Life Cycle Plasticity in Typhula and Pistillaria in the Arctic and the Temperate Zone

Typhulaceae Jülich is one of the cold-adapted fungal families in basidiomycetes. The representative genera, Typhula (Pers.) Fr. and Pistillaria Fr., are distinguished by the discontinuity between stems and hymenia in the former and the continuity in the latter (Fries 1821). This taxonomic criterion is ambiguous, and consequently, the view of Karsten (1882) has been widely accepted: Typhula develops basidiomata from sclerotia, while basidiomata develop directly from substrata in Pistillaris. However, Corner (1970) observed basidiomata of Pistillaria petasitis S. Imai developing from sclerotia in Hokkaido, Japan. We later recognized that P. petasitis basidiomata also emerged directly from substrates on the ground in Hokkaido. An aberrant form of Typhula hyperborea H. Ekstr. was found in Upernavik, West Greenland. This specimen had a stem-like structure on a Poaceae plant, and sclerotia developed on its tip. Similar phenomena were found in other Typhula species in Japan. In this study, we aimed to elucidate the life cycle plasticity in the genera Typhula and Pistillaria through the interactions between their ecophysiological potential and environmental conditions in their localities. We collected and prepared strains of the above fungi from sclerotia or basidiomata, and we elucidated the taxonomical relationship and determined the physiological characteristics of our strains. Our findings imply that both Typhula and Pistillaria have the potential to produce sclerotia as well as the capacity for mycelial growth at ambient air temperatures in each locality where samples were collected. These findings suggest that Typhula spp. develope basidiomata not only from the sclerotia dispersed by the basidiospores but also from mycelia generated by the spore germination, which formed basidiomata multiple times, depending on their growth environments.


Introduction
Several kinds of microorganisms, especially fungi, have been reported in the cryosphere [1][2][3][4][5]. Fungal species were less frequently recorded from the cryosphere than those of the temperate zone, despite the fact that all fungal taxa have already been found in the cryosphere. These records suggest the possibility that various fungi are active in various cold environments. We proposed the term 'cryophilic fungi' to denote fungi adapted to the cryosphere [6]. The concept of cryophilic fungi is defined as fungi that are present in the cryosphere, spend a certain life stage or whole life cycle (sexual and/or asexual reproductive stages), and grow under subzero temperatures where water remains in the solid state, such as snow and ice. The concept of cryophilic fungi also applies to unculturable fungi, such as mycorrhizal fungi in snowbank fungi [7].
In 2007, the first author collected aberrant sclerotia of Typhula sp. ( Figure 1D-F) from Upernavik (72.7869, −56.1472), West Greenland. They resembled those of T. hyperborea ( Figure 1C) but were often formed on the tip of a stem-like structure on the Poaceae host. The stem-like structure was indistinguishable from the stems of Typhula sp. (Figure 1D-F) and seemed identical to the secondary sclerotia of hybrids of T. ishikariensis var. ishikariensis S. Imai × T. ishikariensis var. idahoensis (Remsberg) Årsvoll and J.D. Sm. produced under artificial conditions [26,27].  Pistillaria Fr. is close to the genus Typhula Fr. in Typhulaceae Jülich [28] and saprophytic. The hymenium of Typhula is distinguishable from the stem but indistinguishable from the stem in the genus Pistillaria [29]. However, this criterion proved unclear for separating both genera. Consequently, Karsten's view has been widely accepted; Typhula spp. develop basidiomata from sclerotia, and Pistillaris spp., lacking sclerotia, develop basidiomata directly from substrata [30]. However, Corner [31] observed basidiomata of Pistillaria petasitis S. Imai developed from sclerotia in Hokkaido, Japan.
In this study, we aimed to elucidate the life cycle plasticity in the genera Typhula and Pistillaria through the interactions between their ecophysiological potential and environmental conditions in their localities.

