Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture

Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture

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Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture Yuka Yajima a,*, Tamotsu Hoshino b, Norio Kondo c, Young-Cheol Chang a a

Muroran Institute of Technology, Mizumoto-cho 27-1, Muroran, Hokkaido, Japan National Institute of Advanced Industrial Science and Technology, Kagamiyama 3, Higashi-Hiroshima, Hiroshima, Japan c Research Faculty of Agriculture, Hokkaido University, Kita9, Nishi9, Kita-ku, Sapporo, Hokkaido, Japan b

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abstract

Article history:

The plasmodium of Badhamia alpina thrived at lower temperatures (4  C), and formed

Received 1 December 2016

fruiting bodies at 8  C. The yellow sclerotium and plasmodium were found inside a hollow,

Received in revised form

dead herbaceous stem under melting snow in Apr, and was cultured in moist chambers at

1 April 2017

4  C. The plasmodium did not form fruiting bodies for 6 wk at 4  C. Sporulation was

Accepted 5 April 2017

observed after the incubation temperatures rose to 8  C. Sporulation occurred in the

Available online xxx

morning and cell cleavage at 11 a.m. The order of spore wall formation was observed by TEM for 12 h. The outer spore wall ornamentation was formed first followed by internal

Keywords:

wall layers. Round electron transparent object was observed in the capillitium and

Cryophilic

peridium during the latter part of sporulation.

Physarales

© 2017 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

Slime molds Snowbank species

1.

Introduction

Nivicolous myxomycetes, often referred to as snowbank species are an ecological group of species fructifying under melting snow cover in spring to early summer. The term “nivicolous myxomycetes” is often equated with “alpine myxomycetes”, since these species are collected mainly on high mountain areas (e.g., Fries 1906; Kowalski 1971; Lado 2004). More recently, intensive investigations suggest that the nivicolous species are distributed from low to high

altitudes where heavy snow lasts for several months (reviewed in Ronikier and Ronikier 2010). Nevertheless, the definition of “nivicolous myxomycetes” is still unclear, since some species are considered “strictly nivicolous”, and other species are “non-strictly nivicolous”. These terms are not precisely defined, especially “non-strictly nivicolous” species in the literature, because fresh fruiting bodies are found both under melting snow in spring to early summer, and other times without snow cover. Spore to spore culturing of nivicolous myxomycetes under controlled conditions is lacking (Shchepin et al. 2014), and difficulty in finding fresh plasmodia

* Corresponding author. E-mail address: [email protected] (Y. Yajima). http://dx.doi.org/10.1016/j.myc.2017.04.001 1340-3540/© 2017 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001

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in the field are partly the reason why these terms are still somewhat ambiguous. In the present study, yellow sclerotia and plasmodia were found under melting snow in spring, and identified as Badhamia alpina G. Lister based on the morphology of its fruiting body. The genus Badhamia Berk. is one of the difficult genera to treat taxonomically due to morphological intergrading with the genus Physarum Pers. (Martin and Alexopoulos 1969). There was much discussion about the taxonomic treatment of several nivicolous species relating to B. alpina, and some Badhamia species were transferred to Physarum and vice versa (e.g., Meylan 1925; Kowalski 1975; Singer et al. 2004; Poulain et al. 2011). Since molecular phylogeny does not support separation based on morphology of capillitium of these two genera (Fiore-Donno et al. 2009; Nandipati et al. 2012), a different perspective is needed to recognize the boundary between the physaroid or badhamiaoid species. Therefore, we temporarily treated our specimen with capillitium that approaches the physaroid condition in the present study as B. alpina in Poulain et al. (2011). Badhamia alpina was described based on field collections gathered in Jun or Jul at altitude 2134 m at Arosa, an alpine area in Switzerland (Lister 1914). Although this species is not commonly found in the literature of nivicolous myxomycete research, Poulain et al. (2011) treats this species as nivicolous. This species name is sometimes treated as a synonym of a non-nivicolous species Badhamia foliicola Lister (Martin and Alexopoulos 1969; Lado 2001). If we follow this synonymy, this species might be treated as a “non-strictly nivicolous” myxomycetes. In this study, field collected plasmodia fruited in laboratory culture, and developmental stages were followed during fruiting body formation by the transmission electron microscope. The aims of this study were: 1) to verify the ability of this species to thrive at lower temperatures as a plasmodium and form fruiting bodies in moist chamber culture; 2) to discuss morphological characters and document developmental stages as this relates to physaroid or badhamioid species.

