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Mycological Progress 4(4): 267–271, November 2005
Protostelids from German Beech forests Jens TESMER1, Björn RULIK2, Frederick W. SPIEGEL3, John SHADWICK3, Martin SCHNITTLER1,*
A survey for protostelids from old-growth beech forests of northeastern Germany resulted in 14 species found in both ground and aerial litter, constituting the first survey of these organisms from Central Europe. Additionally the myxomycete Echinostelium bisporum was recorded as new for Germany. A detailed investigation of randomly sampled dead Fagus leaves (16 leaves on ground, 16 aerial leaves, three replicate cultures per leaf) resulted in 7 species; a hyperbolic regression of the respective species accumulation curve gives the theoretical value of 8.3 species. Protostelids occurred especially at aerial leaves with a high frequency (0.94). A program simulating random spore hits was written to calculate the minimum spore fallout necessary to explain this frequency. The resulting average value of about 3 spores per leaf can well be matched by the potential spore productivity estimated to be about 1000 spores per leaf for the most common species, Protostelium mycophaga. Key words: canopy, dispersal abilities, diversity, Myxomycetes
D
iscovered in the early 1960s (OLIVE 1962, 1975), the Protostelia long escaped the attention of mycologists due to their microscopic fructifications, consisting of one to a few spores of 5–40 µm diameter on a delicate 5 to 150 µm long acellular stalk. The 37 described and some undescribed species that have been observed (SPIEGEL 1989; SPIEGEL, LEE & RUSK 1995; SPIEGEL 1998; SPIEGEL et al. 2004) inhabit decaying plant material and develop from mastigote or amastigote amoebae or tiny, multinucleated plasmodia (SPIEGEL 1990). Their trophic cells are quite diverse (SPIEGEL & FELDMAN 1985; SPIEGEL 1990, 1998; SPIEGEL et al. 2004). Nevertheless, diagnostic characters lie mostly in the fructification, e.g., length and thickness of the stalk and stalk tip, spore attachment and spore shape (OLIVE 1975; SPIEGEL 1990; SPIEGEL, SHADWICK & LINDLEY-SETTLEMYRE 2004). The best known protostelid to mycologists is the genus Ceratiomyxa, often classified with the myxomycetes because it forms a large compound fructification. In contrast, the tiniest species of Echinostelium, a myxomycete genus, can hardly be found applying the usual survey methods for plasmodial slime moulds and has long been treated as a protostelid (OLIVE 1975).
In spite of their simple, single-spored fructifications, protostelids seem to be surprisingly abundant in both ground and aerial litter, as shown by a number of studies from the northeastern and central US (BEST & SPIEGEL 1984; MOORE & SPIEGEL 1995, 2000a, 2000c) and the Tropics (STEPHENSON & MOORE 1998; STEPHENSON, LANDOLT & MOORE 1999; MOORE & SPIEGEL 2000b). Although some species seem to have a preference for certain substrata, they are mostly opportunists, able to develop on a broad spectrum of substrata reaching from dead aerial parts of plants, litter, bark of living trees, bark and wood of dead trees and logs, soil and herbivore dung (OLIVE 1975; BEST & SPIEGEL 1984; MOORE & SPIEGEL 1995, 2000a). During a workshop held in August 2004 in Greifswald, protostelids were seen to be abundant in old-growth beech forests. This study presents a more systematically collected species list for old-growth beech forests, the prevailing forest type of the natural vegetation of Central Europe (BOHN et al. 2003).
