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Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701
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ABSTRACT |
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Protostelids and myxomycetes have been isolated from dead plant parts in many different habitats, including tropical rain forests and deserts. However underwater habitats largely have been overlooked. The purpose of this study was to determine whether protostelids do occur in aquatic habitats and to survey the myxomycetes associated with these habitats. Protostelids and myxomycetes were isolated from substrates collected from just above and just below the surface of the water. Several species of both groups were present, and their distributions above and below the water were different. It is not surprising that the trophic cells of slime molds occur in ponds because they are known to grow in films of water. However these findings are significant because this is the first study to demonstrate clearly the occurrence of protostelids in underwater environments and one of the few surveys of myxomycetes from aquatic systems.
Key words: Eumycetozoa, freshwater, fruiting amoebae, Mycetozoa, ponds, streams
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) because their dormant stages, spores and cysts are resistant. However their amoeboid trophic cells must live in a film of liquid water even though it might be temporary in arid environments. Because amoebae are, in effect, aquatic, we thought mycetozoans might be present at the other extreme in wetlands, ponds and lakes. It is possible that these mycetozoans could live for long periods of time in aquatic habitats, resorting to sporulation when the body of water dries or after migrating to substrates at the edge of the water.
To date there have been only a few reports of "aquatic myxomycetes." One recent paper (Kappel and Anken 1992) reports that a plasmodium was found growing on the inside of an aquarium. The plasmodium was placed in culture where it formed the fruiting bodies of a long-stalked Didymium, which the authors identified as Didymium nigripes. An earlier study describes the "nutrition of some aquatic myxomycetes" identified as Physarum gyrosum, Physarum nutans and Fuligo cinerea (Parker 1946). These organisms reportedly were grown submerged in water until the fruiting stages. Myxomycetes have been isolated and identified from substrates submerged in swamps (Shearer and Crane 1986), and an early report of an aquatic myxomycete describes the growth of a strain of Didymium difforme on the roots of Hyacinths being cultured in nutritive solutions (Ward 1886). This myxomycete repeatedly completed its life cycle entirely underwater. In addition Didymium aquatile collected in Brazil is described as living submerged as a phaneroplasmodium until the moment of fructification (Gottsberger and Nannenga-Bremekamp 1971). We are not aware of any published reports of protostelids cultured from submerged substrates. However Tychosporium acutostipes and Schizoplasmodiopsis vulgare have grown from substrates collected from wetlands (Spiegel unpubl obs).
The purpose of this study was to determine whether protostelids could be isolated from submerged substrates and to obtain additional data on myxomycetes in aquatic environments.
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MATERIALS AND METHODS |
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Collection sites.— –
Five collection sites were chosen in northwestern Arkansas and northeastern Oklahoma (TABLE I). Sites 1 (Cincinnati, Arkansas) and 2 (Muskogee, Oklahoma) are farm ponds. Site 3 is near the boat ramp at Carter’s Landing on Tenkiller Ferry Lake, Oklahoma. Sites 4 and 5 are located in Honor Heights Park in Muskogee, Oklahoma. Site 4 is a small (1 m x 1 m x 0.5 m) pond in a rose garden. Site 5 is in the largest pond in Honor Heights Park.
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, Spiegel et al 2004). A small amount of substrate was taken from samples with ethanol-flamed forceps and broken into 6–8 mm pieces, soaked in sterile, distilled water 10 min and plated on weak malt-extract yeast-extract agar (WMY). Approximately eight pieces of substrate were placed on each 100 x 15 mm plate; these pieces were evenly spaced around the plate so that between each there was approximately 1 cm of uninoculated agar. Protostelids observed on the perimeter of one piece that were one species were assumed to be clones. However protostelids on different pieces of substrate on the same plate were considered to be different clones and therefore separate observations. After approximately 10 d the perimeter of each piece was observed under the 10x objective of a compound microscope. Protostelids were identified by characteristics of their fruiting bodies and amoebae. Higher power objectives sometimes were used to aid in the identification of certain patches of fruiting bodies. If a species determination could not be made, then the organism was isolated into monoeukaryotic culture and deposited in the American Type Culture Collection (ATCC).
