Liming Effects on Ecosystem Structure, Function and Trophic Relationships in Lakes

2a:11 Liming effects on ecosystem structure, function and trophic relationships in lakes

FÖRFATTARE Willem Goedkoop, IMA, Institutionen för vatten och miljö, Sveriges lantbruksuniversitet David Angeler, IMA, Institutionen för vatten och miljö, Sveriges lantbruksuniversitet

CONTENTS General introduction 393 Part 1. Analysis of benthic and pelagic community structure 393 Material and Methods 393 Results and Discussion 398 Part 2: Analysis of food web structure using stable isotopes 413 Material and methods 413 Results and Discussion 415 Summary and future perspectives 420 References 420

Ange sidorna 392–422 om du vill skriva ut detta kapitel. GENERAL INTRODUCTION were significantly different between limed lakes and reference lakes. His results provide a basis for a more Freshwater liming programs have been established exhaustive exploration of community structure using in several industrialized countries to alleviate the ef- comparative univariate and multivariate statistics. fects of anthropogenic acid deposition. Sulphur and «>À>ÌÛiÊ>>ÞÃiÃÊÕÃ}Ê`vviÀiÌÊÃÌ>ÌÃÌV>Ê nitrogen deposition can have profound impacts on methods could provide a means to assess whether surface aquatic ecosystems, altering biotic commu- different lake communities in different habitat types nity structure and functional processes in ecosystems of limed lakes converge with those in circumneutral (McKie et al., 2006). Liming as a mitigation measure reference lakes. If this is the case, this could indicate >``ÃÊ > " to anthropogenically acidified waters to 3 that liming has potential to restore lake communities neutralize H+ ions and facilitate precipitation of toxic of anthropogenically acidified lakes to conditions ions, especially aluminum, that become soluble at of circumneutral reference lakes; this could sup- ÜiÀÊ«Ê7i>Ì iÀÞ]Ê£nn®°Ê}Ê >ÃÊÀ>Ãi`Ê«Ê port liming applications as a mitigation measure. in many acidified systems, often allowing recovery Alternatively, if communities in limed lakes are of acid-sensitive organisms (Bradely & Ormerod, structurally different from those in acid and circum- 2002). However, the systematic alkalinization of neutral reference lakes, liming likely fails to achieve streams and lakes itself constitutes a substantial restoration goals, and magnitudes of differences iVÃÞÃÌiiÛiÊ«iÀÌÕÀL>ÌÊ7i>Ì iÀÞ]Ê£nnÆÊ between the structures of different communities may Schreiber, 1996). Liming can increase turbidity and help evaluate to what extent liming can constitute a the inorganic content of particulate matter consumed form of anthropogenic perturbation. In the first part by many invertebrates (Kullberg, 1987), and may of the present report, we test these assumptions by >ÃÊ«ÀiV«Ì>ÌiÊ`ÃÃÛi`ÊÀ}>VÊV>ÀLÊ " ®]Ê means of a standardized comparison of phytoplank- removing it from microbial food webs and reducing ton, zooplankton, and macroinvertebrates in three its buffering potential in humic systems (Kullberg et habitat types (littoral, sublittoral and profundal). Our al., 1993). Furthermore, any additional inflow of acid analysis spans a period from 2000 to 2004, which water from runoff or tributaries into limed systems allows assessing magnitudes of structural differences can create an aluminum chemistry that is potentially between lake categories over several years between more toxic than that of untreated water (Rossland et communities in a standard way. Even though much al., 1992; Teien et al., 2004). larger time series are available in the programme, the The Swedish liming program was established high heterogeneity (missing data, different sampling during the 1970s to mitigate extensive acidification of resolutions) of these data sets precluded their use for poorly buffered freshwaters. In particular, the IKEU a standardized comparison between multiple com- program was initiated in 1989 and comprises lakes munities. In the second part of this report, we focus that have been extensively monitored with regard to on a quantitative analysis of trophic relationships important biological and abiotic variables. Several in food webs of limed acid and circumneutral lakes recent reports (Holmgren, 2008; Persson, 2008a; ÕÃ}ÊÃÌ>LiÊÃÌ«iÃ°Ê7iÊ`iÌiÀiÊÜ iÌ iÀÊLiÌ VÊ Stendera, 2008; Sundbom, 2008a; Östlund, 2008) and pelagic food web structure differs in limed lakes and also previous publications using IKEU data compared to acid and circumneutral references. i°}°]Ê««iLiÀ}]Ê£nÆÊ*iÀÃÃ]ÊÓää£ÆÊ7j]ÊÓääÈÆÊ Persson, 2008b), often only focus on single communities and/or single habitats which limits an overall PART 1. ANALYSIS OF BENTHIC AND assessment of overall biological responses to liming PELAGIC COMMUNITY STRUCTURE from a structural point of view. Only the paper of Persson and Appelberg (2001) provide a comparison of plankton, benthos and fish communities from a Material and Methods production perspective, and Holmgren (2001) studied Data assembly biomass size spectra based on plankton and fish com- 7iÊiÛ>Õ>Ìi`Ê`>Ì>ÊvÊ« ÞÌ«>Ì]Êâ«>ÌÊ munities to provide a means of an integrated struc- in the pelagic and macroinvertebrate communities in tural (community structure) and functional (trophic three benthic habitat types (littoral, sublittoral, and relationships between communities and energy flow) profundal) available in the IKEU and national lake analysis. Finally, Sundbom (2008b) provided a com- monitoring databases. Data have been collected since parative long-term analysis of quantitative structural 1986, but the databases were highly heterogeneous and functional aspects of plankton, benthos and fish with regard to temporal sampling resolution of com- communities. He showed that the biomass levels munities in acid reference lakes (hereafter referred to and temporal trends of different functional groups

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 393 as acid lakes), circumneutral reference lakes (circum- FIGURE 1: Localization of study lakes. Lake catego- neutral lakes) and limed lakes. This was primarily ries: acid reference lakes (A), circumneutral reference due to repeated adjustments of the sampling fre- lakes (N), limed lakes (L). 1 = Ejgdesjön (L), 2 = quencies and differences in sampling methods. For Rotehogstjärnen (A), 3 = Fräcksjön (N), 4 = Härsvatten example, phytoplankton communities were sampled (A), 5 = Stora Härsjön (L), 6 = Gyltigesjön (L), 7 = between 2 and 7 times per year while macroinverte- Stora Skärsjön (N), 8 = Stengårdshultasjön (L), 9 = brates were sampled only once a year during most of Älgarydssjön (A), 10 = Gyslättasjön (L), 11 = Fiolen (N), the program. In order to be able to make standardi- 12 = Storasjö (A), 13 = Brunnsjön (A), 14 = Allgjuttern zed comparisons between communities, all analyses (N), 15 = Stora Envättern (N), 16 = Stensjön (L), 17 = for the present report are based on a single yearly Övre Skärsjön (A), 18 = Västra Skälsjön (L), = Tryssjön sampling occasion (August for phyto- and zooplank- (L), 20 = Bösjön (L), 21 = Stensjön (N), 22 = Källsjön (L), ton communities, October for macroinvertebrates). 23 = Remmarsjön (N), 24 = Lien (L). Encircled numbers Extracting a single yearly value from the databases indicate lakes that were sampled for analysis of stable also helped to avoid potential problems, which would isotopes (i.e. part 2 of this project). arise from the calculation of annual means based on irregular intra-annual sample sizes among lakes and communities. Our final analysis is restricted to the 5-year period between 2000 and 2004 ultimately constrained by methodological differences in the sampling of littoral macroinvertebrate communities in IKEU (limed lakes) and monitoring programs of acid and circumneutral lakes. The lakes summarized in Table 1 and Figure 1 met our final selection criteria for standardized comparisons. Four of these lakes are acid lakes, seven are circumneutral lakes, while eleven lakes are limed lakes. Some of their water quality variables are also shown in Table 1. For the present study all analyses except littoral macroinvertebrates are based on biomass data (mm3/L for phytoplankton, mm3/m3 for zooplankton, g/m2 for sublittoral and profundal macroinvertebrates). Littoral macroinvertebrate samples were collected by standardized kick samples, thus resulting in semi-quantitative abundance data.

Sampling procedures For water quality analysis we used August values of ÃÕÀv>ViÜ>ÌiÀÊÃ>«iÃÊäqÓÊ®]ÊÜ V ÊÜiÀiÊViV- ted, in the open-water mid-lake station in each lake. 7>ÌiÀÊÜ>ÃÊViVÌi`ÊÜÌ Ê>Ê*iÝ}>ÃÊÃ>«iÀÊ>`Ê kept cool during transport to the laboratory. Samples were analyzed for alkalinity, and concentrations of

>]Ê }]Ê >]Ê]Ê-"4]Ê ]Ê]Ê 4-N, NO2-N+NO3-N, total N, PO4*]ÊÌÌ>Ê*]ÊÀi>}Ê*ÊÌÌ>Ê*ÊqÊ*"4-P), -]ÊÌÌ>ÊÀ}>VÊV>ÀLÊ/" ®Ê>`Ê À« ÞÊa. All physicochemical analyses were done at the Secchi depth, water temperature, dissolved oxygen Department of Aquatic Sciences and Assessment fol- concentration, conductivity, and pH were measured lowing international (ISO) or European (EN) stan- in the lakes. These water quality variables helped `>À`ÃÊÜ iÊ>Û>>LiÊ7>`iÀÊiÌÊ>°ÊÓääÎ®°ÊÌÌÀ>Ê to delineate lake types, i.e. while limed lakes clearly macroinvertebrate samples were collected once in comprised one treatment group, we discerned bet- autumn (between September and November) from ween acid and circumneutral reference lakes, chiefly stony habitats (wind exposed littoral regions) using ÊÌ iÊL>ÃÃÊvÊÌ iÀÊ«]Ê Ê>`Ê>>ÌÞÊÛ>ÕiÃÊ standardized kick sampling and a handnet (European (Table 1). ÌÌiiÊvÀÊ-Ì>`>À`Ã>Ì]Ê£{®ÊÜÌ Ê>Êä°xÊ

394 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES NO2+NO3- N μg/l -P 4 μg/l PO TOC mg/l) F mg/l based on summer values (August) ± 1 stan- Cl meq/l SO4 meq/l Alkalinity meq/l Ca meq/lmeq/l Mg Na meq/l K meq/l Max. Depth (m) pH ) 2 Lake area (km SMHI X SMHIY 149526 ±0.10 ±<0.01 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±0.01 ±<0.01 ±4.51 ±0.86 ±11.13 127698 ±0.07 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±<0.01 ±0.23 ±0.24 ±8.22 125783 ±0.03 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±<0.01 ±1.65 ±0.68 ±1.33 148571 ±0.18 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±1.43 ±0.20 ±17.94 151724 ±0.15 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±<0.01 ±0.01 ±0.23 ±0.37 ±1.05 142267 ±0.14 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±0.01 ±<0.01 ±0.96 ±0.24 ±1.29 128665 ±0.07 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±<0.01 ±0.42 ±0.40 ±2.73 162132 ±0.11 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±1.11 ±0.93 ±1.72 154083 ±0.05 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±0.56 ±0.51 ±2.82 158869 ±0.12 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±0.61 ±0.40 ±0.24 133205 ±0.08 ±0.01 ±<0.01 ±<0.01 ±0.01 ±<0.01 ±0.01 ±0.01 ±<0.01 ±0.99 ±0.37 ±1.59 Morphological and water chemistry characteristics of lakes included in the study. Values represent interannual mean values Acid ref. lakes Brunnsjön 627443 0.11 10.60 5.71 <0.01 0.20 0.12 0.21 0.02 0.20 0.17 0.13 20.42 4.20 53.40 Härsvatten 643914 0.19 26.20 4.84 -0.02 0.03 0.06 0.24 0.01 0.09 0.26 0.03 2.38 1.40 55.00 Rotehogstjärnen 652902 0.17 9.40 5.88 0.02 0.07 0.07 0.21 0.01 0.07 0.18 0.05 13.26 2.60 4.60 Övre Skärsjön 663532 1.74 32.00 5.87 0.01 0.07 0.07 0.06 0.01 0.09 0.04 0.11 4.72 1.20 95.20 Circumneutral ref. lakes Allgjuttern 642489 0.19 40.70 6.79 0.07 0.17 0.10 0.13 0.01 0.17 0.09 0.22 7.08 1.80 4.00 Fiolen 633025 1.65 10.50 6.72 0.07 0.15 0.09 0.17 0.04 0.13 0.17 0.07 7.64 1.40 5.40 Fräcksjön 645289 0.28 14.50 6.68 0.08 0.17 0.09 0.26 0.02 0.10 0.26 0.07 9.42 2.40 7.60 Remmarsjön 708619 1.37 14.40 6.40 0.06 0.09 0.04 0.06 0.01 0.03 0.02 0.18 9.08 3.40 6.40 Stensjön 683673 0.57 8.50 6.37 0.04 0.06 0.03 0.05 0.01 0.04 0.02 0.08 6.26 2.40 6.80 Stora Envättern 655587 0.38 11.20 6.70 0.05 0.17 0.07 0.10 0.01 0.12 0.08 0.11 9.40 1.60 3.40 St Skärsjön 628606 0.31 11.50 6.99 0.13 0.18 0.16 0.32 0.01 0.17 0.31 0.07 5.10 1.80 4.20 dard error for the time period 2000–2004. TABLE 1.

