**5. Integrating species and genetic diversity in one experiment: an alternative approach**

The preceding sections provided an overview of benthic experimental studies that alter either species or genotypic diversity separately. It has been argued that genetic diversity is only important in ecosystems that rely on or are dominated by one or a few key or habitat providing species, such as *Zostera marina* ecosystems (Hughes et al., 2008). However, all living organisms are hierarchically organized: genes make up genotypes, genotypes define populations and populations collectively constitute a species (Reusch & Hughes, 2006). If there is variation and heritability, as well as selection in ecologically important traits such as, e.g., growth rate or resistance to parasites, diversity at any level can have important ecological effects (Hughes et al., 2008). Moreover, it has been shown in terrestrial plant communities that genetic diversity can have direct consequences on species diversity: in a long-term experiment, genetically diverse communities reduced the rate at which species diversity declined (Booth & Grime, 2003), and genetic and species diversity maintained each

Integrating Different Organizational Levels in Benthic

Biodiversity – Ecosystem Functioning (BEF) Studies 103

bacterial community. They established closed microcosms with bacterivore nematode species that colonize decaying organic matter, revealing that intraguild species interactions depend on food availability (Dos Santos et al., 2009). They also worked with semi-closed microcosms of sandy beach sediments with more natural diversity levels (up to 24 species). Results showed that bacterivore nematodes did not have a more pronounced effect on the structure of the bacterial community than nematodes from other trophic guilds; however, nematode species richness (across trophic levels) did (Dos Santos, 2009). Moreover, processes such as carbon production increased with species richness, and the most speciesrich nematode assemblage had a positive effect on bacterial diversity (Dos Santos, 2009).

Fig. 1. The experimental system. a) The experimental unit. Microcosms can be setup in a variety of containers; for the illustration we chose a relatively small glass or plastic jar with a thin, water-saturated layer of sediment, covered by a plastic film or lid to avoid desiccation. Experimental microcosms contain increasing levels of nematode taxonomic and/or genetic diversity b) A schematic view of the benthic food web contained in the sediment of the microcosm. Predatory nematodes prey on herbivorous and bacterivore nematodes, which, in turn, graze on diatoms and prokaryotes respectively. In different experimental units, nematode genetic and species diversity are modified. The effects of the different diversity levels on the functioning of the food web are measured in terms of the production of

To address the BEF subject at different organizational levels simultaneously (i.e., species *and* genetic diversity) across different trophic guilds (i.e., grazers, bacterivores and predators), we follow the microcosm approach as applied in the study by Hubas et al. (2010). We suggest assessing components affecting directly the physical integrity of the habitat as a proxy of the functionality of our microcosms, i.e. the production of EPS and its effect on sediment stability (Figure 1b). However, other functions, such as community respiration, prokaryote enzymatic activities or the decomposition of a specific substrate, may be assessed in the same setup. Microbial EPS are increasingly recognized as a major stabilising factor. They are an ubiquitous component of aquatic ecosystems composed

extracellular polymeric substances (EPS) and sediment stability.

other through mutual feedbacks (Lankau & Strauss, 2007). It is therefore fundamental to start to include changes in genetic (population) and species diversity in studies assessing the consequences of biodiversity decline.

Today, *Zostera marina* vegetations probably provide the best systems to investigate species and genetic diversity in concert (Reusch & Hughes, 2006). To our knowledge, it is also the only system where genotypic (seagrass) and species (grazer) diversity have been manipulated simultaneously (Hughes et al., 2010). One big advantage of this system is that seagrasses can reproduce clonally, which means that multiple shoots of one genotype can be handled as one unit when changing genetic (genotypic) richness just like a species is handled as one unit when changing species richness. This makes it possible to alter species and genotypic richness in one experiment. On the other hand, it is a method restricted to clonally reproducing organisms and cannot be applied to sexually reproducing animals. Another major advantage may be that seagrasses consist of relatively few but functionally diverse species. This is very valuable for the manipulation of the experimental units. However, the results cannot be extrapolated to systems with higher species richness, and questions concerning intraguild diversity may be addressed only to a limited extent. Besides, one major disadvantage of the seagrass system is that a lot of space is needed to set up outdoor (or indoor) mesocosms. Moreover, *Zostera marina* has to be available in order to establish the artificial plantings. Yet, its distribution is limited to the northern hemisphere, mainly along European and North American coasts.

