Vol. 74: 121–141, 2015 AQUATIC MICROBIAL ECOLOGY Published online February 12 doi: 10.3354/ame01727 Aquat Microb Ecol

Eutrophication of peatbogs: consequences of P and N enrichment for microbial and metazoan communities in mesocosm experiments

Tomasz Mieczan*, Małgorzata Adamczuk, Barbara Pawlik-Skowronska,´ Magdalena Toporowska

Department of Hydrobiology, University of Life Sciences, Dobrzanskiego´ 37, 20-262 Lublin, Poland

ABSTRACT: Complex interactions between bacteria and protists are essential to the ecosystem ecology of peatbogs. However, little is known about how short-term changes in environmental conditions may influence microbial and metazoan communities. Microbial processes and para - meters may be used as sensitive indicators of eutrophication. We address the hypothesis that an increase in the concentration of nutrients (in an experiment simulating eutrophication) will affect species richness and abundance of microorganisms and small metazoans and will cause changes in food web structure in different types of peatbogs. The experiments were performed in a Sphag- num peatland and a carbonate fen. Four experimental treatments were established (control and fertilised: +P, +N, P+N). An increase in habitat fertility was found to modify the taxonomic compo- sition and functioning of microbial communities. This was reflected in a decrease in the species richness of testate amoebae and a substantial increase in the abundance of bacteria, flagellates and ciliates in both types of peatbogs. A better understanding of which parameters regulate microbial populations in peatbogs is critical for more accurate prediction of how peatbogs will respond to global change or anthropogenic disturbances.

KEY WORDS: Bog · Algae · Bacteria · Protists · Rotifers · Cladocera · Copepoda · Microbial food webs

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INTRODUCTION pH (Kankaala et al. 1996, Mitchell et al. 2000, 2003, Mieczan 2007, 2009, Nguyen-Viet et al. 2007, Miec - Peatbogs are highly sensitive to deposition of nutri- zan et al. 2012, Jiroušek et al. 2013). Little is known ents, acids and other pollutants. Nitrogen and phos- about how short-term (i.e. days to months) variability phorus deposition affects many peatbogs in industri- of environmental conditions may influence protistan alised regions and may shift peatbogs from carbon and metazoan community composition (Mieczan & sinks to sources with important consequences for the Tarkowska-Kukuryk 2013). Previous re search has global carbon cycle (Bragazza et al. 2012, Wardle et generally measured environmental variables on the al. 2012, Jassey et al. 2013b). Understanding the im - day of sampling or used seasonal averages (Jiroušek pact of excessive nutrient contamination on ecologi- et al. 2013). Abiotic factors, however, can change the cal systems such as microbial and metazoan commu- strength of biotic interactions, with wide-ranging nities is challenging. Studies suggest that the most consequences for biological communities. An impor- important factors limiting the occurrence of protists tant consequence of the strength of biotic inter - in peatbog ecosystems are physical and chemical actions is the relative importance of top-down and habitat properties — primarily water table depth and bottom-up effects (Hoekman 2011).

*Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 122 Aquat Microb Ecol 74: 121–141, 2015

Since the publication of the microbial loop concept, The present study supports the hypothesis that an there has been increased interest in protists as a increase in the concentration of nutrients will medi- dominant element of microorganism communities in ate changes in species richness and abundance of peatbog biocoenoses (Gilbert et al. 1998a,b, Mitchell microbial and microfaunal communities. Moreover, et al. 2003, Meyer et al. 2012, Mieczan & Tarkowska- we predicted that due to the considerable sensitivity Kukuryk 2013). Testate amoebae and ciliates are of Sphagnum peatlands to environmental changes important consumers of bacteria and algae. They (Andersen et al. 2013), they would be subject to a participate in transformations of organic matter and substantial decrease in microbial species richness nutrients (Gilbert et al. 1998a,b, Mitchell et al. 2003, accompanied by a considerable change in the food Chróst et al. 2009, Jassey et al. 2013a). The classical web structure. grazer food chain and the microbial food web are linked by several direct and indirect interactions. Matter and energy flow, in part, through a microbial MATERIALS AND METHODS loop consisting of dissolved organic matter, hetero- trophic bacteria, flagellates, testate amoebae and cil- Study area iates (Azam et al. 1983, Gilbert et al. 1998a,b). Meta- zoan grazing is an important process for the recycling Two peatbogs were selected for study: the Sphag- of nutrients and the production of dissolved organic num peatland Krugłe Bagno (pH = 2.89) and the car- substrates for bacteria, but it is also a controlling fac- bonate fen Bagno Bubnów (pH = 7.27; Poleski tor for protozoan community structure (Mieczan & National Park, eastern Poland, 51° N, 23° E). The 2 Tarkowska-Kukuryk 2013). Knowledge is scarcer sites represent various trophic status and vegetation still concerning the regulating mechanisms of micro- types. The Sphagnum peatland is mainly open with bial components, with respect to the species compo- scattered small Pinus sylvestris L. trees. Most of it is sition and abundance of potential predators (small slightly minerotrophic, but small ombrotrophic spots metazoans) and type of peatbogs. are present as well. The vegetation is heavily domi- Only a few studies have monitored the effects of nated by S. angustifolium (C.C.O. Jensen ex Rus- simulated addition of nitrogen and phosphorus on sow), S. cuspidatum Ehrh. ex Hoffm., Polytrichum the microorganism communities of peatbogs (Gilbert sp., Eriophorum vaginatum (L.), Carex acutiformis et al. 1998a,b, Payne et al. 2013). The effect of nutri- Ehr hart and C. gracilis Curt. The carbonate fen is ent fertilisation has been widely discussed among colonised by Cladium mariscus (L.), Phragmites aus- ecologists (Gilbert et al. 1998b, Wright et al. 2009, tralis (Car.), Carex acutiformis Ehrhart, Calliergo - Buosi et al. 2011). It has been demonstrated that nella cuspidata (Hedw.) and Utricularia sp. eutrophication has led to serious economic and envi- ronmental problems (Gilbert et al. 1998b, Buosi et al. 2011). Understanding the impact of nutrients on eco- Enclosure and experimental design logical systems such as microbial communities is challenging. Among microbial organisms, protists In both peatbogs, the experiment consisted of 2 have proved to be useful bioindicators of anthro- groups of treatments (fertilised and control), with 3 pogenic pollution in peatbogs and soil (Gilbert et al. replicates each. ‘Mesocosm’ experimental studies 1998b, Nguyen-Viet et al. 2007, 2008, Wardle et al. were conducted 3 times a year, in the spring, summer 2012, Andersen et al. 2013). Research conducted thus and autumn of 2013, for a period of 21 d in each sea- far has mainly concerned the effect of an increase in son. Microbial communities were examined in situ in the content of biogenic compounds on primary pro- 6 polyethylene enclosures (80 l each, 45 × 45 cm, ducers and protists in the Pradeaux raised bog in 40 cm deep). Three enclosures were gently filled France (Gilbert et al. 1998b). The addition of PKCa with surface water (control treatment) and peat and NPKCa was shown to increase the biomass of mosses. Enrichment with biogenic compounds was heterotrophic bacteria, algae (Bacillariophyceae) and carried out in the following experimental treatments: 3− ciliates, and to decrease the contribution of testate +P = water with P-PO4 enrichment (initial concen- amoebae and microalgae. The effect of an increased tration in control treatment [see Tables 1 & 2] × 10); + concentration of mineral forms of nitrogen and phos- +N = water with N-NH4 enrichment (initial concen- 3− phorus on the occurrence of algae, bacteria, protists tration ×10); and P+N = water with P-PO4 and + and small metazoans in other types of peatbogs has N-NH4 enrichments (N:P = 16:1; Table 1). Each not been investigated. experimental treatment was prepared in 3 enclo- Mieczan et al.: Eutrophication in peatlands 123

