Periphyton as a Habitat for Meiofauna : a case of a neglected community

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Periphyton as a Habitat for Meiofauna — a case of a neglected community

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)

an der

Universität Konstanz

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie

vorgelegt von

Lars Peters

Konstanz, November 2005

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Periphyton as a Habitat for Meiofauna —

a case of a neglected community

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Chapter I

General Introduction

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General Introduction

During the last century, research on benthic meiofauna – defi ned as those animals that pass through a 500-μm sieve and are retained on a 42-μm sieve (Fenchel, 1978) or pass through a 1000-μm and retained on a 63-μm sieve (Giere, 1993) – has increasingly been a focus of marine and freshwater ecologists.

Research on meiobenthic communities of fresh waters has intensifi ed recently, but the basic insights into meiofaunal biology and ecology have been gained from research in marine environments. Over the years, this research has resulted in a considerable number of studies that lead to an enhanced understanding of patterns and processes in meiobenthic assemblages (see Coull & Bell, 1979; Higgins & Thiel, 1988; Giere, 1993). Such studies discuss general meiofaunal community patterns and community development (e.g., Sherman & Coull, 1980; Colangelo & Ceccherelli, 1994), meiofaunal dispersal mechanisms (e.g., Hagerman & Rieger, 1981; Chandler & Fleeger, 1983; Palmer, 1988), and interactions of meiofauna with higher trophic levels (Gee, 1989; Coull, 1990, 1999) or microorganisms (Montagna, 1995; Epstein, 1997). As a whole, these fi ndings illustrate the true nature of meiofauna and point to its importance. Nevertheless, a direct transfer of this knowledge of marine meiofauna to freshwater habitats is diffi cult because of fundamental habitat differences (e.g., the unitary nature of the seas and the lacking tidal activity in fresh waters). However, the transfer of some general processes should be possible, e.g., small- or medium-scaled dispersal mechanisms and interactions between meiofauna and smaller organisms (bacteria, algae, protozoa).

Our knowledge of the various aspects of meiobenthology in freshwater habitats has increased over the past years, but is still far too limited to ascertain whether meiofauna has important ecological functions within the benthos of freshwater ecosystems. A series of introductory and mechanistic studies of freshwater meiofauna published over the past couple of decades, however, indicate that meiofauna is both a diverse and an abundant constituent in freshwater ecosystems. Furthermore, there is growing evidence that meiofauna plays an important role in trophic and functional dynamics in freshwater ecosystems. Meiofauna in benthic freshwater habitats can be integrated into benthic food webs by fulfi lling important roles as prey for larger animals and/or as consumers of bacteria and algae (Schmid-Araya & Schmid, 2000; Schmid & Schmid-Araya, 2002). If meio-

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Within meiofaunal communities, nematodes are very often the dominant group (Traunspurger, 1996a, b; Wu et al., 2004; Witthöft-Mühlmann et al., 2005a) and are widely distributed throughout all types of substrates in aquatic and terrestrial ecosystems.

Within a single lake ecosystem, nematode species can number up to 152 (Lake Ober- see, Germany; Michiels & Traunspurger, 2004), and nematode densities can reach up to 3,464,000 individuals per m² in freshwater sediments (Traunspurger, 2002). Nematodes are not only found ‘everywhere’ and often in high abundance, but are also important in ecosystem processes because of, e.g., grazing on microorganisms (Traunspurger et al., 1997) and benthic algal communities (Montagna, 1995). Nematodes might therefore function as an important link between microbial production and higher trophic levels.

Much of this information on meiofauna in fresh waters has fortunately been brought together in two very important recent contributions to freshwater meiofaunal biology and

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I General Introduction

ecology: in a special issue of the journal Freshwater Biology [vol. 44 (1), 2000] and in the book Freshwater Meiofauna (Rundle et al., 2002). However, it must be mentioned that, as with all meiofaunal studies conducted to date in streams and lakes, almost only soft-bottom (sandy or muddy substrates) meiofauna have been studied. But lakes do not only consist of soft-bottomed areas. They are rather often characterized by large euphotic littoral zones with spatially heterogeneously distributed hard substrates of various size and type. These substrates are often covered with considerable amounts of organic and inorganic material [defi ned as periphyton (Behning, 1924), and also referred to as ‘bio- fi lm’ and ‘Aufwuchs (Seligo, 1915)’] that represent a vital source of primary production and can be important in lake ecosystem processes (Vadeboncoeur & Steinman, 2002;

Vadeboncoeur et al., 2003). Periphyton can roughly be defi ned as a benthic community attached to substrates or surfaces, with benthic algae living in close connection with bacteria, fungi, meiofauna, and organic and inorganic non-living material, embedded in a mucopolysaccharide matrix (Burkholder, 1996) (Fig. 1). Several authors have point out that considerable numbers of non-attached organisms with planktonic or free-swimming lifestyle also occur frequently in the periphyton (Young, 1945; Newcombe, 1950; Welch, 1952). This community is considered to be an independent ecological unit (Ruttner, 1962;

Pieczynska, 1964; Wetzel, 2001) and has been the subject of a large number of studies exploring patterns of periphyton community structure (mainly algal communities) and different processes of periphyton biomass and community structure regulation (see Ste- venson et al., 1996; Wetzel, 2001).

Until relatively recently, however, most ecologists have not described meiofaunal assemblages on (littoral) hard substrates and failed to analyse the role of epilithic meiofauna in ecological processes in freshwater habitats. This lack of information on epilithic meiofauna is all the more astonishing because periphyton, excluding meiofauna, was in- tensively investigated until most recently (see above). However, some researchers during the last 92 years were interested in periphytic (Aufwuchs) meiofaunal communities, and their work resulted in interesting and important contributions to meiofaunal biology and ecology. Most of these early studies dealt with epiphytic communities on reed (Phrag- mites sp.), common clubrush (Scirpus lacustris), or bulrush (Typha latifola), and only two studies dealt with stony hard substrates.