As an alternative approach to test effects of species and genetic diversity on ecosystem functioning, we present a setup allowing experimental manipulation of multitrophic assemblages in very small and easy-to-replicate laboratory microcosms (Figure 1a). These comprise small amounts of sediment containing a benthic microbial food web of diatoms (i.e. primary producers), prokaryotes (i.e. decomposers) and nematodes and/or harpacticoid copepods (i.e. grazers of prokaryotes and of diatoms, as well as predators of other nematodes and copepods) (Figure 1b). These organisms dominate all soft sedimentary habitats such as beaches and mudflats around the world. They typically attain high local diversity, species numbers in 10 ml of sediment typically ranging in the order of hundreds to thousands, and several tens for prokaryotes, diatoms and nematodes, respectively. They are highly relevant to basic benthic ecosystem processes such as the decomposition of organic matter and the mineralization of nutrients. They respond more rapidly to environmental changes than most macrofauna (Bolam et al., 2002).

Former experiments with similar setups have proven to be very useful to study benthic processes. De Mesel et al. (2003, 2004, 2006) studied the impact of bacterivore nematodes on the bacterial community and the decomposition of cordgrass leaves. They found that four different bacterivore nematode species have a significant top-down effect on the structure of the bacterial community (De Mesel et al., 2004). However, species richness within the guild of bacterivore nematodes did not enhance decomposition rates, rather the process depended on species identity and on unexpected inhibitory and facilitative interactions among species within the same guild (De Mesel et al., 2003, 2006). Hubas et al. (2010) investigated the influence of microbial organisms, i.e., bacteria, diatoms and bacterivore nematodes, on the production of extracellular polymeric substances (EPS), a major sediment-stabilizing product (Stal, 2010). The results evidenced that the presence of bacterivore nematodes had a positive impact on microbial abundance and EPS production, which was highest at the highest community complexity, i.e., involving nematodes, diatoms and bacteria (Hubas et al., 2010). Dos Santos (2009) studied bottom-up and top-down controls of nematodes on the

other through mutual feedbacks (Lankau & Strauss, 2007). It is therefore fundamental to start to include changes in genetic (population) and species diversity in studies assessing the

Today, *Zostera marina* vegetations probably provide the best systems to investigate species and genetic diversity in concert (Reusch & Hughes, 2006). To our knowledge, it is also the only system where genotypic (seagrass) and species (grazer) diversity have been manipulated simultaneously (Hughes et al., 2010). One big advantage of this system is that seagrasses can reproduce clonally, which means that multiple shoots of one genotype can be handled as one unit when changing genetic (genotypic) richness just like a species is handled as one unit when changing species richness. This makes it possible to alter species and genotypic richness in one experiment. On the other hand, it is a method restricted to clonally reproducing organisms and cannot be applied to sexually reproducing animals. Another major advantage may be that seagrasses consist of relatively few but functionally diverse species. This is very valuable for the manipulation of the experimental units. However, the results cannot be extrapolated to systems with higher species richness, and questions concerning intraguild diversity may be addressed only to a limited extent. Besides, one major disadvantage of the seagrass system is that a lot of space is needed to set up outdoor (or indoor) mesocosms. Moreover, *Zostera marina* has to be available in order to establish the artificial plantings. Yet, its distribution is limited to the northern hemisphere,

As an alternative approach to test effects of species and genetic diversity on ecosystem functioning, we present a setup allowing experimental manipulation of multitrophic assemblages in very small and easy-to-replicate laboratory microcosms (Figure 1a). These comprise small amounts of sediment containing a benthic microbial food web of diatoms (i.e. primary producers), prokaryotes (i.e. decomposers) and nematodes and/or harpacticoid copepods (i.e. grazers of prokaryotes and of diatoms, as well as predators of other nematodes and copepods) (Figure 1b). These organisms dominate all soft sedimentary habitats such as beaches and mudflats around the world. They typically attain high local diversity, species numbers in 10 ml of sediment typically ranging in the order of hundreds to thousands, and several tens for prokaryotes, diatoms and nematodes, respectively. They are highly relevant to basic benthic ecosystem processes such as the decomposition of organic matter and the mineralization of nutrients. They respond more rapidly to

Former experiments with similar setups have proven to be very useful to study benthic processes. De Mesel et al. (2003, 2004, 2006) studied the impact of bacterivore nematodes on the bacterial community and the decomposition of cordgrass leaves. They found that four different bacterivore nematode species have a significant top-down effect on the structure of the bacterial community (De Mesel et al., 2004). However, species richness within the guild of bacterivore nematodes did not enhance decomposition rates, rather the process depended on species identity and on unexpected inhibitory and facilitative interactions among species within the same guild (De Mesel et al., 2003, 2006). Hubas et al. (2010) investigated the influence of microbial organisms, i.e., bacteria, diatoms and bacterivore nematodes, on the production of extracellular polymeric substances (EPS), a major sediment-stabilizing product (Stal, 2010). The results evidenced that the presence of bacterivore nematodes had a positive impact on microbial abundance and EPS production, which was highest at the highest community complexity, i.e., involving nematodes, diatoms and bacteria (Hubas et al., 2010). Dos Santos (2009) studied bottom-up and top-down controls of nematodes on the

consequences of biodiversity decline.

