individuals were collected from the forests around Tambara Moor (Table 1). For the same reasons as above, 13 Lithobiomorpha individuals were identified as Bothropolys and 2 individuals were identified as Monotarsobius, and all individuals of Scolopendromorpha were identified as S. quadristriatus. In total, 143 Lithobiomorpha excrements and 105 Scolopendromorpha excrements were investigated for the occurrence of M. yatsukahoi (Table 2). Six to twelve (9 on average) excrements were collected from each individual (Supplementary Table S2). Similar to the survey in Sugadaira Research Station, M. yatsukahoi was found in Lithobiomorpha [3 out of 15 individuals (20%), 5 out of 143 excrements (3%)] but not in Scolopendromorpha (Tables 1, 2). Sporophores of M. yatsukahoi were found on excrements from 2 individuals during the first collection and 2 individuals during the second collection. The excrements of 1 individual had sporophores during both collections (Supplementary Table S2). Of note, sporophores of M. yatsukahoi were found on excrements from both Bothropolys (2 individuals) and Monotarsobius (1 individual). Throughout the surveys, an unidentified Coemansia-like fungus was also found on excrements of 2 individuals of Bothropolys collected from Sugadaira Research Station [1 out of 58 individuals (1.7%), 2 out of 425 excrements (0.5%)] and Tambara Moor [1 out of 15 individuals (6%), 4 out of 143 excrements (3%)] (Supplementary Table S2). In total, 34 isolates collected from Lithobiomorpha excrements from Sugadaira Research Station and 5 such isolates from Tambara Moor were successfully established in this study. Unicellular sporocladia with pseudophialides formed on apical vesicles, which are a characteristic of M. yatsukahoi, were observed in the representative isolates obtained from excrements of Lithobiomorpha individuals collected from the Sugadaira Research Station and the Tambara Moor (Fig. 1F-J). These 39 isolates as well as 2 isolates of M. yatsukahoi obtained from unspecified sources, are cryopreserved in the culture collection of the mycology laboratory at the Sugadaira Research Station and are available on request.
Following the findings of M. yatsukahoi from unspecified isolation sources in the type locality and around forests in Tambara Moor, we were able to narrow down the specific isolation source, that is, excrements of Lithobiomorpha. Even though both Lithobiomorpha and Scolopendromorpha co-existed around the habitats investigated in this study, M. yatsukahoi was only found on the excrements of Lithobiomorpha. Centipedes, including Lithobiomorpha, are unlikely to preferably incorporate M. yatsukahoi into their diet because they are predators within the soil fauna food web (Lewis, 2006). One way that centipedes may ingest the sporangiospores of M. yatsukahoi is via grooming, and Lithobiomorpha may be more likely to do this because of their ecomorphotype and foraging habits. Three centipede ecomorphotypes were described by Manton (1977): burrowing, intermediate, and running. Lithobiomorpha and Scolopendromorpha are defined as running and intermediate types, respectively (Manton, 1977; Potapov et al., 2022). The foraging strategies of Lithobiomorpha and Scolopendromorpha also differ: Lithobiomorpha are sit-and-wait predators while Scolopendromorpha are active hunters (Potapov et al., 2022). Lithobiomorpha hunts prey in leaf litter, the upper soil layer, and under stones and bark (Potapov et al., 2022). These behaviors mean that the sporophores of M. yatsukahoi that are in the environment are more likely to attach to Lithobiomorpha, and also that Lithobiomorpha are more likely to re-distribute the sporangiospores of M. yatsukahoi by excretion. Similar to this, Degawa, Ohsawa, Suyama, and Morishita (2014) previously speculated spore dispersal of fungi via grooming by centipedes through the study of Mortierella thereuopodae (Mortierellales) occurring on excrements of Thereuopoda clunifera (Scutigeromorpha, Chilopoda). The observation of sporophores of M. yatsukahoi under a decaying log indicates that sporangiospores of this fungus probably rely on dispersal via attachment to small soil animals rather than by wind. Lithobiomorpha may also indirectly ingest the sporangiospores of M. yatsukahoi, e.g., via the ingestion of fungal-feeding prey. Pollierer, Dyckmans, Scheu, and Haubert (2012) studied the fatty acid composition and δ13C signatures of the individual fatty acids of basal resources and soil animals, including predators such as Lithobius melanops (Lithobiidae). This analysis showed that 50% of fatty acids in L. melanops were derived from plant- and fungal-specific fatty acids and the ratio of plant to fungal fatty acid markers was very low, suggesting that L. melanops mainly consumes fungal-feeding prey. As an example of this food chain, small diplurans feed on fungi and are known to be predated by centipedes (Sendra, Jiménez-Valverde, Selfa, & Reboleira, 2021). Therefore, it is possible that M. yatsukahoi is indirectly ingested by Lithobiomorpha. Further behavioral studies, such as observing grooming under the presence of M. yatsukahoi in a microcosm followed by gut-content analyses, are needed to verify whether M. yatsukahoi is ingested by Lithobiomorpha directly and/or indirectly.
The ecology of M. yatsukahoi still remains largely unknown. The only statement Kurihara et al. (2001) made was that M. yatsukahoi is not an obligate parasite of diplurans. In the present study, 31% of Lithobiomorpha individuals collected from Sugadaira Research Station and 21% of Lithobiomorpha individuals collected from Tambara Moor, were harboring M. yatsukahoi. In contrast, an unidentified Coemansia-like fungus was only observed in 1.7% and 6% of Lithobiomorpha individuals from Sugadaira Research Station and Tambara Moor, respectively. The difference in the occurrence rate between these two kickxellalean fungi indicates that M. yatsukahoi is specifically adapted to Lithobiomorpha. Other kickxellalean species have been isolated from excrements or soil containing excrements. For example, soil containing Dermaptera excrements has been reported as an isolation source for P. coronantispora (Kurihara & Degawa, 2006). Our study provides new insights into the ecology of M. yatsukahoi as a coprophilous fungus and emphasizes the importance of the excrements of predatory or omnivorous soil animals as isolation sources to improve our understanding of the hidden diversity of kickxellalean fungi. Prior to this study, only the type-culture of M. yatsukahoi was available, which restricted research into the variation in phenotypic traits and phylogenetic divergence in the genus. Since Lithobiomorpha is widely distributed throughout the world and in Japan, it is likely that there are more fungi with morphological and phylogenetic traits that differ from the original isolate of M. yatsukahoi. To help clarify whether species delimitation in Myconymphaea corresponds to differences among hosts, locations, and/or climates, we need to collect more isolates from different host species and locations as well as conduct detailed morphological comparisons and phylogenetic analyses.
The authors declare that they have no conflict of interest.
We thank Dr. Tomoko Anezaki for arranging research permits for the Tambara Moor and Mr. Mitsuo Furumi for guiding us around Tambara Moor. This research was supported by a grant from the Institute for Fermentation, Osaka (IFO).
