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Activity and role of the enchytraeid worm Cognettia sphagnetorum (Vejd.) (Oligochaeta: Enchytraeidae) in organic and mineral forest soil
Pedobiologia 47, 303–310, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/pedo
Activity and role of the enchytraeid worm Cognettia sphagnetorum (Vejd.) (Oligochaeta: Enchytraeidae) in organic and mineral forest soil Jari Haimi1* and Anne Siira-Pietikäinen1,2 1 2
Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland Finnish Forest Research Institute, P.O. Box 18, FIN-01301 Vantaa, Finland
Submitted August 25, 2002 · Accepted January 13, 2003
Summary Site preparation following clear felling of coniferous forest creates a mosaic of different kinds of habitats for soil decomposers, ranging from bare mineral soil to thick layers of organic soil and felling residues. To study whether the impact of enchytraeids on nutrient mineralisation processes is different in residues, organic layer, and mineral soil, a microcosm experiment was conducted in the laboratory. Microcosms contained mineral soil (sand) alone or with soil organic layer (humus and F-layer materials) and spruce needles, either separately or together. Enchytraeids (Cognettia sphagnetorum) were introduced to the half of the microcosms. Numbers of enchytraeids, their gut content, and soil pH and mineral nitrogen were determined twice, 8 and 13 weeks from the start. Population increase of C. sphagnetorum was found only in the soil organic layer. In the mineral soil, enchytraeids ingested sand grains together with bacteria and protozoans. In the organic soil enchytraeid intestines were filled with dark coloured humus material in which plant fragments, fungal hyphae and bacterial colonies were recognized. Enchytraeids significantly increased the amount of NH4-N in the bare mineral soil. When a soil organic layer was present on the mineral soil, enchytraeids decreased NH4-N in the presence of needles but had no effect when needles were absent. Enchytraieds did not affect the pH of the mineral soil, but they slightly increased that of the soil organic layer. The results of this experiment revealed that C. sphagnetorum is able to maintain its population and its functional importance also in resource poor mineral soil. Its effect on nitrogen dynamics appears to be dependent on environmental conditions, resource quality and development of a population, i.e. acceleration, no effect and inhibition of nitrogen mineralisation are all possible. Key words: Enchytraeids, forest soil, nitrogen mineralisation, soil pH
Introduction Clear felling is still the main harvesting method used in northern coniferous forests. Usually, harvesting is followed by mechanical site preparation (e.g. harrowing) to ensure favourable conditions for early development of the next, usually planted, tree generation. As a re-
sult, in the forest floor, exposed mineral soil alternates with thick layers of organic material consisting of humus, litter, felling residues, mosses and ground layer vegetation. In many terrestrial ecosystems, earthworms as large
*E-mail corresponding author: [email protected]
0031-4056/03/47/04–303 $15.00/0
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annelids are functionally one of the most important decomposer animal groups comminuting litter and redistributing organic matter in the soil profile (Edwards 2000). In northern coniferous forest soils, however, earthworms are sparse, and their functional role is often taken up, at least to some extent, by enchytraeids. The enchytraeid fauna of coniferous forests is usually strongly dominated by one species, Cognettia sphagnetorum (Vejdovsky´, 1877) (Nurminen 1967; Abrahamsen 1972). This species has been found to be an important animal in the decomposition of organic matter (Standen 1978; Cole et al. 2000) and nutrient mineralisation (Williams & Griffiths 1989; Abrahamsen 1990; Briones et al. 1998a) in soil organic layer of coniferous forests. Its activity has also been shown to enhance primary production in microcosm experiments (Laakso & Setälä 1999; Laakso et al. 2000). On the other hand, although C. sphagnetorum is an abundant and functionally important species, it is sensitive to many kinds of disturbance. Drought (Didden & de Fluiter 1998; Yli-Olli & Huhta 2000) and various kinds of anthropogenic contaminants (Salminen & Sulkava 1996; Salminen & Haimi 1996, 1999) have negatively affected its performance. This species can, however, efficiently increase in numbers – through asexual reproduction – at least after natural disturbances (Nurminen 1967; Lundkvist 1982). In our previous experiment, C. sphagnetorum was among the first decomposer animals increasing in numbers in mineral soil exposed by simulating mechanical site preparation (Siira-Pietikäinen et al. 2003). Several studies have shown that numbers of enchytraeids increase in the soil organic layers after clear felling of boreal coniferous forests (Huhta 1976; Lundkvist 1983; Siira-Pietikäinen et al. 2001b). This population growth may have been caused by increases in the amount of resources for these saprovorous soil animals (felling residues, dying ground layer plants, increasing bacterial growth and biomass) and more favourable moisture and temperature conditions. In our previous field experiment we found, however, that enchytraeids increased significantly only after elimination of root-mycorrhizal connections to the surrounding soil (trenching treatment; resulting in e.g. increases in dead soil organic matter and moisture) but not after addition of slash (Siira-Pietikäinen et al. 2001a). On the other hand, Siira-Pietikäinen et al. (2001b) observed in a large scale field experiment that enchytraeids increased following clear felling and site preparation without any significant increment in soil bacterial biomass. After clear felling enchytraieds increase in patches with untouched forest floor, while tree seedlings are planted and tree seeds germinate most successfully in exposed mineral soil where resources for enchytraeids Pedobiologia (2003) 47, 303–310
are scarce. Conditions and processes, such as nutrient availability, in the vicinity to seedling roots are therefore assumed to be controlled mainly by abiotic factors. Community structure, biomass and the activity of decomposer animals also have potential, however, to significantly affect nutrient acquisition by tree seedlings in exposed mineral soil (e.g. Setälä & Huhta 1991; Laakso et al. 2000). In this study we aimed to test whether functional properties of C. sphagnetorum are similar in mineral soil as repeatedly observed in soil organic layer. We introduced enchytraeids to laboratory microcosms with soil materials from different layers of coniferous forest floor. We applied a full factorial experimental design to find out whether the influence of C. sphagnetorum is related to soil layer, i.e. amount and quality of resources in soil. Soil materials were taken from the same conifer stand where our previous field experiments were performed (Siira-Pietikäinen et al. 2001a, 2003). We hypothesised that the effect of C. sphagnetorum on nitrogen mineralisation is insignificant in soil where its numbers are low and resources rare, i.e. in mineral soil without abundant organic matter and microbial biomass.