mainly along European and North American coasts.

environmental changes than most macrofauna (Bolam et al., 2002).

bacterial community. They established closed microcosms with bacterivore nematode species that colonize decaying organic matter, revealing that intraguild species interactions depend on food availability (Dos Santos et al., 2009). They also worked with semi-closed microcosms of sandy beach sediments with more natural diversity levels (up to 24 species). Results showed that bacterivore nematodes did not have a more pronounced effect on the structure of the bacterial community than nematodes from other trophic guilds; however, nematode species richness (across trophic levels) did (Dos Santos, 2009). Moreover, processes such as carbon production increased with species richness, and the most speciesrich nematode assemblage had a positive effect on bacterial diversity (Dos Santos, 2009).

Fig. 1. The experimental system. a) The experimental unit. Microcosms can be setup in a variety of containers; for the illustration we chose a relatively small glass or plastic jar with a thin, water-saturated layer of sediment, covered by a plastic film or lid to avoid desiccation. Experimental microcosms contain increasing levels of nematode taxonomic and/or genetic diversity b) A schematic view of the benthic food web contained in the sediment of the microcosm. Predatory nematodes prey on herbivorous and bacterivore nematodes, which, in turn, graze on diatoms and prokaryotes respectively. In different experimental units, nematode genetic and species diversity are modified. The effects of the different diversity levels on the functioning of the food web are measured in terms of the production of extracellular polymeric substances (EPS) and sediment stability.

To address the BEF subject at different organizational levels simultaneously (i.e., species *and* genetic diversity) across different trophic guilds (i.e., grazers, bacterivores and predators), we follow the microcosm approach as applied in the study by Hubas et al. (2010). We suggest assessing components affecting directly the physical integrity of the habitat as a proxy of the functionality of our microcosms, i.e. the production of EPS and its effect on sediment stability (Figure 1b). However, other functions, such as community respiration, prokaryote enzymatic activities or the decomposition of a specific substrate, may be assessed in the same setup. Microbial EPS are increasingly recognized as a major stabilising factor. They are an ubiquitous component of aquatic ecosystems composed

Integrating Different Organizational Levels in Benthic

Biodiversity – Ecosystem Functioning (BEF) Studies 105

for over 15 years now (T. Moens, unpublished data). Therefore, the premise is that cultured nematodes have low genetic diversity due to inbreeding, unlike natural populations, which are expected to exhibit higher genetic diversity levels. This approach implies that the level of genetic difference has to be assessed before the experiment, which in view of the substantial cryptic diversity (e.g., Derycke et al., 2005) is not necessarily evident. Alternatively, contrasting genetic diversity levels can be obtained experimentally with cultures of selected nematode species. Cultures of species of the *Rhabditis marina* species complex, for instance, can be raised from single gravid females, yielding highly inbred lineages, which can be maintained easily for many generations. It is therefore possible to raise a culture collection of many inbred lineages and to establish experimental populations of very low to high genetic diversity. The advantage of this approach is that similar to the study by Gamfeldt & Kallstrom (2007), "families" can be obtained, and the level of genetic diversity (i.e. the

The main ecosystem function we concentrate on in this approach is measured in terms of EPS production (concentration and composition), and the stability of the sediments. To assess EPS concentration and composition, sediment samples are mixed with distilled water. Exopolymers are very diverse and complex molecules that are found in a wide variety of forms and structures in aquatic ecosystems. The selection of the protocol for their extraction, purification and subsequently for the measurement of their concentration in any sample will depend ultimately on the type of exopolymer studied (e.g., Panagiatopoulos & Sempéré, 2005; Danovaro, 2010). Exopolymers can be found dissolved in the water column or as gels in biofilms and aggregates so that their extraction and quantification is a primary step in assessing their potential availability (Decho, 1990). A description and evaluation of different methods and protocols is well beyond the scope of this paper, hence we will only briefly mention methods that are commonly used in the literature and which offer an acceptable