Table 1. Nutrient enrichment in different experimental mesocosms (+P: water The abundance and biomass of the 3− + with P-PO4 enrichment; +N: water with N-NH4 enrichment; P+N: water with bacteria were determined using DAPI P-PO 3− and N-NH + enrichments) 4 4 (Porter & Feig 1980). Water samples of 10 ml were preserved in formalde- Treatment Spring Summer Autumn hyde up to a final concentration of 2% Sphagnum peatland and kept in darkness at 4°C. Prepara- 3– −1 +P (mg P-PO4 l ) 3.32 0.22 3.03 tions were made within 24 h after + −1 +N (mg N-NH4 l ) 14.72 6.04 1.01 sampling. Sub-samples of 2 ml were 3– −1 P+N (mg P-PO4 l 0.332 + 23.55 0.022 + 9.66 0.303 + 1.6 condensed on polycarbonate filters + mg N-NH + l−1) 4 dyed with Irgalan black (pore diame- Carbonate fen 3– −1 ter of 0.2 µm). The contribution of +P (mg P-PO4 l ) 1.72 1.11 0.02 + −1 active bacteria with intact mem- +N (mg N-NH4 l ) 10.95 6.06 0.76 3– −1 P+N (mg P-PO4 l 0.172 + 17.52 0.111 + 9.69 0.002 + 1.2 branes (MEM+) was analysed using + −1 + mg N-NH4 l ) LIVE/DEAD BacLight Bacterial Via- bility Kits with 2 dyes, SYTO 9 and propidium iodide (PI), according to sures. A mixture of inorganic nutrients was added to Schumann et al. (2003). SYTO 9 labels all bacteria the 3 mesocosms according to Redfield’s ratio N:P = with intact and damaged membranes, and PI pene- 16:1 (Redfield et al. 1963). trates bacteria with damaged membranes. A mixture The abundance of bacteria, heterotrophic nano- of 2 stains was added (1:1, final concentration of both flagellates (HNF), testate amoebae and ciliates was dyes 0.15%) for a 1 ml subsample, incubated for 15 min measured on Days 0, 7, 14 and 21 of the experiment. at room temperature in the dark, filtered through a Algal, rotifer and crustacean communities were de - 0.2 µm pore size black polycarbonate membrane fil- termined at the beginning and the end of the experi- ter and enumerated by epifluorescence microscopy. ment. Water with Sphagnum moss was sampled The abundance and biomass of HNF were deter- using a plexiglass corer (length 1.5 m, ∅ 50 mm) mined with primuline solution (Caron 1983). Water placed in the bog with mosses. After the tube was samples of 10 ml were collected into dark sterilised filled with water, its lower end was closed with the bottles. The samples were preserved in formalin up bung. The tube was then raised vertically, the lower to a final concentration of 2% and kept in darkness at bung was removed and the water samples were col- 4°C. Four preparations were made from each sample. lected using a glass pipette. To extract the different Sub-samples were condensed on 0.8 µm pore size groups of micro- and macroorganisms from the black Nucleopore filters and enumerated by epifluo- mosses, each sample was first shaken for 1 min with rescence microscopy. a vortex and then filtered through a 250 µm mesh The abundance of testate amoebae and ciliate filter. The volume of water extracted from the plexi- community composition were determined following glass corer ranged from 400 to 500 ml. During each the method of Utermöhl (1958). Testate amoebae and sampling occasion (season), 3 replicate samples were ciliate samples (3 samples; 500 ml each) were sedi- collected from each enclosure. Peatbog water taken mented for 24 h in a cylinder stoppered with Para - from each enclosure was mixed (v/v) together and film, and then the upper volume of 400 ml was gently the integrated sample was treated as representative removed. To determine density and biomass, 3 sam- for the mesocosm. ples were preserved with Lugol’s solution. Addition- The abundance of algae and cyanobacteria was ally, live samples were observed for taxonomic iden- determined using light microscopy in a 1 ml plankton tification. Morphological identification of the testate chamber. Water samples (100 ml) were fixed with a amoebae and ciliates was mainly based on works by formalin-glycerin solution and concentrated by 48 h Foissner & Berger (1996), Foissner et al. (1999), Char- settlement, and then phycoflora was counted; 100 µm man et al. (2000) and Clarke (2003). of filamentous species and 1 colony of coccoid taxa Biovolumes of each microbial community were were recognised as individuals. The biomass of the estimated by assuming geometric shapes and con- microalgae was estimated by cell volume measure- verting to carbon using the following conversion fac- ments (Hillebrand et al. 1999). Taxonomic identifica- tors: heterotrophic bacteria, 1 µm3 = 0.56 × 10−6 µg C; tion of live and fixed material was carried out. Algal flagellates, 1 µm3 = 0.22 × 10−6 µg C; ciliates and tes- systematics were based on Van den Hoek et al. tate amoebae, 1 µm3 = 0.11 × 10−6 µg C (Gilbert et al. (1995). 1998a). 124 Aquat Microb Ecol 74: 121–141, 2015

Rotifers and crustaceans (Cladocera and Cope- ponent analysis (PCA) was used to measure and poda) were collected using a 1 l sampler. Samples illustrate the variability gradients indicated by algae, were sieved through a 40 µm mesh net, dispersed bacteria, protists, rotifers and crustaceans. Because into a 100 ml bottle and fixed with a formalin-glyc- the length of the gradient was <2 standard devia- erin solution. Studies by Ruttner-Kolisko (1974) and tions, we used redundancy analysis (RDA), a method Koste (1978) were used to identify Metazoa. Biomass assuming unimodal species−environment relation- of rotifers was calculated using standard dry weights ships (ter Braak 1988−1992, Kovach 2002). from Bottrell et al. (1976). In the laboratory, crus- taceans were classified and then counted using a Sedgewick-Rafter cell to calculate abundance, ex- RESULTS pressed as ind. l−1. Crustacean biomass was esti- mated using the ratios between the body length and Environmental variables body mass of a given specimen (Dumont et al. 1975, Bottrell et al. 1976) by established mathematic for- Temperature did not differ between the control and mulas. Carbon content of metazoans was calculated the nutrient-enriched experimental treatments (p = using a conversion factor of 0.48 µg C µg−1 dry 0.039). In the peatbogs studied, the highest average weight (Andersen & Hessen 1991). temperature values were noted in summer (21 to 23°C) and the lowest in autumn (10 to 12°C). Irre- spective of the type of peatbog, a substantial de - Physical and chemical variables crease in oxygen concentration was observed in all treatments, as well as an increase in pH and conduc- At the start and end of the experiments, tempera- tivity (p < 0.05 for all). This effect was particularly ture, conductivity, pH, dissolved oxygen (DO), chloro- evident in the P+N treatment. An increase in the con- 3− phyll a (chl a), phosphates (P-PO4 ), ammonium centration of chl a of >900% was recorded in the + nitrogen (N-NH4 ) and dissolved organic carbon mesocosms located in the Sphagnum peatland (from (DOC) were measured in all enclosures. Physical and 16.34 to 150.62 µg l−1). DOC concentrations in all chemical parameters were determined according to treatments increased by 10 to 15%. This was particu- standard methods for hydrochemical analyses (Gol - larly evident in the +N and P+N experiments. The terman 1969). Temperature, conductivity, pH and DO concentrations of ammonium nitrogen showed an were assessed at the sites with a multiparametric increasing trend during the experiment. Similar pat- 3− probe (Hanna Instruments). P-PO4 was determined terns were observed in the case of phosphate con- + by the colorimetric method and N-NH4 by the Kjel- centrations (p < 0.05 for all; Tables 2 & 3). We found dahl method. Chl a was determined by spectro - significant seasonal variations for all abiotic factors photometry following extraction with ethanol. DOC except for DOC (Table 2). analysis was performed using wet potassium persul- phate digestion with an O/I Corporation Model 700 TOC analyser. Phycoflora abundance and composition

In the Sphagnum peatland, enrichment with +P Numerical analyses and +N did not significantly influence the total spe- cies richness of phycoflora (p > 0.005). In contrast, in The mean densities and biomass of the various the carbonate fen, the enrichment caused an in - groups of microorganisms were compared between crease in the total number of taxa of eukaryotic algae experiments and between time periods using 2-way and cyanobacteria (from 74 to 91). The P-enrichment repeated measures ANOVA. The significance level caused an increase in algal abundance in both habi- was set at p < 0.05. Pearson’s correlation coefficients tats, but in the carbonate fen, P-enrichment also were calculated in order to specify the interactions accounted for a strong increase in cyanobacterial between components of the peatbog food webs. The abundance in the spring (p < 0.05; Fig. 1A, Table 4). analysis was performed using STATISTICA 7.0 soft- In the Sphagnum peatland, in the control and +P ware. Ordination methods were used to examine the and P+N treatments, the most abundant algae general structure of the microorganism and meta- were desmids (Cosmarium spp., Zygnematophyceae, zoan data and to test the link between the microbial Chlo ro phyta) and small-sized cyanobacteria (Aphan- communities and environmental data. Principal com- otheceae minutissimum). In +N, alongside the domi- Mieczan et al.: Eutrophication in peatlands 125

3− Table 2. Changes in physical and chemical parameters in 4 experimental mesocosms (control; +P: water with P-PO4 enrich- + 3− + ment; +N: water with N-NH4 enrichment; P+N: water with P-PO4 and N-NH4 enrichments). S: start of experiments (Day 0); E: end of experiments (Day 21); DOC: dissolved organic carbon