Methods Study sites
1
Universität Greifswald, Botanisches Institut und Botanischer Garten, Grimmer Str. 88, D-17487 Greifswald, Germany 2 Staatliche Naturhistorische Sammlungen Dresden, Museum fuer Tierkunde, Koenigsbruecker Landstrasse 159, D-01109 Dresden, Germany 3 Department of Biological Sciences, SCEN 632, University of Arkansas, Fayetteville, AR 72701, USA * corresponding author, email:
[email protected], Fax. +49 3834 864114
Seven pockets of three different old-growth beech (Fagus sylvatica) forests of West-Pomerania (lowlands of northeastern Germany) were sampled for protostelids in 2004, all showing a beech coverage of at least 60 % with a substantial proportion of beech trunks exceeding 40 cm dbh. These are 1: Elisenhain, near Greifswald, 300 m SE of parking lot, 54°04’49“ N, 13°26’44“ E, sampled Oct. 7; 1a: same locality in circumference of 250 m, Aug. 9; 2: Elisenhain, near Greifswald, 500 m SE of parking lot, 54°04’48“ N, 13°26’58“ E, Oct. 7; 3: Rügen, near Lauterbach, Isle of Vilm, large Pleistocene is© DGfM 2005
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land core, 54°19’39“ N, 13°32’24“ E, Oct. 22; 4: Rügen, near Lauterbach, Isle of Vilm, slight depression at the island core, 54°19’45“ N, 13°32’25“ E, Oct. 22; 5: Rügen, near Lauterbach, Isle of Vilm, large island core near the sea-facing cliffs, 54°19’25“ N, 13°32’26“ E, Oct. 22; 6: Gristow, near Greifswald, 400 m NNW of the intersection between road to Gristow and road B 96, 54°09’50“ N, 13°18’25“ E, Nov. 6; 7: Elisenhain, near Greifswald, 450 m SE of parking lot, 54°04’49“ N, 13°26’55“ E, Nov. 10.
Sampling The survey was divided in three parts, with a first series of 41 samples from ground and aerial litter of various dead plant parts to cover a maximum diversity of substrata (loc. 1a). For this and the second series collected in the same manner (49 samples, loc. 1-5) about 0.5–2 g dry material per sample was used, and eight pieces per sample of roughly 1 cm2 area each were placed in one Petri dish. A third series consisted exclusively of dead Fagus leaves (loc. 6 and 7). For each locality, 8 fallen but intact leaves were collected from the ground, further 8 dead but still hanging leaves were sampled between 1.5 and 2 m height. Those originated from trunk sprouts of older trees, which tend to keep their foliage until mid-winter. From each leaf, three circular pieces of 6 mm diameter each were punched out from the tip, the middle part and near its base. To stay comparable with the leaves from the lowermost canopy, only the thinner shade leaves of beech were collected from the ground. For the first series only a species list was compiled, for the two others all species were recorded for every piece of substratum, and for two series of 8 Fagus leaves all observed protostelid sporocarps were counted.
Cultures and species’ identification Using sterile tweezers, substratum pieces were placed on a weak malt yeast agar (0.002 g malt extract, 0.002 g yeast extract, 0.75 g potassium hydrogen phosphate, and 15 g agar/L distilled water) poured 3–5 mm deep in Petri dishes of 9 cm diameter. All cultures were maintained at room temperature (22–23 °C). Cultures were checked, beginning five days after being plated out, at least at 6 occasions over 24 days using the 100× and 200× magnification of a compound microscope. The entire margin of a substratum piece and the surrounding agar surface was systematically scanned for the occurrence of protostelid fructifications. For a subset of 48 leaf cores (loc. 7), all visible protostelid fructifications were identified to species and counted to obtain an estimate of spore productivity. To slow down desiccation, these cultures were kept in a humidity chamber for the first 17 days. The pH values for beach leaves was measured in separate cultures on pre-wetted filter paper adjusted with deionized water and KOH to pH 7.0 for each of the 32 leaves investigated. © DGfM 2005
Data analysis For the cultures from the 96 Fagus leaf cores a species accumulation curve was constructed using a program based on the rarefaction formula (SCHNITTLER 2001). A hyperbolic regression according to the formula y = ax/(b+x), resulting in a curve shape coming very close to a broken-stick model (MAGURRAN 1988) was applied to the data, with the parameter a giving an estimate for the maximum number of species to be expected at this kind of substrate. Assuming, that protostelids can reach a hanging Fagus leaf only via air-borne spores, and one spore is sufficient to establish a protostelid population, the minimum spore fallout necessary to explain the frequencies observed was calculated. A program was written which computes the amount of random-generated numbers between 0 and 1 necessary to fall at least once in that many even-spaced lots between 0 and 1 as given by the proportion of positive on the total number of cultured leaves. As an example, for 8 out of 16 leaves tested positive, 16 lots (0, 0.0625, 0.125, 0.1875,…,1) were defined, and the amount of random numbers to fill 8 of those provides an estimate for the number of spores necessary to explain this frequency. This procedure was repeated 1000 times to obtain a reliable mean estimate. To obtain a figure for the average spore fallout per square centimeter, the estimate was divided by the total surface of all investigated leaves as recorded from leaf scans. As a cross check, putative spore productivities per square centimeter were estimated by counting all fructifications from a set of 48 leaf cores, and this number was divided by the total surface of the cores.