The water-extractable fraction (i.e. dissolved or colloidal EPS which are already present in pore-water and unbound to sediment grains) can be analyzed for carbohydrates and proteins following the phenolsulphuric acid protocol (DuBois et al., 1956) and the modified Lowry procedure (Raunkjaer et al., 1994). Carbohydrate and protein concentrations can be measured with a UV/VIS spectrophotometer and their concentrations are deduced from

Sediment stability can be estimated through the measurement of the erosion shear stress of the biofilm. Typically, it is possible to measure this using flumes (Jonsson et al., 2006) or *in situ* devices such as the Cohesive Strength Meter (CSM). This device has been used extensively in marine ecosystems since the pioneering work of Paterson (1989) and is an efficient way to estimate sediment erodibility. Mechanical properties of the biofilms in terms of surface adhesion have been developed relatively recently. The method is based on the magnetic attraction of magnetic particles (Magnetic Particle Induction - MagPI; Larson et al., 2009) and is both relatively easy to set up and inexpensive. Specifically, a known volume of ferromagnetic fluorescent particles is spread onto a defined area of the sediment surface. The particles are then recaptured by an overlying electromagnet and the force (magnetic flux) needed to retrieve the particles is determined as a measure of the retentive capacity of the substratum, a proxy for adhesion. This method is suitable for sensitive recording of

calibration curves assessed from glucose and bovine serum albumin, respectively.

"number of families") for each experimental group can be determined *a priori*.

**5.1.2 Measuring EPS production and sediment stability** 

ratio of information obtained to amount of time invested.

changes of the surface adhesion of sediments or biofilms.

primarily of carbohydrates and proteins. They have multiple roles and functions: attachment to substrata, flotation and locomotion, feeding, protection against environmental factors such as desiccation, UV radiation and pollution, as well as the development of biofilms (Decho, 1990). Perhaps the most striking feature of these compounds is their ability to bind sediment grains together, enhancing their cohesive strength and thereby increasing resistance to erosion. Particularly diatoms are the major ecosystem engineers of this sediment bio-stabilisation (Stal, 2010), and prokaryotes are increasingly recognised as playing a key role in enhancing sediment surface adhesion and cohesive strength (Gerbersdorf et al., 2008, 2009; Lubarsky et al., 2010). The advantage of this alternative approach is that it offers the possibility of testing both effects of species and genetic diversity on ecosystem functioning.

#### **5.1 Methodology**

#### **5.1.1 Microcosm setup**

To setup the microcosm, a variety of small containers can be suitable, depending on the exact aim of the study. First, a specified amount of non-cohesive sedimentary substrate needs to be placed on the bottom. For example, acid-washed marine sand (Hubas et al., 2010) or non-cohesive glass beads (Lubarsky et al., 2010) can be used. The sediment needs to be fully hydrated by sterile seawater of a specific salinity (which depends on the organisms). Whether or not the seawater needs to cover the sediment depends on the specific aims of the study. Prokaryote and diatom cultures can be established by sampling sediment surface (0 – 5 mm) from natural tidal flats, and re-suspending the sediment slurry in culture media. Alternatively, or additionally, prokaryote and diatom strains can be obtained from established culture collections. Microbial assemblages should then be incubated for several weeks (e.g., Ribalet et al., 2008). The resulting assemblages can then be used for the experiment. An appropriate culture medium, which favours algal rather than bacterial growth, should be applied. F/2 medium is, for instance, widely used for growing marine algae, especially diatoms (Guillard, 1975). If the use of antibiotics cannot be avoided, a proper test of the effect of each antibiotic on the natural assemblage culture should be conducted.

Finally, nematodes can be placed into the microcosms, according to the experimental groups. There are two different approaches in order to obtain different taxonomic (i.e. species richness) diversity levels across trophic groups of nematodes: either from the field or from cultures. For the former, nematode assemblages can be sampled along diversity gradients from, e.g., an intertidal sandy beach (Gingold et al., 2010) or from different beaches (Dos Santos, 2009). They are extracted live through decantation over a 32µm sieve to obtain stock suspensions of different diversity levels. For the latter, different nematode species can be raised under artificial conditions (Moens & Vincx, 1998). Unfortunately, only a limited number of marine nematode species have hitherto been maintained in permanent monospecific cultures, and most of these belong to the same feeding guild, i.e. bacterivores. However, a majority of species from intertidal sediments can be easily extracted from freshly collected sediment and remain active in a variety of laboratory incubation conditions for several weeks (Moens & Vincx, 1998).