Materials and Methods Microcosms made of plastic cylinders with an inner area of 55 cm2 and a height of 9 cm were used in the experiment. There were holes with cotton plugs in the lids allowing proper evaporation and gas exchange. During the experiment, the microcosms were kept in a climate chamber at +15° C, weighed weekly, and when needed, irrigated with de-ionised water. The experiment had a factorial design with three factors: soil organic layer, needles and enchytraeids; each of these were added or not added to the soil mineral layer (sand). This experimental design resulted in eight treatment combinations, each repeated five times. Soil materials (sand and soil organic layer consisting of humus and partly decayed conifer and ground layer vegetation litter together with dead moss shoot bases) for the experiment were taken from the coniferous forest in which our previous studies were conducted (Siira-Pietikäinen et al. 2001a, 2003). The stand is on podzolic sandy soil in central Finland (61°50'N, 24°20'E), and dominated by Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.). The understorey vegetation consists mainly of Vaccinium myrtillus L., Vaccinium vitis-idaea L., Maianthemum bifolium (L.) F. W. Schmidt and Deschampsia flexuosa (L.) Trin. on a thick moss layer dominated by Hylocomium splendens (Hedw.) Bruch
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Table 1. Properties of the soil materials used in the experiment. pH (in water), organic matter content (loss on ignition), NH4+-N and NO3–-N (water extraction). Means ± standard errors are shown. d.m. = dry mass; o.m. = organic matter Mineral soil pH Organic matter-% NH4+-N µg g-1 d.m. NH4+-N µg g-1 o.m. NO3–-N µg g-1 d.m. NO3–-N µg g-1 o.m.
5.3±0.07 1.0±0.04 0.24±0.030 24.4±3.05 0.47±0.013 47.1±1.357
Soil organic layer 4.2±0.02 90.2±0.48 476±9.3 528±10.3 0.70±0.349 0.78±0.041
& Schimp and Pleurozium schreberi (Brid.) Mitt. The organic soil layer is ca. 4 cm thick (Siira-Pietikäinen et al. 2001a). Spruce needles were collected from felling residues in June 2000 in a similar but clear felled stand near Jyväskylä, central Finland (62°20'N, 25°45'E). The stand was felled in the previous autumn, and the needles had lost their green colour, and they dropped down off the twigs when lightly touched. Amongst the felling residues fresh needles are nutrient rich and they are first to reach the soil surface quite soon after felling, coarse material (twigs and branches) entering the decomposer community in the soil only after many years (Hyvönen et al. 2000). Properties of the materials used in the experiment are shown in Table 1. Soil for extracting enchytraeids was taken in a similar forest stand near Jyväskylä as for the materials used to construct the soil profiles. Enchytraeids were extracted using the standard wet funnel method, and individuals of the species Cognettia sphagnetorum (Vejd.) were collected in Petri dishes in water taken from the collecting tubes. More than 95 % of the individuals were found to represent C. sphagnetorum. The worms were allowed to void most of their gut contents in a climate chamber at +15° C overnight. The soil materials were kept for 24 h at +80° C, and frozen for one week at –22° C to eliminate meso- and macrofauna. After this defaunation procedure, 248.9 g (as dry mass) sand was spread on the bottom of each microcosm. On the mineral soil, 9.3 g (d.m.) humus together with 1.58 g (d.m.) partly decayed plant litter and dead moss shoot bases (hereafter called the soil organic layer) and/or 3.61 g (d.m.) spruce needles were added according to the experimental design. After weighing the soil materials in the microcosms each microcosm was inoculated with microflora and -fauna by adding 2 ml soil-water suspension (obtained from 30 g fresh mass of the same organic soil in one litre of water filtered through a 105 µm mesh) into each of them. Finally, on the next day, 55 enchytraeids were transferred to half of the microcosms. Nine weeks after the
Needles 4.7±0.04 95.1±0.61 6.34±0.072 6.67±0.076 0.26±0.103 0.27±0.109
start of the experiment 26 individuals were added to the same microcosms. Microcosms were sampled twice, 8 and 13 weeks after the establishment. One sector of the soil profile from the soil surface to the bottom consisting of ca. 25 % of the total amount of the soil in the first sampling and ca. 50 % of the remaining soil in the second sampling was taken from the microcosms. Soil materials were then divided for different analyses. Number of enchytraeids, contents of mineral nitrogen (ammonium-N and in the first sampling also nitrate-N), soil moisture and pH were separately analysed from sand (soil mineral layer), soil organic layer, and in the first sampling also from needles (in the second sampling needles were included in the soil organic or mineral layer samples). Enchytraeids were extracted from weighed soil subsamples using the standard wet funnel method. After counting the enchytraeids in a sample the gut contents of each specimen were examined under a microscope at an appropriate magnification. First, the proportion of the gut volume filled with any material was assessed. After that the gut content material was qualitatively identified as far as possible. When needed, the specimen was broken, and the gut content was diluted with water to facilitate the recognition of the material. Fresh soil samples for nitrogen and pH analyses were suspended in deionised water (10–20 g mineral soil, 5–7 g organic soil and 1.5 g needles in 100 ml water) and shaken for half an hour. pH was measured and humus-water suspension was then filtered before analyses of ammomium- (NH4) and nitrate-nitrogen (NO3) with a FIA-autoanalyser (Flow Injection Analyser). Soil moisture was measured by drying subsampels at +105° C overnight. The parameters measured were analysed with analysis of variance for repeated measures (repeated measures ANOVA). When the treatment and time interacted with each other, the effect of that treatment was tested separately for both sampling occasions. When interactions between the two treatments were observed, simple effects were examined with repeated measures Pedobiologia (2003) 47,303–310
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ANOVA (no interactions with time) or one-way ANOVA (treatment-time interactions). In addition, correlations between enchytraeid densities and soil nitrogen were analysed using Pearson correlation coefficient. No transformations were needed for the data. For the statistical analyses, a software package SPSS® for Windows™ Release 10.0 was used.