Myxomycete moist chambers.— –
The remainder of each sample was used to set up moist chambers to observe myxomycetes (Stephenson and Stempen 1994a). Moist chambers were prepared by placing a 90 mm diam filter paper inside a sterile 100 x 15 mm polystyrene Petri plate. Substrates were placed so that plates were as full as possible without overlapping pieces of substrate. Samples then were moistened with deionized water. The sample pH was recorded approximately 24 h later. Plates then were observed under a dissecting microscope every 2 or 3 d for approximately 2 mo. Mature fruiting bodies were observed, collected, identified, placed in small boxes for permanent storage and deposited in the myxomycete herbarium at the University of Arkansas (UARK).
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RESULTS |
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). Altogether we made 153 protostelid observations, 67 observations on Above plates and 86 observations of Below plates (TABLE I). Seven species of protostelids were identified on substrates collected just above the surface of the water (nine primary isolation plates, containing 72 pieces of substrate), and nine species were recorded on samples taken from below the surface (13 primary isolation plates, containing 104 pieces of substrate). The most common species found above the surface was Soliformovum irregularis (FIG. 1), which was present on 25% of the pieces of substrate (FIG. 1). Tychosporium acutostipes was present on 11% of the above water pieces and 2% of below water pieces (FIG. 1). Protostelium nocturnum was found on 10% of above water pieces but not at all on subsurface pieces (FIG. 1). Nematostelium ovatum, Protostelium arachisporum and Echinostelium bisporum were not found on samples taken from above the surface, but these species were present on <7% of pieces collected from below the surface of the water (FIG. 1). The most common species (FIG. 1) found below the surface was Schizoplasmodiopsis vulgare, which was found on 19% of the pieces of substrate (FIG. 1). Schizoplasmodiopsis vulgare, Protostelium mycophaga, Schizoplasmodiopsis pseudoendospora, Nematostelium ovatum, Echinostelium bisporum, and Protostelium arachisporum all occurred in higher percentages on pieces from below the surface. Nematostelium gracile was present on 10% of Above and 9% of Below pieces (FIG. 1). One protostelid culture was established from submerged substrates collected from site five. It was identified as Schizoplasmodiopsis vulgare, designated as strain HHPBPB3604-1 and deposited in the American Type Culture Collection "ATCC PRA-158".
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). No samples from site 5 were plated for myxomycetes. Fourteen species of myxomycetes fruited in the 42 moist chambers (FIG. 2). Twenty samples were from above the surface of the water and 22 from below (FIG. 2). Twelve myxomycete species were present on samples from above the water, and four species were present on samples from below. Two species, Licea biforis and Physarum pusillum, were present both above and below the surface (FIG. 2). There were at least six nonfruiting plasmodia, three on the above water plates and three on below water plates (FIG. 2). Comatricha nigra and Licea kleistobolus were present only below the surface (FIG. 2). Arcyria cinerea, Perichaena chrysosperma, Perichaena cf. depressa, Didymium anellus, Didymium iridis, Didymium ochroideum, Didymium squamulosum, Physarum cf. crateriforme, Physarum cf. cinereum and Licea sp. all fruited only on substrates from above the surface (FIG. 2). A few of the typically sessile Didymium ochroideum fruiting bodies have short stalks, an unusual character state in this typically stalkless species (Martin and Alexopoulos 1969). Some of the specimens of Physarum pusillum have lime with a faint yellow pigmentation. All myxomycete voucher specimens have been deposited in the myxomycete herbarium at the University of Arkansas (UARK).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Some of these amoebae could be trophic cells of mycetozoans.