Results Species diversity From a total of 720 pieces from 90 samples cultivated to assess the diversity of protostelids in old-growth beech forests, 14 species of protostelids and 1 protostelid-like myxomycete (E. bisporum) were recorded. The following annotated species list indicates after the species’ name the localities and the number of records (in parentheses, except for loc. 1a). Echinostelium bisporum (Olive & Stoian.) K.D. Whitney & Olive, loc. 1 (1 record) Echinosteliopsis oligospora Reinhardt & Olive, loc. 1a Nematostelium gracile (Olive & Stoian.) Olive & Stoian., loc. 2, 4-6 (5 records) Nematostelium ovatum (Olive & Stoian.) Olive & Stoian., loc. 1a, 1, 5-7 (6 records) Protostelium arachisporum Olive, loc. 3 (1 record) Protosteliopsis fimicola (Olive) Olive & Stoian., loc. 1a Protostelium mycophaga Olive & Stoian., loc. 1a, 1, 2, 6, 7 (46 records) Protostelium nocturnum Spiegel, loc. 1a
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Protostelium pyriformis Olive & Stoian., loc. 1a, 1 (1 record) Schizoplasmodiopsis amoeboidea Olive & K.D. Whitney, loc. 1a, 1, 4, 6, 7 (4 records) Schizoplasmodiopsis pseudoendospora Olive, G.W. Martin & Stoian., loc. 1a, 1-6 (22 records) Schizoplasmodiopsis vulgare Olive & Stoian., loc 1a, 1-7 (13 records) Schizoplasmodium cavostelioides Olive & Stoian., loc. 1a, 5 (1 record) Soliformovum irregularis (Olive & Stoian.) Spiegel, loc. 1a, 1, 2, 4-6 (24 records) Tychosporium acutostipes Spiegel, Moore & Feldman, loc. 1a, 1, 5 (2 records) P. mycophaga is the most abundant species on Fagus leaves (39 records); only 7 records came from the first and second series. It exhibits no preference for a special microhabitat. Soliformovum irregularis is abundant as well (24 records in total) but seems to prefer aerial litter. Schizoplasmodiopsis pseudoendospora is similar common (22 records), with the highest frequency recorded from ground litter (Tab. 1, 2).
