- Email: [email protected]
Nutrient mobilization by plasmodium of myxomycete Physarum rigidum in deadwood
Fungal Ecology 29 (2017) 42e44
Contents lists available at ScienceDirect
Fungal Ecology journal homepage: www.elsevier.com/locate/funeco
Short Communication
Nutrient mobilization by plasmodium of myxomycete Physarum rigidum in deadwood Yu Fukasawa a, b, *, Yasuyuki Komagata a, 1, Shin-ichi Kawakami c a
Laboratory of Forest Ecology, Graduate School of Agricultural Science, Tohoku University, 232-3 Yomogida, Naruko, Osaki, Miyagi 989-6711, Japan School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK c Yamagata Prefectural Museum, 1-8 Kajo-machi, Yamagata-shi, Yamagata 990-0826, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2016 Received in revised form 8 May 2017 Accepted 26 May 2017
To assess the nutrient mobilization ability of myxomycete plasmodia in deadwood, a microcosm experiment was conducted. Alive or dead plasmodia of a common lignicolous myxomycete, Physarum rigidum, were inoculated on field-collected crushed wood powder of white-rot or brown-rot pine wood and incubated for 24 d. The activity of living plasmodia led to increased concentrations of Ca2þ, Kþ, Mg2þ, Naþ, and Cl, but lower PO3e 4 concentrations in the wood powder. For NO3 concentration, the effect of living plasmodia was negative in white-rotted wood but positive in brown-rotted wood. These results suggest that the plasmodium of P. rigidum has the ability to mobilize nutrients in deadwood. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
Corresponding Editor: Martin H. Schnittler Keywords: Calcium Magnesium Mineralization Potassium Slime mold Sodium
1. Introduction The decomposition of dead plant tissue is an essential process that drives nutrient cycling in terrestrial ecosystems (Swift et al., 1979). Although the process is primarily driven by fungi and bacteria, it is becoming increasingly apparent that fauna in the detritus food web plays an important role in nutrient mobilization (de Vries et al., 2013). Previous studies found that amoebae (protozoa) have a € ter et al., central role in nutrient mobilization in forests (Schro 2003). Many field and microcosm studies used ciliates as bacterivorous protozoa to evaluate their nutrient mobilizing abilities (Griffiths, 1986). However, the nutrient mobilization abilities of other protozoa groups are poorly known. Myxomycetes are a group of amoebae with a ubiquitous distribution worldwide (Stephenson et al., 2011), and their abundance
among soil amoebae exceeds 50% (Feest and Madelin, 1988). Their life cycle encompasses two very different trophic stages: microscopic unicellular amoeba and macroscopic giant multinucleate plasmodia (Stephenson et al., 2011). Bacteria apparently represent the main food resource for both trophic stages, but plasmodia are also known to feed on fungi and algae (Stephenson et al., 2011). In the present study, we hypothesized that a myxomycete plasmodium, which feeds on bacteria and fungi, has a potential role in nutrient mobilization. To test this hypothesis, we conducted a microcosm experiment using a cultured strain of Physarum rigidum, a common lignicolous myxomycete species in Japan (Takahashi et al., 2009), on deadwood. We used deadwood as the substrate because its low nutrient concentration (Rayner and Boddy, 1988) would make nutrient mobilization by myxomycetes more critical than in nutrient rich soil. 2. Materials and methods
* Corresponding author. Laboratory of Forest Ecology, Graduate School of Agricultural Science, Tohoku University, 232-3 Yomogida, Naruko, Osaki, Miyagi 9896711, Japan. E-mail address: [email protected] (Y. Fukasawa). 1 Present address: Laboratory of Insect Ecology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyoku, Kyoto 606e8502, Japan. http://dx.doi.org/10.1016/j.funeco.2017.05.005 1754-5048/© 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
A plasmodium of P. rigidum was collected from a polypore sporocarp on a snag at Mt. Ishizuchi, Ehime Pref., Japan (33790 N 133120 E, altitude 1450 m) on October 10, 2010. It was maintained on plain agar (15 g L1, Nacalai, Osaka, Japan) with autoclaved
Y. Fukasawa et al. / Fungal Ecology 29 (2017) 42e44 Table 1 The estimated parameters of the generalized linear model for nutrient concentrations. Nutrient
Parameter estimated 1
Cation Ca2þ Kþ Mg2þ Naþ NHþ 4 Anion Br Cl F NO 2 NO 3 PO3e 4 2e SO4 1
Generalized linear model.