Results Numbers and gut contents of enchytraeids
Enchytraeids’ population sizes decreased in the microcosms by the first sampling. Significant population growth from the first to the second sampling was observed only in the organic soil (repeated measures ANOVA: effect of time: F = 17.5, P = 0.003) (Table 2). Regardless of the treatment extracted specimens were in good condition. In the microcosms without a soil organic layer, the numbers of enchytraeids varied between 1600 and 4100 individuals m-2 (Table 2), and the presence of needles did not affect their numbers. There were significantly fewer enchytraeids in the mineral soil below the organic layer (density varying from 0 to 620 individuals m-2) than in the mineral soil without it (1580 to 4120 individuals m-2; repeated measures ANOVA: F = 32.7, P < 0.001). In the soil organic layer the presence of needles decreased the numbers of enchytraeids (repeated measures ANOVA: F = 8.7, P = 0.019). In the microcosms with a soil organic layer, 0–11 % of enchytraeids were found in the mineral soil. There was no difference in the amount of gut content between the enchytraeids extracted from the mineral and organic soil. On an average, ca. 65 % of the gut volume was filled with soil and/or microbial material. In the mineral soil there was more material in the enchytraeid guts in the presence of needles than without them (72 ± 2.9 % in the presence of needles and 55 ± 3.7 % in the absence of needles; one-way ANOVA: F = 11.8, P = 0.011). A lot of sand grains Table 2. Total numbers of enchytraeids in the microcosms with different structure (all soil layers summed up; individuals m-2: means and standard errors) Microcosm type
Week 8
Week 13
Mineral soil only + needles + soil organic layer + needles and soil orgnanic layer
2819 (1577) 1578 (1141) 4089 (1182)
2646 (934) 4118 (341) 10 649 (2107)
252 (252)
4613 (1663)
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were found in the guts of the enchytraeids extracted from the mineral soil, the diameter of the largest grains being half of the diameter of the gut. The rest of the gut contents was mainly bacteria and protozoans and quite dark coloured unidentified material. Many live bacteria and protozoans were observed in these samples, while only few pieces of fungal hyphae were found. Enchytraeids extracted from the organic soil had ingested mostly dark humus material including decaying plant fragments of different kinds and sizes, fungal hyphae, and bacteria. The presence of needles did not affect the amount or quality of the enchytraied gut contents in the soil organic layer. Soil pH
The pH of the mineral soil varied between 5.4 and 5.6, and enchytraieds did not have any effect on it. Overall, pH slightly increased during the experiment (repeated measures ANOVA: effect of time: F = 6.0, P = 0.020). Presence of the soil organic layer decreased the pH of the mineral soil by ca. 0.1 pH unit. (repeated measures ANOVA: F = 55.6, P < 0.001). Effects of needles on the pH of the mineral soil differed depending on the presence of the soil organic layer (repeated measures ANOVA: organic layer x needles: F = 8.9, P = 0.005). When there was an organic layer (i.e. all soil layers were present) needles had no influence on mineral soil pH, but without the organic layer (needles on the surface of the mineral soil) needles increased the pH of the mineral soil (repeated measures ANOVA for the microcosms without soil organic layer: F = 16.0, P = 0.001). In the soil organic layer pH varied between 4.8 and 5.0, and it did not change from the first to the second sampling. Here, however, enchytraeids increased the pH (repeated measures ANOVA: F = 6.7, P = 0.020), but only 0.5–1.0 pH units. On the other hand, the presence of needles somewhat decreased the pH of the organic layer (repeated measures ANOVA: F = 21.6, P < 0.001). The pH of the needles on the mineral soil was analysed only on the first sampling: pH varied between 5.3 and 5.7, and enchytraeids had no influence on it. Soil mineral nitrogen
Soil NO3-N was analysed only in the first sampling because its levels appeared to be very low in all treatments. The presence of needles clearly decreased the amount of NO3-N in the soil organic layer (0.15 ± 0.065 with and 0.44 ± 0.084 µg g-1 without needles; one-way ANOVA: F = 6.8, P = 0.019). In the mineral soil, NO3-N was measurable only when the soil organic layer was present in the microcosms (NO3-N
Activity and role of Cognettia sphagnetorum
Fig. 1. NH4+-N contents (µg g-1 dry soil; mean ± standard error) of mineral soil of the microcosms with (a) and without (b) organic soil layer. Mineral = mineral soil only; Ne = needles present; En = enchytraeids present
level was ca. 0.2 µg g-1 dry soil). Enchytraeids or the presence of needles had no effects on NO3-N in the mineral soil. Most of the mineral nitrogen was in the form of NH4, and its amount in the mineral soil increased with time (repeated measures ANOVA: effect of time: F = 32.7, P < 0.001) (Fig. 1). There was a strong immobilisation of nitrogen in the needles: NH4-N was always under the detection limit in the needles and in the absence of the soil organic layer also in the mineral soil under the needles (Fig. 1b). The presence of the soil organic layer increased the amount of NH4 in the mineral soil (repeated measures ANOVA: F = 1878.2, P < 0.001). In the presence of the soil organic layer, the effect of needles depended on the presence of enchytraeids (interaction in repeated measures ANOVA: F = 11.0, P = 0.004) (Fig. 1a). Without enchytraeids
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needles did not significantly affect the amount of NH4-N in the mineral soil, but with enchytraieds needles decreased NH4-N (repeated measures ANOVA: F = 7.727, P = 0.024). The influence of enchytraeids on NH4-N was variable in the mineral soil (interactions in repeated measures ANOVA: enchytraeids × soil organic layer: F=7.9, P=0.009; enchytraeids × needles: F = 17.9, P < 0.001; enchytraeids × soil organic layer × needles: F = 5.7, P = 0.023). When the soil organic layer was present and needles absent, enchytraeids had no significant effect on NH4-N. However, when needles were present on the soil organic layer, the presence of enchytraeids decreased NH4-N in the mineral soil (repeated measures ANOVA: F = 9.4, P = 0.016) (Fig. 1a). In the absence of the organic soil and needles, enchytraeids clearly increased the amount of NH4-N in the mineral soil (repeated measures ANOVA: F = 138.9, P < 0.001) (Fig. 1b). In the mineral soil, the only and weak correlation between enchytraeid densities and the amounts of nitrogen was found in the second sampling in NH4-N when the microcosms with soil organic layer and needles were excluded from the analysis (r = 0.614, P = 0.059). In the soil organic layer enchytraeids had no effect on NH4-N (Fig. 2). However, enchytraeid density and NH4-N correlated positively in the first sampling when only the microcosms with enchytraeids were included in the analysis (r = 0.715, P = 0.020). On the other hand, needles decreased NH4-N also in the soil organic layer (repeated measures ANOVA: F = 37.1, P < 0.001). The amount of NH4-N somewhat decreased from the first to the second sampling (repeated measures ANOVA: effect of time: F = 24.3, P < 0.001) (Fig. 2).