In order to establish microcosms containing nematodes of different genetic diversity, lab cultures and field-collected specimens of the same bacteria-feeding species from which permanent cultures exist can be used. Bacteria feeding nematode species such as *Rhabditis marina, Diplolaimelloides meyli, D. oschei,* and *Diplolaimella dievengatensis* have been cultured

primarily of carbohydrates and proteins. They have multiple roles and functions: attachment to substrata, flotation and locomotion, feeding, protection against environmental factors such as desiccation, UV radiation and pollution, as well as the development of biofilms (Decho, 1990). Perhaps the most striking feature of these compounds is their ability to bind sediment grains together, enhancing their cohesive strength and thereby increasing resistance to erosion. Particularly diatoms are the major ecosystem engineers of this sediment bio-stabilisation (Stal, 2010), and prokaryotes are increasingly recognised as playing a key role in enhancing sediment surface adhesion and cohesive strength (Gerbersdorf et al., 2008, 2009; Lubarsky et al., 2010). The advantage of this alternative approach is that it offers the possibility of testing both effects of species

To setup the microcosm, a variety of small containers can be suitable, depending on the exact aim of the study. First, a specified amount of non-cohesive sedimentary substrate needs to be placed on the bottom. For example, acid-washed marine sand (Hubas et al., 2010) or non-cohesive glass beads (Lubarsky et al., 2010) can be used. The sediment needs to be fully hydrated by sterile seawater of a specific salinity (which depends on the organisms). Whether or not the seawater needs to cover the sediment depends on the specific aims of the study. Prokaryote and diatom cultures can be established by sampling sediment surface (0 – 5 mm) from natural tidal flats, and re-suspending the sediment slurry in culture media. Alternatively, or additionally, prokaryote and diatom strains can be obtained from established culture collections. Microbial assemblages should then be incubated for several weeks (e.g., Ribalet et al., 2008). The resulting assemblages can then be used for the experiment. An appropriate culture medium, which favours algal rather than bacterial growth, should be applied. F/2 medium is, for instance, widely used for growing marine algae, especially diatoms (Guillard, 1975). If the use of antibiotics cannot be avoided, a proper test of the effect of each antibiotic on the natural assemblage culture

Finally, nematodes can be placed into the microcosms, according to the experimental groups. There are two different approaches in order to obtain different taxonomic (i.e. species richness) diversity levels across trophic groups of nematodes: either from the field or from cultures. For the former, nematode assemblages can be sampled along diversity gradients from, e.g., an intertidal sandy beach (Gingold et al., 2010) or from different beaches (Dos Santos, 2009). They are extracted live through decantation over a 32µm sieve to obtain stock suspensions of different diversity levels. For the latter, different nematode species can be raised under artificial conditions (Moens & Vincx, 1998). Unfortunately, only a limited number of marine nematode species have hitherto been maintained in permanent monospecific cultures, and most of these belong to the same feeding guild, i.e. bacterivores. However, a majority of species from intertidal sediments can be easily extracted from freshly collected sediment and remain active in a variety of laboratory incubation conditions

In order to establish microcosms containing nematodes of different genetic diversity, lab cultures and field-collected specimens of the same bacteria-feeding species from which permanent cultures exist can be used. Bacteria feeding nematode species such as *Rhabditis marina, Diplolaimelloides meyli, D. oschei,* and *Diplolaimella dievengatensis* have been cultured

and genetic diversity on ecosystem functioning.

**5.1 Methodology 5.1.1 Microcosm setup** 

should be conducted.

for several weeks (Moens & Vincx, 1998).

for over 15 years now (T. Moens, unpublished data). Therefore, the premise is that cultured nematodes have low genetic diversity due to inbreeding, unlike natural populations, which are expected to exhibit higher genetic diversity levels. This approach implies that the level of genetic difference has to be assessed before the experiment, which in view of the substantial cryptic diversity (e.g., Derycke et al., 2005) is not necessarily evident. Alternatively, contrasting genetic diversity levels can be obtained experimentally with cultures of selected nematode species. Cultures of species of the *Rhabditis marina* species complex, for instance, can be raised from single gravid females, yielding highly inbred lineages, which can be maintained easily for many generations. It is therefore possible to raise a culture collection of many inbred lineages and to establish experimental populations of very low to high genetic diversity. The advantage of this approach is that similar to the study by Gamfeldt & Kallstrom (2007), "families" can be obtained, and the level of genetic diversity (i.e. the "number of families") for each experimental group can be determined *a priori*.

