**Integrating Different Organizational Levels in Benthic Biodiversity – Ecosystem Functioning (BEF) Studies**

Ruth Gingold1,2,3, Axayácatl Rocha Olivares1, Tom Moens2 and Cédric Hubas3 *1Department of Biological Oceanography, Centro de Investigación Científica y de Educación Superior de Ensenada, Carretera Ensenada-Tijuana 3918, Apdo. Postal 2732, 22860 Ensenada BC 2Ghent University, Department of Biology, Marine Biology Section, Krijgslaan 281/S8, 9000 Gent 3Muséum National d'Histoire Naturelle, UMR BOREA 7208, MNHN/CNRS/IRD/UPMC, CP 53, 61 rue Buffon, 75231 Paris Cedex 5 1Mexico 2Belgium 3France* 

#### **1. Introduction**

90 Ecosystems Biodiversity

Phelps, W. H. & Phelps, W. H. J. (1950a). Lista de las aves de Venezuela con su distribución,

Phelps, W. H. & Phelps, W. H. J. (1950b). Seven new subspecies of Venezuelan birds, *Proceedings of the Biological Society of Washington*, Vol.63, pp. 115-126, ISSN 0006-324X Ponte, V. (1990). *Recurso trófico utilizado por peces juveniles en dos áreas del delta interior del río* 

Ponte, V. (1995). Contributions of the Warao Indians to the ichtyology of the Orinoco Delta,

Ponte, V. & Lasso, C. (1994). Ictiofauna del caño Winikina, Delta del Orinoco. Aspectos de la

Ponte, V., Machado-Allison, A. & Lasso, C. (1999). La ictiofauna del delta del río Orinoco,

Possingham, H. P., Ball, I. R. & Andelman, S. (2000). Mathematical methods for identifying

Ramos, F., Novoa, D. & Itriago, I. (1982). Resultados de los programas de pesca exploratoria

Rodríguez, J. P. & Rojas-Suárez, F. (Eds.) (2008). *Libro Rojo de la Fauna Venezolana*, 3rd

Schneider, P., Achury, A., Guaiquirián, J. & Cárdenas, J. (2007). Análisis de la estructura y

Vera, B. (1992). Sea grasses of the Venezuelan coast: distribution and community

Zimmer, J. T. & Phelps, W. H. (1950). Three new Venezuelan birds, *American Museum* 

edition, Provita and Shell, Caracas, Venezuela, ISBN 980-210-011-0

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*Orinoco,* Undergraduate thesis, Escuela de Biología, Facultad de Ciencias,

In: *Nature and human ecology in the Neotropics,* H. Dieter Heinen, J. San José & H. Caballero Arias (Eds.), 371-392, Scientia Guaianae, ISSN 0798-1120, Caracas,

ecología de las especies y comunidades asociadas a diferentes hábitats, *Proceedings of the Segundo Congreso Venezolano de Ecología*, Guanare, Venezuela, February 20-26, 1994 Ponte, V. & Mochcco, O. (1997). Evaluación de las actividades pesqueras de la etnia Warao

en el delta del río Orinoco, Venezuela, *Acta Biológica Venezuelica,* Vol.17, No.1, pp.

Venezuela: una aproximación a su diversidad, *Acta Biológica Venezuelica,* Vol.19,

representative reserve networks, In: *Quantitative methods for conservation biology,* S. Ferson & M. Burgman (Eds.), 291-305, Springer-Verlag, ISBN 978-0-387-94322-0,

realizados en el Delta del Orinoco, In: *Los recursos pesqueros del río Orinoco y su explotación,* D. Novoa (Ed.), 162-192, Corporación Venezolana de Guayana, Editorial

comportamiento de las comunidades ícticas en una zona del golfo de Paria con métodos acústicos. *Memoria de la Fundación La Salle de Ciencias Naturales*, Vol.168,

components, In: *Coastal plant communities of Latin America,* U. Seeliger (Ed.), 135-140,