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 395 NO2+NO3- N μg/l -P 4 μg/l PO TOC mg/l) F mg/l Cl meq/l SO4 meq/l Alkalinity meq/l Ca meq/lmeq/l Mg Na meq/l K meq/l Max. Depth (m) pH ) 2 Lake area (km 680235 1.15 17.00 6.75 0.09 0.12 0.02 0.04 0.01 0.03 0.01 0.35 6.72 1.80 5.40 SMHI X SMHIY 141799 ±0.17 ±0.02 ±0.02 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.02 ±0.91 ±0.37 ±2.29 653737 0.83 28.60 7.49 0.25 0.33 0.06 0.28 0.01 0.08 0.28 0.09 5.78 1.80 116.00 125017 ±0.12 ±0.03 ±0.02 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.01 ±<0.01 ±0.75 ±0.58 ±14.52 629489 0.40 20.00 7.04 0.26 0.39 0.11 0.21 0.01 0.10 0.21 0.09 15.68 5.00 154.20 133906 ±0.10 ±0.03 ±0.03 ±0.01 ±0.01 ±<0.01 ±0.01 ±0.01 ±<0.01 ±1.85 ±1.26 ±14.96 633209 0.33 9.80 6.87 0.12 0.29 0.06 0.15 0.01 0.13 0.13 0.08 12.90 1.60 4.20 141991 ±0.06 ±0.01 ±0.01 ±<0.01 ±0.01 ±<0.01 ±0.01 ±0.01 ±<0.01 ±1.01 ±0.24 ±0.92 683582 0.22 17.40 6.78 0.15 0.24 0.04 0.06 0.01 0.04 0.02 0.09 15.24 3.00 4.60 154935 ±0.09 ±0.01 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±1.72 ±0.55 ±1.47 663216 1.53 29.20 6.85 0.12 0.18 0.07 0.10 0.01 0.09 0.07 0.12 7.24 2.40 15.80 148449 ±0.08 ±0.02 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.66 ±0.60 ±7.00 638317 4.98 26.80 7.07 0.18 0.29 0.08 0.15 0.02 0.09 0.16 0.07 10.14 2.40 53.80 138010 ±0.04 ±0.01 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±0.43 ±0.75 ±14.37 656419 0.39 20.60 6.89 0.10 0.22 0.08 0.10 0.02 0.17 0.09 0.07 8.50 1.80 14.20 164404 ±0.06 ±0.01 ±0.02 ±0.01 ±<0.01 ±<0.01 ±0.02 ±0.01 ±<0.01 ±0.48 ±0.37 ±9.04 640364 2.57 42.00 7.36 0.28 0.39 0.08 0.30 0.02 0.12 0.33 0.07 4.80 1.20 133.40 129240 ±0.12 ±0.01 ±0.01 ±<0.01 ±0.01 ±<0.01 ±<0.01 ±0.01 ±<0.01 ±0.19 ±0.20 ±15.53 670275 0.30 19.60 6.52 0.06 0.16 0.03 0.05 0.01 0.03 0.03 0.07 Nd Nd 4.00 146052 ±0.09 ±0.01 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 Nd Nd ±1.38 664620 0.41 18.70 7.00 0.14 0.20 0.03 0.06 0.01 0.09 0.04 0.08 12.58 1.60 3.40 148590 ±0.15 ±0.02 ±0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±<0.01 ±2.37 ±0.40 ±0.51 Limed lakes Bösjön Ejgdesjön Gyltigesjön Gyslättasjön Källsjön Lien Stengårdshultasjön Stensjön Stora Härsjön Tryssjön V. Skälsjön

396 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES mesh size, and preserved in 70% ethanol. Samples of heterotrophic when they contained no photosynthetic sublittoral and profundal invertebrates were sampled apparatus (e.g., euglenozoan flagellates). using an Ekman grab (surface area 0.025 m2), scree- Zooplankton was divided into the functional ned in a 0.5 mm sieve and preserved in 70% ethanol. }ÀÕ«ÃÊ«Ài`>ÌÀÃÊ>`ÊvÌiÀviiìÀÃÊÜVâÊ£È>L®°Ê Five replicate samples were collected and biomasses The latter group not necessarily ingests only bacte- were determined by weighing (ethanol weight); the rioplankton and phytoplantkton, but also preys on average of the five replicates is used for analyses. small animals. However, as filter feeders “passively In the laboratory, samples were sorted under 10x prey” on animals with a smaller body size, they are magnification, identified using dissecting and light ÌÊì>ÌÊÜÌ Ê>ÃÊ«Ài`>ÌÀÃÊÊÌ iÊÃÌÀVÌÊÃiÃi°Ê7iÊ microscopy. Organisms were identified to the lowest considered as predators all cyclopoid copepods, the taxonomic unit possible, generally to the species level, calanoid copepods Heterocope and Eurytemora, the although exceptions occurred with some chironomid cladocerans Bythotrephes and Leptodora, and the larvae and immature oligochaetes. rotifer Asplanchna. The remaining taxa were assigned Zooplankton was sampled quantitatively in as filter-feeders. August using a 55-cm Plexiglas tube (i.d. 10 cm) Functional guilds of macroinvertebrates were equipped with a closing mechanism triggered by a according to the descriptions by Moog (1995). messenger. Samples were generally collected at 2-m Although Moog’s scheme differentiates between 10 intervals from the surface down to 8-m depth. Samp- groups we focused on the broader categories Detriti- les were pooled, screened (40 μm), and preserved in ÛÀiÃ]Ê*Ài`>ÌÀÃ]ÊÌiÀÊiiìÀÃ]Ê>`ÊÀ>âiÀÃ°Ê >ÞÊ acid Lugol’s solution. Taxonomic analyses, enume- species could sometimes be assigned simultaneously ration, and length measurements were done using to several guilds, i.e. due to ontogenetic feeding shifts. an inverted microscope. Biovolumes were calculated 7 iÊÌ ÃÊVVÕÀÀi`ÊÜiÊÕÃi`ÊÌ iÊ`>ÌÊvÕVÌ>Ê from length measurements and known relationships category to characterize a species functional role. for different taxa, life stages and/or size classes. However, if the relative distribution among different «iÌV]ÊÌi}À>Ìi`ÊÃ>«iÃÊäq{Ê®ÊvÊ« Þ- guilds was more even, then we considered this species toplankton samples were collected in August with to have a very broad feeding plasticity during their a tube sampler, usually from 5 sites per lake, poo- life span, and will refer to this group as “Multiple led and preserved in Lugol’s solution. Taxonomic group”, reflecting their functionally multiple roles in analyses and species enumeration was done under an aquatic ecosystems. inverted microscope using the Utermöhl technique Statistical analyses (Olrik et al. 1989). Biovolumes were calculated from geometric shapes following Blomqvist & Herlitz Analysis of variance (ANOVA) was carried out in (1998). -*--ÊÛ°£ÓÊ-*--ÊV]Ê V>}]ÊÃ]Ê1-®ÊÌÊÌiÃÌÊ for differences in selected water quality variables Structural community metrics «]Ê>ÌÞ]Ê >]Ê }]ÊÌÌ>Ê ]ÊÌÌ>Ê*Ê>`Ê-®]Ê and functional groups community metrics (total biomass/abundance, spe- For all communities we used structural metrics ViÃÊÀV iÃÃÊ>`Ê- >7iiÀÊL`ÛiÀÃÌÞ®Ê>`Ê that are routinely used in the analysis of ecological functional groups for each of the studied communi- communities (i.e. total biomass/abundance, species/ ties between lake type (acid lakes, circumneutral lakes Ì>ÝÊÀV iÃÃÊ>`Ê- >7iiÀÊìÝ®°Ê,i}>À- and limed lakes; fixed factor) and over the study ding the functional classification, we followed to a years (random factor). In addition, we contrasted in great extent the schemes used by Sundbom (2008). the same way the Medin Acidity Index calculated for For example, phytoplankton was divided into au- littoral macroinvertebrates (Hendriksson and Medin totrophic, mixotrophic and heterotrophic biomass 1986). The ANOVA models were calculated on the groups following the classification scheme of Jansson basis of Type III sums of squares to take the unbal- iÌÊ>°Ê£È®°Ê >V>À« ÞVi>i]Ê Õ}>Ì« ÞVi>i]Ê anced design into account. Likewise, the Scheffe test, ÀÞ«Ì« ÞVi>i]Ê Þ>« ÞVi>i]ÊÝ« ÞVi>i]Ê*À>Ã- which is a valid, fairly conservative test, sufficiently « ÞVi>i]Ê8>Ì « ÞVi>iVÊ>`Ê À« ÞVi>iÊÜiÀiÊ generalised to be applicable to unequal designs, was considered to be functionally autotrophic, except the used to determine pairwise differences in treatment green algae Polytoma and Polytomella (mixotrophic means. All dependent variables were log (x+1)-trans- taxa). Many groups that are known to have many formed to fulfill the requirements of parametric tests. ÝÌÀ« VÊÃ«iViÃÊ>ÀiÊ ÀÞÃ« ÞVi>i]Ê À>Ã«i`«- Non-metric multidimensional scaling (NMDS) was hyceae, Dinophyceae, Euglenophyceae, Haptophy- done in Primer v.6 (Primer-E Ltd, Plymouth, UK) ceae and Raphidophyceae. Taxa were considered to explore the similarity of community trends over

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 397 the study period across lake types. As a nonlinear total P concentrations of 9.45, 7.43 and 8.18 μg L-1, technique, NMDS ranks points in ordination space respectively, and average total N concentrations of in a way that the distance between sampling points 399.98, 356.97 and 383.50 μg L-1, respectively. The (in this study aquatic communities) reflects commu- concentrations of these nutrients were not signifi- nity similarity (ter Braak, 1995). The ordination is cantly different between lake types (Table 2; Figure L>Ãi`ÊÊ>Ê À>Þ ÕÀÌÃÊ`ÃÃ>ÀÌÞÊ>ÌÀÝÊ`iÀÛi`Ê 2). However, substantial differences in water quality from average values of all replicate lakes and log were observed among lake types with regard to water (x+1)-transformation of the sample by species matrix. quality variables that are most affected by acidifica- In addition, a NMDS analysis was carried out for tion and liming treatments. For example, the mean water quality; in this case the ordination is based on pH of acid reference lakes was always below 6, while a Euclidean distance matrix derived from standardi- circumneutral reference lakes showed pH values zed and log(x+1)-transformed water chemistry data, between 6.4 and 7.0, and some limed lakes showed including Secchi depth, water temperature, dissolved a pH > 7.0. These values were significantly different oxygen concentration, conductivity, pH, Alkalinity, LiÌÜiiÊ>iÊÌÞ«iÃÊ/>LiÊÓÆÊ}ÕÀiÊÓ®°ÊÃÊ >Ê>`Ê

>`ÊVViÌÀ>ÌÃÊvÊ >]Ê }]Ê >]Ê]Ê-"4]Ê ]Ê]Ê 4- Mg concentrations, but not Si concentrations, were

N, NO2-N+NO3-N, total N, PO4-P, total P, remaining significantly different between lake types (Table 2).

*ÊÌÌ>Ê*ÊqÊ*"4*®]Ê-]ÊÌÌ>ÊÀ}>VÊV>ÀLÊ/" ®Ê>`Ê As a result of their ionic composition, lake types had À« ÞÊa. The final solutions for each community also significantly different alkalinity values (Table and the water quality analysis are based on 999 permu- 1). The differences in water chemistry among lake tations. Pearson correlation analyses were carried out types were well captured in multivariate ordination in SPSS to explore the relationship of NMDS dimen- space (Figure 3), and an analysis of similarity showed sions with dominant taxa, community metrics, functio- significant differences in water quality between lake nal groups and water quality variables (Table 1). categories (ANOSIM: global R = 0.996, P < 0.001). Analysis of Similarity (ANOSIM; 999 permuta- Univariate analyses of community structure tions) was also run in Primer to test if significant differences in biomass/abundance of communities Our analyses based on functional categories and tra- occurred among lake types. This analysis is an ap- ditional community metrics, i.e. total biomass or total proximate non-parametric analogue of the standard abundance, species richness and biodiversity indices univariate analysis of variance ANOVA, and it uses ÃÕV Ê>ÃÊÌ iÊ- >7iiÀÊ`iÝ]ÊÛ>ÀÞÊ>}ÊÌ iÊ the R statistic to test differences between groups >>Þâi`ÊVÕÌiÃ°Ê7Ì ÊÀi}>À`ÊÌÊVÕÌÞÊ (R=0, no differences; R=1, all dissimilarities between metrics, phytoplankton showed significant differences groups are larger dissimilarity within groups). In the ÊÃ«iViÃÊÀV iÃÃÊ>`Ê- >7iiÀÊL`ÛiÀÃÌÞ]Ê present study, the ANOSIM analysis was used to while littoral macroinvertebrates showed differences complement the NMDS analyses. As such it was of only in total abundance. Zooplankton, sublittoral prime interest to use the same samples as those used and profundal macroinvertebrates showed no signi- for the ordination. This means that we first calculated ficant differences in community metrics between lake the yearly average for each lake type. This resulted in categories (Table 2, Figures 4-8). 5 replicates (5 study years) x 3 lake types (acid lakes, From a diversity point of view, these results sug- circumneutral lakes, limed lakes) = 15 samples for the gest that phytoplankton has lower species richness analysis. Similarity Percentage routine (SIMPER; also >`Ê- >7iiÀÊL`ÛiÀÃÌÞÊÊ>V`Ê>iÃÊÌ >Ê included in Primer v.6) was used to reveal which taxa in circumneutral lakes and limed lakes (Figure 4). contributed to dissimilarity between lake types. This could be partly due to negative effects of low pH in acid lakes, but also indicate a beneficial ef- Results and Discussion fect of liming on phytoplankton diversity to similar levels as those observed in natural reference lakes. Lake characteristics and water quality 7iÊ } } ÌÊ ÜiÛiÀÊÌ >ÌÊÜiÊÃÃÊ>ÞÊÌÀ>>Õ>Ê Most of the lakes had a surface area <1 km2, but variability and that our findings are limited to sum- some lakes were up to 5-times larger. Brunnsjön was mer conditions. Furthermore, the lack of data before the smallest lake (0.11 km2) while Stengårdshulta- implementing liming as an acidification mitigation sjön was the largest (4.98 km2) (Table 1). The lakes measure in these lakes does not allow us to determine also showed a depth gradient, with circumneutral with certainty to what extent liming increases phyto-