þ
Decay type
Plasmodium
0.21*** 0.40*** 0.05*** 0.06þ 0.002
0.06*** 0.07* 0.01*** 0.07* 0.02
0.002 0.11* 0.01 0.47*** 0.01* 0.04** 0.001
0.01 0.08* 0.004 0.09 0.01* 0.04*** 0.005þ
P < 0.1; *P < 0.05; **P < 0.01; ***P < 0.001.
oatmeal grains at 20 C in darkness until the start of the experiment. No fungal contamination was detected by PCR amplification with primer pair ITS1F/ITS4, but bacteria were detected with primer pair Eubac27F/1492R on the DNA extracted from this plasmodium. Well-decayed pinewood (decay class V, Fukasawa, 2012) was collected at a secondary forest dominated by oak (Quercus serrata) and pine (Pinus densiflora) in Mt. Chitose in Yamagata Pref., northern Japan (38 140 N 140 210 E, altitude 245 m) and was used as the microcosm substrate. Because previous studies suggested that wood decay type has a noticeable effect on microbial communities in deadwood (Fukasawa et al., 2015), we collected two types of deadwood: white- and brown-rotted, distinguished by the macroscopic criteria of Araya (1993). Decayed wood was collected
43
from three different logs for each decay type, and was crushed to pass through a 6 mm screen using a Retsch® SM 300 cutting mill (Verder Scientific Co. Ltd., Germany) and mixed to avoid effects of log-specific differences in microbial communities. The substrates were not sterilized because the aim of the present study was to assess the influence of plasmodium on ion concentrations in the presence of the natural deadwood microbial community. A clear polypropylene case (7.2 7.2 10 cm) (Incu Tissue; SPL Life Sciences, Korea) was filled with approximately 80 mL wood powder (water content, 80% fresh weight base) and capped to make a microcosm. An agar plug (approximately 10 10 5 mm) with a plasmodium of P. rigidum was deposited on the surface of the wood powder in the case. Half of the plugs were frozen at 30 C for more than 2 weeks to kill the plasmodia before placing it into the microcosm. In total, we prepared four experimental categories (two decay types, with dead or alive plasmodium), with five replications. Microcosms were incubated at 20 C in darkness for 24 d until all the plasmodia had become invisible from outside. After the incubation, all wood powder was retrieved, and extracted with 200 mL deionized water in a 250 mL polyethylene bottle by gently shaking for 1 h. Then, the filtrate was used to 2þ 2þ determine the cation (Naþ, Kþ, NHþ 4 , Mg , Ca ) and anion (F , Cl , 2 3 NO , Br , NO , SO , PO ) concentrations of the wood powder, 2 3 4 4 using an ion chromatography system (ICS-1000/2000, Dionex, CA, USA). The ion concentrations were compared among the four experimental categories using a Steel-Dwass test and the effects of plasmodium and wood decay type on the concentrations were tested by generalized linear model (GLM). All the statistical tests were conducted using R version 3.2.1 (R core team, 2015). 3. Results After 24 d, some of the living plasmodia became sclerotia (not recorded), but no fructifications were observed in any of the
Fig. 1. Nutrient ion concentrations in wood powder inoculated with living or dead plasmodia of Physarum rigidum. Box plots display median, 95% confidence interval, and minimum and maximum values and points outside the box plots represent outliers. Different letters indicate significant difference among the four experimental categories (P < 0.05, SteelDwass test).