Fig. 2. NH4+-N contents (µg g-1 dry soil; mean ± standard error) of the soil organic layer of the microcosms. Organic = organic soil layer only; Ne = needles present; En = enchytraeids present Pedobiologia (2003) 47,303–310
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Discussion Population densities of C. sphagnetorum remained at quite low levels in our microcosm study. It is evident that mineral soil without any organic matter input cannot maintain high enchytraeid densities for extended periods. The extracted enchytraeids were, however, active and in good condition. Impact of enchytraeids on soil processes seems to be density dependent (Anderson 1988). Thus, it was assumed that their effects on nitrogen mineralisation would be strong in the presence of high population densities but remaining negligible at low densities. The correlation between enchytraeid density and soil NH4-N was, however, significantly positive in rare cases only. The results of the present experiment demonstrate effects of C. sphagnetorum at low densities that are, in fact, common in northern coniferous forests. It should also be noted, that reduction in the biomass of introduced enchytraeids might have contributed to the nitrogen dynamics in our microcosms. This can not, however, fully explain the differences between the treatments. Despite its low densities, C. sphagnetorum had significant impacts on soil processes in our experiment. The effects were, however, variable: enchytraeids increased pH in the soil organic layer but not in the mineral soil where they, on the other hand, affected nitrogen mineralisation. Enhancement of nitrogen mineralisation by enchytraeids in nutrient poor mineral soil can be important for early development of tree seedlings planted on prepared patches in felled areas. When the soil organic layer was present in the microcosms, the effect of enchytraeids on nitrogen mineralisation in mineral soil was different compared to bare mineral soil. In addition, in the soil organic layer enchytraeids had no influence on mineral nitrogen that contrasts the results of many previous studies in which enchytraeids have clearly increased nitrogen mineralisation (Williams & Griffiths 1989; Abrahamsen 1990; Briones et al. 1998a, b). That enchytraeids decreased NH4-N in the mineral soil in the presence of a soil organic layer and needles could be due to enhancement of nutrient immobilisation by soil microbes induced by enchytraeid activity. Liiri et al. (2001) found that wood ash application turned the positive effects of enchytraieds on plant growth and nitrogen content to negative, and hypothesized that dominating microbes in ash-treated soil may have responded differently to enchytraeid grazing compared to microbes in ash-free soils leading to increased nitrogen immobilisation (see also Visser 1985). Thus, the net impact of enchytraieds on soil nitrogen mineralisation seems to be dependent on several environmental factors, such as soil temperature Pedobiologia (2003) 47, 303–310
and moisture (Abrahamsen 1990), as well as quality of resources and microbial community structure. C. sphagnetorum may also enhance nutrient availability for plants by decreasing soil acidity as observed in our experiment in the soil organic layer. The increase of pH was, however, only small compared to e.g. the observations by Briones et al. (1998a). The difference in the magnitude of the effect may be explained by differences in the enchytraeid densities between these studies, density being much lower in our microcosm experiment. In addition to enchytraeids, spruce needles had a clear effect on nitrogen dynamics in our microcosms: nitrogen became immobilised in the needles both on the mineral and organic soil, most likely due to a high C/N ratio of the needles. Hyvönen et al. (2000) also observed net nitrogen immobilisation during the first years of needle decomposition, and pointed out that needles are the most important component of the felling residues in nitrogen (and also phosphorus) dynamics of the forest soil in the early forest regeneration phase after logging. The importance of needles for soil processes became emphasized in the present study because they also affected the numbers of enchytraeids and their role in nitrogen dynamics. It was interesting to notice that C. sphagnetorum was able to live – although in low numbers – and affect soil processes in the mineral soil with extremely low organic matter content. It clearly could find food, e.g. microbial colonies, in this harsh environment. It has been found in the field that C. sphagnetorum can actively move to mineral soil during unfavourable conditions, such as drought and winter frosts (Lundkvist 1982). Whether these enchytraeids are actively feeding and reproducing during their stay in the mineral soil is not fully known. Our gut content observations revealed that C. sphagnetorum can selectively ingest different kinds of available material and also mineral (sand) particles. When living in mineral soil it has to derive its nutrition mainly from microbes, and significant numbers of microbes were observed in the guts of specimens from mineral soil. It has been shown that enchytraeids can derive nutrition from living microorganisms, but that the digestion is incomplete during passage through the intestine (Brockmayer et al. 1990). On the other hand, Stefan (1990) observed that bacterial cells can easily pass through the enchytraeid’s gut without any damage. The resulting dissemination and concentration of bacteria in their faecal pellets could partly explain the important role of enchytraeids in soil nutrient dynamics (Stefan 1990). Encytraeids have a low assimilation efficiency, and by producing large amounts of faecal pellets they are also important for soil structure, as verified especially in organic soils (see Briones et al.
Activity and role of Cognettia sphagnetorum
1998b). In addition, by mixing inorganic and organic materials enchytraeids can have an impact on soil structure as observed e.g. by Dawod & FitzPatrick (1993). From the results of the present experiment we can conclude that C. sphagnetorum can be functionally important in resource poor mineral soil. Due to its asexual reproduction C. sphagnetorum can effectively increase in numbers as soon as conditions become favourable. Thus, in the field, in exposed mineral soil patches created by mechanical site preparation this species has potential to increase along with accumulating organic matter (see also Siira-Pietikäinen et al. 2003). The effect of C. sphagnetorum on soil nitrogen dynamics appeared to vary in relation to environmental conditions, soil layer and resource quality; i.e. acceleration, no effect and inhibition of nitrogen mineralisation are all possible. The same holds also for the effect of C. sphagnetorum on soil pH. Acknowledgements. We are grateful to Tuomas Lukkari for helping us in practical work in the laboratory. The work of Anne Siira-Pietikäinen was supported by the Maj and Tor Nessling Foundation and Finnish Cultural Foundation.
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Standen, V. (1978) The influence of soil fauna on decomposition by micro-organisms in blanket bog litter. Journal of Animal Ecology 47, 25–38. Stefan, V. (1990) Some studies on the relations between Enchytraeidae and soil microflora. Revue Roumaine de Biologie. Serie de Biologie Animale 35, 101–104. Visser, S. (1985) Role of soil invertebrates in determining the composition of soil microbial communities. In: Fitter, A. H., Atkinson, D., Read, D. J., Usher, B. M. (eds) Ecological interactions in soil: plants, microbes and animals. Blackwell, Oxford, pp. 319–331.