Biodiversity of vertebrate species in all regions of the world has diminished nearly 30% between 1970 and 2005, and 14% when considering only marine species (Humphrey et al., 2008). About 40% of the world's oceans are severely affected by anthropogenic impacts (Humphrey et al., 2008). Among the main reasons for the drastic marine biodiversity decline are habitat loss or fragmentation due to unsustainable urbanization of the coasts (Schlacher et al., 2007), overexploitation of species, especially overfishing (Pauly et al., 1998), pollution resulting from agricultural and urban run-off (Glibert et al., 2006; Paez-Osuna et al., 1999), chronic oil pollution near oil platforms and terminals (Brown & McLachlan, 2002), as well as the invasion of exotic species (Grosholz, 2002). The all-encompassing climate change presents additional environmental challenges. Ecological processes are put at stake due to the loss of species and changes in community patterns, because they depend on the integrity and continuity of communities and ecosystems. These processes guarantee ecological services such as nutrient cycling, stabilization of sediments, and water purification insuring the well-being of plants, animals and humans. Therefore, much scientific effort has turned towards the relationship between biodiversity and ecosystems functioning (BEF) in order to better understand the consequences of the ongoing biodiversity loss.

Most BEF studies consist of experiments, which investigate the relationship between some measure of diversity and some measure of ecosystem functioning. *Diversity* encompasses all levels of biological organization from genes to ecosystems; however, most BEF studies emphasize the consequences of species richness. *Ecosystem functioning* includes all ecological processes such as element cycling, resource use, biomass production, trophic and other

Integrating Different Organizational Levels in Benthic

of eight, and bacteria were chosen from a pool of 12 species.

important interactions among functional groups (Naeem et al., 2000).

Biodiversity – Ecosystem Functioning (BEF) Studies 93

contained autotrophs (algae), decomposers (bacteria) as well as first- and second-order consumers (protists) either of varying numbers of randomly assembled species (1, 2 or 3 species) from a species pool (Naeem & Li, 1997), or of aquatic microbial communities representing a biodiversity gradient as it might occur in nature after species loss across all trophic levels (31 species in the "complete", i.e. most diverse assemblage; McGrady-Steed et al., 1997). Although this approach risks confounding species richness with species composition, additional experimental and statistical controls were applied to separate these factors. The simulated "natural" communities consisted of 4 (McGrady-Steed et al., 1997) or 5 (Petchey et al., 1999) trophic levels: producers (e.g., diatoms), herbivores (e.g. *Brachionus*), bacterivores (e.g. *Rotaria*), predators (e.g. *Heliozoa*), and a bacterial assemblage with 4 to 16 species per functional group (McGrady-Steed et al. 1997; Petchey et al., 1999). The lowest diversity group consisted of fewer species per trophic level or functional groups, simulating diversity loss across trophic levels. As proxies of ecosystem functioning, CO2 concentration (i.e. ecosystem respiration; McGrady- Steed et al., 1997), algae and bacterial biomass (Naeem & Li, 1997) were measured. In line with these studies, Bell et al. (2005) manipulated microcosms with an original pool of 72 species. They argued that manipulating low numbers of species can show which processes are possible, but that manipulating high numbers of species gives an idea about which processes may become important in nature (Bell et al., 2005). Their study system consisted of semi-permanent rain pools from the base of European beech trees. In another approach, algal (producer) and bacterial (decomposer) species diversity were manipulated simultaneously, and algal biomass was measured as a proxy of primary production (Naeem et al., 2000). Algae were chosen randomly from a pool