Ecology All seven species recorded from Fagus leaves (Tab. 2) were found in the first two culture series as well. For both aerial (mean pH 6.21 ± 0.04) and fallen leaves (6.32 ± 0.05) the pH value of the two series did not significantly differ from each other (p = 0.11, t-test). Since all protostelid species for each leaf core were recorded, it was possible to compute a species accumulation curve from the 96 leaf cores of both aerial and ground litter (Fig. 1). All protostelid species develop rather fast; except for one record of P. mycophaga all fructifications appeared during the first 19 days of culture (Fig. 2). No clear successional pattern was found; the peak at day 19 was provoked by removing the cultures from the humidity chamber. The 48 leaf cores from fallen beech leaves (26 records, 4 species, H’ = 0.97) were poorer in both species diversity and
Tab. 1: Frequencies of the more common protostelids (more than 3 records in total) on aerial and ground litter (culture series 1 and 2). Species
Ground
Aerial
0.56 0.13 0.25 0.19 0.25 0.13 0.06
0.13 0.50 0.06 0.19 0.00 0.00 0.06
Schizoplasmodiopsis pseudoendospora Soliformovum irregularis Schizoplasmodiopsis vulgare Protostelium mycophaga Nematostelium ovatum Nematostelium gracile Schizoplasmodiopsis amoeboidea
abundance than those of aerial leaves (40 records, 6 species, H’ = 1.40). Three species were found on aerial leaves only (Tab. 2). For aerial leaves, frequencies seem to increase from the tip to the base of the leaf (n = 48, loc. 6 and 7): tip: 7 of 16 (43.7 %); middle section: 9 of 16 (56.2 %); base: 10 of 16 (62.5 %) leaf cores tested positive. All except one of the 16 leaves tested with three replicates were positive for protostelids. Assuming that the leaves can only be reached by air-borne propagules during the vegetation period (beginning of May to end of November), and one spore is sufficient to found a protostelid population on a leaf, the minimal spore fallout necessary to explain these frequencies can be calculated. With 1000 runs of the program simulating random spore fall (see Methods), a mean of 53.8 ± 0.56 spore hits on the 16 aerial leaves is necessary to reach this frequency. With a total leaf area of 271 cm2 (mean 16.9 ± 1.1 cm2), this equals a minimal spore fallout of 0.2 spores per cm2 over the vegetation period of ca. 6 months. For one set of cultures (loc. 7, aerial litter, n = 24) all protostelid sporocarps were counted. This allowed to calculate a maximum productivity for P. mycophaga as the most common species, recorded ten times with 100, 25, 25, 25, 150, 45,
Tab. 2: Protostelid records on fallen and aerial Fagus leaves from two localities (culture series 3). Species Locality No.
Ground
Aerial
6
7
6
7
Nematostelium gracile (NEMgra) Nematostelium ovatum (NEMova) Protostelium mycophaga (PROmyc) Schizoplasmodiopsis amoeboidea (SCDamo) Schizoplasmodiopsis pseudoendospora (SCDsps) Schizoplasmodiopsis vulgare (SCDvul) Soliformovum irregularis (SOLirr)
1 – 7 – 2 – 7
– – 9 – – – –
– 1 14 1 2 1 5
– 3 10 2 – 4 –
leaf cores positive for protostelids (n = 24) leaves positive for protostelids (n = 8)
11 7
9 6
15 7
10 8
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Fig. 1: Species accumulation curve for protostelids recorded from still attached as well as fallen Fagus leaves, 96 leaf cores cultivated. The hyperbola shown represents the best fit according to the equation y = 8.31 x /(16.25+x), R = 0.981.
2, 7, 8, and 2 sporocarps. With a diameter of 6 mm per leaf core and 24 cores, the 6.79 cm2 total leaf area gave 389 sporocarps, equaling a maximum productivity of 57.9 spores per cm2.