In the fi rst limnological study, Micoletzky (1913) presented a descriptive examina-

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etes, nematodes, and rotifers and found the cladoceran Sida crystallina to be the dominant species (90% of all individuals); within the nematodes, Plectus tenuis and Chromadorina bioculata were the dominant species.

Another qualitative study, done by Schneider (1922), focused on a single meiofaunal group and investigated free-living periphytic nematodes in eight lakes of eastern Holstein (Germany) on reed, rush, and stones. Schneider identifi ed 8387 nematodes to the species level and found a total of 30 species [3–22 (mean ±SD = 8.8 ± 6.2) species per single lake]. In all investigated lakes, Punctodora ratzeburgensis was the dominant species, followed by Chromadorina viridis and C. bioculata. These species were considered to be typical for the periphyton (Aufwuchs) on reed stems and might have adapted to an epiphytic/epilithic life style (e.g., high oxygen demand).

Twelve years later, Meschkat (1934) studied the fauna in the periphyton (‘Bewuchs’) on stems of Phragmites communis in Lake Balaton (Hungary). He also found nematodes to be the dominant faunal group on reed stems (ca. 95% of all fauna), with two species, P. ratzeburgensis and C. bioculata, dominating. From observations of living individuals of these two species, Meschkat deduced that they are able to attach to substrate surfaces and that this is a morphological adaptation for a periphytic lifestyle. He was the fi rst to mention the importance of spatial structure and complexity (derived from the relation- ship between the amount of material attached to the substrate surface and the number of nematodes) of the periphyton mat for meiofaunal organisms.

Two descriptive studies on epilithic and epiphytic communities followed. The fi rst study was conducted in 42 lakes in eastern Holstein (Germany) (Meuche, 1939) and revealed, similarly as the earlier studies, that three species — P. ratzeburgensis, C. viridis and C. bioculata — dominated the nematodes in the periphyton with specifi c commu- nity patterns between lakes. The next study (Young, 1945) revealed that nematodes were much less abundant than oligochaetes, cladocerans, and rotifers.

The fi rst experimental study was conducted about 20 years later by Pieczynska (1964) in Lake Mikolajskie (Poland). Colonisation processes of periphytic nematodes and other meiofauna on reed stems and artifi cial substrates (glass slides) were investigated. The author pointed to the importance of wave action, which enables the dispersal of attached

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(1992) was the fi rst to investigate quantitatively the free-living epilithic nematode species on littoral hard substrates in a lake. Samples were taken at three different depths (2, 8, and 20 m) in the alpine oligotrophic Lake Königssee (Germany). This study revealed a total of 29 epilithic nematode species (excluding Dorylaimidae), with four species (C. bioculata, Rhabdolaimus terrestris, Plectus cirratus, and Eumonhystera simplex) dominating with different distribution patterns at the four different depths.

The few results from the above-mentioned older and more recently conducted studies combined with the information available on soft-bottom meiofauna and periphyton, led to my hypothesis that there is also an abundant and diverse meiofaunal community within the littoral lake periphyton, which certainly can be integrated into ecological processes in shallow littoral zones of lakes. In order to contribute to an advanced understanding of fundamental and mechanistic principles of meiofaunal community structure on hard substrates in the littoral zone, the present study combined fi eld experiments with data collection in the fi eld.

The basis for adequate studies on natural periphyton communities in lakes is an effi cient and reliable method for quantitative and qualitative sampling. Such a sampling device was lacking, and those that have been introduced and described by several authors in the past were ineffi cient, with sampling inaccuracies, or could not be used to sample non- removable substrates (large stones or bedrock). Since aquatic ecologists are increasingly studying littoral hard substrates, and water quality and monitoring programmes utilise periphyton communities for water-quality assessment (Biggs & Kilroy, 2000), there was a general need for a standardised and simple periphyton sampling method. In Chapter 2, I present the construction details and functionality of a new in situ brush sampler, which is based on a previously developed sampling device (Loeb, 1981) and that now allows a simple and quantitative sampling of natural periphyton communities. The brush sampler was tested under ambient conditions in two lake and two stream sites and was compared to the previously developed Loeb sampler and control samples.

This new brush sampler made it possible to investigate natural hard substrates in lakes quantitatively on-site, which is necessary to obtain basic insights into epilithic meiofauna.

Such investigations should focus on a large variety of different lake ecosystems cover- ing different lake types (trophic status, colour, size, depth, etc.) to assess general patterns of meiofaunal community composition and distribution of single nematode species.

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3, meiofaunal group and nematode species composition; the proportion of different feed- ing types, sex, and age; meiofaunal and nematode abundances; and periphyton biomass (algal biomass, organic and inorganic matter content) were analysed. I used multivariate analysis techniques (cluster analyses and analysis of similarity) with different multivariate parameters to test for differences between lakes; a detailed list of identifi ed nematode species and a correlation analysis between meiofaunal and nematode abundance and periphyton biomass are presented.

In the littoral zone of lakes, periodic disturbances (e.g., wave action or water-level fl uctuations) often lead to large-scale changes in the composition of benthic communities or remove most of the organisms from substrates (Barton & Carter, 1982; Dall et al., 1984; Hildrew & Giller, 1994) and will fi nally lead to new space being offered for colonisation. Organisms living on littoral hard substrates must have adapted to these conditions and must respond quickly after the disturbance has passed. The processes of community development and the colonisation pathways of periphytic meiofauna are unknown, but there are data on such processes in meiofaunal communities from soft-bottom communities. In Lake Constance, large water-level fl uctuations occur throughout the year, resulting in large littoral areas that become dry during winter. Substrates in the upper part of the littoral zone are only temporarily fl ooded during summer and are re-colonised anew each spring with increasing water level.