3– + Parameter/ pH O2 Conductivity P-PO4 N-NH4 Chl a DOC −1 −1 −1 −1 −1 −1 mesocosm (mgO2 l ) (µS cm ) (mg l ) (mg l ) (mg l ) (mg l ) SE SE S E S E S E S E SE

Sphagnum peatland SPRING Control 2.89 6.78 8.0 8.2 26.2 32.1 0.332 0.311 4.71 4.61 16.35 62.8 7.0 7.0 +P 3.88 7.88 8.0 6.2 27.4 36.1 3.32 3.52 4.71 4.67 17.24 4.50 7.1 5.9 +N 4.78 8.88 8.0 6.1 29.6 37.2 0.057 0.027 15.829 18.711 16.22 16.61 7.2 6.5 P+N 4.89 8.88 8.0 6.1 31.2 37.2 1.698 1.321 13.591 14.121 16.35 39.17 7.0 9.2 SUMMER Control 3.89 7.87 8.6 5.7 31.2 42.2 0.022 0.067 0.811 0.921 16.34 92.08 8.2 8.9 +P 4.89 8.87 8.6 5.2 33.6 54.1 2.792 1.992 1.405 1.301 16.34 150.62 8.2 9.1 +N 4.89 8.86 8.6 6.2 37.2 58.4 0.061 0.033 4.334 5.894 16.34 112.35 8.2 9.6 P+N 5.89 8.86 8.6 6.2 38.4 58.4 0.073 0.063 1.157 1.987 16.36 118.49 8.3 9.7 AUTUMN Control 2.67 6.76 9.3 8.1 45.2 65.2 0.303 0.039 0.599 0.611 12.1 78.83 6.4 7.2 +P 3.68 6.74 9.4 7.2 51.3 65.3 1.216 1.112 0.466 0.366 12.1 98.84 6.4 6.8 +N 3.69 6.74 9.5 7.4 52.6 65.2 0.066 0.036 2.706 3.116 12.1 68.81 6.4 6.7 P+N 4.70 6.58 9.6 6.8 57.8 65.5 1.588 1.789 8.480 9.563 12.1 78.86 6.4 7.1 Carbonate fen SPRING Control 7.2 8.6 8.6 8.1 66.2 63.3 0.172 0.167 1.095 1.099 18.32 23.2 8.9 9.4 +P 7.8 8.5 8.6 6.2 68.0 71.0 0.864 0.964 1.162 1.011 18.32 54.2 8.9 10.2 +N 7.9 8.4 8.6 5.2 68.0 69.1 0.163 0.179 10.981 12.123 18.32 32.1 8.9 13.1 P+N 8.2 8.6 8.6 5.1 68.0 172.6 1.388 1.437 11.736 13.132 18.32 22.2 8.9 14.2 SUMMER Control 7.6 8.2 8.9 5.7 51.3 52.4 0.111 0.037 1.386 1.471 31.17 54.12 7.3 7.6 +P 7.9 8.7 8.9 5.3 53.4 67.9 1.898 1.698 1.211 1.110 31.17 45.37 7.2 7.7 +N 8.2 8.9 8.9 5.2 59.2 89.2 0.016 0.034 5.484 6.127 31.17 44.69 7.2 7.9 P+N 8.6 9.4 8.9 5.3 61.7 138.2 0.004 0.023 3.795 4.124 31.17 50.46 7.2 7.9 AUTUMN Control 7.2 8.60 8.1 7.8 78.2 82.3 0.002 0.065 0.076 0.691 14.32 15.21 6.2 6.6 +P 7.5 8.63 8.1 6.1 83.3 92.6 0.139 0.129 0.446 0.432 14.32 41.2 6.2 6.9 +N 7.9 8.50 8.1 5.2 84.2 98.3 0.111 0.110 1.846 2.896 14.32 26.2 6.2 6.8 P+N 8.2 8.34 8.1 4.2 98.5 189.2 1.209 1.104 7.838 9.246 14.32 31.6 6.2 6.9

nance of Cosmarium, there was also an increase in and P+N. In P+N, increased abundance of Crypto - the abundance of the raphidiophyte (Vacuolaria-like) monas sp. (Cryptophyceae) was also noted. The con- and large-sized desmid Netrium digitus var. naegeli, tribution of green algae in the +P, +N and P+N exper- but a decrease in cyanobacterial abundance. In the imental treatments decreased to 0.3, 18, and 15%, carbonate fen, enrichment with biogenic compounds res pectively. caused a more complex transformation in the taxo- nomic structure of the phycoflora — from the quan - titative dominance (49%) of diatoms (Bacillario- Bacterial abundance and activity phyceae, mainly Synedra ulna, Fragillaria spp., Nitzschia spp.), Chlorophyta (mainly small-sized In the Sphagnum peatland, the highest density of Chlorophyceae, 17%; filamentous Oedogonium spp., bacteria was recorded in experimental treatments 11%) and cyanobacteria (17%, mainly Planktolyng- +N and P+N (3.6 to 3.9 ± 2.2 [SD] × 106 cells ml−1; bya sp.) in the control to the dominance of pico- Fig. 1B). The highest increase in the total abundance cyanobacteria (e.g. Cyanobium sp., Aphanothece of metabolically active bacteria was observed in spp.) in the +P, +N and P+N treatments, with a high mesocosms +N and P+N. In the carbonate fen, a sub- contribution of Bacillariophyceae (25 to 34%; e.g. stantial increase in the density of bacteria was Fra gillaria spp. and Synedra spp.) in treatments +N recorded only in the P+N mesocosm. In comparison 126 Aquat Microb Ecol 74: 121–141, 2015

Table 3. Results of main effects ANOVA for physical and chemical parameters of water, testing for the effect of the time (season) and the mesocosms. Significant (p <0.05) results are highlighted in bold

Sphagnum peatland Carbonate fen df SS MS F pdfSSMS F p

Temperature Intercept 1 4707.41 4707.41 7381.24 <0.001 1 3201.4 3203.1 6280.44 <0.001 Mesocosms (Me) 1 0.623 0.623 0.978 0.339 1 0.534 0.534 0.966 0.235 Season (Se) 2 665.56 332.7 521.8 <0.001 2 615.5 222.7 420.8 <0.001 Me × Se 2 501.82 250.9 9.75 <0.001 2 501.82 250.9 9.75 <0.001 pH Intercept 1 354.05 354.05 15260 <0.001 1 324.05 324.05 16780 <0.001 Me 1 1.04 5.04 31.98 <0.001 1 1.04 1.04 34.9 <0.001 Se 2 4.67 1.04 44.9 <0.001 2 4.45 3.47 100.91 <0.001 Me × Se 2 4.94 2.34 100.64 <0.001 2 4.14 3.31 12.33 <0.001 Conductivity Intercept 1 24790.2 24790.2 223.96 <0.001 1 23660.1 23660.1 213.15 <0.001 Me 1 648 648 5.85 0.029 1 648 648 6.45 0.020 Se 2 1632.11 816 7.37 0.005 2 1632.1 816.06 6.35 0.005 Me × Se 2 1678.99 6 6.13 0.005 2 1932.2 1036.83 6.36 0.005 + N-NH4 Intercept 1 155440.01 155440.01 237.32 <0.001 1 175420.01 175420.01 337.32 <0.001 Me 1 3 1251 1.91 0.188 1 1251.7 3000 2.21 0.231 Se 2 1251.7 2731 4.17 0.037 2 6321 1251.7 5.11 0.021 Me × Se 2 5462 1368 1.56 0.226 2 4180 2731.01 2.31 0.331 3– P-PO4 Intercept 1 0.93 6.42 232.41 <0.001 1 0.83 5.45 251.33 <0.001 Me 1 0.13 0.25 31.41 <0.001 1 0.13 0.25 30.42 <0.001 Se 2 0.42 0.12 52.56 <0.001 2 0.55 0.11 50.41 <0.001 Me × Se 2 0.19 0.23 3.21 0.050 2 0.13 0.25 4.2 0.050 Chl a Intercept 1 211736 211736 32.21 <0.001 1 211651 211651 32.21 <0.001 Me 1 24738.2 6300 3.76 0.072 1 24738 24500.2 3.66 0.062 Se 2 157068 24738 11.94 <0.001 2 157068 63456.1 14.91 <0.001 Me × Se 2 136915 78534 5.49 0.008 2 136915 63412.5 4.32 0.005 DOC Intercept 1 30041 30041 284.03 <0.001 1 41241 30041 284.03 <0.001 Me 1 44.34 44.34 0.42 0.528 1 47.23 47.31 0.42 0.520 Se 2 180.38 90.19 105.77 0.447 2 113.31 90.11 105.77 0.221 Me × Se 2 66.88 33.44 0.33 0.721 2 46.78 31.42 0.33 0.729 to the control group, an increase in the contribution in the abundance of these organisms was observed in of metabolically active bacteria was observed only in mesocosm +N (3.2 ± 0.2 × 103 cells ml−1). In meso- mesocosm +N (p < 0.001; Fig. 2). The dynamics and cosm N+P, their abundance decreased (2.4 ± 0.3 × density of heterotrophic bacteria in the peatbogs 103 cells ml−1). In the peatbogs studied, the maximum were similar, reaching peak values in spring and abundance of HNF was noted in the spring and summer. The numbers of bacteria in all mesocosms autumn (Fig. 1C, Table 4). were lowest in August. The ANOVA suggested that the species richness and total density of testate amoebae responded to the nutrient treatments (p < 0.05 for all) and changed Protozoan abundance and composition over time (p < 0.001) only in the Sphagnum peatland (Table 3). In this peatbog, in the P+N experimental In the Sphagnum peatland, the highest numbers of treatment, 14 fewer taxa were found than at the flagellates occurred in mesocosm +P (2.8 to 3.3 ± beginning of the experiment (37 species). The den- 0.6 × 103 cells ml−1). In the control samples, the abun- sity was much higher in the control samples (3 ± 1.0 × dance was almost 2 times lower: 1.1 ± 0.1 × 103 cells 102 cells ml−1) than in the +P, +N and P+N samples (p < 0.05). In the carbonate fen, only a slight increase (1.8 to 2.0 ± 1.0 × 102 cells ml−1). In the carbonate fen, Mieczan et al.: Eutrophication in peatlands 127