Fungal Materials
Fungal sclerotia of T. hyperborea were collected from decayed leaves and stems of plants during the surveys. Sclerotium samples were packed in paper envelopes and dried at room temperature during transportation. Sclerotia were surface-sterilized in 70% (v/v) ethanol for 10 s, in 0.5% (as active chlorine) sodium hypochlorite solution for 30 s, and thoroughly rinsed in sterilized distilled water. They were then cut with sterilized steel blades, placed on potato dextrose agar (PDA: Difco, Sparks, MD, USA), and incubated at 4 • C. Mycelia from growing colony margins were transferred to PDA slants and maintained at 0 • C.
The collected basidiomata of Typhula sp. and P. petasitis were placed in plastic cases with wet cotton balls and kept in a refrigerator. Basidiomata were attached to the inside of Petri dish lids with double-sided adhesive tape, and spores were collected on PDA plates to incubate at 4 • C for 1 or 2 days. Basidiospores subsequently germinated and mated to produce heterokaryons and sclerotia. These sclerotia were transferred to fresh PDA plates and incubated at 4 • C for 2 weeks. Isolates were maintained on PDA slants at 0 • C.
These specimens were kept in the mycological herbaria of National Museum of Nature and Science, Tokyo (TNS).

Mating Experiments
Monokaryons of T. ishikariensis var. ishikariensis (strains PR7-6-7 and PR9-4-3 from Japan) and T. canadensis (strains 35-8 and 8-2 from Japan) were designated as testers and paired with dikaryons of collected strains (di-mon mating; [32]) on PDA plates and incubated at 4 • C for one month. A small agar block was cut from monokaryon colonies near the colony junction and transplanted to another PDA plate. Growth from the block was then examined for the presence of clamp connections on hyphae after incubation for 5 to 7 days at 4 • C. The presence of clamp connections on hyphae was the criterion of mating compatibility.

Phylogenic Analyses
Fungal strains were cultured for 1 month at 10 • C on PDA. Sclerotia of T. hyperborea from Upernavik, West Greenland, were harvested, and DNA was extracted by the protocol of DNeasy Plant MiniPrep (QIAGEN GmbH, Hilden, Germany). ITS regions, including the 5.8S gene of genomic rDNA, were amplified using primer pairs ITS1 (5 TCCGTAGGTGAACCTGCGG) and ITS4 (5 -TCCTCCGCTTATTGATATGC), as described by White et al. [33]. PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN GmbH, Hilden, Germany) and sequenced in one direction on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster city, CA, USA) using the ITS1 primer. Multiple alignments of the ITS sequences were performed, and the nucleotide substitution rate (Knuc value) was calculated in CLUSTAL W [34]. A phylogenetic tree was constructed by the maximum likelihood method using the program MEGA X [35].

Morphological Observations
The colors of basidiocarps and sclerotia were described according to the color identification chart of the Royal Botanic Garden Edinburgh (Flora of British Fungi) [36].
For light microscope sections, aberrant sclerotia of Typhula sp. were fixed with 2% glutaraldehyde (Nisshin EM, Co., Ltd., Tokyo, Japan) in 50 mM phosphate buffer and washed in the same buffer. The samples were then post-fixed with 1% osmium tetroxide (Nisshin EM, Co., Ltd., Tokyo, Japan), dehydrated with an ethanol series, and embedded in Quetol 651 (Nisshin EM, Co., Ltd., Tokyo, Japan). The sections (0.8 µm thick) were stained with toluidine blue (Wako Ltd., Osaka, Japan) and observed under a light microscope.

Mycelial Growth Temperature
Mycelial discs of 5 mm diam were cut from the margins of actively growing colonies, inoculated to the centers of PDA plates, and incubated at six different temperatures from 0 to 25 • C in duplicate. After 1, 2, and 3 weeks of inoculation, colony diameters were determined. The linear mycelial growth rate per week was calculated after the initial lag period.

Typhula hyperborea in Greenland
Fungal cultures from aberrant sclerotia from Upernavik, West Greenland, had featherlike mycelia when grown on PDA at 4 • C (Figure 2A), which were typical physiological reactions of T. hyperborea [23], and our strain did not react when paired with both tester monokaryons of T. ishikariensis var. ishikariensis and T. canadensis. Phylogenic analysis of ITS regions also supported this assumption ( Figure S1).  When the cultures were kept at 4 • C for 2 years, sclerotia were found to have stemlike structures with intercalary, small secondary sclerotia, as described by Christen [26,27] ( Figure 2B). Cultures of T. ishikariensis var. ishikariensis from Hokkaido, Japan, also produced similar aberrant sclerotia. Stem-like structures and aberrant sclerotia of T. hyperborea and T. ishikariensis var. ishikariensis on PDA were dark brown (19 bay) as the same color with field samples ( Figure 1D,E). However, the normal stems of both fungi were white (2 B) to pale yellow (6 E) or pale brown (30 clay pink) [23]. Collected stem-like structures of T. hyperborea from Upernavik, West Greenland, were gradually connected with aberrant sclerotia ( Figure 1F), and similar findings were reported at the base of the stem of Typhula sclerotioides (Pers.) Fr. at its origin from the sclerotium [37]. These results suggested that the germination of sclerotia and secondary sclerotium formation were reversible for T. hyperborea and T. ishikariensis var. ishikariensis.
In 1999, T. hyperborea OUP1811 was collected in Nuuk (64.1666, −57.7500) in West Greenland [23]. This strain also showed different culture morphology on PDA plates among the stains of T. hyperborea ( Figure 2C). This strain had weak pathogenicity against the host [23], and they showed normal growth at 4-10 • C and abundant aerial mycelia without sclerotia on PDA. Sclerotium formation was, however, observed on oatmeal agar plates ( Figure 2D).