44
Y. Fukasawa et al. / Fungal Ecology 29 (2017) 42e44
microcosms. Frozen plasmodia disappeared without any evidence of survival structures (like sclerotia). Living plasmodia of P. rigidum significantly increased Ca2þ, Kþ, Mg2þ, Naþ and Clconcentrations in the wood powder, although it reduced PO3e 4 concentration in white-rot wood (Table 1). Living plasmodia increased Cl concentration in white-rot wood, but not in brown-rot wood (Fig. 1). For NO 3 concentrations, the effect of living plasmodium was negative in white-rot wood but positive in brown-rot wood. There were no 2 significant effects of alive plasmodium on NHþ 4 , Br , F , NO2 , SO4 concentrations.
analysis, and to Willow Smallbone for her help in making graphics by R. We also thank two anonymous reviewers and the handling editor, Martin Schnittler, for their valuable comments that greatly improved the draft manuscript. This work was supported by the Institute for Fermentation, Osaka.
References 4. Discussion Feest and Madelin (1985, 1988) reported that the abundance of myxomycete plasmodia in soil positively correlated with soil Mg and K levels and negatively with P level. In addition to their findings, results of the present study suggest that increase in Mg and K availability and a reduction of P in the substrate is attributable to the activity of the plasmodium. It is known that Ca is important for the protoplasmic streaming of a plasmodium (Ridgway and Durham, 1976), and thus, an active plasmodium has a rich content of oscillating Ca (Gustafson and Thurston, 1974), which may affect the ambient Ca concentrations. Gray and Alexopoulos (1968) also reported that K and Na concentrations fluctuate when a plasmodium is migrating. In contrast, P is necessary for constructing adenosine triphosphate to promote protoplasmic streaming (Gray and Alexopoulos, 1968), and thus, may be absorbed by the plasmodium from the environment. On the other hand, plasmodium of P. rigidum did not show any apparent mobilization ability for nitrogen in this study. These results suggest that the feature of nutrient mobilization by myxomycete plasmodium may be different from that of other protozoa, such as ciliates, which are known to be primarily effective in €ter et al., 2003). Although the abundance mobilizing nitrogen (Schro of plasmodium, rather than microscopic amoebae, in deadwood is still in debate (Taylor et al., 2015), our preliminary results showed the unique abilities of myxomycete plasmodium in nutrient mobilization with their bacterial associates. Acknowledgments We are grateful to Kanako Kimura for ion chromatography
Araya, K., 1993. Relationship between the decay types of dead wood and occurrence of Lucanid beetles (coleoptera: lucanidae). Appl. Entomol. Zool. 28, 27e33. bault, E., Liirie, M., Birkhofer, K., Tsiafouli, M.A., Bjørnlund, L., de Vries, F.T., The Jørgensen, H.B., Brady, M.V., Christensen, S., de Ruiter, P.C., d’Hertefeldt, T., Frouz, J., Hedlund, K., Hemerik, L., Gera Hol, W.H., Hotes, S., Mortimer, S.R., Set€ al€ a, H., Sgardelis, S.P., Uteseny, K., van der Putten, W.H., Wolters, V., Bardgett, R.D., 2013. Soil food web properties explain ecosystem services across European land use systems. PNAS 110, 14296e14301. Feest, A., Madelin, M.F., 1985. Numerical abundance of myxomycetes (myxogastrids) in