Stensjön being the shallowest (Zmax = 8.5 m) and the plankton diversity. limed Stora Härsjön being the deepest (Zmax = 42 m). Functional group characteristics also seemed 7Ì ÊÀi}>À`ÊÌÊÌÀ« VÊÃÌ>ÌiÊV >À>VÌiÀÃÌVÃÊ>V`Ê>iÃ]Ê to vary as a function of lake type. Higher hete- circumneutral lakes and limed lakes showed average rotrophic phytoplankton biomass was found in

398 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES attributes of phytoplankton, zooplankton, s that share various functional characteristics according tral lakes, and limed lakes). Significance levels: * P < 0.05, Shannon-Wiener Shannon-Wiener diversity Autotrophic biomass Heterotrophic biomass Mixotrophic biomass Biomass Species richness pH Alkalinity Ca Mg Si Total P N Total Biomass Species richness diversity Shannon-Wiener Filter feeders Predators df MS F MS F MS F MS F MS F MS F df MS F MS F MS F MS F MS F MS F MS F df MS F MS F MS F MS F MS F ANOVA results showing degrees of freedom, mean squares, F-ratios and significance levels structural functional community Error <0.01 <0.01 <0.01 0.03 0.06 0.02 Interaction 8, 95 <0.01 NS <0.01 NS <0.01 NS <0.01 NS 0.01 NS <0.01 NS <0.01 NS Years 4, 11.26 <0.01 NS <0.01 NS <0.01 NS <0.01 NS 0.11 7.58** 0.02 NS 0.05 6.27** Lake Type 2, 8 0.05 364.4*** 0.03 350.01*** 0.03 797.0*** <0.01 307.8*** 0.03 NS 0.09 NS <0.01 NS Lake Type 2, 8 0.01 NS 0.58 70.52*** 0.14 46.11*** 0.01 11.85** <0.01 38.60*** 0.01 NS Years 4, 11.26 0.01 NS 0.02 NS 0.01 NS <0.01 NS <0.01 NS 0.01 NS Lake TypeYears 2, 8InteractionError 8, 95 4, 11.26 0.31 0.76 0.11 NS 6.25** NS 0.23 0.01 <0.01 <0.01 NS NS NS <0.01 0.01 0.01 0.01 NS NS NS <0.01 0.92 1.17 0.12 7.39** 9.68** 0.38 0.74 1.44 0.12 0.16 NS NS NS Interaction 8, 95 <0.01 NS 0.01 NS <0.01 NS <0.01 NS <0.01 NS <0.01 NS Error 0.02 0.01 0.01 <0.01 <0.01 <0.01 and macroinvertebrates in three habitat types (littoral, sublittoral, profundal) of lake (acid lakes; circumneu ** P < 0.01, *** 0.001, NS not significant. Note for macroinvertebrates that the “Multiple group” represents those organism to Moog (1995). See Material and Methods for details. TABLE 2. Water quality Phytoplankton Zooplankton

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 399 Multiple groups biomass Multiple group biomass Multiple group biomass Predator biomass Predator biomass Predator biomass Grazer biomass Grazer biomass Filter feeder biomass Grazer biomass Filter feeders biomass Filter feeders biomass Detritivore biomass Detritivore biomass Detritivore biomass Shannon-Wiener Shannon-Wiener diversity Shannon-Wiener Shannon-Wiener diversity Shannon-Wie- ner diversity Acidity index Species richness Species richness Species richness Biomass Biomass Biomass df MS F MS F MS F MS F MS F MS F MS F MS F df MS F MS F MS F MS F MS F MS F MS F MS F df MS F MS F MS F MS F MS F MS F MS F MS F MS F Continued. Error 0.10 0.11 0.01 0.05 0.03 0.02 0.06 0.02 NS Interaction 8, 95 0.05 NS 0.05 NS 0.01 NS 0.01 NS 0.02 NS 0.01 NS 0.02 NS 0.01 NS Years 4, 11.26 0.08 NS 0.11 NS 0.02 NS 0.03 NS 0.01 NS 0.01 NS 0.04 NS 0.04 NS Lake Type 2, 8 0.26 NS 0.26 NS 0.07 NS 0.62 88.10*** 0.16 10.32** 0.07 9.36** 0.57 28.19** 0.03 NS Lake TypeYears 2, 8InteractionError 8, 95 4, 11.26 0.26 0.05 0.04 NS NS 0.29 NS 0.06 NS 0.04 0.18 NS 0.05 NS 0.01 NS 0.01 NS 0.13 NS 0.19 0.01 0.01 NS NS NS 0.02 0.01 <0.01 NS <0.01 NS NS 0.01 0.08 <0.01 NS <0.01 NS NS 0.94 0.06 0.02 53.96** NS <0.01 NS <0.01 NS <0.01 <0.01 NS <0.01 NS 0.15 <0.01 NS Lake Type 2, 8 0.13 8.86** 0.66 NS <0.01 NS 0.01 11.85*** 0.28 NS 2.80 63.05*** 4.34 84.35*** 0.43 NS 1.09 7.84* Years 4, 11.26 0.02 NS 0.08 NS <0.01 NS <0.01 NS 0.20 NS 0.16 NS 0.18 NS 0.14 NS 0.04 NS Interaction 8, 95 0.01 NS 0.22 NS <0.01 NS <0.01 NS 0.32 NS 0.04 NS 0.05 NS 0.10 NS 0.14 NS Error 0.03 0.19 <0.01 0.21 0.36 0.31 0.12 0.24 Littoral macroinvertebrates TABLE 2. Sublittoral macroinvertebrates Profundal macroinvertebrates

400 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES FIGURE 2. Comparison of selected water quality variables between acid lakes, circumneutral lakes and limed lakes. Shown are the means ± standard errors of 4 (acidic ref. lakes), 7 (circumneutral ref. lakes) and 11 (limed lakes) replicates. The letters highlight significant differences between lakes from a pairwise comparison (Scheffe test).

FIGURE 3. Nonmetric multidimensional scaling ordination showing temporal trends of water quality in acid lakes, circumneutral lakes and limed lakes between 2000 [00] and 2004 [04].

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 401 circumneutral lakes and acid lakes than in limed groups), varied among lake types, with the only signi- lakes (Table 2; Figure 4). Predator biomass among ficant difference occurring in the littoral community zooplankton was not significantly different across where circumneutral lakes had a higher biomass than lake types but filter feeder biomass was higher in i`Ê>iÃÊ>`Ê>V`Ê>iÃÊ/>LiÊÓÆÊ}ÕÀiÃÊÈqn®°Ê circumneutral lakes than in limed lakes. Filter feeder 7 iÊÃÌÊ`vviÀiViÃÊÜiÀiÊvÕ`ÊÊÌ iÊÃÌÀÕV- biomass had similar values in circumneutral lakes tural and functional composition of planktonic and and acid lakes but was higher in limed lakes (Table 2; benthic communities, the effect of time was only Figure 5). The biomass/abundance of detritivores in significant for total biomass and filter feeder bio- the macroinvertebrate communities differed accor- mass for zooplankton, which could be due to chance `}ÊÌÊ >LÌ>ÌÊV >À>VÌiÀÃÌVÃ°Ê7 iÊÊÃ}vV>ÌÊ events. However, the interaction term (lake type x differences of detritivores were found in littoral and time) was not significant in any ANOVA model, profundal habitats across lake types, the sublittoral which suggests that the community characteristics habitat showed a significantly lower biomass of this observed on a single sampling data were temporally group in limed and circumneutral lakes (Table 2; stable in all lake types during the study period. This Figures 6-8). The biomass of filter feeders and grazers finding suggests that liming does not substantially seemed to be consistently lower in acid references impact on community characteristics of limed lakes compared to circumneutral references and limed relative to circumneutral and acid lakes. lakes, but a significant difference could only be found Structural and functional responses to liming varied for littoral and sublittoral habitats (Table 2; Figures between the different communities. This suggests that Èqn®°Ê*Ài`>ÌÀÊL>ÃÃÊÃ Üi`Ê««ÃÌiÊ«>ÌÌiÀÃÊÊ environmental monitoring and assessment of impacts the different lake types. The predator guild in littoral of liming as a mitigation measure based on a single habitats was significantly lower in acid lakes. The organism group and community (i.e. planktonic algae, opposite was observed in the sublittoral and profun- littoral macroinvertebrates) is not straightforward. dal habitats, were predator biomass was significantly Even within a single organism group responses can higher in circumneutral lakes and limed lakes (Table vary as a function of habitat characteristics. This calls ÓÆÊ}ÕÀiÃÊÈqn®°Ê>Þ]Ê>ÃÊÌ iÊL>ÃÃÊvÊÃ«iViÃÊ for studies that combine the information of multiple which showed a broad feeding plasticity (i.e, multiple organism groups and habitats to unravel the whole

FIGURE 4. Comparison of phytoplankton community FIGURE 5. Comparison of zooplankton community met- metrics and the biomass of functional guilds between rics and the biomass of functional guilds between acid acid lakes, circumneutral lakes and limed lakes. Shown lakes, circumneutral lakes and limed lakes. Shown are are the means ± standard errors 4 (acidic ref. lakes), 7 the means ± standard errors of 4 (acidic ref. lakes), 7 (circumneutral ref. lakes) and 11 (limed lakes) replicates. (circumneutral ref. lakes) and 11 (limed lakes) replicate The letters highlight significant differences between lakes lakes. The letters highlight significant differences between in a pairwise comparison (Scheffe test). lakes in a pairwise comparison (Scheffe test).

402 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES TABLE 3. Results of ANOSIM (Analysis of Similarity) tests showing the R statistic and P levels of global models and pairwise comparisons of different communities across lake types (acid lakes, circumneutral lakes and limed lakes).

Macroinvertebrates Phytoplankton Zooplankton Littoral Sublittoral Profundal RPRPRP RPRP Global model 0.83 0.001 0.67 0.001 0.68 0.001 0.80 0.001 0.82 0.001 !CIDçXç.EUTRAL 1 0.008 0.90 0.001 0.94 0.008 0.99 0.008 1 0.008 !CIDçXç,IMED 0.47 0.008 0.67 0.001 1 0.008 0.99 0.008 0.74 0.016 #IRCUMNEUTRALçXç Limed 0.93 0.008 0.57 0.001 0.35 0.06 0.41 0.016 0.74 0.008

FIGURE 6. Comparison of littoral macroinvertebrate FIGURE 7. Comparison of sublittoral macroinvertebrate community metrics and the biomass of functional guilds community metrics and the biomass of functional guilds between acid lakes, circumneutral lakes and limed lakes. between acid lakes, circumneutral lakes and limed lakes. Shown are the means ± standard errors of 4 (acidic ref. Shown are the means ± standard errors of 4 (acidic ref. lakes), 7 (circumneutral ref. lakes) and 11 (limed lakes) lakes), 7 (circumneutral ref. lakes) and 11 (limed lakes) replicate lakes. The letters highlight significant differenc- replicate lakes. The letters highlight significant differences between lakes in a pairwise comparison (Scheffe test). es between lakes in a pairwise comparison (Scheffe test).

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 403 magnitude of environmental impacts in general in ordinations was < 0.1 which according to >ÀiÊ boreal lake ecosystems and specifically that of liming. (1993) can be regarded as a good ordination with no Stendera and Johnson (2008) reached similar con- real risk of drawing false inferences. Most communi- clusions in their study of community recovery from ties in limed lakes seemed to occupy intermediate anthropogenic acidification of several Swedish lakes positions compared with acid and circumneutral (some of them included in the present study), medi- lakes (Figure 9). Analysis of similarity indicated that ated in part by habitat characteristics. these differences were significant for all groups except for littoral macroinvertebrates, which did not differ Multivariate analyses of community structure between circumneutral lakes and limed lakes (Table The analyses based on nonmetric multidimensional 3). This suggests that the majority of communities of scaling show a clear separation of all communities limed lakes do not converge with those in circumneu- among acid lakes, circumneutral lakes and limed la- tral lakes as a result of liming. kes. For each analysis the stress value of the obtained Similarity percentages (SIMPER) analyses allowed

TABLE 4. Results of SIMPER (Similarity Percentages) analysis, showing the percentage of phytoplankton species contributing to community structure, based on their biomass.