These characteristics of Lake Constance and the questions on colonisation processes and pathways of epilithic meiofauna led to an experiment which focused on the development of an epilithic meiofauna (chapter 4) and nematode community (chapter 5) and their colonisation pathways. In this fi eld experiment, direct colonisation of artifi cial hard substrates was prevented by elevating the substrates in the water column. The community development was followed over a 57-day period and was compared to non-elevated artifi cial substrates, natural hard substrates, and sediment trap samples. Chapter 4 focuses on the means of general colonisation processes of meiofauna and periphyton and presents results on the temporal development of the periphyton biomass and meiofaunal taxonomic-group composition and colonisation pathways of meiofauna. I used the non-metric multi-dimensional scaling technique and analysis of similarity to test for differences in

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colonisation means, and present data on nematode abundance and species composition, feeding type, and age/sex distribution.

As already mentioned, periphyton has been the subject of a large number of studies, including consumer–resource interactions at the periphyton–herbivore interface. The presence of grazers often leads to a reduction of periphyton biomass and can alter algal species composition (e.g., Feminella & Hawkins, 1995; Hillebrand, 2002; Hillebrand et al., 2002). Since meiofauna is present in the periphyton and interactions with macrofauna on hard substrates are not known, it remained to be tested whether meiofaunal organisms are also affected by macrofaunal grazing activity. Therefore, I investigated the impact of macrograzers on meiofaunal groups and periphyton biomass, integrating the aspect of the spatial heterogeneous distribution of both grazing effects and resource (prey) availability (Chapter 6). In a fi eld experiment in Lake Erken (Sweden), the grazer access to periphyton was manipulated in a hierarchical nested design at three sites (kilometre scale) consisting of three subsites (metre scale).

This thesis brings together two aspects of benthic ecology in lakes, periphyton and meiofauna, which can be considered as a detailed introduction to epilithic (periphytic) meiofauna. A general description of the meiofaunal and nematode communities and some fundamental mechanisms affecting meiofauna on hard substrates in different lake ecosystems are presented.

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Chapter II

An effi cient in situ method for sampling periphyton in lakes and streams

Lars Peters, Nicole Scheifhacken, Maria Kahlert & Karl-Otto Rothhaupt Archiv für Hydrobiologie (2005) Vol. 163(1), 133–141

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Arch. Hydrobiol. 163 1 133–141 Stuttgart, May 2005

Note

An efficient in situ method for sampling periphyton in lakes and streams

Lars Peters^{1, 3}, Nicole Scheifhacken¹, Maria Kahlert² and Karl-Otto Rothhaupt¹

With 2 figures and 1 table

Abstract: We present an efficient in situ sampling device that allows a simple and quantitative sampling of natural periphyton communities. The Brush Sampler is based on a previously developed sampler, which was improved by the addition of an external water supply, ball valve closing mechanism, and special exchangeable stiff brushes to solve previously reported problems of biomass overestimation and underestimation.

The Brush Sampler was tested under ambient conditions in the field at four different sampling sites in a lentic and a lotic system and compared to the old sampler and control samples. The tests revealed a high inaccuracy of the old sampler and showed that biomass estimates (ash-free dry mass and chlorophyll-a content) obtained with the improved Brush Sampler did not significantly differ from biomass values determined from scrapings of control samples collected at the same sites. Therefore, our modified sampling device can be used as a tool for quantitative and qualitative epilithon community analyses.

Key words: periphyton, chlorophyll-a, sampling method, Brush Sampler, epilithon, littoral hard substrates, microbenthos, meiofauna.

Introduction

In the past years, many studies in streams and the littoral zones of lakes have focused on the periphyton – the assemblage of autotrophic and heterotrophic organisms attached to surfaces (Stevenson et al. 1996). However, a standar-

1 Authors’ addresses: Limnological Institute, University of Konstanz, 78457 Kon- stanz, Germany.

2 Erken Laboratory, Department of Limnology, Evolutionary Biology Centre, Uppsala University, Norr Malma 4200, SE-76173 Norrtälje, Sweden.

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dised sampling method that provides simple and consistent qualitative and quantitative analyses of periphyton communities on different types of stony substrates is still lacking. Artificial substrates have often been used to simplify algal quantification (e. g. Slàdeckovà 1962, Austin et al. 1981, Aloi 1990, Cattaneo & Amireault 1992), but the algal community on artificial substrates does not always mirror the natural algal community (Cattaneo &

Amireault 1992, Wetzel2001), and therefore artificial substrates cannot be used when comparing different ecosystems.

Aloi (1990) critically reviewed field methods for natural periphyton sampling in freshwater and described a large variety of different methods that have been commonly used for investigations of periphyton in the field, e. g.

scraping areas of rock surfaces or using more complex periphyton samplers. A simple periphyton sampler, consisting of a 55-ml plastic syringe with a toothbrush glued on the end of the plunger to sample a defined area, was developed by Stockner &Armstrong (1971). This device was modified and improved by Perkins & Kaplan (1978) and by Loeb (1981), who added a second syringe to the sampler.

The efficiency of Loeb’s sampler was tested by Cattaneo & Roberge (1991) who compared chlorophyll-a analyses of epilithon (benthic algal communities on stones) collected by Loeb’s sampler with direct extraction of chlorophyll-a from cobblestones. The sampler was shown to have several me- thodological drawbacks as it overestimates the chlorophyll-a content in lakes and underestimates the chlorophyll-a content in streams. Cattaneo & Ro- berge (1991) explained these discrepancies by the sampler’s difficulties in re- moving tightly attached algae in streams, yielding the underestimation, and the enrichment of the sample by neighbouring algal material in lakes, combined with possible loss of loosely attached algal material during the lifting of the control stone from the lake.