Fig. 1. Seasonal changes in the numbers of (A) phy- coflora, (B) bacteria, (C) heterotrophic nanoflagellates, (D) testate amoebae, (E) ciliates, (F) rotifers and (G) crustaceans in 4 experimental mesocosms (C: control 3− treatment; +P: water with P-PO4 enrichment; +N: + water with N-NH4 enrichment; P+N: water with P- 3− + PO4 and N-NH4 enrichments) in Sphagnum peatland and a carbonate fen. Error bars are SD 128 Aquat Microb Ecol 74: 121–141, 2015

Table 4. Results of main effects ANOVA on density of algae, bacteria, heterotrophic nanoflagellates (HNF), testate amoebae, ciliates, rotifers and crustaceans, testing for the effect of the time (season) and the mesocosms. Significant (p <0.05) results are highlighted in bold

Sphagnum peatland Carbonate fen df SS MS F pdfSSMS F p

Phycoflora Intercept 1 480.50 480.50 961.00 <0.001 1 480.50 480.50 961.00 <0.001 Mesocosms (Me) 1 144.50 144.50 289.00 <0.001 1 233.10 123.50 289.00 <0.001 Season (Se) 7 21.41 13.51 9.43 0.042 7 23.45 15.54 9.43 0.031 Me × Se 2 13.11 15.11 12.32 0.047 2 13.11 15.11 12.32 0.047 Bacteria Intercept 2 70.02 70.01 90.28 <0.001 2 67.21 60.04 80.11 <0.001 Me 2 21.00 11.03 14.97 <0.001 2 23.11 12.01 15.14 <0.001 Se 7 21.46 14.51 9.43 0.050 7 21.45 14.55 9.35 0.050 Me × Se 2 2.18 9.12 17.35 0.033 2 4.11 9.12 18.31 0.031 HNF Intercept 2 70.02 60.01 90.28 <0.001 2 70.02 62.02 90.23 <0.001 Me 2 20.00 11.03 18.97 <0.001 2 20.00 11.01 18.90 <0.001 Se 7 21.41 13.51 9.43 0.032 7 22.42 15.14 9.23 0.031 Me × Se 2 3.18 12.12 17.35 0.033 2 3.18 12.12 16.31 0.041 Testate amoebae Intercept 1 53.74 53.74 27.85 <0.001 1 71.23 64.71 28.84 <0.001 Me 2 14.72 19.32 5.81 <0.001 2 14.72 20.22 7.12 <0.001 Se 8 5.76 7.21 3.52 0.092 8 5.71 7.22 4.51 0.092 Me × Se 2 2.36 1.53 4.62 0.081 2 2.66 1.21 3.61 0.081 Ciliates Intercept 2 80.02 80.01 120.28 <0.001 2 70.02 80.01 103.14 <0.001 Me 2 21.00 11.03 14.97 <0.001 2 21.00 10.14 15.32 <0.001 Se 7 0.46 0.54 7.43 0.055 7 0.56 0.67 8.11 0.069 Me × Se 2 2.18 9.12 17.35 0.030 2 3.11 8.14 19.23 0.020 Rotifers Intercept 1 27.11 26.22 68.12 <0.001 1 24.12 24.21 65.11 <0.001 Me 2 5.92 2.95 50.75 <0.001 2 5.96 3.11 48.13 <0.001 Se 9 8.81 11.11 18.52 0.050 9 8.80 15.12 19.14 0.050 Me × Se 2 0.57 3.31 8.19 0.047 2 0.64 3.27 9.14 0.041 Crustaceans Me 1 43.24 53.74 27.85 <0.001 1 42.22 55.66 24.41 <0.001 Se 2 23.32 19.32 55.81 0.087 2 23.32 39.35 16.81 0.057 Me × Se 8 8.72 12.21 3.52 0.092 8 8.75 14.43 3.52 0.093

a slight increase in the testate amoebae abundance dominance of H. elegans. In the carbonate fen, the was observed in the treatments with added nutrients maximum value was noted in the spring, with domi- (Fig. 1D). The dominance structure of the testate nance of A. discoides. The contribution of empty amoebae underwent relatively insignificant changes. shells in comparison to the control sample also in - The control samples from the Sphagnum peatland creased. Living amoebae constituted from 56 to 60% were primarily dominated by Hyalosphenia elegans, in mesocosms +P and P+N, while in the control sam- Amphitrema flavum and Nebela tincta. In the control ples they reached up to 76%. samples in the carbonate fen, Arcella discoides and The addition of nutrients caused an increase in the Corythion dubium were dominant. In the samples species richness of ciliates (p < 0.05). It was particu- with added nutrients, we observed only a slight in - larly evident in the Sphagnum peatland, where 18 crease in the contribution of species from the genera taxa were recorded in the control sample and as Arcella and Nebela. The density of testate amoebae many as 28 ciliate taxa in mesocosms +N and P+N. In did not vary significantly between seasons. In the the carbonate fen the species richness of ciliates was Sphagnum peatland, only a small peak of testate also much higher in the +N and P+N mesocosms. In amoebae density was noted in the summer, with both peatbogs, significantly higher ciliate density Mieczan et al.: Eutrophication in peatlands 129