Species Acid Neutral Limed Species Acid Neutral Limed Bacillariophyceae Rhodomonas lacustris 7.18 3.3 Aulacoseira alpigena 2.63 2.95 Chrysophyceae Aulacoseira distans 1.81 Chrysidiastrum catenatum 0.64 Aulacoseira distans var. Chrysochromulina parva 1.5 1.22 tenella 1.7 0.53 Chrysococcus sp. 0.51 Cyclotella spp. (10-15 μm) 1.13 Monosigales spp. 0.55 Tabellaria flocculosa var. Pseudopedinella sp. 2.25 3.46 asterionelloides 0.81 1.01 Stichogloea doederleinii 0.89 Chlorophyta Spiniferomonas sp. 0.93 Botryococcus terribilis 3.28 6.63 Synurophyceae Botryococcus spp. 0.91 Mallomonas allorgei 0.6 Monoraphidium dybowskii 2.21 2.48 1.61 Mallomonas caudata 1.06 0.45 Oocystis sp. 0.71 0.46 Mallomonas sp. 1.2 Uroglena sp. 1.76 1.57 Mallomonas crassisquama 0.8 Unidentified Raphidophyceae chlorococcales 2.4 2.54 1.02 Gonyostomum semen 54.47 41.97 Dinophyta Cyanoprokaryota Ceratium furcoides 0.6 Merismopedia tenuissima 2.12 5.32 0.48 Ceratium hirundinella 2.25 1.59 Woronichinia naegeliana 0.72 Gymnodinium fuscum 0.6 Woronichinia naegeliana 0.72 Gymnodinium spp. (10-14 μm) 1.5 Colourless flagellates Gymnodinium uberrimum 4.13 3 Katablepharis ovalis 3.15 1.44 Peridinium inconspicuum 2.14 3.43 1.93 5NIDENTIFIEDçTAXA Unidentified monads Peridinium willei 2.17 (<3 μm) 0.67 Peridinium sp. 0.47 Unidentified monads Rhodophyceae (3-5 μm) 4.2 7.53 2.33 Cryptomonas marssonii Unidentified monads (<20 μm) 1.62 (5-7 μm) 2.1 4.56 3.09 Cryptomonas spp. Unidentified monads (<20 μm) 6.34 3.05 (7-10 μm) 2.46 2.28 Cryptomonas spp. Unidentified monads (<20-40 μm) 9.83 15.76 3.73 (>10 μm) 1.45

404 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES us to identify species that characterize the communi- FIGURE 8. Comparison of profundal macroinvertebrate ties in the different lake types, i.e. in reference lakes community metrics and the biomass of functional guilds >`Êi`Ê>iÃ°Ê7Ì ÊÀi}>À`ÊÌÊ« ÞÌ«>Ì]Ê>V`Ê between acid lakes, circumneutral lakes and limed lakes. reference lakes were characterized by fewer species Shown are the means ± standard errors of 4 (acidic ref. relative to circumneutral lakes and limed lakes. This lakes), 7 (circumneutral ref. lakes) and 11 (limed lakes) was due to the relatively high occurrence of the lakes. The letters highlight significant differences between raphidophycean flagellate Gonyostomum semen that lakes in pairwise comparisons (Scheffe test), i.e. lakes comprised more than 50% of phytoplankton biomass having no letters in common are significantly different. Ê>V`Ê>iÃ°Ê->ÀÞ]ÊÊ>V`Ê>iÃ]Ê ÀÞÃ« ÞVi>iÊ >`Ê-ÞÕÀ« ÞVi>iÊÜiÀiÊ>LÃiÌÊÜ iÊ ÀÞ«Ì« ÞVi>iÊ biomass was markedly lower than in the other lake ÌÞ«iÃ°Ê ÀVÕiÕÌÀ>Ê>iÃÊ>`Êi`Ê>iÃÊÃ >Ài`Ê many species, but their relative contribution varied between both lake types, underlying the significant difference in community structure found in the multivariate analyses (Figure 9, Table 4) The correlation analysis relating the NMDS dimensions to phytoplankton taxa was in good agreement with the SIMPER analysis (Table 5). The positive correlation of many phytoplankton taxa with NMDS dimension 1 (horizontal dimension) suggests that these species become more abundant towards the right side of the ordination coincident with the location of limed lakes and circumneutral lakes in the ordination. Among these species we find e.g., Katablepharis ovalis, Gymnodinium sp., Rhodo- monas lacustris and Chrysochromulina parva. The negative correlation of Gonyostomum semen with NMDS 1 clearly supports the high dominance of this species in acid lakes, while the negative correlation of autotrophic biomass with NMDS further suggests that this functional group was less important in these >iÃ°Ê7Ì ÊÀi}>À`ÊÌÊiÛÀiÌ>ÊÛ>À>LiÃ]ÊÌ iÊ

negative correlation of NO2-N+NO3 Ê>`Ê/" Ê with NMDS 1 suggests that limed lakes and circumneutral references had a lower water color and a more abundant in acid lakes than in circumneutral lower amount of nitrogen compounds relative to acid and limed lakes, respectively, while Holopedium lakes. NMDS 2 (vertical dimension) was positively gibberum, absent from acid lakes, contributed on average at least 10% to zooplankton biomass in correlated with Na, SO4]Ê }Ê>`Ê Ê>`Êi}>ÌÛiÞÊ VÀÀi>Ìi`ÊÜÌ Ê«]ÊV`ÕVÌÛÌÞ]Ê>>ÌÞÊ>`Ê >°Ê circumneutral and limed lakes. Also, Daphnia cristata The correlations also reflect nicely the separation was more abundant in limed and circumneutral of limed lakes from the other lake categories along lakes, respectively, than in acid lakes. There were also this dimension in the ordination, with limed lakes several indifferent species that had similar biomasses showing positions in the ordination where e.g., pH, in all lake types, e.g., Diaphanosoma brachyurum >>ÌÞÊ>`Ê >ÊVViÌÀ>ÌÃÊÜiÀiÊ } Êi}>ÌÛiÊ and Eubosmina coregoniÊ >`ViÀ>®Ê>`ÊConochilus correlations with NMDS 2). unicornis (Rotifera) This could indicate their broad Zooplankton communities in acid, circumneutral ecological plasticity and/or tolerance to acid condi- and limed lakes were clearly separated along NMDS tions or liming treatments of lakes (Table 6). 1, with limed lakes occupying an intermediate posi- ÀÀi>ÌÊvÊ -Ê`iÃÃÊÜÌ ÊÜ>ÌiÀÊ tion to acid and circumneutral lakes (Figure 9). The chemistry variables, zooplankton taxa, community SIMPER analysis showed that acid lakes had a higher metrics and functional guilds further helped to eva- share of rotifer species than circumneutral and limed luate forcing functions for community structure in the lakes. Other notable differences were found in the three lake types (Table 7). Being in agreement with cladocerans, where Ceriodaphnia quadrangula was the SIMPER analysis, NMDS dimension 1 correlated

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 405 TABLE 5. Pearson correlations between NMDS dimen- TABLE 6. Results of SIMPER (Similarity Percentages) SIONSçANDçWATERçQUALITYçVARIABLES çPHYTOPLANKTONçTAXA ç analysis, showing the percentage of zooplankton species and phytoplankton functional groups. For phytoplankton contributing to community structure based on their bio- TAXAçONLYçCORRELATIONçCOEFFICIENTSçççAREçSHOWNçWHEREç mass. all correlations were significant at P< 0.001.

MDS 2 Circum- Species Acid neutral Limed Water quality variables Cladocera F 0,71** Ceriodaphnia K 0,87*** quadrangula 14.92 2.4 11.31 NO -N+NO -N -0,88*** 2 3 Daphnia cristata 7.99 15.75 14.08 TOC -0,64** Daphnia cucullata 3.86 Na 0,84*** Daphnia galeata 2.81 3.39 10.85 SO 0,73** 4 Daphnia sp. 13.97 14.89 15.79 Mg 0,71** Diaphanosoma Cl 0,68** brachyurum 7.11 6.27 5.61 pH -0,63* Eubosmina coregoni 17.49 15.29 14.35 Conductivity -0,71** Holopedium gibberum 11.33 9.95 Alkalinity -0,80*** Limnosida frontosa 2.69 Ca -0,82*** Copepoda Taxa Diaptomus graciloides 1.99 Katablepharis ovalis 0,86 Rotifera Gymnodinium sp. (10-14 μm) 0,84 Conochilus unicornis 3.56 3.52 3.15 Unidentified monads Kellicottia bostoniensis 8.03 (<3 μm) 0,80 Keratella cochlearis Spiniferomonas sp. 0,78 f. typica 2.6 2.09 Chrysochromulina parva 0,77 Kellicottia longispina 2.15 Rhodomonas lacustris 0,75 Ploesoma hudsoni 2.89 Gonyostomum semen -0,90 Polyarthra remata 4.88 Cyclotella sp. (15-20 μm) -0,77 Polyarthra vulgaris 3.29 6.98 4.83 Functional groups Trichocerca capucina 2.77 Autotrophic biomass -0,78***

positively with Daphnia cristata, D. cucullata, Daph- limed lakes than in acid lakes, while the opposite was nia sp., Holopedium gibberum, Kellicottia lonigspina, true for predator biomass. The three lake types also Limnosida frontosa and Polyarthra vulgaris. The differed with respect to several water quality variab- analysis therefore shows that these species are more les. According to the zooplankton ordination, lake abundant in circumneutral and limed lakes than in types were separated chiefly on the basis of nitrogen >V`VÊ>iÃ°Ê ÛiÀÃiÞ]ÊÌ iÊi}>ÌÛiÊVÀÀi>ÌÊvÊ compounds (negative correlation with NMDS 1; Ceriodaphnia quadrangula, Kellicotia bostoniensis, ÀiviVÌ}Ê>V`VÊV`ÌÃ®Ê>`Ê]Ê«]Ê>`Ê Ê«- Trichocerca capucina, and Polyarthra remata with sitive correlation with NMDS 1; reflecting conditions NMDS 1 indicates a more “acidophilic” charac- ÊVÀVÕiÕÌÀ>Ê>`Êi`Ê>iÃ®°Ê>ÌÞ]Ê >]Ê teristics of these species. Furthermore, the positive and phosphates correlated negatively with NMDS 2 correlation of species richness with NMDS 1 further reflecting conditions in limed lakes, while the positive suggests that circumneutral and limed lakes had hig- correlation of sulphates and Mg with NMDS 2 reflect her species richness than acid lakes. These trends are conditions of reference lakes. shown in Figure 5 even though there was no signifi- Littoral macroinvertebrates were also clearly cant treatment effect. The biomass of filter feeding separated along NMDS dimension 1 in the multiva- zooplankters was also higher in circumneutral and riate ordination, even though communities of limed

406 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES lakes clustered closer to those of circumneutral lakes TABLE 7. Pearson correlations between NMDS dimensions than those of acid lakes (Figure 9). The separation in ANDçENVIRONMENTALçCHARACTERISTICS çZOOPLANKTONçTAXA çCOMMU- ordination space of communities of limed lakes and nity metrics and zooplankton functional groups. Correlation circumneutral lakes was not significant according to coefficients and corresponding P values are given. the ANOSIM analysis. However, it must be high- * P < 0.05, ** P < 0.001, *** P< 0.001. lighted that the analysis of littoral macroinvertebrate communities is based on abundance data while that MDS 1 MDS 2 for the other organism groups was done on biomass Environmental variables data. Possibly, biomass-based analyses more accura- Fluoride 0,90*** tely detect differences among lake types as it includes pH 0,77*** a quantitative dimension rather than information on K 0,74** abundance. Cl -0,57* The SIMPER analysis shows that circumneutral NO2-N + NO3-N -0,76*** and limed lakes share many macroinvertebrate taxa SO4 0,68** that equally contributed to community composition (Table 8). Indeed, the ANOSIM analysis did not Mg 0,63* detect significant differences in macroinvertebrate Alkalinity -0,54* community structure between these lake types. Se- Ca -0,54* veral species were relatively important in these lake PO4-P -0,52* types while they were absent from acid lakes (e.g., Taxa the ephemeropterans Caenis horaria, C. lucturosa, Daphnia cristata 0,87*** Centroptilium luteolum and Ephemera vulgaris, the Daphnia cucullata 0,70** coleopteran Oulimnius troglodytes-tuberculatus or the plecopteran Nemoura avicularis, and several Daphnia sp. 0,83*** dipterans). Other species were exclusively found in Holopedium gibberum 0,92*** acid lakes; however, each comprised a relatively low Kellicottia longispina 0,55* 0,58* individual share in community structure, e.g. the Limnosida frontosa 0,70** chironomids Phaenospectra sp., Stenochironomus Polyarthra vulgaris 0,72** sp. and unidentified Tanypodinae, the trichopterans Ceriodaphnia quadrangula -0,77*** Cyrnus insolutus and Holocentropus sp., the odonate Kellicottia bostoniensis -0,70** Erythromma najas, and the coleopteran Hygrotus Trichocerca capucina -0,82*** sp. Yet other species were more or less indifferent and occurred in all three lake types, e.g. the bivalve Polyarthra remata -0,57* 0,63* Pisidium sp., the chironomid Psectrocladius sp., the Eubosmina coregoni 0,77* epheropteran Leptophlebia vespertina, water mites Conochilus unicornis 0,61* (Hydracarina, Hydrachnida), and the isopod Asellus Diaphanosoma brachyurum 0,73** aquaticusÊ ÀÕÃÌ>Vi>®°Ê Diaptomus graciloides 0,62* Results of the Pearson correlation analysis of Keratella cochlearis f. typica 0,59* water chemistry variables and littoral macroinverte- Community metrics brate taxa with the NMDS dimensions are shown in Species richness 0,60* Table 9. The positive correlation of phosphate and ammonium-N concentrations with NMDS 1 sug- Functional groups gests that limed and circumneutral lakes had higher Biomass filter feeders 0,52* 0,75* nutrient concentrations than acid lakes. By contrast, Biomass predators -0,59* the analysis suggests that acid lakes were characterized by higher Si contents, indicated by the negative correlation of Si with NMDS 1. All lakes were This contrast with the analyses for phytoplankton, Ãi«>À>Ìi`ÊÜÌ ÊÀi}>À`ÊÌÊ ÊVViÌÀ>ÌÃÊ>}Ê zooplankton and sublittoral and profundal macroin- NMDS 2, which suggests that all lakes generally vertebrates, and highlights that other factors than >`Ê } iÀÊ ÊVViÌÀ>ÌÃÊ`ÕÀ}Êi>ÀiÀÊÞi>ÀÃÊ acidity/acidification alone can be relevant for struc- vÊÌ iÊÃÌÕ`Þ°Ê Ã«VÕÕÃÞ]Ê>ÞÊÜ>ÌiÀÊV iÃÌÀÞÊ turing the communities in boreal lakes. Stendera and variables indicative of acidity/acidification (e.g., pH, Johnson (2008) made similar observations regarding alkalinity) were not significant in the correlation the recovery of benthic communities from anthropo- analysis with littoral macroinvertebrate communities. genic acidification. However, rather than in littoral