Here, we describe an improved Brush Sampler based on Loeb’s sampler with several important modifications. We equipped our sampler with an external water supply with an exchangeable gauze filter and added a ball valve as closing mechanism to the brush to minimise sample losses after brushing.

These modifications prevent contamination of the sample by adjacent loose organic material solving the problem of overestimation of epilithic biomass.

Additionally, we replaced the toothbrush with an easily exchangeable, stiff- bristled strip brush to ensure complete removal of algal material from the sub-

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Sampling periphyton in lakes and streams 135

grammes utilise periphyton communities for water quality assessment (Biggs

& Kilroy2000).

Methods

Design of the new Brush Sampler

The sampling device is made of a cylindrical Plexiglas^® tube (internal diameter 20 mm, wall thickness 5 mm, length 197mm) with two screw threads at both ends and a stainless steel plunger with an exchangeable strip brush (Fig. 1 A). The plunger is mounted on a guide rail with a star-shaped knob used to rotate the brush on the substrate surface. Several ring-shaped spacers can be installed on the guide rail between the upper collar and the knob to regulate brush pressure on the substrate surface. Two O-ring seals placed at the guide rail end of the plunger create a slight vacuum to suction up water when the plunger is raised after brushing the substrate. The knob is screwed onto the guide rail and can be screwed off. A Plexiglas^® collar with internal screw thread is screwed on one end of the main cylinder and can be screwed off to remove the plunger for maintenance and cleaning. The upper collar has a centred bore for plunger guidance and three additional small bores to guarantee an easy run when the plunger is pulled to the top of the sampler. A conventional ³⁄⁴inch brass ball valve is screwed onto the other end of the main cylinder. The lower metal collar is screwed into the end of the ball valve, which determines the sampling area (3.14 cm²) and car- ries a rubber seal to guarantee a tight connection to the substrate surface (Fig. 1 A, B).

Special strip brushes (transparent, PA6 polyamide bristles; bristle thickness 0.3 mm, bristle height 10 mm, August Mink KG, Germany) with four tufts were used; the brushes can be mounted at the end of the plunger and fixed with two hexagonal socket set screws (Fig. 1 C). To ensure a supply of water as the plunger moves up after brushing, the Brush Sampler is equipped with a lateral tube with a filter element. For this purpose, a tangential hole in the ball valve was drilled close to the sampling area and a plastic elbow joint with a flexible tube and the screw thread of a 50-ml dropper bottle were used as a filter mount. A small piece of 30-μm gauze was clamped into the screw thread of the filter element. The tangentially drilled hole for water supply leads to an eddy inside the cylinder when water is sucked up after brushing. This effect additionally cleans the sampled substrate surface and transports the sampled material upwards.

This new Brush Sampler can be adjusted for various individual purposes, e. g. the size (sampling area) or the external water supply can be customised with finer gauze or an external water tank with filtered water, respectively.

In the field, the plunger is moved down towards the aperture and the sampler is carefully placed onto the substrate. If the water depth is lower than the height of the sampler, the filtering element can be turned around to dip into the water. The Brush Sampler must be fixed with one hand on the substrate, while the other hand pushes

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Fig. 1.The modified Brush Sampler device:A, location drawing;B, front view of sam- pling area and brush without rubber ring sealing; and C, detail of the plunger with brush retainer. (Bv) Ball valve, (Bvh) Ball valve handle, (Cyl) Plexiglas^® cylinder, (Fe) Filtering element, (Fs) Fixation screws, (Ft) Flexible tube, (Gr) Guide rail, (Lc)

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Field test

The Brush Sampler was tested in August 2004 in Upper (upper lake) and Lower (lower lake) Lake Constance, two basins of a large pre-alpine meso-oligotrophic lake in southern Germany. The lake site “lower lake” is characterised by a large inflow of nutrients due to an agriculture drainage, which has led to thick periphyton layers on the stones in the surroundings. The periphyton layer on the stones in Upper Lake Con- stance was much thinner with higher amounts of inorganic sediments. We also conducted a test at two different sites in the Mühlenbach, a small stream between Lake Mindelsee and Lower Lake Constance with different flow velocities (stream fast:

0.45 m/s; stream slow: 0.23 m/s).

We constructed a periphyton sampler (Loeb Sampler) following the descriptions in Loeb (1981) and Biggs & Kilroy (2000). Therefore, we used 60 ml plastic syringes with a toothbrush screwed on the plunger. Total sampling area was 6.16 cm². Eight samples of epilithon were taken with our Brush Sampler and the Loeb Sampler on different stones at each site in close vicinity to each other. Sampling depth in the lake littoral was 30 to 40 cm, in the “stream fast” and “stream slow” sites 11cm and 20 cm, respectively. Additionally, eight cobblestones were sampled at each site as controls using a sampler based onSnoeijs&Snoeijs(1993) device. We modified this sampler by using the same lower collar with rubber sealing (sampling area 3.14 cm², Fig. 1 B) as for the Brush Sampler. This lower collar was screwed into the centre of an alumin- ium plate. Sampling was done by clamping a cobble stone between the upper alumin- ium plate with collar and a second base plate directly in the water, to minimise periphyton losses. All material within the sampling area of the control stones was scraped off outside the water with metal spatula of different size and small brushes to ensure complete removal of all attached material. All sampled cobblestones were chosen ran- domly and were similar in size (5 – 9 cm in diameter).