Discussion Although this study represents the first published survey of protostelids from Europe, the relatively similar species assemblage found in studies from other parts of the world lets us assume, that most if not all species hitherto known can be found in Europe as well. Consequently, the species list presented herein is a very preliminary one. Some species, abundant in the present study, such as Protostelium mycophaga, Soliformovum irregularis, Schizoplasmodiopsis pseudoendospora, and Nematostelium spp. are very common worldwide (MOORE & SPIEGEL 1995, STEPHENSON & MOORE 1998, STEPHENSON, LANDOLT & MOORE 1999, SPIEGEL & STEPHENSON 2000, MOORE et al. 2000, MOORE & SPIEGEL 2000a, 2000b, 2000c, MOORE & STEPHENSON 2003). Besides the 14 protostelid taxa recorded, the first German record for the smallest known myxomycete, the two-spored E. bisporum, found at loc. 1 on dead hanging leaves of Urtica dioica in 30 cm height, enlarges the checklist of German myxomycetes (SCHNITTLER et al. 1996) to 321 species. E. bisporum is a species uncommon in temperate litter microhabitats but abundant in the Tropics (MOORE & STEPHENSON 2003). As assessed from the still fragmentary surveys, temperate habitats seem to have the greatest species diversity (MOORE & SPIEGEL 2000a), with tropical rainforests (MOORE & SPIEGEL 2000b) next, and boreal forests and tundra (MOORE et al. 2000) having the lowest species diversity. As indicated by the high frequency of aerial beech leaves inhabited by protostelids, these organisms seem © DGfM 2005
to have a rather efficient mode of dispersal by airborne spores. Each of the investigated leaves was hanging free on a stalk of ca. 0.5 mm diameter and 0.4–1 cm length, thus it should be a habitat island, comparable with the straws introduced as an artificial habitat by MOORE & SPIEGEL (1995). The proportion of straws colonized over a period from 1 to 9 weeks exposure in Puerto Rican forest communities was between 27 and 44 % (MOORE & SPIEGEL 2000b) and 74 % in NW Arkansas (MOORE & SPIEGEL 2000a); we found 94 % for the Fagus leaves exposed much longer (up to 36 weeks). Over the entire vegetation period of ca. 6 months, the leaves can work as spore traps. If we assume, that one spore is sufficient to form a protostelid population, and the leaves are subjected to random spore fallout, about 1 spore hit per 5 cm2 (or 3.4 per leaf) would explain the high frequency found. The same calculation for the straws (6.5 × 0.3 cm) used in palm forests of Puerto Rico (MOORE & SPIEGEL 2000b), 44 % positive, homothally assumed) would result in 1 spore hit per 10 cm2 (or 0.6 per straw). These figures can well be matched by the potential productivity of the leaves, as indicated by a value of about 58 spores per cm2 (or 981 per leaf). Obviously, the optimum conditions of our culture will hardly be realized in nature, but here the leaves probably produce fructifications at several occasions during the vegetation period. It would be well imaginable, that the tiny plasmodia and amoebae of protostelids are quite susceptible to competition from myxomycetes, having much larger plasmodia and fructifications. However, in many plates with both protostelids and myxomycetes, some species of protostelids, e.g. S. pseudoendospora, appear to grow better in the presence of myxomycetes. This can not explain the higher frequency of protostelids on aerial leaves, a nutrient-poor habitat where myxomycetes hardly can form large plasmodia (with 19 times in 48 ground litter leaf cores and with only 8 times in 48 aerial leaf cores), and three tiny, 5–30 spored fructifications of Licea sp. occurred in one of the protostelid cultures. Among environmental conditions, differences in pH can be ruled out, but the much more fluctuating moisture gradient of aerial litter could well favor protostelids with their short live cycles (compare Fig. 2) over myxomycetes. At day 5, almost one half of all fructifications on beech leaves (33 out of 69 records) had been developed, a figure found also by MOORE & SPIEGEL (1995). Among myxomycetes, only some species of Echinostelium seem to be able to develop within 5 days (SCHNITTLER 2001). For most parts of lowland Central Europe beech forests are assumed to be the natural vegetation (BOHN et al. 2003), and this was the reason to choose tracts of old-grown beech forests for sampling. Although Fagus leaves, producing a quite nutrient-poor, slowly decaying litter layer, seem to have a less diverse protostelid assemblage than other decaying plant materials, these organisms are very efficient settlers of both ground and aerial Fagus litter. For the latter substratum, the vegetation season of six months seems to be
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MAGURRAN AE (1988) Ecological diversity and its measurement. Princeton University Press, Princeton, N.J, 179 p.