2.

Materials and methods

Field work was carried out in Sapporo city, Hokkaido, Japan (lat. 43 040 N, long. 141 200 E, alt. 15 m), where heavy snow cover lasts at least for 3 mo every year. The yellow sclerotia and plasmodia were found inside hollow dead herbaceous stems under melting snow cover on 12 Apr. At the collecting site, abundant fresh fruiting bodies of nivicolous myxomycetes (e.g., Lamproderma spp., Didymium dubium Rostaf., Diderma alpinum (Meyl.) Meyl., Trichia alpina (R.E.Fr.) Meyl., Lepidoderma chailletii Rostaf.) are observed every year. The substratum of fresh plasmodia and sclerotia was cut in half vertically, then cut again in half horizontally, to obtain the same amount of plasmodium and sclerotium on each piece of the substratum. These pieces were placed in the moist chamber cultures, and one culture was completely covered with aluminum foil to simulate dark conditions, and the other was left uncovered for exposure to light conditions. These two moist chamber cultures were kept at 4  C. The cultures were examined at weekly intervals. After 6 wk, the plasmodia were moved to a 8  C

incubator. When the early immature fruiting body formation was observed, the developing sporocarps were picked off the substratum and chemically fixed at 1-h intervals for the next 12 h. The sporocarps were fixed in 2% gultaraldehyde in 50 mM HEPES buffer, overnight, and rinsed with the same buffer. After the rinse, the specimens were secondarily fixed with 1% OsO4 in the same buffer, rinsed, and then dehydrated with serial EtOH, then substituted in Aceton. The resin infiltration was done with a series of Aceton: Epon 812 Resin ¼ 1:2, 1:1, 2:1, and 100% resin, and polymerized at 60  C for 3 d. Thin sections were cut with a Leica UC7 (Leica Microsystems, Wetzlar), transferred to slot grids, and then stained with UA and lead. The thin sections were observed under a transmission electron microscope (TEM, Hitachi H-800, Hitachi, Tokyo). Scanning electron microscopic observations were made on dried fruiting bodies with a JSM-5310LV (JEOL, Tokyo). A dried fruiting body specimen was deposited in the Hokkaido University Museum, Sapporo, as SAPA 100015. Identification of fruiting bodies was made using pictures and keys in Poulain et al. (2011).

3.

Results

3.1.

Moist chamber culture

The light and dark moist chamber cultures with the combination of sclerotia (Fig. 1A) and plasmodia (Fig. 1B) were started on 12 Apr, and kept at 4  C. Although active pale yellow plasmodia (Fig. 1C) emerged from the stem of the dead herbaceous plant in both light and dark cultures, disappearance of the plasmodium under dark conditions was found on 2 May and there was no sclerotium in the moist chamber culture. The plasmodium cultured under light conditions was observed creeping in the moist chamber, and did not form fruiting bodies for 6 wk at 4  C. Cultures were changed to 8  C on 24 May, and sporulation occurred on 26 May at 10 a.m. The sporophore gradually changed from pale yellow to orange, to light reddish brown, to a dark brown (Fig. 1DeG). The lid of the moist chamber was slightly opened the next day to let the fruiting bodies dry, and the surface of fruiting body became white (Fig. 1HeI). Morphology of the dry specimen was as follows; Sporangium sessile, pulvinate to subglobose, 0.5e0.8 mm diam, or forming short plasmodiocarps, light gray to white. Stalk absent. Hypothallus pale, membranous. Peridium single, thin, sparingly calcareous, hyaline under the light microscope when the calcareous granules are sparse or absent. Columella absent. Capillitium consisting of a delicate net of calcareous tubules to nearly noncalcareous. Spores black in mass, violet brown by LM, 10e11 mm diam, spinulose with groups of darker spinules.