3 in the carbonate fen). In both peatbog types, the densities decreased significantly (p < 0.001) with the added nutrients (Table 3, Fig. 1G). In the Sphagnum peatland, in all mesocosms, cladocerans Alonella excisa and Ceriodaphnia setosa were dominant. In the carbonate fen, small-sized cladoceran Chydorus sphaericus predominated in the control samples and cyclopoid copepods Megacyclops viridis and Eucy- clops macruroides in +P, +N and P+N. In both peat- lands studied, the maximum abundance of Metazoa was noted in autumn, except in the control samples from the Sphagnum peatland, where the highest density was seen in spring. Fig. 2. Seasonal changes in percentage contribution of active cells (those with intact membranes, MEM+) to the total num- bers of bacteria in 4 experimental mesocosms (C: control 3− treatment; +P: water with P-PO4 enrichment; +N: water Biomass and relations between food web + 3− with N-NH4 enrichment; P+N: water with P-PO4 and N- components + NH4 enrichments) in 2 investigated peatbogs (Sphag num peatland and carbonate fen). Error bars are SD Our results show that the biomass of algae, bacte- ria, flagellates, ciliates and rotifers increased (on was observed in the +P and P+N experimental treat- average by 15%), while that of testate amoebae and ments, where small Scuticociliatida and large species crustaceans remained unaffected or even decreased. belonging to the group Peritrichida predominated N-enrichment accounted for a significant increase in (Table 4, Fig. 1E). The dynamics of ciliate communi- the biomass of algae only in the Sphagnum peatland ties in the peatbogs were very similar, reaching (Table 5). The biomass of bacteria was significantly maxi mum values in spring and summer during the higher in experimental treatments +N and P+N than mass development of small bacterivorous Scuticocili- in the control sample. The relative biomass of HNF, atida and Colpodea. The density was lowest through- ciliates and rotifers significantly increased from out the autumn. 4−7% in the control treatments to 8−20% in experi- mental treatments +P, +N and P+N. At the same time, the relative biomass of testate amoebae and crus- Metazoan abundance and composition taceans decreased from 25−30% in the control sam- ples to 7−10% in the samples with added nutrients The species richness of rotifers increased together (Fig. 3A,B). The biomass of each microbial commu- with the enrichment of the water with nutrients. This nity varied seasonally. In all peatbogs, the biomass of was particularly evident in the carbonate fen, where heterotrophic bacteria was very low in summer and in the experimental treatment P+N, 5 more taxa were autumn, and reached its maximum in spring. The recorded than at the beginning of the experiment peaks of the HNF biomass were noted in spring and (6 taxa). Irrespective of the type of peatbog, signifi- autumn. HNF biomass throughout the summer was cantly higher densities of rotifers were observed in very low. The biomass of testate amoebae was char- the experimental treatments +P and +N (p < 0.001; acterised by 2 peaks occurring in spring and autumn Table 4, Fig. 1F). In the control samples in the Sphag- in the Sphagnum peatland, while in the carbonate num peatland, the rotifer community was dominated fen the highest peaks were recorded in summer and by Bdelloidea. In mesocosms +P, +N and P+N, the autumn. The highest biomass of ciliate and metazoan contribution of the genera Lecane and Keratella communities was noted in spring and autumn. In the increased. In the carbonate fen, Keratella and Filinia Sphagnum peatland, the strength of the correlation were dominant in all mesocosms. between DOC and bacteria, as well as between bac- The highest number of species of Crustacea was teria and protists, increased in mesocosms with found in the control samples (6 species in the Sphag- added nutrients (Table 6). In the carbonate fen, in num peatland and 8 in the carbonate fen), and lower mesocosms +P and P+N, no significant correlations in the nutrient-enriched treatments. The lowest num- were observed between the biomass of bacteria and ber of species was found in the experimental treat- DOC. A very strong correlation was found between ment P+N (4 species in the Sphagnum peatland and the biomass of Metazoa, ciliates and HNF (Table 6). 130 Aquat Microb Ecol 74: 121–141, 2015

Table 5. Results of main effects ANOVA on biomass of algae, bacteria, heterotrophic nanoflagellates (HNF), testate amoebae, ciliates, rotifers and crustaceans, testing for the effect of the time (season) and the mesocosms. Significant (p <0.05) results are highlighted in bold

Sphagnum peatland Carbonate fen df SS MS F pdfSSMS F p

Phycoflora Intercept 1 410.40 470.30 151.00 <0.001 1 410.40 480.10 153.00 <0.001 Mesocosms (Me) 1 123.20 124.60 179.30 <0.001 1 122.20 123.70 188.40 <0.001 Season (Se) 7 24.22 11.11 9.43 0.050 7 24.26 11.11 9.43 0.050 Me × Se 2 15.11 17.11 12.36 0.050 2 16.21 16.14 11.23 0.030 Bacteria Intercept 2 62.04 72.01 80.28 <0.001 2 61.07 70.01 80.23 <0.001 Me 2 21.00 11.03 13.98 <0.001 2 21.00 12.00 14.67 <0.001 Se 7 21.46 15.53 9.43 0.050 7 22.42 16.25 8.53 0.050 Me × Se 2 2.18 9.12 7.15 0.031 2 2.46 8.17 8.13 0.040 HNF Intercept 2 90.05 50.12 80.32 <0.001 2 70.00 40.15 70.23 <0.001 Me 2 20.00 11.03 17.91 <0.001 2 19.21 15.12 18.95 <0.001 Se 7 31.41 14.31 9.43 0.031 7 45.11 23.43 18.34 0.041 Me × Se 2 3.18 12.12 16.33 0.032 2 4.12 14.13 18.34 0.040 Testate amoebae Intercept 1 460.40 430.32 731.03 <0.001 1 378.50 441.12 647.02 <0.001 Me 1 144.50 144.50 299.00 <0.001 1 145.50 145.50 211.00 <0.001 Se 2 17.00 6.00 12.00 0.137 2 18.00 7.14 14.65 0.121 Me × Se 2 4.00 2.00 4.00 0.064 2 5.00 2.00 5.00 0.089 Ciliates Intercept 2 80.02 80.01 120.28 <0.001 2 80.34 80.32 140.12 <0.001 Me 2 21.00 11.03 14.97 <0.001 2 23.00 14.21 15.18 <0.001 Se 7 9.43 17.21 12.23 0.050 7 12.14 23.14 16.12 0.050 Me × Se 2 3.18 14.12 15.21 0.030 2 4.23 16.12 17.32 0.020 Rotifers Intercept 1 27.11 26.22 68.12 <0.001 1 32.12 36.21 76.12 <0.001 Me 2 5.92 2.95 50.75 <0.001 2 6.67 3.43 42.12 <0.001 Se 9 8.81 11.11 18.52 0.050 9 8.21 10.23 20.14 0.050 Me × Se 2 0.57 3.31 8.19 0.050 2 1.24 4.27 7.22 0.050 Crustaceans Intercept 1 43.24 153.74 27.85 0.123 1 44.21 42.75 59.14 0.050 Me 2 23.32 39.32 15.81 0.234 2 76.65 32.14 6.21 0.050 Se 8 8.72 12.21 3.52 0.088 8 9.32 14.22 4.55 0.077 Me × Se 2 3.14 1.13 5.72 0.079 2 2.15 2.11 6.74 0.088

Ordination analyses The direct effect of the treatments on abundance of the communities studied is seen on the ordination PCA axis 1 (λ = 0.516) and axis 2 (λ = 0.351) ex- plot; as a result, the 4 treatments are distributed sepa- plained 49.6% of the total variance in community rately on the ordination diagram (Fig. 5B). However, composition (Fig. 4). In the Sphagnum peatland (RDA RDA performed for each taxonomic group separately analyses) all of the analysed variables together ex- showed that environmental variables explained be- plained 57.4% of the total variance. Variables tween 54.1% (Crustacea) and 87.8% (algae) of total that significantly explained variance in community variance in their distribution in the mesocosms. De- + abundance included N-NH4 , DOC and chl a. On spite high variance in the algal taxa and environmen- + the ordination plot, control samples are clearly sepa- tal factors, only 2 variables, DOC and N-NH4 , showed rated from treatments +P, +N and P+N (Fig. 5A). RDA significant importance, and both variables corre- for the carbonate fen showed that all variables ex- sponded with Chrysophyceae, Zygnematophy ceae, plained 74.1% of the total variance. The Monte Carlo Dinophyceae, Bacillariophyceae, Ulvophyceae and 3− permutation test showed the significance of 5 vari- filamentous Chlorophyta (Fig. 6A). pH, DOC, P-PO4 + 3− + ables: N-NH4 , P-PO4 , DOC, pH and O2 (Table 7). and N-NH4 appeared to be the variables best ex- Mieczan et al.: Eutrophication in peatlands 131