soils in the West of England. FEMS Microbiol. Ecol. 1, 353e360. Feest, A., Madelin, M.F., 1988. Seasonal population changes of myxomycetes and associated organisms in five non-woodland soils, and correlations between their numbers and soil characteristics. FEMS Microbiol. Ecol. 4, 141e152. Fukasawa, Y., 2012. Effects of wood decomposer fungi on tree seedling establishment on coarse woody debris. For. Ecol. Manage 266, 232e238. Fukasawa, Y., Takahashi, K., Arikawa, T., Hattori, T., Maekawa, N., 2015. Fungal wood decomposer activities influence community structures of myxomycetes and bryophytes on coarse woody debris. Fung. Ecol. 14, 44e52. Gray, W.D., Alexopoulos, C.J., 1968. Biology of the Myxomycetes. The Ronald Press Company, New York. Griffiths, B.S., 1986. Mineralization of nitrogen and phosphorus by mixed cultures of the ciliate protozoan Colpoda steinii, the nematode Rhabditis sp. and the bacterium Pseudomonas flourescens. Soil Biol. Biochem. 18, 637e641. Gustafson, R.A., Thurston, E.L., 1974. Calcium deposition in the myxomycete Didymium squamulosum. Mycologia 66, 397e412. R core team, 2015. R: a Language and Environment for Statistical Computing R Foundation for Statistical Computing. Vienna, Austria. Rayner, A.D.M., Boddy, L., 1988. Fungal Decomposition of Wood (Wiley, Chichester). Ridgway, E.B., Durham, A.C.H., 1976. Oscillations of calcium ion concentrations in Physarum polycephalum. J. Cell Biol. 69, 223e226. €ter, D., Wolters, V., de Ruiter, P.C., 2003. C and N mineralisation in the Schro decomposer food webs of a European forest transect. Oikos 102, 294e308. Stephenson, S.L., Fiore-Donno, A.M., Schnittler, M., 2011. Myxomycetes in soil. Soil Biol. Biochem. 43, 2237e2242. Swift, M.J., Heal, O.W., Anderson, J.M., 1979. Decomposition in Terrestrial Ecosystems (Blackwell, Oxford). Takahashi, K., Hada, Y., Mitchell, D.W., 2009. Substrate preference of lignicolous myxomycetes relative to wood types in temperate Japanese forests. Hikobia 15, 287e298. Taylor, K.M., Feest, A., Stephenson, S.L., 2015. The occurrence of myxomycetes in wood. Fung. Ecol. 17, 179e182.
Contents lists available at ScienceDirect
Fungal Ecology journal homepage: www.elsevier.com/locate/funeco
Short Communication
Nutrient mobilization by plasmodium of myxomycete Physarum rigidum in deadwood Yu Fukasawa a, b, *, Yasuyuki Komagata a, 1, Shin-ichi Kawakami c a
Laboratory of Forest Ecology, Graduate School of Agricultural Science, Tohoku University, 232-3 Yomogida, Naruko, Osaki, Miyagi 989-6711, Japan School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK c Yamagata Prefectural Museum, 1-8 Kajo-machi, Yamagata-shi, Yamagata 990-0826, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2016 Received in revised form 8 May 2017 Accepted 26 May 2017
To assess the nutrient mobilization ability of myxomycete plasmodia in deadwood, a microcosm experiment was conducted. Alive or dead plasmodia of a common lignicolous myxomycete, Physarum rigidum, were inoculated on field-collected crushed wood powder of white-rot or brown-rot pine wood and incubated for 24 d. The activity of living plasmodia led to increased concentrations of Ca2þ, Kþ, Mg2þ, Naþ, and Cl, but lower PO3e 4 concentrations in the wood powder. For NO3 concentration, the effect of living plasmodia was negative in white-rotted wood but positive in brown-rotted wood. These results suggest that the plasmodium of P. rigidum has the ability to mobilize nutrients in deadwood. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