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Williams, B. L., Griffiths, B. S. (1989) Enhanced nutrient mineralization and leaching from decomposing Sitka spruce litter by enchytraeid worms. Soil Biology and Biochemistry 21, 183–188. Yli-Olli, A., Huhta, V. (2000) responses of co-occurring populations of Dendrobaena octaedra (Lumbricidae) and Cognettia sphagnetorum (Enchytraeidae) to soil pH, moisture and resource addition. Pedobiologia 44, 86–95.
Activity and role of the enchytraeid worm Cognettia sphagnetorum (Vejd.) (Oligochaeta: Enchytraeidae) in organic and mineral forest soil Jari Haimi1* and Anne Siira-Pietikäinen1,2 1 2
Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland Finnish Forest Research Institute, P.O. Box 18, FIN-01301 Vantaa, Finland
Submitted August 25, 2002 · Accepted January 13, 2003
Summary Site preparation following clear felling of coniferous forest creates a mosaic of different kinds of habitats for soil decomposers, ranging from bare mineral soil to thick layers of organic soil and felling residues. To study whether the impact of enchytraeids on nutrient mineralisation processes is different in residues, organic layer, and mineral soil, a microcosm experiment was conducted in the laboratory. Microcosms contained mineral soil (sand) alone or with soil organic layer (humus and F-layer materials) and spruce needles, either separately or together. Enchytraeids (Cognettia sphagnetorum) were introduced to the half of the microcosms. Numbers of enchytraeids, their gut content, and soil pH and mineral nitrogen were determined twice, 8 and 13 weeks from the start. Population increase of C. sphagnetorum was found only in the soil organic layer. In the mineral soil, enchytraeids ingested sand grains together with bacteria and protozoans. In the organic soil enchytraeid intestines were filled with dark coloured humus material in which plant fragments, fungal hyphae and bacterial colonies were recognized. Enchytraeids significantly increased the amount of NH4-N in the bare mineral soil. When a soil organic layer was present on the mineral soil, enchytraeids decreased NH4-N in the presence of needles but had no effect when needles were absent. Enchytraieds did not affect the pH of the mineral soil, but they slightly increased that of the soil organic layer. The results of this experiment revealed that C. sphagnetorum is able to maintain its population and its functional importance also in resource poor mineral soil. Its effect on nitrogen dynamics appears to be dependent on environmental conditions, resource quality and development of a population, i.e. acceleration, no effect and inhibition of nitrogen mineralisation are all possible. Key words: Enchytraeids, forest soil, nitrogen mineralisation, soil pH
Introduction Clear felling is still the main harvesting method used in northern coniferous forests. Usually, harvesting is followed by mechanical site preparation (e.g. harrowing) to ensure favourable conditions for early development of the next, usually planted, tree generation. As a re-
sult, in the forest floor, exposed mineral soil alternates with thick layers of organic material consisting of humus, litter, felling residues, mosses and ground layer vegetation. In many terrestrial ecosystems, earthworms as large
*E-mail corresponding author: [email protected]
0031-4056/03/47/04–303 $15.00/0
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annelids are functionally one of the most important decomposer animal groups comminuting litter and redistributing organic matter in the soil profile (Edwards 2000). In northern coniferous forest soils, however, earthworms are sparse, and their functional role is often taken up, at least to some extent, by enchytraeids. The enchytraeid fauna of coniferous forests is usually strongly dominated by one species, Cognettia sphagnetorum (Vejdovsky´, 1877) (Nurminen 1967; Abrahamsen 1972). This species has been found to be an important animal in the decomposition of organic matter (Standen 1978; Cole et al. 2000) and nutrient mineralisation (Williams & Griffiths 1989; Abrahamsen 1990; Briones et al. 1998a) in soil organic layer of coniferous forests. Its activity has also been shown to enhance primary production in microcosm experiments (Laakso & Setälä 1999; Laakso et al. 2000). On the other hand, although C. sphagnetorum is an abundant and functionally important species, it is sensitive to many kinds of disturbance. Drought (Didden & de Fluiter 1998; Yli-Olli & Huhta 2000) and various kinds of anthropogenic contaminants (Salminen & Sulkava 1996; Salminen & Haimi 1996, 1999) have negatively affected its performance. This species can, however, efficiently increase in numbers – through asexual reproduction – at least after natural disturbances (Nurminen 1967; Lundkvist 1982). In our previous experiment, C. sphagnetorum was among the first decomposer animals increasing in numbers in mineral soil exposed by simulating mechanical site preparation (Siira-Pietikäinen et al. 2003). Several studies have shown that numbers of enchytraeids increase in the soil organic layers after clear felling of boreal coniferous forests (Huhta 1976; Lundkvist 1983; Siira-Pietikäinen et al. 2001b). This population growth may have been caused by increases in the amount of resources for these saprovorous soil animals (felling residues, dying ground layer plants, increasing bacterial growth and biomass) and more favourable moisture and temperature conditions. In our previous field experiment we found, however, that enchytraeids increased significantly only after elimination of root-mycorrhizal connections to the surrounding soil (trenching treatment; resulting in e.g. increases in dead soil organic matter and moisture) but not after addition of slash (Siira-Pietikäinen et al. 2001a). On the other hand, Siira-Pietikäinen et al. (2001b) observed in a large scale field experiment that enchytraeids increased following clear felling and site preparation without any significant increment in soil bacterial biomass. After clear felling enchytraieds increase in patches with untouched forest floor, while tree seedlings are planted and tree seeds germinate most successfully in exposed mineral soil where resources for enchytraeids Pedobiologia (2003) 47, 303–310
are scarce. Conditions and processes, such as nutrient availability, in the vicinity to seedling roots are therefore assumed to be controlled mainly by abiotic factors. Community structure, biomass and the activity of decomposer animals also have potential, however, to significantly affect nutrient acquisition by tree seedlings in exposed mineral soil (e.g. Setälä & Huhta 1991; Laakso et al. 2000). In this study we aimed to test whether functional properties of C. sphagnetorum are similar in mineral soil as repeatedly observed in soil organic layer. We introduced enchytraeids to laboratory microcosms with soil materials from different layers of coniferous forest floor. We applied a full factorial experimental design to find out whether the influence of C. sphagnetorum is related to soil layer, i.e. amount and quality of resources in soil. Soil materials were taken from the same conifer stand where our previous field experiments were performed (Siira-Pietikäinen et al. 2001a, 2003). We hypothesised that the effect of C. sphagnetorum on nitrogen mineralisation is insignificant in soil where its numbers are low and resources rare, i.e. in mineral soil without abundant organic matter and microbial biomass.