positively related to O2. A number of spe- cies, including Colpoda steinii, Askenasia volvox, Para dileptus elephantinus, Pla- giopyla nasuta, Spa thi dium sensu lato, Spirostomum ambigum, Paramecium pu- trinum, Aspidisca cos tata, Holosticha pul- laster, Oxytricha sp., Stylonychia mytilus, Codonella cratera, Halteria gradinella, Amphileptus cleparedei , Amphileptus pleurosigma, Coleps hirtus and Prorodon 3− sp., were related to P-PO4 (Fig. 6C). Three of the variables significantly af- fected the assemblages of rotifers: DOC, + N-NH4 and pH (Fig. 6D). On the ordina- tion plot, a group of species including the genera Lecane (L. intrasinuata, L. luna, L. lunaris) and Lepadella (L. ovalis, L. patella), as well as Colurella hindenburgi, Dicranophorus hercules and Keratella serrulata, showed a correlation with N- + NH4 . Lecane closterocerca, Collotheca wiszniewski, Cephalodella gibboides and Keratella cochlearis were related to pH, while Cephalodella gibba was most influ- enced by DOC (Fig. 6D). For crustaceans, pH and DOC showed significant impor- tance (Fig. 6E). pH was the variable that most strongly influenced the distribution Fig. 3. Relative contribution of microbial groups to the total microbial com- of the copepods Macrocyclops fuscus and munity in 4 experimental mesocosms (C: control treatment; +P: water with 3− + Thermocyclops crassus, whereas the P-PO4 enrichment; +N: water with N-NH4 enrichment; P+N: water with 3− + cladoceran Chy dorus sphaericus and the P-PO4 and N-NH4 enrichments) over 3 seasons in (A) Sphagnum peat- land and (B) carbonate fen nauplius stage of Cope poda were related to DOC (Fig. 6E). plaining the variability of testate amoebae in all ex- perimental treatments (Fig. 6B). The species Cory- DISCUSSION thion dubium and Nebela carinata corresponded with the rising gradient of pH. A large group of species (in- Irrespective of the type of peatbog, a decrease in cluding Amphitrema flavum, A. wrightianum, Arcella dissolved oxygen concentrations and an increase in vulga ris, Assulina muscorum, Centropyxis acu leata pH and conductivity were observed. Moreover, the type, Cyclopyxis arcelloides type, Cypho deria am- concentration of chl a and DOC in the water pulla, Difflugia elegans, Euglypha ciliata, E. compressa, increased. This effect was particularly evident in Heleoptera petricola, H. sphagnii, Hyalo sphenia ova - experimental treatments +P and P+N. An increase in lis and Nebela militaris) showed a positive relation- pH and a decrease in oxygen concentration in the 3− ship with the rising gradient of nutrients (P-PO4 , N- water were also observed in the Pradeaux peatland + NH4 ) and DOC (Fig. 6B). In the case of ciliates, the in France (Gilbert et al. 1998b). According to Bob- Monte Carlo permutation test showed the sig - bink et al. (1998) the increase in nutrient deposition 3− nificance of 4 variables: DOC, pH, P-PO4 and O2 is a major concern in northern ecosystems, which are (Fig. 6C). On the RDA biplot, the species Cineto - nutrient-limited with slow rates of decomposition and chilum margaritaceum showed a positive relationship N mineralisation. Some studies have suggested that with DOC, Paramecium bursaria and Vorticella com- N deposition may account for a proportion of the panula corresponded with the rising gradient of pH, missing CO2 sink in the carbon budget (Bubier et al. and Euplotes sp. and Drepanomonas revoluta were 2007). Alternatively, recent studies suggest higher 132 Aquat Microb Ecol 74: 121–141, 2015

rates of peat decomposition and CH4 bold emission with N deposition and global – – climate change (Bragazza et al. 2006). A

– recent study of peat decomposition 0.50 across a natural gradient of N deposi-

0.52 tion in Europe found enhanced CO2 emissions and DOC release from peat accumulated under higher atmospheric 0.56 0.56 N supplies (Bragazza et al. 2006). These 0.01) are highlighted in ≥ authors predicted reductions in peat- 0.56 0.59 0.52 land C accumulation with nutrient

0.23 – addition as a result of increased decom-

values (p position with changes in peat litter B MEM+: bacteria with intact membranes; quality and plant species composition. Many studies have shown how changes 0.50 in concentrations of nutrients alter plant community structure (Bragazza et al. 2006, Bubier et al. 2007). The increase in vascular plants and the decrease in Sphagnum in response to warming have probably modified the quantity of plant-

0.57 0.59 derived organic matter, which in turn has affected microbial communities. 0.44 –

– Effects of nutrient addition on 0.52 – 0.51 0.62 – 0.56 0.59 – abundance and composition of –

0.50 phycoflora

Experimental fertilisation of peatbogs resulted in changes in the species rich- ––– ––– ness and taxonomic composition of individual groups of phycoflora. Enrich- 0.51 0.51 ment with nutrients affected the auto- trophs of the Sphagnum peatland and 0.54 0.54 the carbonate fen differently. In the Sphagnum peatland, desmids (Zygne- matophyceae) were a dominant and typical component of phycoflora. This is consistent with other re ports (Mataloni 1998, Stepankova et al. 2012). Nutrient enrichment did not significantly change phycoflora species richness (55 to 60

Control +P +Nspecies), but it did influence P+N species 0.36 0.34 0.36 0.34 composition within the same taxonomic groups. For example, N-enrichment 0.59 0.55 0.53 0.55 caused a change from dominance of the small-sized Cosmarium to the large- DOC B B MEM+ HNF TA Cl M DOC B B MEM+ HNF TA Cl M DOC B B MEM+ HNF TA Cl M DOC B B MEM+ HNF TA Cl M sized Netrium (desmids). Differences in

peatland the species composition of desmids have also been observed in Argentina in several acidic peatbogs with varying content of P and N (Mataloni 1998). In ClColpodea 0.37 0.35 – HNFTACyrtophoridaNassulidaOligotrichidaScuticulociliatida 0.23 0.27 0.33 0.29 0.33 – – 0.28 0.29 0.33 – 0.25 0.37 0.33 – 0.29 0.47 0.39 – BB MEM+ 0.29 0.28 – – M–––– Carbonate fen DOCB – 0.27 – 0.41 – – 0.21 – 0.34 – 0.45 – Sphagnum DOC – – 0.31 0.42 HNFTAClColpodea 0.33 – 0.31 – – 0.45 – 0.43 – – 0.37 0.47 – – – 0.47 – B MEM+ 0.27CyrtophoridaNassulidaOligotrichida – Scuticulociliatida 0.23 0.27 0.33 0.29 0.33 0.28 0.29 0.33 0.25 0.33 0.27 0.39 M 0.33 0.43 – 0.47 Table 6. Linear correlationTable coefficients between microbial components and metazoans in the investigated peatbogs. B: bacteria; HNF: heterotrophic testate amoebae; Cl: ciliates; M: metazoans; DOC: dissolved organic carbon. Significant nanoflagellates; TA: Mieczan et al.: Eutrophication in peatlands 133

the Sphagnum peatland, with typical acidic waters, phycoflora was more abundant than in the carbonate fen. The abundance values were higher than those obtained by Duthie (1965). An increase in the contri- bution of cyanobacteria in the case of experimental

Table 7. Summary of Monte Carlo permutation test of dis- tinct communities and environmental variables from the studied peatlands. Only variables with p < 0.05 are included. DOC: dissolved organic carbon. λ: amount of variance explained by variable factor

λ F p

Phycoflora DOC 0.71 10.86 0.002 + N-NH4 0.21 3.65 0.028 Testate amoebae pH 0.30 28.88 0.002 DOC 0.22 29.71 0.002 3– P-PO4 0.06 9.16 0.002 + N-NH4 0.01 2.57 0.020 Ciliata DOC 0.25 23.36 0.002 pH 0.20 25.33 0.020 3– Fig. 4. Principal component analysis biplots for axes 1 and 2 P-PO4 0.07 10.51 0.002 showing taxonomic communities and experimental treat- O2 0.05 6.54 0.004 ments (C: control treatment, triangles; +P: water with P- Rotifera DOC 0.25 23.36 0.002 3− + N-NH + 0.07 7.89 0.008 PO4 enrichment, circles; +N: water with N-NH4 enrich- 4 3− + pH 0.07 7.27 0.004 ment, rectangles; P+N: water with P-PO4 and N-NH4 en richments, diamonds). SP: Sphagnum peatland, CF: car- Crustacea pH 0.44 2.77 0.006 bonate fen DOC 0.30 2.07 0.016

Fig. 5. Redundancy analysis triplots for (A) Sphagnum peatland and (B) carbonate fen showing studied communities, environ- mental variables and samples collected in 4 distinct experimental treatments (C: control treatment, triangles; +P: water with 3− + 3− + P-PO4 enrichment, circles; +N: water with N-NH4 enrichment, rectangles; P+N: water with P-PO4 and N-NH4 enrichments, diamonds). DOC: dissolved organic carbon 134 Aquat Microb Ecol 74: 121–141, 2015

Fig. 6. Biplots of redundancy analyses of the studied com- munities. (A) Phycoflora, (B) testate amoebae, (C) ciliates, (D) rotifers and (E) crustaceans. Solid arrows indicate signif- icant parameters in Monte Carlo permutation tests at p < 0.05. Experimental treatments were C: control treatment; 3− + +P: water with P-PO4 enrichment; +N: water with N-NH4 3− + enrichment; P+N: water with P-PO4 and N-NH4 enrich- ments. DOC: dissolved organic carbon. Species codes are given in Table 8 fertilisation of Sphagnum peatlands was also ob- abundance of some pennate Bacillariophyceae and served by Gilbert et al. (1998b). In the carbonate fen, Chlorophyta. The results obtained revealed that with water of circumneutral pH, both P and N enrich- enrichment with biogenic compounds caused signifi- ment caused an increase in the contribution of pico- cant changes in the phycoflora of both the oligo - cyanobacteria and Cryptophyceae, and a decrease in trophic Sphagnum peatland and the eutrophic car- Mieczan et al.: Eutrophication in peatlands 135