Each Brush, Loeb and control sample was transferred into a 100-ml polyethylene bottle immediately after sampling and stored in the dark at 4 ˚C until all processing was completed within 5 h. The sampled epilithon was resuspended and adjusted to a defined volume and algal conglomerates were carefully separated with scissors and forceps. One half was filtered on pre-combusted filters (Schleicher & Schuell GF6, ∅ 25 mm) for the analysis of ash-free dry mass (AFDM), and the other half was filtered on the same glass fibre filters for chlorophyll-a determination. Chlorophyll-a was extracted directly after filtration with acetone (90 %) in the dark for 24 and concentrations were measured spectrophotometrically, correcting the values for pheopigment content (Lorenzen 1967). Ash-free dry mass was measured by drying the filters at 80 ˚C for 24 h, followed by combustion at 550 ˚C for 5 h.

One-way ANOVA and Tukey HSD test were used to analyse differences of biomasses between sampling methods within each site. Log-transformed data were homosce- dastic.

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Fig. 2. Median box plot of the results obtained with the Brush Sampler (black), Loeb Sampler (unshaded) and the control stones (grey). Upper plots show autotrophic biomass (chlorophyll-a) and lower plots show total organic matter (ash-free dry mass) at the four sites (stream fast & slow, upper & lower lake). Boxes represent 25 –75 % values; the error bars indicate minimum and maximum values. Letters indicate significant differences between sampling methods within each sampling site (Tukey HSD test).

yses in lakes and streams under different environmental conditions. The results of the field tests showed that the Brush Sampler is accurate and provides data that mirrors the biomass data (chlorophyll-a and AFDM) obtained from

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Table 1. One-way ANOVA (shaded) and Tukey HSD Post-hoc comparison (unshaded) for each of the four sampling sites (stream fast, stream slow, upper lake &

lower lake) on log-transformed chlorophyll-a and ash-free dry mass (AFDM) values, with sampling method (Control, Loeb & Brush) as independent factor. The table gives the significance levels (*** p<0.001) and the F-ratio of the One-way ANOVA and the p-level for the Post-hoc comparison (as matrix) for each sampling site and biomass pa- rameter.

Chlorophyll-a AFDM

Control Loeb Control Loeb

stream fast *** (F_2,20=11.13) n. s. (F_2,18=3.53)

Loeb 0.008 0.123

Brush 0.619 <0.001 0.912 0.052

stream slow *** (F2,21=24.54) *** (F2,21=14.59)

Loeb <0.001 <0.001

Brush 0.182 <0.001 0.682 0.001

upper lake *** (F2,21=13.62) *** (F2,21=20.09)

Loeb 0.005 <0.001

Brush 0.332 <0.001 0.236 <0.001

lower lake *** (F2,21=21.26) *** (F2,21=24.30)

Loeb <0.001 <0.001

Brush 0.409 <0.001 0.574 <0.001

parison using a Tukey HSD test of the chlorophyll-a contents obtained with the three methods revealed no significant differences between the Brush Sam- pler and the controls at all sampling sites, but highly significant differences in chlorophyll-a contents between the Loeb Sampler and the Brush Sampler and controls at all four sampling sites (Table 1, Fig. 2).

The AFDM values obtained with the Brush Sampler and from the controls also did not significantly differ, but highly significant differences between the Loeb Sampler and the Brush Sampler and controls were found at the sampling sites “stream slow”, “upper lake”, and “lower lake” (Table 1, Fig. 2). The chlorophyll-a and AFDM contents obtained with the Loeb Sampler were always significantly lower, i. e., 2 –7-fold lower (Fig. 2), than those of our Brush Sampler and controls and did not confirm the overestimation of biomasses as described in Cattaneo & Roberge (1991). We attribute these large differences between Loeb Sampler and controls in our test to an inaccurate func- tioning of the affixed toothbrushes and to sample losses after brushing when

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the sampling device, but is rather a result of differences between stones, which has also been observed in previous studies on the epilithon (e. g. Harrison &

Hildrew1998,2001, Kahlertet al. 2002,Peterset al., unpubl. results).

The test of the sampler developed byLoeb(1981) conducted byCattaneo

& Roberge(1991) showed an overestimation of algal biomass in lakes and an underestimation of algal biomass in streams. In contrast, the data obtained with our Brush Sampler did not show a similar pattern presumably because of the specific modifications made to the sampler, which have been proposed earlier (Flower1985, Turneret al. 1987, Cattaneo&Roberge1991). With the new Brush Sampler, loosely attached algal material of the surroundings can be excluded from the samples, and tightly attached sub-communities of epilithon can be quantitatively sampled. For use in streams, the efficiency can be additionally enhanced by using different types of brushes, e. g., stiffer plastic or metal bristles, which makes the Brush Sampler very flexible in use. The use of the Snoeijs & Snoeijs (1993) device is more complicated and time- consuming than our Brush Sampler. This device restricts sampling to movable substrates of small to medium size and may lead to sample losses since the stones will be removed from the water for sampling. Therefore, we suggest that our improved sampling device should be used as a standard method for in situ investigations on epilithic communities.

Acknowledgements

We thank M. Wolf for helpful comments on the construction details and manufactur- ing the Brush Sampler. M. Hamitou helped with the Brush Sampler test and the biomass analyses. W. Traunspurgergave helpful comments on an earlier version of the manuscript and K. A. Bruneedited the English of the manuscript. We thank two ano- nymous reviewers for their helpful comments on the manuscript. This study was sup- ported by the German Research Foundation (DFG) within the Collaborative Research Center 454 “Littoral Zone of Lake Constance”.

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Slàdeckovà, A.(1962): Limnological investigation methods for the periphyton (“aufwuchs”) community. – Bot. Rev.28:286 – 350.

Snoeijs, P.&Snoeijs, F.(1993): A simple sampling device for taking quantitative mi- croalgal samples from stone surfaces. – Arch. Hydrobiol.129:121–126.

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Stockner, J. G. &Armstrong, F. A. J. (1971): Periphyton of the experimental lakes area, northwestern Ontario. – J. Fish. Res. Bd. Can.28:215 – 229.