#### **5.1.2 Measuring EPS production and sediment stability**

The main ecosystem function we concentrate on in this approach is measured in terms of EPS production (concentration and composition), and the stability of the sediments. To assess EPS concentration and composition, sediment samples are mixed with distilled water. Exopolymers are very diverse and complex molecules that are found in a wide variety of forms and structures in aquatic ecosystems. The selection of the protocol for their extraction, purification and subsequently for the measurement of their concentration in any sample will depend ultimately on the type of exopolymer studied (e.g., Panagiatopoulos & Sempéré, 2005; Danovaro, 2010). Exopolymers can be found dissolved in the water column or as gels in biofilms and aggregates so that their extraction and quantification is a primary step in assessing their potential availability (Decho, 1990). A description and evaluation of different methods and protocols is well beyond the scope of this paper, hence we will only briefly mention methods that are commonly used in the literature and which offer an acceptable ratio of information obtained to amount of time invested.

The water-extractable fraction (i.e. dissolved or colloidal EPS which are already present in pore-water and unbound to sediment grains) can be analyzed for carbohydrates and proteins following the phenolsulphuric acid protocol (DuBois et al., 1956) and the modified Lowry procedure (Raunkjaer et al., 1994). Carbohydrate and protein concentrations can be measured with a UV/VIS spectrophotometer and their concentrations are deduced from calibration curves assessed from glucose and bovine serum albumin, respectively.

Sediment stability can be estimated through the measurement of the erosion shear stress of the biofilm. Typically, it is possible to measure this using flumes (Jonsson et al., 2006) or *in situ* devices such as the Cohesive Strength Meter (CSM). This device has been used extensively in marine ecosystems since the pioneering work of Paterson (1989) and is an efficient way to estimate sediment erodibility. Mechanical properties of the biofilms in terms of surface adhesion have been developed relatively recently. The method is based on the magnetic attraction of magnetic particles (Magnetic Particle Induction - MagPI; Larson et al., 2009) and is both relatively easy to set up and inexpensive. Specifically, a known volume of ferromagnetic fluorescent particles is spread onto a defined area of the sediment surface. The particles are then recaptured by an overlying electromagnet and the force (magnetic flux) needed to retrieve the particles is determined as a measure of the retentive capacity of the substratum, a proxy for adhesion. This method is suitable for sensitive recording of changes of the surface adhesion of sediments or biofilms.

Integrating Different Organizational Levels in Benthic

temperature of the environment.

**6. Conclusions** 

Biodiversity – Ecosystem Functioning (BEF) Studies 107

experiment would not reflect absolute temporal scales of climate change, it may well reflect and therefore be representative for the rates of temperature change long-lived organisms might experience (Petchey et al., 1999). Knowing that genetic diversity enhanced resistance of seagrass *Zostera marina* to climate extremes (Reusch et al., 2005), it would be particularly interesting to plan hypotheses involving genetic diversity of nematodes and increased

A further advantage of our system is that it includes different trophic levels, from decomposers (prokaryotes) and producers (diatoms) to grazers (bacterivorous and herbivorous nematodes) and their predators (predatory nematodes), some of the latter being omnivorous rather than strictly predacious (Moens et al., 2004). Moreover, for some setups, we can integrate species of a natural community and not only the dominant species of a given habitat. This is important because even weak interactions can have important stabilizing effects on communities (Berlow, 1999). Contrary to the seagrass system, which alters diversity at the primary-producer level, we can assess the effect of species and genetic diversity also at the consumer level. This is important, as it has been suggested that the stronger top-down control in the sea relative to terrestrial habitats (Shurin et al., 2002) implies that in marine systems ecosystem functions such as primary production may be influenced more by herbivores and predators than by plant diversity (Duffy, 2003; Paine, 2002). In order to track and quantify trophic pathways, pulse-chase experiments which label a particular component and trace transfer to consumers can be designed (Middelburg et al., 2000; Moens et al., 2002; Van Oevelen et al., 2006). Such a characterization and quantification of the relationships among the different components of the community would be an additional improvement, since experimental research on multi-level food webs should not only assess the consequences of species richness and identity on ecosystem processes, but also evaluate trophic cascades and the distribution of interaction strengths within natural

communities and how they change with community composition (Duffy, 2002).

after which treatment effects are likely to become confounded by bottling effects.