Corresponding Editor: Martin H. Schnittler Keywords: Calcium Magnesium Mineralization Potassium Slime mold Sodium
1. Introduction The decomposition of dead plant tissue is an essential process that drives nutrient cycling in terrestrial ecosystems (Swift et al., 1979). Although the process is primarily driven by fungi and bacteria, it is becoming increasingly apparent that fauna in the detritus food web plays an important role in nutrient mobilization (de Vries et al., 2013). Previous studies found that amoebae (protozoa) have a € ter et al., central role in nutrient mobilization in forests (Schro 2003). Many field and microcosm studies used ciliates as bacterivorous protozoa to evaluate their nutrient mobilizing abilities (Griffiths, 1986). However, the nutrient mobilization abilities of other protozoa groups are poorly known. Myxomycetes are a group of amoebae with a ubiquitous distribution worldwide (Stephenson et al., 2011), and their abundance
among soil amoebae exceeds 50% (Feest and Madelin, 1988). Their life cycle encompasses two very different trophic stages: microscopic unicellular amoeba and macroscopic giant multinucleate plasmodia (Stephenson et al., 2011). Bacteria apparently represent the main food resource for both trophic stages, but plasmodia are also known to feed on fungi and algae (Stephenson et al., 2011). In the present study, we hypothesized that a myxomycete plasmodium, which feeds on bacteria and fungi, has a potential role in nutrient mobilization. To test this hypothesis, we conducted a microcosm experiment using a cultured strain of Physarum rigidum, a common lignicolous myxomycete species in Japan (Takahashi et al., 2009), on deadwood. We used deadwood as the substrate because its low nutrient concentration (Rayner and Boddy, 1988) would make nutrient mobilization by myxomycetes more critical than in nutrient rich soil. 2. Materials and methods
* Corresponding author. Laboratory of Forest Ecology, Graduate School of Agricultural Science, Tohoku University, 232-3 Yomogida, Naruko, Osaki, Miyagi 9896711, Japan. E-mail address: [email protected] (Y. Fukasawa). 1 Present address: Laboratory of Insect Ecology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyoku, Kyoto 606e8502, Japan. http://dx.doi.org/10.1016/j.funeco.2017.05.005 1754-5048/© 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
A plasmodium of P. rigidum was collected from a polypore sporocarp on a snag at Mt. Ishizuchi, Ehime Pref., Japan (33790 N 133120 E, altitude 1450 m) on October 10, 2010. It was maintained on plain agar (15 g L1, Nacalai, Osaka, Japan) with autoclaved
Y. Fukasawa et al. / Fungal Ecology 29 (2017) 42e44 Table 1 The estimated parameters of the generalized linear model for nutrient concentrations. Nutrient
Parameter estimated 1
Cation Ca2þ Kþ Mg2þ Naþ NHþ 4 Anion Br Cl F NO 2 NO 3 PO3e 4 2e SO4 1
Generalized linear model.
þ
Decay type
Plasmodium
0.21*** 0.40*** 0.05*** 0.06þ 0.002
0.06*** 0.07* 0.01*** 0.07* 0.02
0.002 0.11* 0.01 0.47*** 0.01* 0.04** 0.001
0.01 0.08* 0.004 0.09 0.01* 0.04*** 0.005þ
P < 0.1; *P < 0.05; **P < 0.01; ***P < 0.001.