Materials and Methods Microcosms made of plastic cylinders with an inner area of 55 cm2 and a height of 9 cm were used in the experiment. There were holes with cotton plugs in the lids allowing proper evaporation and gas exchange. During the experiment, the microcosms were kept in a climate chamber at +15° C, weighed weekly, and when needed, irrigated with de-ionised water. The experiment had a factorial design with three factors: soil organic layer, needles and enchytraeids; each of these were added or not added to the soil mineral layer (sand). This experimental design resulted in eight treatment combinations, each repeated five times. Soil materials (sand and soil organic layer consisting of humus and partly decayed conifer and ground layer vegetation litter together with dead moss shoot bases) for the experiment were taken from the coniferous forest in which our previous studies were conducted (Siira-Pietikäinen et al. 2001a, 2003). The stand is on podzolic sandy soil in central Finland (61°50'N, 24°20'E), and dominated by Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.). The understorey vegetation consists mainly of Vaccinium myrtillus L., Vaccinium vitis-idaea L., Maianthemum bifolium (L.) F. W. Schmidt and Deschampsia flexuosa (L.) Trin. on a thick moss layer dominated by Hylocomium splendens (Hedw.) Bruch
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Table 1. Properties of the soil materials used in the experiment. pH (in water), organic matter content (loss on ignition), NH4+-N and NO3–-N (water extraction). Means ± standard errors are shown. d.m. = dry mass; o.m. = organic matter Mineral soil pH Organic matter-% NH4+-N µg g-1 d.m. NH4+-N µg g-1 o.m. NO3–-N µg g-1 d.m. NO3–-N µg g-1 o.m.
5.3±0.07 1.0±0.04 0.24±0.030 24.4±3.05 0.47±0.013 47.1±1.357
Soil organic layer 4.2±0.02 90.2±0.48 476±9.3 528±10.3 0.70±0.349 0.78±0.041
& Schimp and Pleurozium schreberi (Brid.) Mitt. The organic soil layer is ca. 4 cm thick (Siira-Pietikäinen et al. 2001a). Spruce needles were collected from felling residues in June 2000 in a similar but clear felled stand near Jyväskylä, central Finland (62°20'N, 25°45'E). The stand was felled in the previous autumn, and the needles had lost their green colour, and they dropped down off the twigs when lightly touched. Amongst the felling residues fresh needles are nutrient rich and they are first to reach the soil surface quite soon after felling, coarse material (twigs and branches) entering the decomposer community in the soil only after many years (Hyvönen et al. 2000). Properties of the materials used in the experiment are shown in Table 1. Soil for extracting enchytraeids was taken in a similar forest stand near Jyväskylä as for the materials used to construct the soil profiles. Enchytraeids were extracted using the standard wet funnel method, and individuals of the species Cognettia sphagnetorum (Vejd.) were collected in Petri dishes in water taken from the collecting tubes. More than 95 % of the individuals were found to represent C. sphagnetorum. The worms were allowed to void most of their gut contents in a climate chamber at +15° C overnight. The soil materials were kept for 24 h at +80° C, and frozen for one week at –22° C to eliminate meso- and macrofauna. After this defaunation procedure, 248.9 g (as dry mass) sand was spread on the bottom of each microcosm. On the mineral soil, 9.3 g (d.m.) humus together with 1.58 g (d.m.) partly decayed plant litter and dead moss shoot bases (hereafter called the soil organic layer) and/or 3.61 g (d.m.) spruce needles were added according to the experimental design. After weighing the soil materials in the microcosms each microcosm was inoculated with microflora and -fauna by adding 2 ml soil-water suspension (obtained from 30 g fresh mass of the same organic soil in one litre of water filtered through a 105 µm mesh) into each of them. Finally, on the next day, 55 enchytraeids were transferred to half of the microcosms. Nine weeks after the
Needles 4.7±0.04 95.1±0.61 6.34±0.072 6.67±0.076 0.26±0.103 0.27±0.109
start of the experiment 26 individuals were added to the same microcosms. Microcosms were sampled twice, 8 and 13 weeks after the establishment. One sector of the soil profile from the soil surface to the bottom consisting of ca. 25 % of the total amount of the soil in the first sampling and ca. 50 % of the remaining soil in the second sampling was taken from the microcosms. Soil materials were then divided for different analyses. Number of enchytraeids, contents of mineral nitrogen (ammonium-N and in the first sampling also nitrate-N), soil moisture and pH were separately analysed from sand (soil mineral layer), soil organic layer, and in the first sampling also from needles (in the second sampling needles were included in the soil organic or mineral layer samples). Enchytraeids were extracted from weighed soil subsamples using the standard wet funnel method. After counting the enchytraeids in a sample the gut contents of each specimen were examined under a microscope at an appropriate magnification. First, the proportion of the gut volume filled with any material was assessed. After that the gut content material was qualitatively identified as far as possible. When needed, the specimen was broken, and the gut content was diluted with water to facilitate the recognition of the material. Fresh soil samples for nitrogen and pH analyses were suspended in deionised water (10–20 g mineral soil, 5–7 g organic soil and 1.5 g needles in 100 ml water) and shaken for half an hour. pH was measured and humus-water suspension was then filtered before analyses of ammomium- (NH4) and nitrate-nitrogen (NO3) with a FIA-autoanalyser (Flow Injection Analyser). Soil moisture was measured by drying subsampels at +105° C overnight. The parameters measured were analysed with analysis of variance for repeated measures (repeated measures ANOVA). When the treatment and time interacted with each other, the effect of that treatment was tested separately for both sampling occasions. When interactions between the two treatments were observed, simple effects were examined with repeated measures Pedobiologia (2003) 47,303–310
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ANOVA (no interactions with time) or one-way ANOVA (treatment-time interactions). In addition, correlations between enchytraeid densities and soil nitrogen were analysed using Pearson correlation coefficient. No transformations were needed for the data. For the statistical analyses, a software package SPSS® for Windows™ Release 10.0 was used.