However, there are probably three major disadvantages that have to be taken in account when planning the experiments: first, only a limited number of species of nematodes can be cultured with the present methods. Second, the identification of nematodes and diatoms needs considerable expertise, especially when working with field samples containing many different species. And third, the design essentially relies on closed microcosms, hence excluding the role of immigration and emigration. Moreover, by bottling aqueous sediments, a system is created which accumulates end products of decomposition and depletes oxygen. As a consequence, the microcosms can only be used for a limited duration,

Our experimental system provides a good alternative to investigate effects of species and genetic diversity in concert. It is easy to setup and contains organisms that are easily accessible. Moreover, it provides the possibility to be extended and address current challenges of BEF experiments. One of these challenges consists in that future BEF studies must go beyond experiments relating some selected species, singly and in combination, to some ecosystem process. Rather, they must address environmental heterogeneity in space or time, which can be captured in long-term studies on one hand, and working with natural communities on the other. Our experimental units as described here are closed systems, however, they can possibly be converted to semi-closed systems and be setup outdoors. Like

#### **5.1.3 Assessing community composition**

We suggest assessing nematode, prokaryote, and diatom community diversity and composition at a genetic and taxonomic level. Most studies identify nematode species morphologically, nematodes first being mounted on permanent slides (Platt & Warwick, 1988). Species are identified with a microscope, using pictorial (Platt & Warwick 1983, 1988; Warwick et al., 1998) taxonomic keys and primary literature on species descriptions, which is becoming increasingly available in online public databases such as NeMys (Deprez et al., 2005). Morphology based identification of diatoms is achieved by fixing the sampling cores in 4% glutaraldehyde and then embedding in Naphrax after chemically removing organic matter using potassium permanganate. The species composition of the microalgal community is assessed by light microscopy using identification keys (Krammer & Lange-Bertalot, 1986-1991; Lange-Bertalot, 1997; Pankow, 1990; Simonsen, 1962; Underwood et al., 1998; Witkowski et al., 2000). However, species diversity can be assessed using a metagenetic approach as well. The three sets of taxa (nematodes, prokaryotes and diatoms) may be pyrosequenced after distinct sampling procedures. Nematodes are extracted from sediment by sieving (45 µm mesh) and floatation techniques and genotyped using both small and large rDNA subunits (Creer et al., 2010; Porazinska et al., 2009, 2010) and/or mitochondrial genes such as part of the cytochrome oxidase subunit I gene (Derycke et al., 2010). Diatoms are collected on a filter before DNA extraction and amplification using the small rDNA subunit (Cuvelier et al., 2010; Medinger et al., 2010; Quaiser et al., 2010). Prokaryotes are assessed from direct DNA extractions of sediment samples and genotyped using 16S rDNA (Coolon et al., 2010; Xu, 2006). When studying well-documented communities, where sequence data is available for a majority of species, such an approach can yield information on species composition, relative abundances and diversity. In the absence of such a reference database (currently unavailable for any marine assemblage, but this will undoubtedly change in the near future), it can still provide an estimate of diversity in terms of both species richness and evenness. Metagenetic analyses yield information on the different genotypes present, and thus allow simultaneous assessment of species as well as intraspecific diversity, pending proper knowledge on barcoding gaps in the taxa of interest.

#### **5.1.4 Advantages/disadvantages of our system**

As a model system for BEF research, our setup combines many advantages. One major advantage from the practical point of view may be that it can be manipulated in small containers and yet contain diversity levels representative of natural communities. The microcosms can easily be incubated under controlled conditions and sufficiently replicated. Nematodes, diatoms and prokaryotes are key players in almost any coastal or stream soft sediment, performing similar essential ecosystem functions across a range of habitats. Therefore they are easily collectable, either at a sandy beach or tidal flat at seashore or from a stream or lake when working with freshwater organisms. A whole experimental setup fits in any average-sized laboratory. If enough space is available, it can even be expanded to bigger mesocosm units including macrofauna as an additional trophic and functional level, which is likely to impact on nematodes, diatoms and prokaryotes through trophic as well as physical interactions (Austen & Widdicombe, 1998; Braeckman et al., 2011). Other extensions are also possible, for example a temperature treatment can be applied to simulate temperature rise as a consequence of climate change. Although the duration of such a short