oatmeal grains at 20 C in darkness until the start of the experiment. No fungal contamination was detected by PCR amplification with primer pair ITS1F/ITS4, but bacteria were detected with primer pair Eubac27F/1492R on the DNA extracted from this plasmodium. Well-decayed pinewood (decay class V, Fukasawa, 2012) was collected at a secondary forest dominated by oak (Quercus serrata) and pine (Pinus densiflora) in Mt. Chitose in Yamagata Pref., northern Japan (38 140 N 140 210 E, altitude 245 m) and was used as the microcosm substrate. Because previous studies suggested that wood decay type has a noticeable effect on microbial communities in deadwood (Fukasawa et al., 2015), we collected two types of deadwood: white- and brown-rotted, distinguished by the macroscopic criteria of Araya (1993). Decayed wood was collected
43
from three different logs for each decay type, and was crushed to pass through a 6 mm screen using a Retsch® SM 300 cutting mill (Verder Scientific Co. Ltd., Germany) and mixed to avoid effects of log-specific differences in microbial communities. The substrates were not sterilized because the aim of the present study was to assess the influence of plasmodium on ion concentrations in the presence of the natural deadwood microbial community. A clear polypropylene case (7.2 7.2 10 cm) (Incu Tissue; SPL Life Sciences, Korea) was filled with approximately 80 mL wood powder (water content, 80% fresh weight base) and capped to make a microcosm. An agar plug (approximately 10 10 5 mm) with a plasmodium of P. rigidum was deposited on the surface of the wood powder in the case. Half of the plugs were frozen at 30 C for more than 2 weeks to kill the plasmodia before placing it into the microcosm. In total, we prepared four experimental categories (two decay types, with dead or alive plasmodium), with five replications. Microcosms were incubated at 20 C in darkness for 24 d until all the plasmodia had become invisible from outside. After the incubation, all wood powder was retrieved, and extracted with 200 mL deionized water in a 250 mL polyethylene bottle by gently shaking for 1 h. Then, the filtrate was used to 2þ 2þ determine the cation (Naþ, Kþ, NHþ 4 , Mg , Ca ) and anion (F , Cl , 2 3 NO , Br , NO , SO , PO ) concentrations of the wood powder, 2 3 4 4 using an ion chromatography system (ICS-1000/2000, Dionex, CA, USA). The ion concentrations were compared among the four experimental categories using a Steel-Dwass test and the effects of plasmodium and wood decay type on the concentrations were tested by generalized linear model (GLM). All the statistical tests were conducted using R version 3.2.1 (R core team, 2015). 3. Results After 24 d, some of the living plasmodia became sclerotia (not recorded), but no fructifications were observed in any of the
Fig. 1. Nutrient ion concentrations in wood powder inoculated with living or dead plasmodia of Physarum rigidum. Box plots display median, 95% confidence interval, and minimum and maximum values and points outside the box plots represent outliers. Different letters indicate significant difference among the four experimental categories (P < 0.05, SteelDwass test).
44
Y. Fukasawa et al. / Fungal Ecology 29 (2017) 42e44
microcosms. Frozen plasmodia disappeared without any evidence of survival structures (like sclerotia). Living plasmodia of P. rigidum significantly increased Ca2þ, Kþ, Mg2þ, Naþ and Clconcentrations in the wood powder, although it reduced PO3e 4 concentration in white-rot wood (Table 1). Living plasmodia increased Cl concentration in white-rot wood, but not in brown-rot wood (Fig. 1). For NO 3 concentrations, the effect of living plasmodium was negative in white-rot wood but positive in brown-rot wood. There were no 2 significant effects of alive plasmodium on NHþ 4 , Br , F , NO2 , SO4 concentrations.
analysis, and to Willow Smallbone for her help in making graphics by R. We also thank two anonymous reviewers and the handling editor, Martin Schnittler, for their valuable comments that greatly improved the draft manuscript. This work was supported by the Institute for Fermentation, Osaka.