Turner, M. A., Jackson, M. B., Findlay, D. L., Graham, R. W., De Bruyn, E. R.

&Vandermeer, E. M.(1987): Early response of periphyton to experimental lake acidification. – Can. J. Fish. Aquat. Sci.44(Suppl. 1): 135 –149.

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Submitted: 30 August 2004; accepted: 15 November 2004.

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Chapter III

Species distribution of free-living nematodes and other meiofauna in littoral periphyton

communities of lakes

Lars Peters & Walter Traunspurger Nematology (2005) Vol. 7(2), 267-280

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Nematology, 2005, Vol. 7(2), 267-280

Species distribution of free-living nematodes and other meiofauna in littoral periphyton communities of lakes

Lars PETERS¹^,∗and Walter TRAUNSPURGER²

1University of Konstanz, Limnological Institute, 78457 Konstanz, Germany

2University of Bielefeld, Animal Ecology, Morgenbreede 45, 33615 Bielefeld, Germany Received: 18 January 2005; revised: 7 March 2005 Accepted for publication: 7 March 2005

Summary –Recent studies on meiofaunal and nematode communities have focused on soft sediments in streams, lakes and marine environments. Despite a large number of studies dealing with periphyton, meiofaunal and nematode communities, on littoral hard substrates in lakes have not yet been investigated in detail. Therefore, epilithic communities with particular emphasis on nematode species composition, were analysed in 17 Swedish lakes differing greatly in size, depth, trophic status and epilithic biomass. Nematode abundance ranged from 2.3 to 161.5 cm⁻², and the abundance of nematodes relative to total meiofauna ranged from 20 to 77%

(mean 53%). Fifty-eight nematode species were identified; species numbers varied from eight to 34 species per lake. The dominant species wereRhabdolaimus aquaticus,Punctodora ratzeburgensis,Eumonhystera disparandCrocodorylaimus flavomaculatus. Deposit feeders dominated (71% of total fauna), followed by suction feeders (14%), epistrate feeders (12%) and chewers (3%). Of 3624 nematodes examined, 54% were juveniles, 35% females, 6% males and 5% gravid females. Multivariate analysis of the nematode species composition revealed significant differences in the community structures among lakes. This is the first study to show that meiofauna is a numerically abundant group within littoral periphyton communities in lakes, with nematodes representing the dominant group.

Keywords –community structure, diversity, epilithon, feeding types, freshwater, geographical distribution, hard substrates, meiofauna, Swedish lakes.

Stony substrates in euphotic littoral zones of lakes are often covered with a considerable amount of organic and inorganic material. This assemblage – the periphyton – represents a complex community, with heterotrophic bacteria, fungi, protozoa and autotrophic components, as well as small metazoans (meiofauna) in close spatial vicinity (sensuWetzel, 2001), and is an important source of production in aquatic habitats (Vadeboncoeur et al., 2003). Meiofaunal organisms are of major importance for aquatic ecosystem function, as has been shown in a considerable number of studies that have focused on distribution, feeding, colonisation, production or their role within benthic food webs. These studies were mainly conducted in soft sediments of streams and lakes (e.g., Borchardt & Bott, 1995; Montagna, 1995; Traunspurger, 1996a, b; Hakenkamp & Morine, 2000; Robertson, 2000;

Schmid-Araya & Schmid, 2000; Beier & Traunspurger, 2003a, b; Bergtold & Traunspurger, 2004).

∗Corresponding author, e-mail: lars.peters@uni-konstanz.de

Within meiofaunal communities, nematodes are often the numerically dominant group (e.g., Heipet al., 1985;

Strayer, 1985; Traunspurger, 1996a, b), widely distributed throughout all types of substrates in aquatic and terrestrial ecosystems. Nematodes can reach densities of up to 3 464 000 m⁻²in freshwater sediments (Traunspurger, 2002) and are known to inﬂuence important ecological processes by grazing on microorganisms (Traunspurger et al., 1997) and algal communities (Montagna, 1995;

Epstein, 1997). The ﬁrst investigations on nematodes, in the ‘Aufwuchs’, had a low species and spatial resolution (Micoletzky, 1922; Schneider, 1922). More recent investigations on such communities have reported a diverse species community with nematodes as the dominant group (Traunspurger, 1992; Löhlein, 1998). However, detailed information on epilithic meiofaunal communities is still lacking, and mechanisms structuring such communities are still under debate. Effects (e.g., grazing, physical disturbance) that directly inﬂuence the periphyton could indi-

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rectly affect the associated meiofauna by consumption or by altering habitat structure and/or resource availability.

Grazing and nutrients have opposing effects on periphyton (Hillebrand, 2003) by increasing or reducing algal diversity, respectively, and grazing generally tends to reduce periphyton biomass (Feminella & Hawkins, 1995; Hille- brand, 2002) and, therefore, habitat complexity. Other mechanisms, such as physical disturbance due to low or high exposure to wind (Cattaneo, 1990) or differences in the community structure of macrobenthic grazers (e.g., snails, insect larvae), might inﬂuence epilithic communities. Considering that these factors are likely to vary between lake ecosystems, large differences in meiofaunal abundance and nematode species distribution between lakes are most likely to be found. However, information on epilithic meiofauna is still lacking.

In this study, we present data on nematode species distribution in epilithic communities in the littoral zone of various lakes that differ in morphology and trophic status.

Therefore, we investigated distribution patterns of meiofaunal groups and nematode species in lakes of differing trophy. Nematode species composition, the proportion of different feeding types, sex and age, meiofaunal and nematode abundances, and periphyton biomass were analysed.