In all the above-mentioned studies, biodiversity had a positive stabilizing effect: ecosystem respiration became more predictable (McGrady-Steed et al., 1997), community biomass as well as density measures were more consistent (Naeem & Li, 1997), and bacterial respiration increased with species richness (Bell et al., 2005). A higher predictability and lower variability of measures of ecosystem functioning underpin the idea that species diversity enhances ecosystem stability, whereas the stimulatory effect on respiration *per se* demonstrates a positive relationship between diversity and ecosystem process rates. On the other hand, extinction risk in warming environments remained unaffected by biodiversity, but instead depended on the trophic position (Petchey et al., 1999). However, diverse communities were more likely to contain temperature-tolerant species and therefore retained more species than depauperate ones (Petchey et al., 1999). Further, the simultaneous manipulation of two trophic levels showed that neither algal nor bacterial species richness alone could explain the significant differences among microcosms, but that the algal biomass was a joint function of both algal and bacterial diversity implying

Studies on microbial communities are very useful as they can be carried out at very high species richness levels (Bell et al., 2005). Moreover, microbial communities are omnipresent, and play fundamental roles in virtually all ecosystems. Besides, experiments allow changing diversity across trophic levels. This is an important issue since species loss is not random; it rather depends on the trophic position, with top predators being especially prone to extinction (Petchey et al., 1999). However, all microbial organisms of those studies are unicellular, and it remains unclear to what extent the results can be extrapolated to communities comprising metazoans. A meta-analysis linking metazoan species diversity

relationships among the organisms, as well as any form of resistance or resilience of the community and the system. The ways in which ecosystem functioning is measured experimentally vary greatly among studies. Among others, nutrient flux (Emmerson et al., 2001), primary and secondary production (Duffy et al., 2003), as well as food web dynamics (Duffy et al., 2007), but also resistance to and resilience from disturbance (e.g., Hughes & Stachowicz, 2004) and invasion success (Stachowicz et al., 1999) have been assessed.

In this chapter, we aim at giving a succinct overview of the seminal BEF studies and presenting a new experimental approach in this field of research. In the following sections, we present various experimental systems and organisms with special emphasis on benthic communities. For this purpose we scanned the reviews by Covich et al. (2004), Duffy et al. (2007), Hooper et al. (2005), Loreau et al. (2001), Stachowicz et al. (2007), and Worm et al. (2006,) and selected more than 30 of the most cited studies. To extend the survey to the present, we selected relevant studies from those citing the above-mentioned reviews. We focus on the different experimental approaches, highlight advantages and shortcomings of the applied methodologies or systems, and we briefly summarize the most important results. As will be shown, benthic BEF studies have hitherto investigated the effects of species and genetic diversity separately. Against this background, we present a new experimental model system of a benthic food web, which allows investigating the effects of species and genetic diversity in combination.

#### **2. The pioneering BEF study systems**

#### **2.1 Terrestrial plants**

The pioneering BEF studies were conducted on terrestrial plant systems (e.g., Tilman & Downing, 1994; Tilman, 2006). They established plots of different plant species richness and measured resilience and resistance from a major draught in terms of primary productivity (i.e., plant biomass; Tilman & Downing, 1994). Others applied similar methods and repeated the same experiment at a number of sites differing in climate and major environmental factors in order to search for general (global) patterns (Hector et al., 1999). These studies were among the first to provide clear experimental evidence about the positive relationships between taxonomic and functional diversity and productivity, substantiating the need for biodiversity conservation. However, they also demonstrated the difficulties with the experimental setups, such as the problem of scales (space and time), or the "sampling effect", i.e., the probability that the assemblage of the highest diversity is the most likely to contain the most productive species (Benedetti-Cecchi, 2004). Further, although these studies included different "functional groups" by including functionally different plants (i.e., nitrogen-fixing legumes, grasses and herbs), they only considered the basic trophic level of primary producers.