We suggest assessing nematode, prokaryote, and diatom community diversity and composition at a genetic and taxonomic level. Most studies identify nematode species morphologically, nematodes first being mounted on permanent slides (Platt & Warwick, 1988). Species are identified with a microscope, using pictorial (Platt & Warwick 1983, 1988; Warwick et al., 1998) taxonomic keys and primary literature on species descriptions, which is becoming increasingly available in online public databases such as NeMys (Deprez et al., 2005). Morphology based identification of diatoms is achieved by fixing the sampling cores in 4% glutaraldehyde and then embedding in Naphrax after chemically removing organic matter using potassium permanganate. The species composition of the microalgal community is assessed by light microscopy using identification keys (Krammer & Lange-Bertalot, 1986-1991; Lange-Bertalot, 1997; Pankow, 1990; Simonsen, 1962; Underwood et al., 1998; Witkowski et al., 2000). However, species diversity can be assessed using a metagenetic approach as well. The three sets of taxa (nematodes, prokaryotes and diatoms) may be pyrosequenced after distinct sampling procedures. Nematodes are extracted from sediment by sieving (45 µm mesh) and floatation techniques and genotyped using both small and large rDNA subunits (Creer et al., 2010; Porazinska et al., 2009, 2010) and/or mitochondrial genes such as part of the cytochrome oxidase subunit I gene (Derycke et al., 2010). Diatoms are collected on a filter before DNA extraction and amplification using the small rDNA subunit (Cuvelier et al., 2010; Medinger et al., 2010; Quaiser et al., 2010). Prokaryotes are assessed from direct DNA extractions of sediment samples and genotyped using 16S rDNA (Coolon et al., 2010; Xu, 2006). When studying well-documented communities, where sequence data is available for a majority of species, such an approach can yield information on species composition, relative abundances and diversity. In the absence of such a reference database (currently unavailable for any marine assemblage, but this will undoubtedly change in the near future), it can still provide an estimate of diversity in terms of both species richness and evenness. Metagenetic analyses yield information on the different genotypes present, and thus allow simultaneous assessment of species as well as intraspecific diversity, pending proper

As a model system for BEF research, our setup combines many advantages. One major advantage from the practical point of view may be that it can be manipulated in small containers and yet contain diversity levels representative of natural communities. The microcosms can easily be incubated under controlled conditions and sufficiently replicated. Nematodes, diatoms and prokaryotes are key players in almost any coastal or stream soft sediment, performing similar essential ecosystem functions across a range of habitats. Therefore they are easily collectable, either at a sandy beach or tidal flat at seashore or from a stream or lake when working with freshwater organisms. A whole experimental setup fits in any average-sized laboratory. If enough space is available, it can even be expanded to bigger mesocosm units including macrofauna as an additional trophic and functional level, which is likely to impact on nematodes, diatoms and prokaryotes through trophic as well as physical interactions (Austen & Widdicombe, 1998; Braeckman et al., 2011). Other extensions are also possible, for example a temperature treatment can be applied to simulate temperature rise as a consequence of climate change. Although the duration of such a short

**5.1.3 Assessing community composition** 

knowledge on barcoding gaps in the taxa of interest.

**5.1.4 Advantages/disadvantages of our system** 

experiment would not reflect absolute temporal scales of climate change, it may well reflect and therefore be representative for the rates of temperature change long-lived organisms might experience (Petchey et al., 1999). Knowing that genetic diversity enhanced resistance of seagrass *Zostera marina* to climate extremes (Reusch et al., 2005), it would be particularly interesting to plan hypotheses involving genetic diversity of nematodes and increased temperature of the environment.

A further advantage of our system is that it includes different trophic levels, from decomposers (prokaryotes) and producers (diatoms) to grazers (bacterivorous and herbivorous nematodes) and their predators (predatory nematodes), some of the latter being omnivorous rather than strictly predacious (Moens et al., 2004). Moreover, for some setups, we can integrate species of a natural community and not only the dominant species of a given habitat. This is important because even weak interactions can have important stabilizing effects on communities (Berlow, 1999). Contrary to the seagrass system, which alters diversity at the primary-producer level, we can assess the effect of species and genetic diversity also at the consumer level. This is important, as it has been suggested that the stronger top-down control in the sea relative to terrestrial habitats (Shurin et al., 2002) implies that in marine systems ecosystem functions such as primary production may be influenced more by herbivores and predators than by plant diversity (Duffy, 2003; Paine, 2002). In order to track and quantify trophic pathways, pulse-chase experiments which label a particular component and trace transfer to consumers can be designed (Middelburg et al., 2000; Moens et al., 2002; Van Oevelen et al., 2006). Such a characterization and quantification of the relationships among the different components of the community would be an additional improvement, since experimental research on multi-level food webs should not only assess the consequences of species richness and identity on ecosystem processes, but also evaluate trophic cascades and the distribution of interaction strengths within natural communities and how they change with community composition (Duffy, 2002).

However, there are probably three major disadvantages that have to be taken in account when planning the experiments: first, only a limited number of species of nematodes can be cultured with the present methods. Second, the identification of nematodes and diatoms needs considerable expertise, especially when working with field samples containing many different species. And third, the design essentially relies on closed microcosms, hence excluding the role of immigration and emigration. Moreover, by bottling aqueous sediments, a system is created which accumulates end products of decomposition and depletes oxygen. As a consequence, the microcosms can only be used for a limited duration, after which treatment effects are likely to become confounded by bottling effects.