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 407 FIGURE 9. Nonmetric multidimensional scaling ordinations showing temporal trends of phytoplankton, zooplankton, and macroinvertebrate (in littoral, sublittoral and profundal habitats) communities in acid reference lakes, circumneutral lakes and limed lakes between 2000 [00] and 2004 [04]. Note that the ORDINATIONSçAREçBASEDçONçBIOMASSçVALUESçOFçINDIVIDUALçTAXA çEXCEPTçFORçLITTORALçMACROINVERTEBRATES çWHICHç are based on abundance

habitats, they found community responses to be as indicated by the ANOSIM analysis, these sublit- uncoupled from acidification in profundal habitats of toral communities were significantly different. The Ì iÀÊÃÌÕ`ÞÊ>iÃ°Ê ÀÀi>ÌÃÊvÊ>VÀÛiÀÌiLÀ>ÌiÊ SIMPER analysis showed that circumneutral and taxa with the NMDS dimensions is in good agree- limed lakes had a relatively high biomass of Pisidium ment with the SIMPER analysis, with Endochirono- sp. (Mollusca, Bivalvia), Asellus aquaticus (Isopoda, mus sp. and Sialis lutaria seemed to be representative ÀÕÃÌ>Vi>®Ê>`ÊEphemera vulgata (Ephemeroptera) of acidic lakes, while Oulimnius sp. and Mystacides (Table 10). On the other hand, the higher relative longicornis/nigra apparently being more abundant in biomass of Cyrnus flavidus and Sialis lutaria in the limed and circumneutral lakes than in acid lakes. The sublittoral of acid lakes is in good agreement with positive correlation of several species with NMDS 2 the results found for the littoral macroinvertebrate is a consequence of the fact that these species were VÕÌiÃ°Ê Ã«VÕÕÃÊÃÊÌ iÊ>ÃÃÛiÊVVÕÀÀiViÊ more abundant during earlier years of the study, as of the predatory dipteran Chaoborus flavicans in acid the distribution patterns of study years in the NMDS lakes and limed lakes (Table 10). ordinations indicate (Figure 9). The correlation analysis of NMDS dimensions The NMDS ordination of sublittoral macroinverte- with water chemistry variables and community brates is similar to that obtained for littoral macroin- structural and functional metrics is in agreement with vertebrates (Figure 9) with the communities in limed observations made for phytoplankton and zooplank- and natural lakes clustering close together. However, ÌÊVÕÌiÃ°Ê7>ÌiÀÊµÕ>ÌÞÊÛ>À>LiÃÊÀi>Ìi`ÊÌÊ

408 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES TABLE 8. Results of SIMPER (Similarity Percentages) analysis, showing the percentage of littoral macroinvertebrate species contributing to community structure (based on their abundance) in acid reference lakes, circumneutral reference lakes, and limed lakes.

Species Acid Circumneutral Limed Diptera Ablabesmyia longistyla 0.71 1.41 Ablabesmyia monilis 0.55 Ceratopogonidae 2.1 2.77 3.9 Cladopelma sp. 0.6 0.56 Cladotanytarsus sp. 2.37 3.92 2.12 Cricotopus sp. 0.99 0.69 1.24 Demicryptochironomus vulneratus 0.97 0.52 Dicrotendipes sp. 3.6 1.64 1.45 Empididae 0.65 Endochironomus sp. 2.23 1.57 0.96 Epoicocladius ephemerae 0.6 Glyptotendipes sp. 0.57 Heterotanytarsus apicalis 0.43 Lauterborniella agrayloides 1.46 1.73 Microtendipes sp. 0.8 Pagastiella orophila 0.83 2.02 1.79 Parakiefferiella sp. 0.64 0.54 Paramerina sp. 2.83 0.58 1.16 Paratanytarsus sp. 2.09 1 Phaenopsectra sp. 0.76 Polypedilum breviantennatum 0.98 0.56 Polypedilum sp. 0.41 Procladius sp. 3.51 2.1 2.4 Psectrocladius sp. 8.62 4.03 5.35 Pseudochironomus prasinatus 1.23 1.41 0.75 Stenochironomus sp. 0.67 Tanypodinae, unidentified 0.56 Tanytarsus sp. 3.9 4.69 3.55 Thienemannimyia sp. 1.32 1.44 Trichoptera Cyrnus flavidus 2.14 1.35 0.88 Cyrnus insolutus 1.21 Cyrnus trimaculatus 1.23 Ecnomus tenellus 0.94 Holocentropus sp. 0.72 Hydroptila sp. 1.98 Lepidostoma hirtum 0.51 1 Limnephilus sp. 0.91 0.41 Limnephilidae unidentified 1.31 0.4 Molannodes tinctus 0.5 Mystacides azurea 1.03 1.6 Mystacides longicornis/nigra 0.87 1.09 1.58 Oecetis testacea 0.85

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 409 TABLE 8. Continued.

Species Acid Circumneutral Limed Oxyethira sp. 0.67 2.02 0.6 Tinodes waeneri 0.5 Ephemeroptera Caenis horaria 3.9 4.23 Caenis luctuosa 4.5 5.42 Centroptilum luteolum 1.79 1.18 Cloeon dipterum 1.48 2.28 2.01 Ephemera vulgata 0.65 2.31 Kageronia fuscogrisea 4.63 1.74 3.15 Leptophlebia marginata 0.59 2.93 2.8 Leptophlebia vespertina 14.4 6.78 6.88 Hydrachnida Argyroneta aquatica 0.53 0.78 Hydracarina 4.11 1.83 1.65 Crustacea Asellus aquaticus 12.77 8.66 7.64 Annelida Erpobdella octoculata 1 Odonata Erythromma najas 0.57 Platycnemis penn.-Pyrrhosoma nymph. 0.57 Zygoptera 0,99 Mollusca Gyraulus albus 1.36 Pisidium sp. 2.5 4.62 5.39 Coleoptera Hygrotus sp. 0.76 Oulimnius sp. 0.5 Oulimnius troglodytes-tuberculatus 0.79 0.81 Heteroptera Micronecta sp. 2.69 Plecoptera Nemoura avicularis 0.88 0.55 Megaloptera Sialis lutaria 1.62 0.85 0.57 Plathelminthes Turbellaria 0.74

acidity reflecting the distribution of lakes types in the (negative correlation of these variables with NMDS ordinations. For example, alkalinity, and concentra- 1; Table 11). Also with this community, the results ÌÃÊvÊ >]Ê]ÊÝÞ}iÊ>`ÊÊVÀÀi>Ìi`Ê«ÃÌÛiÞÊ from the correlation analysis are in good agreement with NMDS dimension 1, indicating that circumneu- with the SIMPER analysis regarding the distribu- tral lakes and limed lakes have higher values of these tion of taxa. Species with negative correlations with variables in comparison with acid lakes which had NMDS 1 (e.g. Cyrnus flavidus, Sialis lutaria) seemed >Ê } iÀÊ>ÕÌÊvÊ >]Ê Ê>`ÊÌÀ}iÊV«Õ`ÃÊ to have relatively higher biomasses in acid lakes,

410 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES TABLE 9. Pearson correlations between NMDS dimen- particularly abundant during the study years 2003 sions and environmental characteristics, littoral macroin- Ê>V`Ê>iÃÊ>`Ê`ÕÀ}ÊÓääÓqÓää{ÊÊi`Ê>iÃÊ VERTEBRATEçTAXAç3HOWNçAREçCORRELATIONçCOEFFICIENTSçANDç (Figure 9). The latter shows how large interannual corresponding P values. * P < 0.05, **, P < 0.001, *** variation in species biomass/abundance can affect the P< 0.001. Community metrics and functional groups analysis. were not significant. NMDS dimensions of the sublittoral macroinvertebrate community ordination also showed significant MDS 1 MDS 2 correlations with structural community metrics and Water quality variables functional feeding groups (Table 11). These correla- PO4-P 0,83*** tions are in good agreement with the results obtained NH4 0,54* vÀÊÌ iÊÕÛ>À>ÌiÊ>>ÞÃÃÊ}ÕÀiÊÇ®°Ê- >7i- Si -0,58* ner biodiversity seemed to be higher in circumneutral Cl 0,52* and limed lakes (positive correlation with NMDS 1), Taxa while total biomass and species richness was higher in acid lakes (negative correlations with NMDS 1) Endochironomus sp. -0,61* (Table 11). Also with regard to functional groups, the Sialis lutaria -0,56* biomass of filter feeder was higher in limed lakes (po- Oulimnius sp. 0,58* sitive correlation with NMDS 1 and negative correla- Mystacides longicornis/nigra 0,67** tion with NMDS 2), whilst the biomass of predators Erythromma najas 0,70** and detritivores was higher in acid lakes (Figure 7, Paramerina sp. 0,63* Table 11). Procladius sp. 0,61* Finally, profundal macroinvertebrate community structure also showed a clear separation by lake type, Dicrotendipes sp. 0,58* with limed lakes occupying an intermediate position Cyrnus flavidus 0,56* between acid and circumneutral lakes in the ordination (Figure 9). SIMPER analysis revealed a high while species that positively correlated with NMDS1 contribution of Chaoborus flavicans to the profundal (e.g. Asellus aquaticus, Ephemera vulgata) seemed benthos communities in all lake types, while Pisidium more abundant in circumneutral and limed lakes. also contributed to explain community structure in Interestingly, Chaoborus flavicans, which strongly circumneutral lakes (Table 12). Despite Chaoborus contributed to the biomass of sublittoral macroinver- also being dominant in limed lakes, other rare species tebrates in acid and limed lakes correlated negatively (Pisidium sp, Hydracarina), that do not appear due to with NMDS 2. This suggests that this species was the 90%-cutoff level applied in statistical analysis (see

TABLE 10. Results of SIMPER (Similarity Percentages) analysis, showing the percentage of sublittoral macroinvertebrate species contributing to community structure (based on their abundance) in acid reference lakes, circumneutral reference lakes, and limed lakes.