Typhula sp. and Pistillaria Petasitis in Hokkaido, Japan
Basidiomata of Typhula sp., whose hymenium was distinguishable from the stem, were found on the dead petioles of  Pistillaria petasitis basidiomata were found on various substrates such as Petasites japonicus, Conioselinum filicinum, and Cirsium kamtschaticum. Basidiomata of P. petasitis were found on substrates on the ground surface up to 10 cm on 3 August 2010 ( Figure 4D). Up to 80 cm on 23 August 2010 ( Figure 4E). All P. petasitis isolates produced sclerotia under culture conditions ( Figure 4C). The sclerotia of our isolates were similar to those described by Corner [37], ca. 2.5-22.5 × 2.5-5.5 mm, fusiform, somewhat flattened, and light brown (15 brick).

Effect of Temperature on Mycelial Growth
Optimal mycelial growth of T. hyperborea from Upernavik, West Greenland, Typhula sp. from Ashoro, and P. petasitis from Higashikawa occurred at 5, 15, and 20 • C, respectively ( Figure 5). Maximal growth temperatures were 15, 25, and 30 • C, respectively. The mycelial growth range of T. hyperborea in Upernavik, West Greenland, was psyhrophilic and typical of this fungus [23]. According to the ranges of their mycelial growth temperatures, Typhula sp. from Ashoro and P. petasitis from Higashikawa were psychrotolerant. This is the first record of the mycelial growth range of P. petasitis.  These growth temperature relations agreed with the ambient temperatures when basidioma samples were collected in each locality. These results suggested that the mycelia of these three fungi could potentially and be active without snow in their localities.