MOORE DL, SPIEGEL FW (2000b) Microhabitat distribution of protostelids in tropical forests of the Caribbean National Forest, Puerto Rico. – Mycologia 92: 616-625. MOORE DL, SPIEGEL FW (2000c) The effect of season on protostelid communities. – Mycologia 92: 599-608. MOORE DL, SPIEGEL FW (2000a) Microhabitat distribution of protostelids in temperate habitats in northwestern Arkansas. – Canadian Journal of Botany-Revue Canadienne de Botanique 78: 985-994. MOORE DL, STEPHENSON SL (2003) Microhabitat distribution of protostelids in a Tropical Wet Forest in Costa Rica. – Mycologia 95: 11-18. MOORE DL, STEPHENSON SL, LAURSEN GA, WOODGATE WA (2000) Protostelids from boreal forest and tundra ecosystems in Alaska. – Mycologia 92: 390-393. OLIVE LS (1962) The genus Protostelium. – American Journal of Botany 49: 297-303. OLIVE LS (1975) The Mycetozoans. Academic Press, New York, San Franzisco, London. SCHNITTLER M (2001) Ecology of Myxomycetes from a winter-cold desert in western Kazakhstan. – Mycologia 93: 653-669. SCHNITTLER M, KRIEGLSTEINER LG, MARX H, FLATAU L, NEUBERT H, NOWOTNY W, BAUMANN K (1996) Vorläufige Rote Liste der Schleimpilze (Myxomyceten) Deutschlands. – Schriftenreihe für Vegetationskunde 28: 481-525. SPIEGEL FW (1998) Key to genera of Protostelids bases on sporocarp morphology, and based on trophic cell morphology. University of Arkansas. http://comp.uark.edu/~fspiegel/protist.html. SPIEGEL FW (1990) Phylum plasmodial slime molds, Class Protostelida. In Margulis L, Corliss JO, Melkonian M, Chapman DJ (eds) Handbook of Protoctista, pp. 484-497. Jones & Bartlett Publishers, Boston, Massachusetts. SPIEGEL FW, FELDMAN J (1985) Obligate amoebae of the protostelids: Significance for the concept of Eumycetozoa. – Biosystems 18: 377-386. SPIEGEL FW, LEE SB, RUSK SA (1995) Eumycetozoans and Molecular Systematics. – Canadian Journal of Botany-Revue Canadienne de Botanique 73: 738-746. SPIEGEL FW, SHADWICK JD, LINDLEY-SETTLEMYRE LL (2004) A beginner’s guide to identifying the common Protostelids. University of Arkansas, Fayetteville. http://cavern.uark.edu/ua/ mycetozo. SPIEGEL FW, STEPHENSON SL (2000) Protostelids of Macquarie Island. – Mycologia 92: 849-852. SPIEGEL FW, STEPHENSON SL, KELLER HW, MOORE DL, CAVENDER JC (2004) Mycetozoans. In Mueller GM, Bills GF, Foster MS (eds) Biodiversity of Fungi. Inventory and monitoring methods, pp. 547-576. Elsevier Acad. Press, Amsterdam. STEPHENSON SL, LANDOLT JC, MOORE DL (1999) Protostelids, dictyostelids, and myxomycetes in the litter microhabitat of the Luquillo Experimental Forest, Puerto Rico. – Mycological Research 103: 209-214. STEPHENSON SL, MOORE DL (1998) Protostelids from tropical forests of Costa Rica. – Mycologia 90: 357-359.
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Accepted: 15.9.2005
Fig. 2: Successional sequence of protostelid species recorded from Fagus leaves. The size of the circles indicates the relative increase in records at each of the six times where cultures were checked (smallest circle: one record). For abbreviations of species names compare Tab. 2.
sufficient to reach almost every leaf by air-borne spores, indicating a high effectiveness of spore dispersal. Further studies, especially from the forest canopy, are needed to reveal the biodiversity and ecological importance of these organisms for Central Europe.
Acknowledgements We owe thanks to all participants of the workshop on the taxonomy and ecology of protostelids for help with the first series of cultures, held from Aug. 13-18, 2004 at Greifswald, funded by a grant from the US National Science Foundation project DEB-0316284 „PBI: Global Biodiversity of Eumycetozoans“.
References BEST SC, SPIEGEL FW (1984) Protostelids and other simple mycetozoans of Hueston State Park and Nature Preserve. In Willeke G (ed) Hueston Woods State Park and Nature Preserve Proceedings of Symposium, April 16-18,1982, pp. 116-121. Miami Univ., Miami, Ohio. BOHN U, NEUHÄUSL R, GOLUB G, HETTWER C, NEUHÄUSLOVÁ Z, SCHLÜTER H, WEBER H (2003) [Map of the natural vegetation of Europe, part 1: text with CD-Rom; part 2: legend; part 3: maps]. Landwirtschaftsverlag, Münster.
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