Results Numbers and gut contents of enchytraeids
Enchytraeids’ population sizes decreased in the microcosms by the first sampling. Significant population growth from the first to the second sampling was observed only in the organic soil (repeated measures ANOVA: effect of time: F = 17.5, P = 0.003) (Table 2). Regardless of the treatment extracted specimens were in good condition. In the microcosms without a soil organic layer, the numbers of enchytraeids varied between 1600 and 4100 individuals m-2 (Table 2), and the presence of needles did not affect their numbers. There were significantly fewer enchytraeids in the mineral soil below the organic layer (density varying from 0 to 620 individuals m-2) than in the mineral soil without it (1580 to 4120 individuals m-2; repeated measures ANOVA: F = 32.7, P < 0.001). In the soil organic layer the presence of needles decreased the numbers of enchytraeids (repeated measures ANOVA: F = 8.7, P = 0.019). In the microcosms with a soil organic layer, 0–11 % of enchytraeids were found in the mineral soil. There was no difference in the amount of gut content between the enchytraeids extracted from the mineral and organic soil. On an average, ca. 65 % of the gut volume was filled with soil and/or microbial material. In the mineral soil there was more material in the enchytraeid guts in the presence of needles than without them (72 ± 2.9 % in the presence of needles and 55 ± 3.7 % in the absence of needles; one-way ANOVA: F = 11.8, P = 0.011). A lot of sand grains Table 2. Total numbers of enchytraeids in the microcosms with different structure (all soil layers summed up; individuals m-2: means and standard errors) Microcosm type
Week 8
Week 13
Mineral soil only + needles + soil organic layer + needles and soil orgnanic layer
2819 (1577) 1578 (1141) 4089 (1182)
2646 (934) 4118 (341) 10 649 (2107)
252 (252)
4613 (1663)
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were found in the guts of the enchytraeids extracted from the mineral soil, the diameter of the largest grains being half of the diameter of the gut. The rest of the gut contents was mainly bacteria and protozoans and quite dark coloured unidentified material. Many live bacteria and protozoans were observed in these samples, while only few pieces of fungal hyphae were found. Enchytraeids extracted from the organic soil had ingested mostly dark humus material including decaying plant fragments of different kinds and sizes, fungal hyphae, and bacteria. The presence of needles did not affect the amount or quality of the enchytraied gut contents in the soil organic layer. Soil pH
The pH of the mineral soil varied between 5.4 and 5.6, and enchytraieds did not have any effect on it. Overall, pH slightly increased during the experiment (repeated measures ANOVA: effect of time: F = 6.0, P = 0.020). Presence of the soil organic layer decreased the pH of the mineral soil by ca. 0.1 pH unit. (repeated measures ANOVA: F = 55.6, P < 0.001). Effects of needles on the pH of the mineral soil differed depending on the presence of the soil organic layer (repeated measures ANOVA: organic layer x needles: F = 8.9, P = 0.005). When there was an organic layer (i.e. all soil layers were present) needles had no influence on mineral soil pH, but without the organic layer (needles on the surface of the mineral soil) needles increased the pH of the mineral soil (repeated measures ANOVA for the microcosms without soil organic layer: F = 16.0, P = 0.001). In the soil organic layer pH varied between 4.8 and 5.0, and it did not change from the first to the second sampling. Here, however, enchytraeids increased the pH (repeated measures ANOVA: F = 6.7, P = 0.020), but only 0.5–1.0 pH units. On the other hand, the presence of needles somewhat decreased the pH of the organic layer (repeated measures ANOVA: F = 21.6, P < 0.001). The pH of the needles on the mineral soil was analysed only on the first sampling: pH varied between 5.3 and 5.7, and enchytraeids had no influence on it. Soil mineral nitrogen
Soil NO3-N was analysed only in the first sampling because its levels appeared to be very low in all treatments. The presence of needles clearly decreased the amount of NO3-N in the soil organic layer (0.15 ± 0.065 with and 0.44 ± 0.084 µg g-1 without needles; one-way ANOVA: F = 6.8, P = 0.019). In the mineral soil, NO3-N was measurable only when the soil organic layer was present in the microcosms (NO3-N
Activity and role of Cognettia sphagnetorum
Fig. 1. NH4+-N contents (µg g-1 dry soil; mean ± standard error) of mineral soil of the microcosms with (a) and without (b) organic soil layer. Mineral = mineral soil only; Ne = needles present; En = enchytraeids present
level was ca. 0.2 µg g-1 dry soil). Enchytraeids or the presence of needles had no effects on NO3-N in the mineral soil. Most of the mineral nitrogen was in the form of NH4, and its amount in the mineral soil increased with time (repeated measures ANOVA: effect of time: F = 32.7, P < 0.001) (Fig. 1). There was a strong immobilisation of nitrogen in the needles: NH4-N was always under the detection limit in the needles and in the absence of the soil organic layer also in the mineral soil under the needles (Fig. 1b). The presence of the soil organic layer increased the amount of NH4 in the mineral soil (repeated measures ANOVA: F = 1878.2, P < 0.001). In the presence of the soil organic layer, the effect of needles depended on the presence of enchytraeids (interaction in repeated measures ANOVA: F = 11.0, P = 0.004) (Fig. 1a). Without enchytraeids
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needles did not significantly affect the amount of NH4-N in the mineral soil, but with enchytraieds needles decreased NH4-N (repeated measures ANOVA: F = 7.727, P = 0.024). The influence of enchytraeids on NH4-N was variable in the mineral soil (interactions in repeated measures ANOVA: enchytraeids × soil organic layer: F=7.9, P=0.009; enchytraeids × needles: F = 17.9, P < 0.001; enchytraeids × soil organic layer × needles: F = 5.7, P = 0.023). When the soil organic layer was present and needles absent, enchytraeids had no significant effect on NH4-N. However, when needles were present on the soil organic layer, the presence of enchytraeids decreased NH4-N in the mineral soil (repeated measures ANOVA: F = 9.4, P = 0.016) (Fig. 1a). In the absence of the organic soil and needles, enchytraeids clearly increased the amount of NH4-N in the mineral soil (repeated measures ANOVA: F = 138.9, P < 0.001) (Fig. 1b). In the mineral soil, the only and weak correlation between enchytraeid densities and the amounts of nitrogen was found in the second sampling in NH4-N when the microcosms with soil organic layer and needles were excluded from the analysis (r = 0.614, P = 0.059). In the soil organic layer enchytraeids had no effect on NH4-N (Fig. 2). However, enchytraeid density and NH4-N correlated positively in the first sampling when only the microcosms with enchytraeids were included in the analysis (r = 0.715, P = 0.020). On the other hand, needles decreased NH4-N also in the soil organic layer (repeated measures ANOVA: F = 37.1, P < 0.001). The amount of NH4-N somewhat decreased from the first to the second sampling (repeated measures ANOVA: effect of time: F = 24.3, P < 0.001) (Fig. 2).