Table 8. Species/taxon codes used in Fig. 6

Species/taxon Abbreviation Species/taxon Abbreviation

Phycoflora Ciliata (continued) Bacillariophyceae Bacillar Coleps spetai Col.spe Chlorophyceae Chlorop Colpoda cucullus Col.cuc Chrysophyceae Chrysop Colpoda steinii Col.ste Cryptophyceae Cryptop Cyrtohymena muscorum Cyr.mus Cyanobacteria Cyanobac Drepanomonas revoluta Dre.rev Dinophyceae Dinop Euplotes sp. Eup.sp Euglenophyceae Euglenop Halteria gradinella Hal.gra Filamentous Chlorophyta fil.Chlo Holosticha pullaster Hol.pul Raphidiophyceae Raphid Oxytricha sp. Oxy.sp Ulvophyceae Ulvop Paradileptus elephantinus Par.ele Zygnematophyceae Zygnem Paramecium bursaria Par.bur Testate amoebae Paramecium putrinum Par.put Amphitrema flavum Amp.fla Plagiopyla nasuta Pla.nas Amphitrema wrightianum Amp.wri Podophyra sp. Pod.sp Arcella catinus type Arc.cat Prorodon sp. Pro.sp Arcella discoides type Arc.dis Spathidium sensu lato Spa.sen Arcella vulgaris Arc.vul Spirostomum ambigum Spi.amb Assulina muscorum Ass.mus Strombidium viride Str.vir Assulina seminulum Ass.sem Stylonychia mytilus complex Sty.myt Centropyxis aculeata type Cen.acu Tokophyra sp. Tok.sp Centropyxis aerophila Cen.aer Vorticella companula Vor.com Centropyxis platystoma type Cen.pla Rotifera Corythion dubium Cor.dub Cephalodella gibba Cep.giba Corythion trinema type Cor.tri Cephalodella gibboides Cep.gibo Cryptodifflugia oviformis Cry.ovi Collotheca wiszniewski Col.wis Cyclopyxis arcelloides type Cyc.arc Colurella hindenburgi Col.hin Cyphoderia ampulla Cyp.amp Dicranophorus hercules Dic.her Difflugia elegans Dif.ele Keratella cochlearis Ker.coc Difflugia globulosa Dif.glo Keratella serrulata Ker.ser Euglypha ciliata Eug.cil Lecane closterocerca Lec.clo Euglypha compressa Eug.com Lecane elasma Lec.ela Euglypha rotunda type Eug.rot Lecane flexilis Lec.fle Euglypha strigosa Eug.str Lecane intrasinuata Lec.int Euglypha tuberculata type Eug.tub Lecane luna Lec.lun Heleoptera petricola Hel.pet Lecane lunaris Lec.luna Heleoptera rosea Hel.ros Lecane scutata Lec.scu Heleoptera sphagnii Hel.sph Lepadella ovalis Lep.ova Hyalosphenia elegans Hya.ele Lepadella patella Lep.pat Hyalosphenia ovalis Hya.ova Rotaria sordida Rot.sor Hyalosphenia papilio Hya.pap Crustacea Hyalosphenia subflava Hya.sub Acantholeberis curvirostris Aca.cur Nebela bohemica Neb.boh Alonella excisa Alo.exc Nebela carinata Neb.car Ceriodaphnia setosa Cer.set Nebela collaris Neb.col Chydorus sphaericus Chy.sph Nebela flabellulum Neb.fla Cyclops scutifer Cyc.scu Nebela griseola type Neb.gri Diacyclops bicuspidatus Dia.bic Nebela militaris Neb.mil Ectocyclops phaleratus Ect.pha Nebela vitraea type Neb.vit Eucyclops macruroides Euc.mac Placocista spinosa type Pla.sci Eucyclops speratus Euc.spe Trigonopyxis arcula Tri.arc Copepoda copepodites copep Ciliata Leydigia leydigi Ley.ley Amphileptus cleparedei Amp.cle Macrocyclops fuscus Mac.fus Amphileptus pleurosigma Amp.ple Megacyclops viridis Meg.vir Askenasia volvox Ask.vol Copepoda nauplii naup Aspidisca costata Asp.cos Scapholeberis microcephala Sca.mic Cinetochilum margaritaceum Cin.mar Simocephalus serrulatus Sim.ser Codonella cratera Cod.cra Streblocerus serricaudatus Str.ser Coleps hirtus Col.hir Thermocyclops crassus The.cra 136 Aquat Microb Ecol 74: 121–141, 2015

bonate fen. Although phosphorus has been consid- total DOC are frequently absent. Irrespective of the ered to be the primary nutrient limiting phycoflora type of peatbog, the contribution of metabolically growth in fresh water (Smith 1982), other studies active bacterial cells decreased with the addition of have suggested that N limitation and co-limitation by nutrients. According to a study by del Giorgio et al. N and P are more common than previously thought (1996), the rate of consumption of metabolically (Biddanda et al. 2008). Aerts et al. (1992) showed that active bacteria is 4 times higher than the consump- vegetation in bogs with high N deposition becomes tion of inactive or dead bacteria. On the other hand, P-limited. Malmer & Wallèn (2005) reported that an increase in the contribution of inactive or dormant increased N deposition might increase mineralisa- bacteria may also be one of the mechanisms of pro- tion, increasing the supply of P to plants; therefore, tection against the pressure of potential grazers. growth of plants may be as much due to the indirect effect of increased availability of P as to the direct effect of N deposition. In the Sphagnum peatland, chl Effects of nutrient addition on abundance a data indicated N limitation in spring and co-limita- of HNF tion in summer and autumn. In the carbonate fen, the chl a data indicated co-limitation of N and P in spring In the peatbogs investigated, the highest numbers and summer, while no limitation was ob served in of flagellates were noted in mesocosm +P. In the car- autumn. Similar results were observed in a eutrophic bonate fen, a decrease in their numbers was ob - lake (Biddanda et al. 2008). Bioassay studies have served in mesocosm P+N. The significant decrease in been used to determine which nutrients limit algal the number of flagellates with the addition of nutri- growth. Typically, total N to total P ratios <20 are ents is quite surprising considering the positive cor- indicative of N limitation, and ratios >50 are indica- relations reported in the literature between the tive of P limitation, with co-limitation occurring abundance of bacteria and flagellates and the con- between ratios of 20 and 50 (Dzialowski et al. 2005). centrations of total phosphorus. Riemann (1985) demonstrated that the addition of nutrients had no significant effect on the abundance of HNF in Effects of nutrient addition on bacterial eutrophic systems. However, we found a modest in - abundance and activity crease in chrysomonads with nutrient addition. Com- parison with the few available data sets (Auer & In the Sphagnum peatland, the highest density of Arndt 2001) concerning taxonomic composition of bacteria and contribution of metabolically active bac- HNF seems to confirm this trend. A strong decline in teria were recorded in experimental treatments +N HNF populations in summer has frequently been and P+N. In the carbonate fen, a substantial increase reported in connection with strong predation pres- in the density of bacteria was recorded only in the sure by testate amoebae, rotifers and crustaceans. P+N mesocosm. An increase in the contribution of metabolically active bacteria in comparison to the control group was observed only in mesocosm +N. Effects of nutrient addition on abundance and Their biomass was positively correlated with the composition of testate amoebae concentrations of nutrients and DOC. As reported by Chróst & Siuda (2006), the biomass and abundance of In the Sphagnum peatland, the number of species of bacteria are positively correlated with the concentra- testate amoebae decreased with nutrient enrichment. tions of DOC. The correlations between DOC and the The decrease was particularly pronounced in the ex- biomass of bacteria were more evident in the carbon- perimental treatment P+N. A decrease in the species ate fen (treatments +P, +N, P+N) than in the Sphag- richness of testate amoebae as fertility of habitats in- num peatland. This probably resulted from the fact creased was also observed in peatlands located in that the composition and availability of DOC are north-eastern France (Gilbert et al. 1998b, Jassey et more important than its amount for the development al. 2012). The contribution of living forms of testate of heterotrophic bacteria. According to the study by amoebae also decreased in comparison to the control. Chróst & Siuda (2006), particles included in DOC, This may have resulted from less favourable oxygen known as refractors, are only available after their conditions. As reported by Mazei et al. (2007), in con- enzymatic decomposition. Moreover, significant cor- ditions of decreasing oxygen concentration the diver- relations occur between the concentrations of DOC sity of testate amoebae decreased by 50 to 65%, and secreted by algae, whereas such correlations with the contribution of empty shells increased. The abun- Mieczan et al.: Eutrophication in peatlands 137