Species Acid Neutral Limed Mollusca Pisidium sp. 27.36 24.51 Crustacea Asellus aquaticus 11.04 9.33 Trichoptera Cyrnus flavidus 12.88 2.18 Molanna angustata 1.99 Ephemeroptera Ephemera vulgata 21.2 11.06 Diptera Chaoborus flavicans 59.63 18.34 42.81 Megaloptera Sialis lutaria 21.92 12.66

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 411 TABLE 11. Pearson correlations between NMDS dimen- TABLE 12. Results of SIMPER (Similarity Percentages) sions and environmental characteristics, sublittoral analysis, showing the percentage of profundal macroin- MACROINVERTEBRATEçTAXA çCOMMUNITYçMETRICSçANDçSUBLIT- vertebrate species contributing to community, based on toral macroinvertebrate functional groups. Shown are cor- their biomass. relation coefficients and corresponding P values. Taxa Acid Neutral Limed * P < 0.05, **, P < 0.001, *** P< 0.001. Mollusca MDS 1 MDS 2 Pisidium sp. 14.91 Water quality variables Diptera F 0,96*** Chaoborus flavicans 99.95 85.01 92.02 Alkalinity 0,75*** Ca 0,61** K 0,59* TABLE 13.Pearson correlations between NMDS dimen- O2 0,58* sions and environmental characteristics, profundal NO2-N + NO3-N -0,58* MACROINVERTEBRATEçTAXA çCOMMUNITYçMETRICSçANDçPROFUN- Na -0,76*** dal macroinvertebrate functional groups. Shown are Cl -0,77*** correlation coefficients and corresponding P values. Mg 0,59* * P < 0.05, ** P < 0.001, *** P< 0.001. Taxa MDS 1 MDS 2 Asellus aquaticus 0,69** -0,52* Ephemera vulgata 0,62* -0,53* Water quality variables Cyrnus flavidus -0,62* F 0.86*** Sialis lutaria -0,65** K 0.73** Chaoborus flavicans -0,58* pH 0.69** Community metrics NO2-N+NO3-N -0.83*** Shannon-Wiener diversity 0,72** Conductivity 0.55* Species richness -0,62* Taxa Total biomass -0,72** -0,65** Pisidium sp. 0.81*** Functional groups Chaoborus flavicans -0.93*** Filter feeders 0,70** -0,57* Community metrics Predators -0,76*** Shannon-Wiener diversity 0.59* 0.53* Detritivores -0,90*** Species richness -0.53* Total biomass -0.64** -0.72** Functional groups methods section), have contributed to explain some Detritivores 0.86*** 8% of community structure. Filter feeders 0.82*** The correlation analysis of NMDS dimensions with environmental variables, profundal macroin- Grazers 0.81*** vertebrate taxa, community structural attributes and Predators -0.94*** functional guilds shows both similarities and differences with regard to the macroinvertebrate communities in shallower habitat types (littoral and sublittoral). The correlation of Pisidium and Chaoborus are in the higher biomass of predators in acid lakes agree good agreement with the results from the SIMPER with the results from the analysis of sublittoral com- analysis. Also the correlation of structural commu- munities. The only notable difference is the positive nity metrics agrees with the results observed in the correlation of detritivores which seem to be higher ÃÕLÌÌÀ>ÊLiÌ ÃÊVÕÌÞÊÜÌ Ê- >7iiÀÊ in circumneutral lakes, thereby contrasting with the diversity being higher in circumneutral lakes and li- lower biomass found in the sublittoral of acidic lakes. med lakes (positive correlation with NMDS 1), while Notwithstanding, the overall results of the multivari- total species richness and total biomass was higher in ate analysis of profundal macroinvertebrates matches acid lakes (negative correlation with NMDS 2). Also again well the results from the univariate analysis. with regard to functional groups the higher biomass of filter feeders in circumneutral and limed lakes and

412 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES PART 2: ANALYSIS OF FOOD WEB a whole water-column sample during unstratified STRUCTURE USING STABLE ISOTOPES conditions. DOM in this water sample was trapped by adding 1 g/L of DAEA-cellulose (dietylaminoeth- 7iÊÕÃi`Ê>ÌÕÀ>ÊÃÌ>LiÊÃÌ«iÃÊÌÊiÃÌ>ÌiÊÌ iÊ ylcellulose, Aldrich) during repeated agitation for at sources of carbon and nitrogen assimilated by dif- least one hour. During this time DOM sorbes to the ferent trophic levels in benthic and pelagic food webs DAEA- cellulose, resulting in a clear overlying water of limed, acid and circumneutral lakes. Analysis of phase. DAEA was then allowed to settle and regained carbon stable isotopes is frequently carried out to Ê>Ê«>«iÀÊvÌiÀ]Ê>`ÊÃÌÀi`ÊVÊÓqÎÊc ®ÊÕÌÊ discriminate between carbon originating from dif- desorption in the lab. After a washing step (deion- ferent sources (Peterson and Fry, 1987; Post, 2002). ized water) desorption of DOM was accomplished by The basis is that physical and biological turnover of adding 0.3 M NaOH and subsequent filtration to re- elements affects the proportions of light and heavy move the DAEA. Desorbed, concentrated DOM was isotopes. Both photosynthesis and diffusion in water collected, frozen, and lyophilized (i.e. the material is 13 12 reduce the carbon isotope ( É ®ÊV«>Ài`ÊÌÊ rapidly frozen and dehydrated). >ÌÃ« iÀVÊ " °Ê ÃiµÕiÌÞ]ÊÌ iÊÜiÃÌÊÀ>ÌÊ 2 Samples of epilithic biofilms were collected by is found among pelagic primary producers and less LÀÕÃ }ÊÌ iÊÃÕÀv>ViÊvÊÎqxÊÃÌiÃÊViVÌi`ÊvÀÊÌ iÊ reduced ratios in terrestrial primary production. littoral zone with minor additions of ambient lake Because both enzymatic metabolism and excretion water in a tray. The suspension of epilithic biofilms discriminate against the heavy nitrogen isotope, the was transferred to 50-mL polyethene flasks and 15 14 nitrogen ratio ( N/ N) will increase with trophic frozen in dry ice. A subsample was preserved in 70% level in the food chain. The analyses of the stable ethanol for taxonomic analysis of algae. ÃÌ«iÊV«ÃÌÊvÊ Ê>`Ê Ê>ÀiÊÌiÌi}À>Ìi`Ê Samples of littoral macroinvertebrates were col- representations of the food sources assimilated by lected with hand nets (0.5 mm-mesh) in stony habitat the consumers (Lajtha and Michener, 1995; Vander äq£ÊÊ`i«Ì ®Ê>`Ê>}ÊÌ iÊÃ«>ÀÃiÊÛi}iÌ>Ì°Ê- Zanden et al. 1999). Standard fractionation with dividuals of dominant taxa were sorted, identified to ÌÀ« VÊiÛiÊ>ÀiÊ>LÕÌÊ£Ê²ÊvÀÊ Ê>`Ê>LÕÌÊÎ°{Ê²Ê the lowest possible taxonomic unit (i.e. species, genus vÀÊ Ê*iÌiÀÃÊ>`ÊÀÞ]Ê£nÇÆÊ >L>>Ê>`Ê,>ÃÕÃ- or in some cases family-level), transferred to cryonic sen, 1994), but several studies have shown that these vials, and snap-frozen in liquid nitrogen. Profundal values show large variation (see review by Vander invertebrates were sampled from the deepest part of Zanden and Rasmussen 2001. the lake using an Ekman grab and subsequent sieving (0.5 mm). Also here several individuals of abundant Material and methods chironomid taxa and the phantom midge Chaoborus Study design flavicans were transferred to cryonic vials and frozen A subset of nine monitoring lakes, three each of limed as described above. lakes and acid and circumneutral reference lakes (Ta- Fish samples were obtained either from ongoing ble 14), respectively, were visited three times during fish-monitoring programs run by the Swedish Fish- ÓääÈqÓääÇÊÊÃÕiÀ]Êv>]Ê>`ÊÃ«À}®ÊÌÊViVÌÊ eries Board, or from own sampling trips. From fish samples of seston, epilithic biofilms, zooplankton, collected from monitoring programs, a sample of dor- benthic invertebrates, and fish for stable isotope (SI) sal muscle was dissected in the field and frozen within analyses. Life samples were filtered/sorted and snap- ÕÀÃÊÊVÀÞVÊÛ>Ã°Ê `}ÊvÊÌ iÊÛ>ÃÊ}Õ>À>Ìii`Ê frozen in the field. linking to fish data (species, age, length, weight, etc.). Seston samples (<65 μm) were collected by Fish samples collected during our own sampling vÌiÀ}ÊÉ ]Ê«ÀiVLÕÃÌi`®Ê>ÊÝi`Êi«iÌVÊ events consisted of whole individuals that were frozen sample, or a whole water-column sample during in the field. From selected individuals a sample of unstratified conditions. An appropriate volume of dorsal muscle was prepared from partly thawed fish water was filtered in the field, but on a few occasions, preventing the loss of water and effects in stable iso- due to extreme weather conditions, this was done in Ì«iÊV«ÃÌÊi°}°ÊiÕV Ì>ÞÀÊ>`ÊÀiÞÊÓääÎ®°Ê the lab within 8 hours after sampling. Filters contain- Samples of Perch (Perca fluviatilis) were collected for ing seston were wrapped in aluminum-foil and snap Ì ÀiiÊ`vviÀiÌÊÃâiÊV>ÃÃiÃ]Ê°i°Ê{qÈÊV]Ênq£ÓÊV]Ê>`Ê frozen in liquid nitrogen. A subsample of the water >15 cm, to reflect the species’ ontogenetic feeding was fixed in Lugol’s solution for taxonomic analyses. shifts from planktivorous to benthivorous, and Additionally, humic and fulvic acids (collectively further to piscivorous size classes. Unfortunately, no referred to as dissolved organic matter; DOM) were fish samples were collected in Lake Storasjö. extracted from a 25-L mixed epilimnetic sample, or

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 413 TABLE 14. Lake and predominant catchment characteristics and mean values of selected water chemistry variables for limed lakes (L), acid lakes (A), and circumneutral lakes (C) that were part of the stable isotope study. Abbreviations: Agri=Agriculture; Alk=Alkalinity; TOC,=Total Organic Carbon.

Lake name Lake Size Catchment % % % Zmean pH Alk Tot-P TOC and coordinates type (ha) (ha) Forest Agri Water (m) (meq/l) (μg/l) (mg/l) Stora Härsjön L 257 2270 65.4 3.0 23.0 14 6.8 0.195 8 5 640364, 129240 Gyltigesjön L 40 17200 60.7 7.7 6.0 9.1 6.5 0.108 9 6 629489, 133906 Gyslättasjön L 32 280 70.4 6.2 10.3 2.8 7.7 0.178 15 12 633209,141991 Älgarydssjön A 32 345 86.9 3.0 9.3 1.6 5.5 -0.002 18 15 633989, 140731 Rotehogtjärnen 652902, 125783 A 16 380 93.2 0.5 4.4 3.6 5.5 0,0004 14 12 Storasjö 631360, 146750 A 35 212 83.2 0.0 16.7 3.5 5,4 -0.003 17 8 Fiolen 633025, 142267 C 155 548 40.2 16.0 28.2 3,9 6.5 0,05 11 6 Fräcksjön 645289, 128665 C 27 398 88.5 0.0 6.7 4.1 6,4 0.06 10 8 St. Skärsjön 628606, 133205 C 33 246 86.3 0.0 13.5 3,9 6.8 0.11 8 5

For stable isotope analyses, all samples were associated with the quantification of algal or detrital lyophilized and appropriate amounts of dry weight food resources and provide better predictions of the were transferred to tin capsules, packed, and sent off trophic position of organisms, molluscan primary ÌÊÌ iÊÃÌ>LiÊÃÌ«iÊv>VÌÞÊ>ÌÊ1 >ÛiÃÊ >vÀ>]Ê consumers have been suggested as baseline indicator USA). Note that composite samples were analyzed organisms in stable isotope studies (e.g. Post, 2002; for invertebrates, while muscle tissue collected from Vadeboncoeur et al., 2003). However, as these taxa single individuals of fish were analyzed. The D13 Ê>`Ê are largely absent from the lake types studied here we D15N, as well as contents of nitrogen and carbon were used epilithic biofilms, seston (<65 μm) to characteri- analyzed on a gas chromatograph coupled to a mass ze the base of the food webs. These trophic pathways spectrometer. D13 ÊÛ>ÕiÃÊ>ÀiÊÀi«ÀÌi`ÊÀi>ÌÛiÊÌÊÌ iÊ ÜiÀiÊL>Ãi`ÊÊÌ iÊV«>ÀÌiÌÃÊÕÌi`ÊLiÜ°Ê7iÊ 6q*ii iiÊ iiÌiÊ* ]ÊiÃÌi®Ê-Ì>`>À`]Ê°i°Ê wish to point out that temporal variability in species D13 ÊÊ²]ÊÃÊÌ iÊ`iÛ>ÌÊvÊÌ iÊÃÌ«VÊÀ>ÌÊvÊÌ iÊ occurrences and the lack of acid sensitive taxa in acid 13 sample from that of the standard; D Ê²®ÊrÊRsample/ and limed lakes (i.e. differences among communities, 13 12 13 Rstandardq£®ÊÝÊ£äää]ÊÜ iÀiÊR = É °ÊÊ«ÃÌÛiÊD Ê see also part 1) in some cases limit statistical compa- value means that the sample has more of the heavier risons of lake categories. isotope (13 ®ÊÌ >ÊÌ iÊÃÌ>`>À`°ÊD15N in ‰, is the Benthic food web: deviation of the isotopic ratio of the sample from that EPILITHON: Algae, protozoa, fungi, bacteria and other of the atmospheric nitrogen standard; D15N (‰) = microbes growing on the surfaces of stones were con- (R /R q£®ÊÝÊ£äää]ÊÜ iÀiÊR = 15N/14N. sample standard sidered to build the base of the benthic food web. Classification of functional groups and food webs GRAZERS: This trophic level represents the “herbivo- 7iÊ`ÃViÀi`ÊLiÌÜiiÊÌ iÊLiÌ VÊ>`ÊÌ iÊ«i>}VÊ ÀiÃ»ÊÊÌ iÊLiÌ VÊv`ÊÜiL°Ê7iÊÕÃi`ÊÌ iÊÃÌ«iÊ trophic pathways, to assess whether liming im- signatures of the mayflies Heptagenia fuscogrisea and pacts food web structure and trophic relationships Heptagenia sp. (Ephemeroptera) which are expected in contrasting lake habitats. These pathways were to feed on epilithon. Although a few specimens of treated separately, based on the evidence that these these taxa were found in acidic lakes because of their food webs are separated in terms of their carbon acid sensitivity, these were the only grazer species flows (France 1995). In order to circumvent problems available for the comparative study.