Naked amoebae (gymnamoebae, amoebae that do not secrete a hard covering or test) (Page 1988) are observed commonly in marine and freshwater habitats. Because it is unlikely that protostelids or myxomycetes would fruit under the conditions that are used to observe and study naked amoebae, it is possible that many mycetozoans and close relatives have been observed in aquatic habitats without being recognized as mycetozoans because of their failure to fruit. Aquatic habitats were found to be both more species rich and biocomplex than terrestrial habitats when surveyed for amoeboid protists (Anderson 2003). Naked amoebae also have been shown to vary in abundance because of localized nutrient enrichment, particle size, earthworm activity and season (Anderson 2003, Anderson 2000, Bass and Bischoff 2001). Naked amoebae from terrestrial environments have been shown to increase dramatically in numbers in response to moist conditions, which suggests that a truly aquatic environment might be suitable for their growth and reproduction, and some species are reported to be both aquatic and terrestrial (Anderson 2000). This also might be true for some eumycetozoan amoebae.
These results do not show absolutely that trophic cells of protostelids and myxomycetes were actively growing in aquatic habitats. However several lines of evidence show that these eumycetozoans will grow submerged. As mentioned above there are a few reports of submerged myxomycete plasmodia (Gottsberger and Nannenga-Bremekamp 1971, Kappel and Anken 1992, Parker 1946, Stephenson and Stempen 1994a, Ward 1886). Also myxomycetes have been isolated from the air into liquid culture (Miller 1898). In this case fruiting was induced reliably by the addition of partially submerged solid support (sterilized hay) (Miller 1898). Protostelids have regularly been observed to grow submerged in films of water, then to penetrate the surface film to fruit in both primary isolation plates and in culture (Olive 1975, Spiegel unpubl obs). Protostelid and myxomycete trophic cells grow readily in liquid culture (Olive 1975, Spiegel 1982, Miller 1898). It could be suggested that the eumycetozoans we observed from submerged substrates might have come from airborne propagules that were floating on the film or from aerial contamination as the substrates were brought out of the water. Because it is likely that such propagules would be dilute in nature, it would be expected that, although present, these mycetozoans would be much less abundant in the plates of substrates collected from below the water. We observed no obviously lower abundance of myxomycetes and protostelids on plates of substrates from below the water when compared with those from above.
Myxomycetes and protostelids might differ in their likelihood to exploit the aquatic environment. Although our sample sizes were small, fruiting bodies of myxomycetes were more common on samples collected from just above the surface of the water, which could indicate that many species do not generally exploit the submerged habitat. However they might be present in aquatic environments as populations of amoeboflagellates that do not progress through the life cycle under our culture conditions. In fact eumycetozoan trophic cells have been isolated repeatedly from aquatic environments and erroneously described as novel organisms despite the overwhelming ultrastructural and molecular phylogenetic evidence showing that these are myxomycete and/or protostelid amoeboflagellates (Rolf et al 2003, Walker et al 2003, Walochnik et al 2004). Protostelids on the other hand do not appear to be depauperate in freshwater habitats, but our data do suggest that the amoebae of some species might exploit this habitat more than others. For instance the high numbers of Schizoplasmodiopsis vulgare from submerged substrates suggest that this species might be well suited to aquatic environments (FIG. 1).
In this study some species (Licea kleistobolous and Comatricha nigra) were present only below the water. We attribute this difference to small sample size rather than habitat preference. In addition some protostelid species (Protostelium nocturnum, Endostelium sp., Soliformovum expulsum) probably are underestimated systematically in biogeographic surveys because they often fruit en mass. When grown in monoeukaryotic culture only amoebae are observed during the day, while fruiting bodies often can be observed at night. In this study all the Protostelium nocturnum fruiting bodies were observed early in the morning (on the first few plates checked for the day). It was conspicuously absent from samples checked later in the day.
Based on SSU rRNA sequences, freshwater gymnamoebae are reported to be phylogenetically divergent from marine amoebae of the same or similar species (Sims et al 2002). It would be interesting to make intraspecific comparisons in species of myxomycetes and protostelids that occur on both terrestrial and submerged substrates to see whether they represent large populations or if the aquatic and terrestrial examples are habitat specific.
While it is not surprising that eumycetozoans do inhabit lakes and ponds, few researchers have looked for them in these ecological situations; therefore it is likely that some species have been overlooked or underestimated because of a bias toward thinking of mycetozoans as terrestrial.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: lalindl{at}uark.edu
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LITERATURE CITED |
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