Discussion
Snow molds representing cryophiles resume growth typically by carpogenic germination of sclerotia (sexual cycle) in autumn. Mycelia prevail on dormant plants under the snow to produce sclerotia in late winter before dormancy (asexual cycle). The life cycle of the genus Typhula is illustrated in Figure 6. Basidiospores germinate to develop into monokaryons, which subsequently mate with their counterparts differing in mating incompatibility alleles to produce dikaryons. Dikaryons are capable of sexual recombination through carpogenic germination of sclerotia, which is critical to generating diversity to cope with fluctuating environments and flexibility, as we found in this study.
Kawakami et al. [38] elucidated the conditions required for stem elongation from the sclerotium and fertile head development in T. ishikariensis. Stem elongation occurred at low temperatures and high humidity, but light was not essential. In contrast, light and moderate day length (8 h/day) were prerequisite for fertile head development. Several strains of T. hyperborea also produced basidiomata under Kawakami's conditions [23]. T. hyperborea in the Arctic also acts to produce the asexual formation of basidiomata dispersing basidiospores under light conditions (red dashed line in Figure 6). Mycelia of T. hyperborea did not produce basidiomata under snow or dark conditions. T. ishikariensis complex formed sclerotium on the top of the remaining stems ( Figure 1D,E). Our observation of sclerotia with stems was similar to secondary sclerotia described by Christen [26,27].
In addition, Tkachenko [39] reported another type of secondary sclerotia in T. ishikariensis var. ishikariensis on tulip bulbs in Russia. One to seven secondary sclerotia were found within original sclerotia. We also observed this type of secondary sclerotia from bloated original sclerotia from T. hyperborea from Upernavik ( Figure 2B) and other strains of the T. ishikariensis complex (Hoshino et al., unpublished results).  Corner [37] suggested that the aggregation of hyphae of Typhula gyrans (Batsch) Fr. developed into the tissue of the stem and sclerotium. Hyphal aggregation of Macrotyphula phacorrhiza (Reichard) Olariaga, Huhtinen, Laessøe, J.H. Petersen, and K. Hansen also extended throughout the head. However, the texture of the hyphal walls in the head and central part of the stem was never as tough as in the sclerotium or on the surface of the stem. In addition, the mycelia of M. phacorrhiza and other Macrotyphula spp. on PDA plates were stroger than those of Typhula spp. (Hoshino et al., unpublished results). M. phacorrhiza was the type species of Typhula. However, it has presently been changed to T. incarnata [40]. These points suggested that stem and sclerotium had common properties in Typhula and related genera. According to Lind [41], rusts, smuts, and species of Dothideales Lindau in the Arctic have perennial mycelia in the host, enabling them to grow as soon as the season starts. Previously, we reported that several strains of T. hyperborea in West Greenland had weak pathogenic activity [23] and abundant aerial mycelium with less productivity of sclerotium ( Figure 2C,D). These physiological characteristics supported adaptation to the Arctic summer climate (blight and cold conditions).
Similar phenomena were also recorded from other Typhula sp. in the Arctic (loss of sclerotium-forming ability under cultural conditions) [22] and T. cf. subvariavilis in Antarctica (no sclerotia at the field survey) [25]. Basidiomata of T. cf. subvariavilis in Antarctica emerged directly from substrates, and this fungus had high homology in the ITS region with Typhula sp. Wh-1 in Iran [42] and Typhula variabilis Riess, which is rather ubiquitous in the Northern Hemisphere, including areas with rare snow cover such as the Azores [43]. Snow mold symptoms of T. cf. subvariavilis in Antarctica and Typhula sp. Wh-1 in Iran were not observed in sclerotia just after the snow melts.
Sclerotia of Typhula sp. in Iran were formed ca. 2 weeks to 1 month after the snowmelt (Figure 7), remained immature as mycelial aggregations after snow melt, and matured without snow. T. canadensis in Norway also had sclerotia with aerial mycelia in the field, which were considered to facilitate dispersal by the wind [44,45]; however, they matured under the snow cover. Therefore, it was different from the ecophysiological characteristics of sclerotia from T. cf. subvariavilis in Antarctica and T. canadensis in Norway. These results suggested that T. cf. subvariavilis in Antarctica and other related species could still act after the snow melts in their localities.  Most of the T. hyperborea strains showed irregular growth on PDA at more than 10 • C [23]. However, these strains showed normal growth at the same temperature on corn meal agar or PDA with free radical scavengers such as ascorbic acid or β-carotene [23]. When T. ishikariensis complex, T. incarnata, and Typhula trifolii Rostr. from Canada were first exposed to the maximum growth temperature (20 or 25 • C) and then incubated at their optimal growth temperatures, the T. ishikariensis complex formed a "fan-like" irregular colony that was similar to the colony morphology of typical T. hyperborea [46]. Oxygen uptake of the T. ishikariensis complex was optimal at 20 • C (maximum growth temperature), about 15 • C higher than its optimal growth temperature (5 to 10 • C). Typical strains of T. hyperborea have strong pathogenicity against host plants. Therefore, they obtained free radical scavengers from hosts.
On the other hand, several strains of T. hyperborea from West Greenland obtained saprophytic activity (we did not collect such strains from East Greenland). Probably, they lost pathogenicity and acquired resistance to oxidative stress near their maximum growth temperature. There is a positive correlation between virulence and psychrophily. However, this significance is unclear [47,48]. Most species belonging to Typhulaceae are psychrotolerant and saprophytic. The pathogenic species of Typhula spp. found the new resource of overwintering plants and evolved in a cold environment under snow cover.
Many rusts in the Arctic have a simplified life cycle, only producing one kind of spore (micropuccinia) instead of a life cycle with three spore forms (eupuccinia) more commonly seen in warmer areas [48,49]. It is the first finding that the new life cycle stage of the T. ishikariensis complex and similar phenomena were observed in Typhula sp. in Ashoro, Hokkaido ( Figure 3) and P. petasitis in Higashikawa and Sapporo, Hokkaido (Figure 4). Many types of research on the T. ishikariensis complex were carried out in temperate or frigid zones where air temperatures were higher than those of their psychrophily. Therefore, dikaryons of the T. ishikariensis complex grew only under the snow cover and formed sclerotia for the passing spring and summer seasons.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11082028/s1, Figure S1: A phylogenetic tree of partial ITS sequences of selected typhuloid fungi including Typhula ishikariensis complex constructed by the ML method, inferred by using K2 model.