3.2.

SEM observations of the mature fruiting body

Calcareous granules were observed on the outer surface of the peridium (Fig. 2A, B). At higher magnification, SEM showed the embedded (Fig. 2B, arrowhead), and membrane covered (Fig. 2B, arrow) calcareous granules. The smooth outer surface of the peridium was also observed where calcareous granules

Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001

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Fig. 1 e Badhamia alpina (SAPA 100015). A, B: Sclerotium (white arrowhead) and plasmodium (white arrow) in hollow stem of dead herbaceous plant. C: Plasmodium in moist chamber culture (white arrow). The plasmodium emerged from a substrate (black arrow) and climbed up an inside wall of the chamber. DeG: Time sequence of fruiting body. D: 13 p.m., 1st day. E: 16 p.m. F: 20 p.m. G: 13 p.m., 2nd day. H, I: Mature fruiting bodies. J, K: Two spores. Black arrowhead: groups of darker spinules. Bars: A, B, H, I 0.5 mm; C 1 cm; D‒G 4 mm; J, K 10 mm.

Fig. 2 e SEMs of mature fruiting body of Badhamia alpina (SAPA 100015). A, B: Outer surface of peridium. Arrow: Membrane covered calcareous granules. Arrowhead: Embedded calcareous granules. C: Inner surface of peridium. D: Capillitium. Arrow: Slender thread. Arrowhead: Narrow flat plate. E: Mature spores with ornamentation. Bars: A 10 mm; B, D 3 mm; C, E 5 mm. Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001

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were not apparent. In contrast to the outer surface, the inner surface of the peridium was slightly roughened with no calcareous granules (Fig. 2C). The capillitium surface was also slightly roughened with no calcareous granules, and it was composed of slender threads and narrow flat plates (Fig. 2D). The spore was spinulose, 0.2e0.3 mm high on the surface (Fig. 2E).

3.3.

TEM observations of the fruiting body formation

Fruiting bodies of Badhamia alpina were examined during development with transmission electron microscopy. At the

beginning of sequential fixation (11 a.m.), the protoplasm was in the cleavage stage (Fig. 3A), and the surface of the cell membrane was smooth (Fig. 4A). One hour later, the spore initial became a rounded shape (Fig. 3B), and faint fibrous material was observed on the surface of cell membrane (Fig. 4B). After 5 h, the spore wall ornamentation gradually developed on the cell membrane (Figs. 3CeG, 4C‒G). Eight hour later after cleavage (16 p.m., Fig. 3H), a faint fibrous layer was observed on the surface of the cell membrane at high magnification (Fig. 4H), and the fibrous layer gradually thickened after 4 h (Fig. 4IeL). Although the fibrous electron dense layer was basically adjacent to the cell membrane,

Fig. 3 e Badhamia alpina (SAPA 100015). AeL: Spore formation in time sequence. A: 11 a.m. B: 12 p.m. C: 13 p.m. D: 14 p.m. E: 16 p.m. F: 17 p.m. G: 18 p.m. H: 19 p.m. I: 20 p.m. J: 21 p.m. K: 22 p.m. L: 23 p.m. White allowhead: Spore wall ornamentation. White arrow: Separation of fibrous electron dense layer and cell membrane. N: Nucleus. Bars: A 4 mm; B‒L 2 mm. Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001

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separations were sometimes observed (Figs. 3K, 4K, 6E). The contents in the separations were also variable, almost electron transparent (Figs. 4K, 6E), with various shapes of vesicles (Fig. 7F), and projections of the cell surface (Fig. 7E). The capillitium and peridium were already formed at the beginning of sequential fixation (11 a.m.). The peridium was constructed from three basic structures: the thinnest inner most layer (Fig. 5, black arrows), electron dense layer (Fig. 5, black arrowhead), and the fibrous material layer with various thickness (Fig. 5, white arrowhead). Although the thinnest inner most layer of the spore initial was sometimes difficult to