Materials and methods

SAMPLING SITES AND PROCEDURE

The meiofaunal taxonomic composition, nematode species composition and biomass parameters (chlorophyll a, ash-free dry mass) of samples from 17 lakes around Uppsala, Sweden were analysed (Fig. 1). The lakes sampled differed greatly in size (0.2-36 km²), depth (mean depth: 1-9 m; max. depth: 1.8-21 m) and trophic status (oligotrophic-eutrophic) (Table 1). Three replicates of each sample were taken from the littoral zone at one, two or three subsites within each lake on hard substrates (stones or bedrock). Per replicate, two samples of one stone were pooled (sample area: 6.28 cm²) to yield enough material and to minimise small-scale heterogeneity. All samples were taken at 40 cm depth in October 2003 using a brush sampler (Peters et al., 2005). This syringe-like sampler scrapes off a deﬁned circular area (3.14 cm²) on hard substrates and collects all sampled material without loss and without contamination by adjacent loose material.

Table 1.Characteristics of the lakes sampled. Morphometric parameters and trophic status of each lake are given. Numbers in parentheses indicate the number of subsites within each lake.

Lakes are sorted in ascending order within each trophic status according to the chlorophyllacontent.^*(n.e.= not estimated.)

Lake Surface Mean Max. Trophic

area depth depth status (km²) (m) (m)

Largen (2) 1.5 8.3 21.0 Oligotrophic Långsjön (2) 2.5 6.5 13.0

Storsjön (1) 0.8 n.e. 13.0

Viren 2 (2) 1.4 n.e. 10.0 Mesotrophic Trehörningen (2) 0.7 2.6 5.1

Tomtasjön (2) 0.5 3.3 6.7 Österby Stordamm (2) 3.1 1.6 3.2 Gimodamm (2) 3.1 1.7 2.9 Viren 1 (1) 1.4 n.e. 3.5 Exarbysjön (1) 0.2 3.0 6.1

Erken (3) 24.0 9.0 21.0

Gavel-Långsjön (2) 5.5 4.0 9.4 Eutrophic Vendelsjön (2) 4.4 1.1 2.0

Hosjön (1) 2.4 1.0 2.4

Söder-Giningen (1) 3.1 1.5 2.8 Tämnaren (2) 36.0 1.3 1.8 Limmaren (3) 5.4 4.6 7.8

Each sample was transferred into a 100 ml polyethylene bottle immediately after sampling and stored in the dark at 4^◦C until all processing was completed within 12 h. The sampled epilithic material was resuspended, the total sample volume (95-150 ml) was determined and algal conglomerates were carefully separated with scissors and forceps. One aliquot (10 ml) was ﬁltered on pre-combusted ﬁlters (GF6, diam. 25 mm, Schleicher &

Schuell Microscience GmbH, Dassel, Germany) for the analysis of ash-free dry mass, and one aliquot (10 ml) was filtered on the same type of filter for chlorophylla determination. Additionally, a 10 and 20 ml aliquot was used for photopigment analysis (HPLC) and algal species analysis, respectively. All filtered chlorophylla samples were stored frozen at−20^◦C until analysis.

BIOMASS AND SPECIES ANALYSES

Chlorophylla was extracted with ethanol (Markeret al., 1980; Nusch, 1980), and concentrations were mea- sured spectrophotometrically; values were corrected for pheopigment content (Lorenzen, 1967). Organic and inorganic matter were determined by measuring the ash-

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Nematodes in periphyton communities of lakes

Fig. 1.Location of sampling sites in Sweden. Abbreviations of lakes: Erk, Erken; Exa, Exarbysjön; Gav, Gavel-Långsjön; Gim, Gimodamm; Hos, Hosjön; Lån, Långsjön; Lar, Largen; Lim, Limmaren; Öst, Österby-Stordamm; Söd, Söder-Giningen; Sto, Storsjön;

Täm, Tämnaren; Tom, Tomtasjön; Tre, Trehörningen; Ven, Vendelsjön; Vir1, Viren 1; Vir2, Viren 2. Map modiﬁed from Weinelt (2004).

free dry mass; filters were dried at 105^◦C for 24 h, followed by combustion at 550^◦C for 5 h. The remainder (45-100 ml) of each sample was sieved through a 30μm mesh net and fixed with formaldehyde (4% final concen- tration). All meiofaunal organisms were counted and classified into taxonomic groups (copepods, gastrotrichs, nematodes, oligochaetes, ostracods, rotifers and tardigrades) using a stereomicroscope (40×magnification) after stain- ing with Rose Bengal. When possible, 50 nematodes per replicate were isolated using a stereomicroscope, transferred to glycerol (Seinhorst, 1959, 1962), mounted on slides and identified to the species level. Nematodes were classified according to age (juvenile, adult), feeding type (following Traunspurger, 1997: deposit feeder, epistrate feeder, suction feeder and chewer) and sexual category (female, gravid female, male).

STATISTICAL ANALYSIS

To analyse diversity, the Shannon-Wiener species diversity index (H; log base 2) and Pielou’s evenness (J) were calculated. Differences in periphyton biomass, meiofaunal and nematode abundance, and species diversity be-

tween lakes were analysed by one-way analysis of vari- ance (ANOVA) with log-transformed data. Meiofauna and nematode abundance were correlated with periphyton biomass (chlorophylla, organic and inorganic matter) using the Spearman rank correlation coefﬁcient. Statistical tests were conducted using Statistica 6 (StatSoft Inc., Tulsa, OK, USA).