References 4. Discussion Feest and Madelin (1985, 1988) reported that the abundance of myxomycete plasmodia in soil positively correlated with soil Mg and K levels and negatively with P level. In addition to their findings, results of the present study suggest that increase in Mg and K availability and a reduction of P in the substrate is attributable to the activity of the plasmodium. It is known that Ca is important for the protoplasmic streaming of a plasmodium (Ridgway and Durham, 1976), and thus, an active plasmodium has a rich content of oscillating Ca (Gustafson and Thurston, 1974), which may affect the ambient Ca concentrations. Gray and Alexopoulos (1968) also reported that K and Na concentrations fluctuate when a plasmodium is migrating. In contrast, P is necessary for constructing adenosine triphosphate to promote protoplasmic streaming (Gray and Alexopoulos, 1968), and thus, may be absorbed by the plasmodium from the environment. On the other hand, plasmodium of P. rigidum did not show any apparent mobilization ability for nitrogen in this study. These results suggest that the feature of nutrient mobilization by myxomycete plasmodium may be different from that of other protozoa, such as ciliates, which are known to be primarily effective in €ter et al., 2003). Although the abundance mobilizing nitrogen (Schro of plasmodium, rather than microscopic amoebae, in deadwood is still in debate (Taylor et al., 2015), our preliminary results showed the unique abilities of myxomycete plasmodium in nutrient mobilization with their bacterial associates. Acknowledgments We are grateful to Kanako Kimura for ion chromatography
Araya, K., 1993. Relationship between the decay types of dead wood and occurrence of Lucanid beetles (coleoptera: lucanidae). Appl. Entomol. Zool. 28, 27e33. bault, E., Liirie, M., Birkhofer, K., Tsiafouli, M.A., Bjørnlund, L., de Vries, F.T., The Jørgensen, H.B., Brady, M.V., Christensen, S., de Ruiter, P.C., d’Hertefeldt, T., Frouz, J., Hedlund, K., Hemerik, L., Gera Hol, W.H., Hotes, S., Mortimer, S.R., Set€ al€ a, H., Sgardelis, S.P., Uteseny, K., van der Putten, W.H., Wolters, V., Bardgett, R.D., 2013. Soil food web properties explain ecosystem services across European land use systems. PNAS 110, 14296e14301. Feest, A., Madelin, M.F., 1985. Numerical abundance of myxomycetes (myxogastrids) in soils in the West of England. FEMS Microbiol. Ecol. 1, 353e360. Feest, A., Madelin, M.F., 1988. Seasonal population changes of myxomycetes and associated organisms in five non-woodland soils, and correlations between their numbers and soil characteristics. FEMS Microbiol. Ecol. 4, 141e152. Fukasawa, Y., 2012. Effects of wood decomposer fungi on tree seedling establishment on coarse woody debris. For. Ecol. Manage 266, 232e238. Fukasawa, Y., Takahashi, K., Arikawa, T., Hattori, T., Maekawa, N., 2015. Fungal wood decomposer activities influence community structures of myxomycetes and bryophytes on coarse woody debris. Fung. Ecol. 14, 44e52. Gray, W.D., Alexopoulos, C.J., 1968. Biology of the Myxomycetes. The Ronald Press Company, New York. Griffiths, B.S., 1986. Mineralization of nitrogen and phosphorus by mixed cultures of the ciliate protozoan Colpoda steinii, the nematode Rhabditis sp. and the bacterium Pseudomonas flourescens. Soil Biol. Biochem. 18, 637e641. Gustafson, R.A., Thurston, E.L., 1974. Calcium deposition in the myxomycete Didymium squamulosum. Mycologia 66, 397e412. R core team, 2015. R: a Language and Environment for Statistical Computing R Foundation for Statistical Computing. Vienna, Austria. Rayner, A.D.M., Boddy, L., 1988. Fungal Decomposition of Wood (Wiley, Chichester). Ridgway, E.B., Durham, A.C.H., 1976. Oscillations of calcium ion concentrations in Physarum polycephalum. J. Cell Biol. 69, 223e226. €ter, D., Wolters, V., de Ruiter, P.C., 2003. C and N mineralisation in the Schro decomposer food webs of a European forest transect. Oikos 102, 294e308. Stephenson, S.L., Fiore-Donno, A.M., Schnittler, M., 2011. Myxomycetes in soil. Soil Biol. Biochem. 43, 2237e2242. Swift, M.J., Heal, O.W., Anderson, J.M., 1979. Decomposition in Terrestrial Ecosystems (Blackwell, Oxford). Takahashi, K., Hada, Y., Mitchell, D.W., 2009. Substrate preference of lignicolous myxomycetes relative to wood types in temperate Japanese forests. Hikobia 15, 287e298. Taylor, K.M., Feest, A., Stephenson, S.L., 2015. The occurrence of myxomycetes in wood. Fung. Ecol. 17, 179e182.