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observe, it was completely separated from (Fig. 5B, I), or adjacent to the electron dense layer (Fig. 5D, G). The electron dense layer was ca. 500 nm in thickness. Part of this layer was directly connected to capilltium (Fig. 5F), or sometimes split into a few layers (Fig. 5G). The outer most part of the peridium with the fibrous material layer was very variable in thickness and electron density, from ca. 500 nm (Fig. 5E and F) to ca. 12 mm (Fig. 5D). It sometimes contained bacteria (Fig. 5D). A round shaped electron transparent object was also observed in the peridium (Fig. 5, white arrow). There were two types of capillitium observed under TEM (Fig. 6), 1) a large inner space

Fig. 4 e Badhamia alpina (SAPA 100015). AeL: Formation of spore wall and ornamentation in time sequence. A: 11 a.m. B: 12 p.m. C: 13 p.m. D: 14 p.m. E: 15 p.m. F: 16 p.m. G: 17 p.m. H: 18 p.m. I: 19 p.m. J: 20 p.m. K: 21 p.m. L: 22 p.m. White arrowhead: Spore wall ornamentation. White arrow: Separation of fibrous electron dense layer and cell membrane. Black arrow: Fibrous material. Black arrowhead: Fibrous layer. CM: cell membrane. Bars: A‒L 0.5 mm. Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001

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with fibrous materials and electron translucent round material (Fig. 6A white arrowhead, B, C); 2) tight and with less inner space type (Fig. 6A black arrowhead, D). The connection of these two types of capillitium was observed (Fig. 6E). A thin layer covered the capillitium that was sometimes observed (Fig. 6C, D black arrow). Through fruiting body development, food (Fig. 7D) and autophagic vacuoles (Fig. 7AeC) were observed.

4.

Discussion

The definition of nivicolous myxomycetes, formerly treated as ‘alpine’ species of myxomycetes, has long been under discussion. Paradoxically, their ecological strategy is obvious because their fresh fructifications are constantly observed

under melting snow in spring. No complete method of culture has been established (Shchepin et al. 2014), and there are some reports on moist chamber cultures under laboratory conditions, however, fructification processes of nivicolous species were not observed (Kowalski 1971; Marx 1998; Dulger et al. 2007; Ronikier et al. 2010; Ronikier et al. 2013). Preference for snow strongly suggests that nivicolous species might be cryophilic, in fact, microplasmodia of some species are observed when agar cultures were kept at 2  C (Shchepin et al. 2014). However, for plasmodium and fruiting body formation, moist chambers were kept at a temperature of 12e25  C (Marx 1998; Dulger et al. 2007; Ronikier et al. 2013) and at 5  C they did not observe the plasmodium (Ronikier et al. 2010). Although there is one report of spore-to-spore culture of a nivicolous myxomycete Lepidoderma carestianum (Rabenh.) Rostaf. (Kowalski 1971), he did not mention any detail about

Fig. 5 e Badhamia alpina (SAPA 100015). AeI: Peridium in fruiting body formation. A: 11 a.m. B, C: 12 p.m. D: 15 p.m. E: 16 p.m. F: 17 p.m. G: 18 p.m. H: 14 p.m. I: 20 p.m. Black arrowhead: Thinnest innermost layer. Black arrowhead: Electron dense layer. White arrowhead: Fibrous material layer. White arrow: Round shape electron transparent object. Ba: Bacteria. Ca: Part of capillitium. Bars: A, B, D 2 mm; C, E‒I 500 nm. Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001