#### **2.2 Microbial communities**

In order to investigate experimentally the relationship between diversity and ecosystem functioning at multiple trophic levels, BEF studies on aquatic microbial communities followed. These studies allowed new insights into functional diversity, because they considered species diversity within several trophic levels from producers to predators (McGrady-Steed et al., 1997; Naeem & Li, 1997; Petchey et al., 1999). Replicated aquatic microbial microcosms were established with varying numbers of species per functional group (McGrady-Steed et al., 1997; Naeem & Li, 1997; Petchey et al., 1999). Each microcosm

relationships among the organisms, as well as any form of resistance or resilience of the community and the system. The ways in which ecosystem functioning is measured experimentally vary greatly among studies. Among others, nutrient flux (Emmerson et al., 2001), primary and secondary production (Duffy et al., 2003), as well as food web dynamics (Duffy et al., 2007), but also resistance to and resilience from disturbance (e.g., Hughes &

In this chapter, we aim at giving a succinct overview of the seminal BEF studies and presenting a new experimental approach in this field of research. In the following sections, we present various experimental systems and organisms with special emphasis on benthic communities. For this purpose we scanned the reviews by Covich et al. (2004), Duffy et al. (2007), Hooper et al. (2005), Loreau et al. (2001), Stachowicz et al. (2007), and Worm et al. (2006,) and selected more than 30 of the most cited studies. To extend the survey to the present, we selected relevant studies from those citing the above-mentioned reviews. We focus on the different experimental approaches, highlight advantages and shortcomings of the applied methodologies or systems, and we briefly summarize the most important results. As will be shown, benthic BEF studies have hitherto investigated the effects of species and genetic diversity separately. Against this background, we present a new experimental model system of a benthic food web, which allows investigating the effects of

The pioneering BEF studies were conducted on terrestrial plant systems (e.g., Tilman & Downing, 1994; Tilman, 2006). They established plots of different plant species richness and measured resilience and resistance from a major draught in terms of primary productivity (i.e., plant biomass; Tilman & Downing, 1994). Others applied similar methods and repeated the same experiment at a number of sites differing in climate and major environmental factors in order to search for general (global) patterns (Hector et al., 1999). These studies were among the first to provide clear experimental evidence about the positive relationships between taxonomic and functional diversity and productivity, substantiating the need for biodiversity conservation. However, they also demonstrated the difficulties with the experimental setups, such as the problem of scales (space and time), or the "sampling effect", i.e., the probability that the assemblage of the highest diversity is the most likely to contain the most productive species (Benedetti-Cecchi, 2004). Further, although these studies included different "functional groups" by including functionally different plants (i.e., nitrogen-fixing legumes, grasses and herbs), they only considered the basic trophic

In order to investigate experimentally the relationship between diversity and ecosystem functioning at multiple trophic levels, BEF studies on aquatic microbial communities followed. These studies allowed new insights into functional diversity, because they considered species diversity within several trophic levels from producers to predators (McGrady-Steed et al., 1997; Naeem & Li, 1997; Petchey et al., 1999). Replicated aquatic microbial microcosms were established with varying numbers of species per functional group (McGrady-Steed et al., 1997; Naeem & Li, 1997; Petchey et al., 1999). Each microcosm

Stachowicz, 2004) and invasion success (Stachowicz et al., 1999) have been assessed.

species and genetic diversity in combination.

**2. The pioneering BEF study systems** 

**2.1 Terrestrial plants** 

level of primary producers.