dance of testate amoebae was seasonally variable in observed in a lake ecosystem where low pH clearly all of the mesocosms studied, but the differences were limited the number of ciliate taxa (Mieczan 2007). In not significant. A seasonal change in testate amoebae the case of ciliates, their species richness has been assemblages has also been noted (Warner et al. 2007). observed to accompany an increase in the trophic In the present study, their highest abundance oc- status of habitats (Buosi et al. 2011). Our results curred during spring and/or summer and could have revealed that ciliates responded to changes in the resulted from higher concentrations of nutrients and environmental conditions much more intensively an increase in pH. Spring or summer abundance than testate amoebae. Their generation times are peaks were also noted by Heal (1964). The hypothesis short, and they are only protected from the environ- concerning a decrease in species diversity as habitat ment by a delicate cell membrane. Therefore, the fertility increases in eutrophic systems was only con- potential response time to pollution events is fast firmed in the case of testate amoebae. In the carbonate (Buosi et al. 2011, Andersen et al. 2013). The highest fen, only a slight decrease in the species diversity of abundance of ciliates occurred in spring and summer the organisms was observed, with a simultaneous in- and coincided with higher concentrations of DOC crease in their abundance. The dominance structure and nutrients. Spring and summer abundance peaks of testate amoebae underwent relatively small were also noted by Gilbert et al. (1998a). In the changes, which is probably linked to the duration of Sphagnum peatland, a substantial transformation in the experiment. In the Sphagnum peatland, the con- the dominance structure of ciliates occurred at the trol samples were primarily dominated by Hyalo- end of the experiment — from Col podea, dominant in sphenia elegans, Amphitrema flavum and Nebela the control sample and at the beginning of the exper- tincta. The carbonate fen was dominated by Arcella iment, to small bacterivorous Scuticociliatida. In the discoides and Corythion dubium. The highest density carbonate fen, a decrease was noted in the contribu- of H. elegans and A. flavum seems to be partly ex- tion of Oligotrichida, and an increase in the contribu- plained by the ecological behaviour of these species. tion of Scuticociliatida and Peritrichida. Oligotrichida Hyalosphenia and Amphitrema are mixotrophic spe- occurred in large numbers in a micro-environment cies living in symbiosis with some microalgae present with an appreciable level of nutrients (Mieczan in their cytoplasm. These species were primarily ob- 2009). However, the contribution of Scuticociliatida served in the top part of mosses and/or in surface wa- (Cinetochilum margaritaceum) increased with the ter, where their endosymbionts can photosynthesise level of nutrients and pH. Species of the genera (Gilbert & Mitchell 2006, Mieczan 2010, Jassey et al. Colpodea and Cyrtophorida occur in large numbers, 2011, Mieczan & Tarkowska-Kukuryk 2013). In the in both oligo trophic and eutrophic environments. mesocosms with added nutrients, only a slight in- Scuticociliatida as a rule occur numerously in crease in the contribution of species from the genera eutrophic waters and are also observed in humic Arcella and Nebela was recorded. This may be linked lakes, as well as in peatbog ecosystems (Järvinen to highly variable food conditions. In the treatments 1993, Mieczan 2007). The results of the RDA analysis +P and P+N, a substantial increase in the abundance showed that Askenasia volvox, Paradileptus ele- of algae and protists was observed. Nebela collaris phantinus, Plagiopyla nasu ta, Spathidium sensu lato, sensu lato mainly consumes algae, particularly di- Spirostomum ambigum and Paramecium putrinum atoms (45%), spores and mycelia of fungi (36%), and characterised the community composition at the end also large ciliates, rotifers and small amoebae (Gilbert of the experiment in the fertilised treatment. Such et al. 2003). An increase in the contribution of species species can potentially be considered indicators of from the genus Arcella may result from their relatively eutrophic environments. wide ecological tolerance. As evidenced by a study by Nicolau et al. (2005), species of this genus also occur in sewage with a high content of biogenic compounds. Effects of nutrient addition on metazoan abundance and composition

Effects of nutrient addition on abundance and Metazoans were predominated by small rotifers composition of ciliates ranging in size from 40 to 200 µm. An increase in the abundance of these organisms was observed in all The number of species of ciliates increased together treatments with nutrient addition and probably with the increase in nutrients, DOC concentrations resulted from increased abundance of their potential and chl a. The opposite tendency, however, was food (particularly bacteria). Rotifers usually consume 138 Aquat Microb Ecol 74: 121–141, 2015

particles ranging in size from 0.5 to 20 µm (Rothhaupt nutrient enrichment the low-pH indicatory species 1990), including bacteria and flagellates. According Alonella excisa and Ceriodaphnia setosa were domi- to Arndt (1993), rotifers play a slightly smaller role nant. The worsening environmental conditions for than crustaceans in controlling the number of bacte- dominant species of Crustacea probably favoured ria and flagellates. Ordination analysis showed that rotifers. An increase in the abundance of these nutrients and DOC are dominant controls on distri- organisms was observed together with nutrient addi- bution patterns in rotifer assemblages. These factors, tion and may have resulted from an increased abun- however, show distinct variation in individual meso- dance of their potential food (particularly bacteria) cosms. In the control treatment, species characteristic and diminished competitive influence of crustaceans. of environments with low nutrient concentration (e.g. The occurrence of high-trophy indicators in the Bdelloidea) were recorded in high numbers. These control samples as well as at the initial time of the groups have typically been found in oligotrophic nutrient-enriched treatments positively affected the habitats (Arndt 1993). Sphagnum acidifies its habi- increase in rotifer communities that accompanied tats, so rotifer diversity is limited to species that toler- nutrient enrichment. Until now there have been no ate low pH. Bdelloids tolerate a wide range of ambi- studies on the influence of nutrients on metazoan ent pH. Such a high tolerance may be associated with species richness in peatbogs. This problem has been their mode of reproduction, i.e. obligatory partheno- studied thoroughly in lakes, where the species num- genesis (Ricci 1987). Therefore in the nutrient addi- ber of Metazoa has been found to decrease with tion treatments, rotifer species characteristic of high increasing eutrophication (Jeppesen et al. 2011). In nutrient conditions, such as Lecane and Keratella, our experiment, an increase in the number of rotifer were recorded in high numbers. Similar results have species and a decrease in the number of species and been reported from lakes, where rotifers responded density of Crustacea were found to accompany nutri- to shifts in trophic status (from oligotrophic to meso- ent enrichment. The Monte Carlo permutation test trophic or eutrophic; Arndt 1993). We have presented showed that in addition to nutrients, DOC and pH the first data on the fate of crustaceans in peatbogs also influenced metazoan communities. The relation- affected by fertilisation. Our results showed a ships with DOC resulted from top-down influences decrease in the density of Crustacea with the nutri- between metazoans and microbial communities that ent enrichment, irrespective of the season. Some showed positive correlations to this variable. The role reports have indicated that an increase in trophic of pH in developing metazoan communities is well status may increase the total count of crustaceans known. (Adamczuk 2004, Pinto-Coelho et al. 2005). In the experiment, the opposite phenomenon (a decrease in total abundance of crustaceans) was found. This Effects of nutrient addition on the biomass of could be explained by the very rapid increase in microbial and metazoan communities nutrients in comparison to the control mesocosm. The nutrient enrichment probably affected crustaceans Experimental enrichment with biogenic com- indirectly by changing the quantity and quality of pounds affected the analysed groups of organisms their food (Elser et al. 2000, Plath & Boersma 2001). In with varying intensity, depending on the type of the carbonate fen, the high-trophy indicator Chy- peatbog. In general, an increase in the biomass of dorus sphaericus dominated in the control samples, most organisms was observed, which was particu- but in the nutrient-enriched treatments, their density larly evident in the Sphagnum peatland. The contri- diminished despite improving conditions, in favour of bution of total biomass of bacteria and ciliates in the crustacean cyclopoids. A decrease in cladoceran Sphagnum peatland increased significantly in all dominance in favour of cyclopoids together with experimental treatments. In the carbonate fen, algal eutrophication has also been observed in lakes biomass clearly dominated. A substantial decrease in (Karabin 1985). However, the diminishment of Chy- the contribution of flagellates was recorded, as well dorus sphaericus in the experiment may have re - as an increase in the contribution of ciliates and a sulted from the unconscious predatory influence of decrease in the contribution of testate amoebae and cyclopoids. Crustacean populations in the Sphagnum crustaceans to the total biomass of the organisms. peatland were quite resistant to nutrient elevation, as Nutrient addition also seemed to affect mutual rela- changes in their biomasses in the experimental treat- tions between the groups of organisms studied. In ments were insignificant. Moreover, no shifts in the both peatbogs, in all experimental treatments, the dominance structure occurred, as irrespective of maximum biomass of ciliates corresponded to an Mieczan et al.: Eutrophication in peatlands 139

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Editorial responsibility: Karel Sˇimek, Submitted: June 30, 2014; Accepted: October 10, 2014 Cˇeské Budeˇjovice, Czech Republic Proofs received from author(s): January 12, 2015