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his culture conditions nor plasmodial activity. The lack of evidence for plasmodial activity and fruiting body formation of nivicolous species in lower temperature conditions is one of the reasons for this ambiguous definition of this ecological group. In the present study, our observation on moist chamber cultures of B. alpina suggests that the plasmodium of this species prefers cryophilous conditions (4  C), and rising temperatures might be a trigger to start fruiting body formation. It is noteworthy that the plasmodium cultured in dark condition appeared dead and completely disappeared, and when cultured in light conditions fruiting bodies developed. On the other hand, Ronikier et al. (2010) reported that Lamproderma argenteobrunneum A. Ronikier, Lado & Mar. Mey. made fruiting bodies under dark conditions at 5  C. It is well known that snow may influence physical conditions, e.g., radiation, humidity, gas, and temperatures for various organisms that survive under snow cover (Pomeroy and Brun 2001). Although the results of the present paper based on a one-time fieldcollected plasmodium, and responses to such field conditions might be considered in defining nivicolous myxomycetes. Badhamia alpina is treated as a nivicolous species (Poulain et al. 2011), but also is one of the doubtful species as being “strictly” nivicolous. Martin and Alexopoulos (1969) and Lado (2001) treat this species as a synonym of a non-nivicolous species B. foliicola. The original description of B. alpina is based on a field collection from an alpine area in Switzerland in Jun or Jul, and on the substrata of hollow scapes of Crisium spinosissimum (L.) Scop. and Senecio alpinus (L.) Moench. (Lister 1914). She did not mentioned whether the specimen was collected near melting snow, but wrote that snow remains throughout the summer at heights of Arosa. She also collected ‘cold-loving’ species, now treated as “late autumn species”

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(Schnittler and Novozhilov 1998) during the same period. Based on these facts, B. alpina at least prefers colder environmental conditions. Badhamia foliicola, on the other hand, is described based on specimens collected in Sep 1896 at Waustead Park, Essex, England (Lister 1897). In the original description, he mentioned other specimens collected at Highcliff, Lyme Regis, England on Feb 1897. Badhamia foliicola might prefer cooler environments, but may not be cryophilic. Our specimen was found inside a hollow stem of a dead herbaceous plant under melting snow in a region where a heavy snow layer lasts 3e4 mo and apparently the plasmodium prefers cryophilic conditions that coincide with the environmental preference of the original description of B. alpina. In the original description of the genus Badhamia, Berkeley (1853) defines species with clustered spores and has a line drawing explained as “Mother-cell, with young spores”. According to the drawing and in the main body of the text, he believed that immature clustered spores are enclosed by a membrane and it disappears when the spores mature. Many researchers expanded the concept of Badhamia to enclose both clustered and non-clustered spore species. A TEM study of matured clustered spores of myxomycetes (Demaree and Kowalski 1975) showed that the compact clustered spores of B. nitens Berk. have completely fused spore walls, in contrast, the loosely clustered spores have hollow spaces in the clustered spores of B. versicolor Lister with fusion points at the tip of ornamentation. Badhamia alpina is not a clustered spored species, and even B. foliicola, rarely has small spore clusters (Lister 1897, 1914; Poulain et al. 2011). This present study failed to find a membrane surrounding each spore as Berkeley mentioned. Instead of the membrane, immature spores were closely connected with tip to tip spore ornamentation as seen

Fig. 6 e Badhamia alpina (SAPA 100015). AeD: Capillitium in fruiting body formation. E: Capillitium and spores. A: Large inner space with fibrous material and electron translucent round material containing type (white arrowhead) and tight and less inner space type (black arrowhead), 17 p.m. B: Large inner space with fibrous material and electron translucent round material containing type, 11 a.m. C: Large inner space with fibrous material and electron translucent round material containing type, 13 p.m. D: Tight and less inner space type, 13 p.m. E: These two types connected, 20 p.m. Black arrow: Thin fibrous layer. White arrow: Separation of fibrous electron dense layer and cell membrane. Bars: A, B 2 mm; C, D 500 nm; E 5 mm. Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001

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by TEM. This has been reported during myxomycete sporulation in many species in the literature (e.g., Mims 1972; Bechtel 1977; Spiegel and Feldman 1988). However, a thinner layer was observed next to the inner surface of the peridium, and it occasionally separated from the peridium. This layer is not the membrane Berkeley mentioned, because it was observed only under the electron dense layer and it covered the entire spore initials, not each spore. The spore wall ornamentation of Badhamia alpina was formed first followed by wall layers. This sequence is same as previous reports on spore formation of myxomycetes as mentioned above. Before the formation of the transparent second layer, known as inner (non-melanized) wall (Raub and Aldrich 1982), the secretion by fused vesicles with cell membranes was reported (Mims 1972; Bechtel 1977). Various shapes of vesicles between the cell membrane and the outer wall of spores were observed here.