414 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES SHREDDERS: This functional feeding group is expected the acidic Storasjö, limiting the comparison between to convert coarse organic matter into finer fractions lake categories. of organic matter. The crustacean Asellus aquaticus PISCIVOROUS FISH: Pike (Esox lucius) and adult perch is an acid-tolerant species, and it could be sampled in (body lengths > 15 cm) comprised the top predators all study lakes. Leaf litter serves as a food source of in the pelagic food webs. As was the case with perch this species and we used the stable isotope fractiona- fry, the occurrence of individuals of predatory fish tion values provided by Bohman (2005) for assessing varied substantially even within a single lake cate- the importance of leaf litter as a food source in the gory and no data were available for Storasjö, thereby benthic food web. limiting comparisons between acid, circumneutral SHREDDERS/GRAZERS: This group is comprised of and limed lakes. species that share functional characteristics of grazers Statistical analyses and shredders, and which can be regarded to graze either on epilithon or to transform coarse organic 7iÊÕÃi`ÊÌ iÊ7VÝÊÌiÃÌÊÌÊV«>ÀiÊÌ iÊìÌ>Ê Ê matter. For this study, Limnephilidae were well repre- >`ÊìÌ>Ê ÊvÀ>VÌ>ÌÊÊÌ iÊ`vviÀiÌÊÌÀ« VÊ sented in all lake categories and were used to repre- levels composing the benthic and the pelagic food sent this group of broader feeding plasticity. webs between limed, circumneutral and acid lakes. 7iÊ«i`Ê`>Ì>ÊvÀÊÌ iÊÌ ÀiiÊÃ>«}Ê«iÀ`ÃÊ INVERTEBRATE PREDATORS: Members of this group because the food web bases (epilithon in the benthic prey on other invertebrates. In this study we used food web, and seston in the pelagic food web) were larvae of Sialis lutaria (Megaloptera), Cordulia aenea not significantly different between sampling dates. (Odonata), Aeshna sp. (Odonata), Erythromma najas Furthermore, pooling of the data was necessary be- and other unidentified Zygoptera, and predatory cause species of selected higher trophic levels showed leeches (Hirudinea). strong temporal variability, i.e. were missing from our samples due to ontogenetic reasons (e.g. small life VERTEBRATE (FISH) PREDATORS: In this study we con- stages that were not caught by our hand nets). Some sidered benthivorous perch (Perca fluviatilis) as the size classes of macroinvertebrate species and fish were top predator in the benthic food web. Size classes of present in very low abundances during some of the 8-12 cm of perch are known to feed preferentially on samplings or absent from some lakes (particularly benthic invertebrates (Quevedo and Olsson 2006). acid-sensitive taxa in acid lakes). Although, tempo- Pelagic food web: ral within-taxa variability in stable isotope com- DISSOLVED ORGANIC MATTER (DOM) AND SESTON: In position may occur in selected macroinvertebrates this study we measured the stable isotope signatures (e.g. Bohman 2005), we assume that environmental of dissolved organic matter. Humic matter can be constraints inherent to each lake category ultima- “channeled” through the microbial loop to consume- tely dictate the feeding behavior of organisms in the rs at higher trophic levels (herbivorous zooplankton, longer term. Thus, if liming as a mitigation measure invertebrate and fish predators). Seston (< 65 μm), by significantly affects lake ecosystem structure and contrast, can be assimilated directly by grazers, which function, variability among lake categories should themselves serve as food source for invertebrate and override temporal variability within a set of specific fish predators. Seston and DOM can therefore be lakes. Hence we assume that food web structure in regarded as the base of the pelagic food web. limed, acid and circumneutral lakes can be depicted in a confident way. INVERTEBRATE PREDATORS: The phantom midge (Cha- oborus flavicans: Diptera) was well represented in Results and Discussion all lakes, and its intermediate position in the pelagic A number of recent studies have claimed that correc- food web allows us to assess whether energy from the tions of the isotopic signal (i.e. D13 ®Ê>ÀiÊiiì`ÊvÀÊ food web base (dissolved organic matter and seston) lipid-rich tissue or whole animals in order to make is transferred to top predators. sound interpretations of stable isotope data, in parti- PLANKTIVOROUS FISH: In this study we considered cular concerning the origin of their food (Mateo et al. perch fry (young life history stages; 4-6 cm size class 2008, Logan et al. 2008). The reason behind this is [Magnus Dahlberg, The Swedish Board of Fisheries, that lipids have more negative D13 Û>ÕiÃÊÌ >Ê«À- personal communication]) and roach (Rutilius ruti- teins due to kinetic isotope effects that occur during lus) to prey preferentially on zooplankton. Unfortu- lipid synthesis. Variability in lipid content of tissue or nately, the occurrence of perch fry was highly hetero- whole animals may affect D13 Ê>`ÊV>ÊÌ ÕÃÊiÀÀi- geneous between lakes and no data were available for usly be interpreted as habitat shifts or food sources

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 415 originating from terrestrial and aquatic sources, Also other functional feeding groups in acid lakes respectively. This effect is most pronounced for lipid- tended to have lower D13 ÊÛ>ÕiÃÊÊ>V`Ê>iÃÊÌ >Ê rich tissues in fish (e.g. liver) and for zooplankters in the other lake categories, although the differen- Ì >ÌÊ>ÀiÊÀi>ÌÛiÞÊÀV ÊÊ«`Ã°Ê/ iÊ>ÌVÊ É ÊÀ>ÌÊ ces were not significant (P > 0.05). However, these of samples, analyzed simultaneously with carbon and patterns are not a consequence of the acid status of nitrogen isotopes, have been suggested as a proxy for lakes in general, but rather strongly dependent on the the lipid contents of samples. However, Logan et al. catchment characteristics of the different study lakes. Óään®ÊVVÕìÊÌ >ÌÊÌ iÊ É ÊÀ>ÌÊ>VVÕÌÃÊvÀÊiÃÃÊ For example, organisms belonging to the same fun- of the variation in invertebrate samples, with D13 Ê VÌ>Êvii`}Ê}ÀÕ«ÊÀÊÃ>ÀÊÌ>Ý>ÊÊ>iÊ}>- signatures increasing only slightly more than 1 ‰ rydssjön had substantially more negative D13 ÊÛ>ÕiÃÊ Õ«Ê«`ÊiÝÌÀ>VÌÊvÀÊÃ>«iÃÊÜÌ ÊLÕÊ É ÊLiÌ- than their counterparts in the other two acidic lakes, ween 4 and 8 (their Figure 4). In our study, samples Rotehogstjärnen and Storasjö. Organisms belonging vÊvÃ ÊÌÃÃÕiÊ >`Ê É ÊÀ>ÌÃÊÌ >ÌÊÜiÀiÊVÃÃÌiÌÞÊ to the same functional feeding groups or taxonomic ÜiÀÊÌ >Ê{°£]ÊÜ iÊÌ iÊä«iÀViÌiÊvÀÊ É ÊÀ>ÌÃÊ units in the latter lakes showed D13 ÊÛ>ÕiÃÊÌ >ÌÊÜiÀiÊ of invertebrate samples was 7.9. For these reasons similar to those in limed and circumneutral lakes. we have chosen to present uncorrected data in this These differences are likely a consequence of the report. v>VÌÊÌ >ÌÊÌ iÊ } ÞÊ ÕVÊ>iÊ}>ÀÞ`ÃÃÊ >ÃÊ>Ê food web where assimilation of respired carbon (by Benthic food web microbes) is quantitatively more important than in In general epilithon, grazers, grazers/shredders, and the other lakes. The assimilation of respired carbon 15 13 benthivorous perch showed similar D N and D Ê implies several fractionation steps, resulting in more values in acid, limed and circumneutral lakes (Figure depleted (more negative) D13 ÊÛ>ÕiÃÊi°}°ÊìÊÀ}Ê 10), which suggests that their carbon sources and and Peters, 1994). Differences in the degree of assimi- trophic structure are not affected by liming. Signifi- lation of respired carbon are also apparent for D13 Ê cant differences were detected only for shredders and signatures in the epilithic samples, which tended to be invertebrate predators that showed a significantly higher in neutral lakes than that in the other lake ty- 15 lower D N in acid lakes than in limed and circum- pes. This is probably due to a higher incorporation of neutral lakes, respectively (Figure 10). This suggests - À}>VÊV>ÀLÊvÀÊV>ÌV iÌÊiÀÃÊ "3 ) in that these functional groups occupy lower trophic these lakes (that have a higher alkalinity) and a lower positions in the food web of acid lakes compared degree of assimilation of carbon originating from lake with circumneutral and limed lakes. In other words, internal processes (i.e. respiration). Repeated liming there is less trophic fractionation (fewer intermediate should theoretically result in higher D13 ÊÛ>ÕiÃ]Ê>ÃÊ trophic levels) below shredders and invertebrate the D13 ÊvÊiÃÌiÊÃÊâiÀ°ÊÜiÛiÀ]ÊÌ iÊiÝÌiÌÊvÊ predators in acid lakes. This may be a result of the in- this cannot be extracted from our results. hibition of microbial degradation of leaf litter and the Several interesting patterns emerge from a schema- disappearance of small, acid-sensitive invertebrates tic benthic food web using isotopic data (Figure 12). at intermediate trophic levels. Also predators showed Most notably are the low D15N values for grazers, 13 D ÊÛ>ÕiÃÊÌ >ÌÊÜiÀiÊÕµÕiÊÌÊi>V Ê>iÊV>Ìi}ÀÞ]Ê being in the same range or lower than D15N values 13 with the lowest D ÊÛ>ÕiÃÊvÕ`ÊÊi`Ê>iÃÊ>`Ê for their presumed food source, epilithon. These the highest in acid lakes. This suggests that inver- data suggest a very low degree of isotopic fractiona- tebrate predators in limed lakes rely more on lake tion, i.e. the difference between consumers and their internal carbon production, while those in acid lakes food source, between epilithon and grazers. Isotopic depend more on terrestrial sources of carbon. The ob- fractionation between epilithon and invertebrate ÃiÀÛ>ÌÊÌ >ÌÊÌ iÀiÊÃÊÃÕLÃÌ>Ì>Ê vÀ>VÌ>ÌÊÊ grazers was much less than the frequently assumed limed and acid lakes whereas all trophic levels are in D15N of 3.4‰ across trophic levels (Minagawa and Ì iÊÀ>}iÊvÊqÓÇÊÌÊqÓÊÊVÀVÕiÕÌÀ>Ê>iÃÊÃÊ>ÃÊ 7>`>]Ê£n{®°Êìi`Ê } Êv`µÕ>ÌÞÊÀiÃÕÀViÃÊ>ÞÊ interesting. These differences are likely a consequence be transferred to grazer/detritivores with only low of the fact that our acid and limed lakes were more N-fractionation (e.g. Vanderklift and Ponsard 2003, humic-rich and thus presumably more heterotrophic, i`«ÊiÌÊ>°ÊÓääÈ®°ÊÜiÛiÀ]ÊÌÊÕÃÌÊLiÊi« >Ã- resulting in a relatively larger assimilation of respired zed that only a single sample for grazers was avai- V>ÀLÊi°}°ÊìÊÀ}Ê>`Ê*iÌiÀÃÊ£{®°Ê iÃ«ÌiÊ >LiÊvÀÊ>iÊ}>ÀÞ`ÃÃ]Ê>ÊÃ>«iÊÌ >ÌÊ>ÞÊÌÊ these differences between lake types, the results sug- be representative of the other acid lakes. This calls for gest that liming does not have systematic effects on some caution in the interpretation of the results. the stable isotope ratios of taxa within functional guilds in benthic habitats.

416 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES FIGURE 10. Stable isotope signatures (means ± 1 SE) of compartments of the littoral benthic food web (i.e. epilithon, invertebrate grazers, grazers/shredders, shredders and predators, as well as benthivorous perch) INçLIMED çCIRCUMNEUTRALçANDçACIDçLAKESç3IGNIFICANTçDIFFERENCESç7ILCOXONçTEST çINçAçPAIRWISEçCOMPARISONçAREç highlighted (p values are shown). Note the differences in scales.