Fig. 2. NH4+-N contents (µg g-1 dry soil; mean ± standard error) of the soil organic layer of the microcosms. Organic = organic soil layer only; Ne = needles present; En = enchytraeids present Pedobiologia (2003) 47,303–310
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Discussion Population densities of C. sphagnetorum remained at quite low levels in our microcosm study. It is evident that mineral soil without any organic matter input cannot maintain high enchytraeid densities for extended periods. The extracted enchytraeids were, however, active and in good condition. Impact of enchytraeids on soil processes seems to be density dependent (Anderson 1988). Thus, it was assumed that their effects on nitrogen mineralisation would be strong in the presence of high population densities but remaining negligible at low densities. The correlation between enchytraeid density and soil NH4-N was, however, significantly positive in rare cases only. The results of the present experiment demonstrate effects of C. sphagnetorum at low densities that are, in fact, common in northern coniferous forests. It should also be noted, that reduction in the biomass of introduced enchytraeids might have contributed to the nitrogen dynamics in our microcosms. This can not, however, fully explain the differences between the treatments. Despite its low densities, C. sphagnetorum had significant impacts on soil processes in our experiment. The effects were, however, variable: enchytraeids increased pH in the soil organic layer but not in the mineral soil where they, on the other hand, affected nitrogen mineralisation. Enhancement of nitrogen mineralisation by enchytraeids in nutrient poor mineral soil can be important for early development of tree seedlings planted on prepared patches in felled areas. When the soil organic layer was present in the microcosms, the effect of enchytraeids on nitrogen mineralisation in mineral soil was different compared to bare mineral soil. In addition, in the soil organic layer enchytraeids had no influence on mineral nitrogen that contrasts the results of many previous studies in which enchytraeids have clearly increased nitrogen mineralisation (Williams & Griffiths 1989; Abrahamsen 1990; Briones et al. 1998a, b). That enchytraeids decreased NH4-N in the mineral soil in the presence of a soil organic layer and needles could be due to enhancement of nutrient immobilisation by soil microbes induced by enchytraeid activity. Liiri et al. (2001) found that wood ash application turned the positive effects of enchytraieds on plant growth and nitrogen content to negative, and hypothesized that dominating microbes in ash-treated soil may have responded differently to enchytraeid grazing compared to microbes in ash-free soils leading to increased nitrogen immobilisation (see also Visser 1985). Thus, the net impact of enchytraieds on soil nitrogen mineralisation seems to be dependent on several environmental factors, such as soil temperature Pedobiologia (2003) 47, 303–310
and moisture (Abrahamsen 1990), as well as quality of resources and microbial community structure. C. sphagnetorum may also enhance nutrient availability for plants by decreasing soil acidity as observed in our experiment in the soil organic layer. The increase of pH was, however, only small compared to e.g. the observations by Briones et al. (1998a). The difference in the magnitude of the effect may be explained by differences in the enchytraeid densities between these studies, density being much lower in our microcosm experiment. In addition to enchytraeids, spruce needles had a clear effect on nitrogen dynamics in our microcosms: nitrogen became immobilised in the needles both on the mineral and organic soil, most likely due to a high C/N ratio of the needles. Hyvönen et al. (2000) also observed net nitrogen immobilisation during the first years of needle decomposition, and pointed out that needles are the most important component of the felling residues in nitrogen (and also phosphorus) dynamics of the forest soil in the early forest regeneration phase after logging. The importance of needles for soil processes became emphasized in the present study because they also affected the numbers of enchytraeids and their role in nitrogen dynamics. It was interesting to notice that C. sphagnetorum was able to live – although in low numbers – and affect soil processes in the mineral soil with extremely low organic matter content. It clearly could find food, e.g. microbial colonies, in this harsh environment. It has been found in the field that C. sphagnetorum can actively move to mineral soil during unfavourable conditions, such as drought and winter frosts (Lundkvist 1982). Whether these enchytraeids are actively feeding and reproducing during their stay in the mineral soil is not fully known. Our gut content observations revealed that C. sphagnetorum can selectively ingest different kinds of available material and also mineral (sand) particles. When living in mineral soil it has to derive its nutrition mainly from microbes, and significant numbers of microbes were observed in the guts of specimens from mineral soil. It has been shown that enchytraeids can derive nutrition from living microorganisms, but that the digestion is incomplete during passage through the intestine (Brockmayer et al. 1990). On the other hand, Stefan (1990) observed that bacterial cells can easily pass through the enchytraeid’s gut without any damage. The resulting dissemination and concentration of bacteria in their faecal pellets could partly explain the important role of enchytraeids in soil nutrient dynamics (Stefan 1990). Encytraeids have a low assimilation efficiency, and by producing large amounts of faecal pellets they are also important for soil structure, as verified especially in organic soils (see Briones et al.
Activity and role of Cognettia sphagnetorum
1998b). In addition, by mixing inorganic and organic materials enchytraeids can have an impact on soil structure as observed e.g. by Dawod & FitzPatrick (1993). From the results of the present experiment we can conclude that C. sphagnetorum can be functionally important in resource poor mineral soil. Due to its asexual reproduction C. sphagnetorum can effectively increase in numbers as soon as conditions become favourable. Thus, in the field, in exposed mineral soil patches created by mechanical site preparation this species has potential to increase along with accumulating organic matter (see also Siira-Pietikäinen et al. 2003). The effect of C. sphagnetorum on soil nitrogen dynamics appeared to vary in relation to environmental conditions, soil layer and resource quality; i.e. acceleration, no effect and inhibition of nitrogen mineralisation are all possible. The same holds also for the effect of C. sphagnetorum on soil pH. Acknowledgements. We are grateful to Tuomas Lukkari for helping us in practical work in the laboratory. The work of Anne Siira-Pietikäinen was supported by the Maj and Tor Nessling Foundation and Finnish Cultural Foundation.
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