Differences between lakes were analysed using different multivariate parameters (meiofaunal groups (9), nematode species (58), and feeding types (4)) using cluster analysis (single linkage) and analysis of similarity (ANOSIM) based on Bray-Curtis similarity with untrans- formed data (PRIMER 5, Clarke & Warwick, 2001). Sim- ilarity between samples was measured on matrices with absolute species/group abundance cm⁻². Bray-Curtis coefficient S was used for computing similarity, with a coefficient value of 100% for completely similar samples and a value of 0% when two samples had no species in common. The Bray-Curtis coefficient has the advantage of not being influenced by joint absences of species in two samples, a circumstance under which many other coeffi- cients fail (Clarke & Green, 1988). The ANOSIM pro-

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cedure compares the ranked similarities for differences within and between groups. The resulting R-value usu- ally lies between 0 and 1, but can lie within a range of

−1 to+1. AnR-value of approximately 0 suggests an ac- ceptance of the null hypothesis, whereas largeR-values indicate separation of the groups and small values close to zero imply little or no separation (Clarke & Warwick, 2001).

Results

EPILITHIC BIOMASS

The amount of photoautotrophic, organic and inorganic material varied largely among the lakes (Table 2). The amount of algal biomass on stones measured as chlorophylla ranged from 1.2±0.3μg cm⁻²in Lake Gavel- Långsjön to 34.6±10.9μg cm⁻²in Lake Erken. Total organic matter measured as ash-free dry mass was lowest (0.7±0.2 mg cm⁻²) in Lake Gavel-Långsjön and highest (11.7±1.5 mg cm⁻²)in Lake Exarbysjön. The mean of inorganic matter ranged from 188.5±51.9 mg cm⁻² in Lake Storsjön to 1.5±0.9 mg cm⁻² in Lake Tom- tasjön. The proportion of organic matter relative to inorganic matter ranged from 3.9±0.6% to 68.5±12.8%.

Highly signiﬁcant differences in algal biomass (ANOVA:

F(16;73) = 8.76;P < 0.001), total organic matter (ash- free dry mass) (F(16;73)=3.40;P <0.001) and inorganic matter (F(16;73) =9.35;P <0.001) between lakes were found.

MEIOFAUNAL COMPOSTION

Nine meiofaunal groups were identiﬁed: nematodes, rotifers, harpacticoid, cyclopoid and calanoid copepods, ostracods, oligochaetes, tardigrades and gastrotrichs. Meio- faunal density reached an overall mean value of 114.5± 12.2 cm⁻² for all lakes. Lowest values were found in Lake Gavel-Långsjön (9.0±3.4 cm⁻²), and highest values were found in Lake Exarbysjön (258.2±112.0 cm⁻²).

Nematodes were the most abundant group in most lakes, with the exception of Lake Gavel-Långsjön (21.5±6.2%), Lake Vendelsjön (27.4±9.4%) and Lake Viren 2 (20.1± 8.1%). The mean nematode proportion was 53.9±2.4%

for all lakes, with a range from 20.1±8.0% in Lake Viren 2 to 77.4±4.9% in Lake Gimodamm. Rotifers were the second largest group, with a mean proportion of 26.8±1.7%, followed by crustaceans with 13.7±1.6%

and oligochaetes, tardigrades, gastrotrichs with 5.5±0.6%

(Fig. 2).

NEMATODE SPECIES COMPOSITION AND DIVERSITY Nematode abundance ranged from 2.3 ±1.4 cm⁻² in Lake Gavel-Långsjön to 161.5±70.7 cm⁻² in Lake Exarbysjön, with an overall mean across lakes (N =17) of 72.0±12.7 cm⁻²(Table 3).

In all 17 lakes, a total of 58 nematode species were identiﬁed; six species represented 66% of all nematodes (Table 4). The most common species wereRhab- dolaimus aquaticusrepresenting 30% of all nematodes;

Punctodora ratzeburgensis, 10%;Eumonhystera dispar, 8%;Crocodorylaimus ﬂavomaculatus, 7%;Aphelenchoi- des parietinus, 6% and Rhabdolaimus terrestris, 5%.

All other species comprised <5% of the total number of nematodes. Twenty-nine percent of all species were found in more than 50% of all lakes. Twenty-seven rare species were only found in one or two lakes (47% of all species); only 9% of all species were found in 14 or more lakes (Table 4). The most abundant species,R. aquaticus, was found in all 17 lakes, and E. dispar, E. ﬁliformis, E. pseudobulbosaandE. vulgariswere found in at least 14 lakes.

The number of species (S) varied greatly between lakes and resulted in signiﬁcant differences between lakes (ANOVA:F(16;71) = 3.36, P < 0.001). Species numbers were highest in Lake Tämnaren (34) and Lake Österby-Stordamm (33) (Table 3); lowest numbers were found in Lake Gavel-Långsjön (8) and Lake Viren 2 (11).

A comparison of lakes using the Shannon-Wiener diversity index (H) and Pielou’s evenness (J) also resulted in signiﬁcant differences (ANOVA: H:F(16;71) = 3.31, P < 0.001; J:F(16;70) = 2.32, P < 0.01). Shannon- Wiener diversity index values ranged from 3.07±0.15 in Lake Österby-Stordamm to 0.96±0.29 in Lake Gavel- Långsjön. Pielou’s evenness ranged from 0.68 in Lake Viren 1 to 0.93 in Lake Viren 2 (Table 3).

FEEDING TYPES AND AGE STRUCTURE

There was a clear dominance of deposit feeders, which comprised 71.2±4.4% of all nematodes (overall mean, n = 17), except in Lake Limmaren (25%) and Lake Trehörningen (46%) (Table 4; Fig. 3). Deposit feeders comprised from 25% of all nematodes in Lake Limmaren to 96% of all nematodes in Lake Exarbysjön. Suction feeders comprised 14% (range: 0-52%), epistrate feeders comprised 12% (0-70%) and chewers comprised 3% (0- 17%) of all nematodes. The age structure of nematodes was as follows (overall mean, n=17): juveniles, 54.8± 1.4% (range: 44-64%); females, 36.9±1.5% (19-48%);