FIGURE 11. Stable isotope signatures (means ± 1 SE) of compartments of the pelagic food web (i.e. dissolved humic matter, seston, roach, perch and pike) in limed, circumneutral and acid lakes. Note that no significant DIFFERENCESçWEREçFOUNDçINçPAIRWISEçCOMPARISONSç7ILCOXONçTESTS ç.OTEçALSOçTHEçDIFFERENCEçINçSCALES

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 417 Ã«VÕÕÃÊ>ÀiÊ>ÃÊÌ iÊÀiVVÕÀÀ}Ê«>ÌÌiÀÃÊvÊ VÀVÕiÕÌÀ>Ê>iÃ]ÊqÎä°ÎÎÊ>ÛiÀ>}i®Ê´£°nÊ- ®]Ê increasing D15N values across gradients of acidifica- qÓn°££Ê´Êä°ÎÈÊ>`ÊqÓn°äxÊ´Êä°£Ç]ÊÀiÃ«iVÌÛiÞÊvÀÊÌ iÊ tion (i.e. acid to limed to circumneutral lakes) for three lake types. These values are in the same range grazers, shredders and invertebrate predators (Figure as D13 ÊÃ}>ÃÊÀi«ÀÌi`ÊvÀÊ " ÊÊvÕÀÊÕÌÀiÌ 10). These patterns show that similar taxonomic or «ÀÊvÀiÃÌÊ>iÃÊÊ ÀÌ iÀÊ-Üiì]ÊÀ>}iÊqÓÇ°ÇÊ functional groups have lower D15N values in acid ÌÊqÓ°Ê>ÀÃÃÊiÌÊ>°ÊÓääÎ®°Ê ÃÌÊì«iÌi`ÊÊ lakes than in circumneutral lakes, while limed lakes D13 ÊÜiÀiÊ`ÃÃÛi`ÊÀ}>VÊ>ÌÌiÀÊvÀÊ}>ÀÞ`Ã- have an intermediate position. This increase was most ÃÊqÎ{°Î®]ÊÌ >ÌÊ>ÃÊV>ÕÃi`ÊÌ iÊ } ÊÛ>À>ÌÊÊ pronounced for shredders (i.e. Asellus aquaticus) that D13 Ê>}Ê>V`Ê>iÃ°Ê/ iÊÀi>ÃÊvÀÊÌ iÊÜiÀÊD13 Ê had a much higher D15N in circumneutral lakes than Û>ÕiÃÊvÀÊ`ÃÃÛi`ÊÀ}>VÊ>ÌÌiÀÊÊ>iÊ}>- in acid lakes. The differences may be a consequence ryddssjön is unclear. Possibly, a relatively large share of the differences in leaf processing between lake of exudates from the Raphidophycean Gonyostomum types and/or the utilization of other food sources. For semen may have contributed to this observation. example, the reduced competition due to the disap- Lysis of Gonyostomum cells upon handling and ad- pearance of acid-sensitive taxa (in acid lakes, but dition of the ion exchanger may have contributed to partly also in limed lakes) may allow species with a a large pool of algal exudates in the sample obtained high functional plasticity to feed on alternative food from this lake. Algal carbon and thus excreted sugars sources. This conjecture is supported by 13 `>Ì>Ê and polymers will be more depleted in D13 ÊÌ >Ê`Ã- showing that shredders (i.e. Asellus aquaticus) are solved organic matter (e.g. Karlsson et al. 2003). more depleted in 13 ÊÊ>V`Ê>iÃÊÌ >ÊÊi`Ê>`Ê Seston samples in our lakes had D13 ÊÛ>ÕiÃÊLiÌ- circumneutral lakes, suggesting a larger share of aut- ÜiiÊqÓÊ>`ÊqÎäÊ>`ÊÜiÀiÊVÃiÊÌÊÌ iÊÛ>ÕiÃÊvÀÊ ochthonous benthic algal production in their food in forest lakes reported by Karlsson et al (2003). Seston limed and circumneutral lakes (and likely microfauna samples consisted of a mixture of bacteria and algal associated with these biofilms). Indeed, the acid lakes cells, largely reflecting the size fraction available sampled in this study were much more humic than for zooplankton. Zooplankton samples have been the other lake types, thus causing light-limitation collected, divided into claderocerans, herbivorous and lower autochthonous production of benthic copepods and carnivorous copepods, and will be algae in acid lakes. Moreover, epilithic samples analyzed during the winter of 2009. These samp- from circumneutral lakes were less depleted in 13 ]Ê les of zooplankton, although not specifically part further supporting the conjecture that alternative of this project, will provide additional information food sources were utilized in these lakes. As expected, about the D13 ÊÃ}>ÌÕÀiÊvÊÌ iÊi`LiÊvÀ>VÌÊvÊÌ iÊ 15N-enrichment was higher for invertebrate predators seston and the transfer of nutrients and energy from and fish, due to repeated fractionation at consecutive seston to the invertebrate predator Chaoborus and to lower trophic levels. planktivorous fish. Chaoborus larvae were some 2‰ more depleted than seston samples, which is in line Pelagic food web with an assumed 1‰ fractionation per trophic level The various compartments of the pelagic food webs (Petersen and Fry, 1987). Interestingly, Chaoborus of limed, acid and circumneutral lakes showed dif- larvae were much more depleted than the samples 15 13 ferent degrees of variability in their D N and D Ê- of invertebrates, planktivores and other fish (Figures gure 11). Partly due to the relatively high variability 11 and 12), suggesting a strong link to food sources in some taxonomic and/or functional feeding groups obtained in the benthic habitat where consecutive it was not possible to detect significant differences fractionations by microbes and small invertebrates between lakes. For example, for the invertebrate provide a 13 ì«iÌi`Êv`ÊÃÕÀViÊi°}°ÊÀiÞÊiÌÊ>°Ê predator Chaoborus and fish predators no significant 2004). Many of the relatively small lakes investiga- differences were detected as a consequence of the low ted here, in particular the more dystrophic (humic) sample size and high variability within some of the lakes, have long periods of hypoxia during summer groups (e.g., planktivorous perch and roach in acid and fall, likely resulting in a potentially important lakes). Again, the low sample size was a consequence role of methanogenic bacteria iÃÊ>`ÊÀiÞÊÓää{]Ê of a lack of acid sensitive taxa/groups in acid lakes. ÀiÞÊiÌÊ>°ÊÓää{®. Deposit-feeding chironomid larvae Despite the lack of significant differences among of the genus Chironomus, collected in the profundal lake categories, the data reveal some interesting sediments of some of our lakes had low D13 Û>ÕiÃÊ trends in stable isotope composition between lake vÊqÎ£°£ÊÌÊqÎÈ°£]Êi`}ÊÃÕ««ÀÌÊÌÊÌ ÃÊViVÌÕÀi°Ê types. For example, at the base of pelagic trophic pat- No significant differences in D15N between lake hways, isolated dissolved organic matter were more types were found for any of the investigated samples, depleted in D13 ÊÊ>V`Ê>iÃÊÌ >ÊÊi`Ê>iÃÊ>`Ê

418 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES FIGURE 12. Schematic presentation of the benthic and pelagic trophic pathways in limed, acid and circumneutral lakes based on the stable isotope composition of their biota. Data from leaf litter, obtained from Bohman (2005), are marked with an asterisk. Plots show means ± standard errors.

i.e. seston dissolved organic matter, invertebrates these slightly acidic, humic lakes and/or differences in or fish. However, some interesting observations are stratification patterns among lake types (longer strati- worth commenting upon. The apparent decreasing fication in acid brown-water lakes) may affect the dif- trend in seston D15N across the gradient from acid to ferences in D15N for these zooplankton-feeding larvae. limed to circumneutral lakes may indicate that the For example, although mixotrophy generally implies base of the food web in the acid lakes (that are more an increase in D15N (i.e. enrichment, see above), the humic than the other types of lakes) is dominated mass occurrence of Gonyostomum during the most more than in the other lake types by mixotrophic productive months of the year may be efficiently species. Mixotrophy implies trophic fractionation transferred to higher trophic levels by yet unknown and thus enrichment in D15 °Ê ÛiÀÃiÞ]Ê>ÃÊ} ÌÊ ÌÀ« VÊ«>Ì Ü>ÞÃ°Ê7iÊV>ÊÞÊÃ«iVÕ>ÌiÊ>LÕÌÊ«Ã- conditions are better, seston in the clearer limed and sible causes for this observation, but possibly future circumneutral to a larger share may include auto- analyses of stable isotopes in preserved samples of trophic species, likely explaining the lower D15N- different zooplankton taxa/groups may help to reveal values. Also the lower mean D15N in the predator-/ the mechanism behind these observations. phantom midge Chaoborus flavicans in acid lakes Pike in acid and limed lakes was more enriched in (by more than 2 ‰) are conspicuous. These data 15N than was piscivorous (large) perch (Figure 12), (near-significant at P = 0.07) suggest a more efficient whereas these two fish groups had similar D15N in transfer of nutrients to Chaoborus larvae in acid circumneutral lakes. However, rather than resulting lakes than in the other two lake types. Interestingly, from liming, we attribute this difference to ontoge- a similar tendency of lower mean D15N in acid lakes netic characteristics of the sampled pike populations. is found for planktivorous perch (Figure 11). Pos- One of the sampled individuals in the limed lakes sibly the occurrence of repeated summer blooms of was very small (136 cm) leading to a slightly lower the mixotrophic flagellate Gonyostomum semen in average size of pike in limed lakes (483 ± 126.6 cm)

2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES 419 relative to acid (528 ± 26.7 cm) and circumneutral sediment archive) and fish (samples from banking). references (546 ± 115 cm). In other words, we ascribe The temporal change of stable isotope patterns could this result to the differences in size (age) of the few be compared with genetic and autecological traits pikes that were caught, as larger pike is know to be of organisms hatched from different sediment strata more depleted in 15N than smaller individuals (e.g. }iiÀÊÓääÇ®°Ê7iÊ>ÌV«>ÌiÊÌ >ÌÊÃÕV Ê>Ê«>i- Meili et al. 1993). limnological approach can elucidate structural and functional shifts, particularly in isotopic signatures of key taxa (cladocerans, reflecting also seston isotopic SUMMARY AND changes, chironomids) during both acidification and FUTURE PERSPECTIVES liming. Our study of communities of different organism Determining an appropriate isotopic signature of groups, almost consistently based on biomass data phytoplankton, bacterioplankton, or epilithic algae is (part 1) points at systematic and significant differen- tricky, as a pure sample of cells is difficult to obtain ces, with communities in limed lakes repeatedly being (but see Vuorio et al 2006). Our samples of seston the intermediate between acid and circumneutral and epilithic communities were also a mixture of lakes. These results likely reflect that community algal and bacterial cells, likely also “contaminated” compositions and biomass in limed lakes are kept at with protozoans and ciliates. In other words, these some intermediate state due to liming. Alternatively, composite samples may not give good estimates the intermediate state indicate how far these lakes of the isotopic composition of the food source of have come in the transition from an acidified to a grazers. Future analyses of zooplankton samples will 13 fully restored state since the liming began. In acid la- provide further information, as the D ÊÃ}>ÊvÊ kes, acid-sensitive taxa have disappeared and biomass herbivorous zooplankton should be close to that of production is frequently lower, while circumneutral their food source. lakes have more taxa and higher biomasses within During 2009 we will continue to work on the da- these taxa. Univariate analysis of structural (total bio- taset generated in this study. The inclusion of isotopic >ÃÃ]ÊÃ«iViÃÊÀV iÃÃÊ>`Ê- >7iiÀÊ`ÛiÀÃÌÞ®Ê data for zooplankton samples will further improve and functional (feeding guilds) characteristics showed the data set and allow for a more thorough analysis. community and lake-type-specific responses. 7iÊ>Ê>ÌÊ«À`ÕV}Ê>ÌÊi>ÃÌÊÌÜÊÃViÌvVÊ«ÕLV>- Our study of the isotopic composition of orga- tions. Reprints will be sent to the Swedish EPA as a nisms in acid, circumneutral and limed lakes (part 2) means of additional reporting. does not point at systematic changes in the isotopic signature of fish, pelagic and benthic invertebrates, REFERENCES and their food sources as a result of liming. However, some interesting differences between lakes types were }iiÀ]Ê °°ÊÓääÇ®Ê,iÃÕÀÀiVÌÊiV}ÞÊ>`Ê found. For example, our study showed that shredders global climate change research in freshwater ecosys- (i.e. Asellus aquaticus) had a much higher D15N in tems. Journal of the North American Benthological circumneutral lakes than in acid lakes, suggesting dif- Society, 26, 12-22. ferences in leaf processing between lake types and/or Appelberg, M. (1998) Restructuring of fish assem- the utilization of other food sources due to ecological blages in Swedish lakes following amelioration of release as a consequence of the disappearance of acid acid stress through liming. Restoration Ecology, 6, sensitive taxa. Repeated liming should theoretically 343-352. result in higher D13 ÊÛ>ÕiÃÊ>ÃÊÌ iÊD13 ÊvÊlimestone Blomqvist,P och Herlitz, E. 1998. Methods for quan- is zero. Such lime-induced modifications of the D13 Ê signature of organisms should primarily occur in phy- titative assessment of phytoplankton in freshwaters, - part 2.Naturvårdsverket, Rapport 4861. Ì«>ÌÊ>`Êi«Ì VÊ>}>iÊÌ >ÌÊ>ÃÃ>ÌiÊ "3 originating from lime stone additions. However, our >]Ê°ÊÓääx®Ê >ÀÃiÊìÌÀÌÕÃÊÊ}ÌÀ« VÊ results did not show significant differences in the D13 Ê >iÊÌÌÀ>ÊâiÃÊqÊÕÌâ>ÌÊLÞÊÛiÀÌiLÀ>ÌiÃÊ>`Ê of seston and epilithon between lake types, partly due contribution to carbon flow. Dissertation, Faculty of to relatively high variability of lakes within each type. Natural Sciences, University of Kalmar, Dissertation The question whether liming has affected the isotopic Series Nº 19, pp. 1-38. signature of seston and epilithon (and higher trophic À>ìÞ]Ê ° °ÊEÊ"ÀiÀ`]Ê-°Ê°ÊÓääÓ®Ê}ÌiÀÊ iÛiÃ®ÊV>ÃÊvÀÊ>Ê>ÌiÀ>ÌÛiÊÃÌÕ`ÞÊìÃ}°Ê7iÊÌ iÀi- effects of catchment liming on invertebrates in upland fore will propose the hind casting of changes in stable streams. Freshwater Biology, 47, 161-171. isotope composition of faunal elements (from the

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422 2A:11 – LIMING EFFECTS ON ECOSYSTEM STRUCTURE, FUNCTION AND TROPHIC RELATIONSHIPS IN LAKES