The deposition mechanisms of calcium carbonate in the Physaraceae and Didymiaceae were discussed and reviewed many times (e.g., Gustafson and Thurston 1974; Bechtal and Horner 1975; Collins 1979; Schoknecht and Keller 1989; Clark and Haskins 2014), and several techniques were used to observe the deposition. In the present study, conventional chemical fixation techniques and UA and lead staining were used without any specific staining. As a result, electron translucent round materials ca. 500 nm diam were found inside the fibrous material of both immature capillitium and peridium. SEM observations of calcareous granules on mature fruiting bodies were ca. 500 nm to 1 mm diam. Hatano and Keller (2008) also showed that Badhamia species have calcareous granules 300 nm to 1.9 mm diam. Therefore, the translucent round material observed might be a calcareous granule, and it is possible that calcareous granules would be in the fibrous material. The number of peridial layers has also been

Fig. 7 e Badhamia alpina (SAPA 100015). A, B: Autophagic vacuoles in spore initial. A: 14 p.m. B: 23 p.m. C: Autophagic vacuole containing a degraded nucleus. 21 p.m. D: Food vacuole containing a bacterium, 11 a.m. E, F: Projection of the surface of cell (E) and vesicles (F) in the spore wall intervals, 21 p.m. Black arrowhead: Autophagic vacuole. White arrowhead: Spore wall ornamentation. White arrow: Projection of the surface of cell. CM: Cell membrane. N: Nucleus. Bars: A, B 2 mm; C‒F 500 nm. Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001

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discussed in the myxomycete literature (Martin and Alexopoulos 1969). TEM observations showed that the thinnest layer is inner most, the thickness of fibrous material layer is highly variable, and sometimes the electron dense layer split into a few layers. These results suggest that the number of peridial layers is highly variable, even in a single fruiting body. The morphological continuity of the genera Badhamia Berk. and Physarum Pers. have long been a topic for discussion since its original description (Berkeley 1853), and several Badhamia species have been transferred to the genus Physarum, and vice versa (Lado 2001). These transfers were often based on the morphology of capillitium of these genera, as either called “Badhamioid” or “Physaroid” capillitium (Martin and Alexopoulos 1969; Keller and Braun 1999). In fact, there is a lot of discussion about the taxonomic treatment of several nivicolous species of Badhamia and Physarum (e.g., Meylan 1925; Kowalski 1975; Singer et al. 2004; Poulain et al. 2011; Ronikier and Lado 2013). Molecular phylogeny shows a polyphyletic origin of Badhamia with Physarum that does not support separation based on capilltium of these two genera, and reevaluation of the current taxonomy is required (FioreDonno et al. 2009; Nandipati et al. 2012). The capillitium of our specimen of B. alpina was composed of slender threads and narrow flat plates under SEM, and electron translucent round material was observed in narrow flat plates by TEM. The specimen studied here could be considered physaroid, and a possible species of Physarum. However, Keller and Schoknecht (1989) mentioned that fructifications developed under laboratory conditions sometimes lead to premature drying with suppression of calcium carbonate formation in the capillitium based on their spore-to-spore culture of Physarum spinisporum Eliasson & N. Lundq., and they transferred the species to Badhamia. They also mentioned that the premature drying of fruiting bodies can cause spore mass aggregation. Since some fruiting bodies of our specimen were hard, and SEM observations on fruiting bodies showed a wrinkled peridium and somewhat aggregated spores, the specimen may have represented a “premature” drying of fruiting bodies. Our observation suggests that the difference between Badhamia and Physarum is ambiguous, and to evaluate the taxonomy of these genera more detailed research is needed on the cellular behavior of capillitial formation and its maturation.

Acknowledgments We thank Prof. Hideki Takahashi and Dr. Takahito Kobayashi (The Hokkaido University Museum), for their valuable help to deposit and examine the specimen.

references

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Please cite this article in press as: Yajima Y, et al., Fruiting body formation of the nivicolous myxomycete Badhamia alpina in moist chamber culture, Mycoscience (2017), http://dx.doi.org/10.1016/j.myc.2017.04.001