**2.2 Microbial communities** 

contained autotrophs (algae), decomposers (bacteria) as well as first- and second-order consumers (protists) either of varying numbers of randomly assembled species (1, 2 or 3 species) from a species pool (Naeem & Li, 1997), or of aquatic microbial communities representing a biodiversity gradient as it might occur in nature after species loss across all trophic levels (31 species in the "complete", i.e. most diverse assemblage; McGrady-Steed et al., 1997). Although this approach risks confounding species richness with species composition, additional experimental and statistical controls were applied to separate these factors. The simulated "natural" communities consisted of 4 (McGrady-Steed et al., 1997) or 5 (Petchey et al., 1999) trophic levels: producers (e.g., diatoms), herbivores (e.g. *Brachionus*), bacterivores (e.g. *Rotaria*), predators (e.g. *Heliozoa*), and a bacterial assemblage with 4 to 16 species per functional group (McGrady-Steed et al. 1997; Petchey et al., 1999). The lowest diversity group consisted of fewer species per trophic level or functional groups, simulating diversity loss across trophic levels. As proxies of ecosystem functioning, CO2 concentration (i.e. ecosystem respiration; McGrady- Steed et al., 1997), algae and bacterial biomass (Naeem & Li, 1997) were measured. In line with these studies, Bell et al. (2005) manipulated microcosms with an original pool of 72 species. They argued that manipulating low numbers of species can show which processes are possible, but that manipulating high numbers of species gives an idea about which processes may become important in nature (Bell et al., 2005). Their study system consisted of semi-permanent rain pools from the base of European beech trees. In another approach, algal (producer) and bacterial (decomposer) species diversity were manipulated simultaneously, and algal biomass was measured as a proxy of primary production (Naeem et al., 2000). Algae were chosen randomly from a pool of eight, and bacteria were chosen from a pool of 12 species.

In all the above-mentioned studies, biodiversity had a positive stabilizing effect: ecosystem respiration became more predictable (McGrady-Steed et al., 1997), community biomass as well as density measures were more consistent (Naeem & Li, 1997), and bacterial respiration increased with species richness (Bell et al., 2005). A higher predictability and lower variability of measures of ecosystem functioning underpin the idea that species diversity enhances ecosystem stability, whereas the stimulatory effect on respiration *per se* demonstrates a positive relationship between diversity and ecosystem process rates. On the other hand, extinction risk in warming environments remained unaffected by biodiversity, but instead depended on the trophic position (Petchey et al., 1999). However, diverse communities were more likely to contain temperature-tolerant species and therefore retained more species than depauperate ones (Petchey et al., 1999). Further, the simultaneous manipulation of two trophic levels showed that neither algal nor bacterial species richness alone could explain the significant differences among microcosms, but that the algal biomass was a joint function of both algal and bacterial diversity implying important interactions among functional groups (Naeem et al., 2000).

Studies on microbial communities are very useful as they can be carried out at very high species richness levels (Bell et al., 2005). Moreover, microbial communities are omnipresent, and play fundamental roles in virtually all ecosystems. Besides, experiments allow changing diversity across trophic levels. This is an important issue since species loss is not random; it rather depends on the trophic position, with top predators being especially prone to extinction (Petchey et al., 1999). However, all microbial organisms of those studies are unicellular, and it remains unclear to what extent the results can be extrapolated to communities comprising metazoans. A meta-analysis linking metazoan species diversity

Integrating Different Organizational Levels in Benthic

environmental conditions (Bell, 1991).

et al., 2010; Phillips & Hickey, 2010).

**3.2.1 Macrophyte vegetations** 

phosphorus (Engelhardt & Ritchie, 2001).

Biodiversity – Ecosystem Functioning (BEF) Studies 95

abiotic and biotic factors (Hughes & Stachowicz, 2009). Similar results were obtained when genotypes of the green alga *Chlamydomonas reinhardtii* were set up in culture tubes in monocultures and all possible pair wise combinations: the mixtures were consistently more productive and less variable in their productivity under simulation of different

To our knowledge, there are very few experimental studies on benthic animals investigating the role of genetic diversity on ecosystem function, two of which assessed the effects on intertidal invertebrates. Within-species diversity was manipulated in the barnacle *Balanus improvisus* by altering the number of parental broods (Gamfeldt et al., 2005). In a similar way, the number of maternal families was controlled in the amphipod *Gammarus duebeni* from intertidal rock pools (Gamfeldt & Kallstrom, 2007). Both experiments found positive effects of increased genetic diversity: larval settlement of *B. improvisus* increased and the more diverse amphipod assemblages exhibited more predictable (but not higher on average) survival when exposed to multiple stressors (Gamfeldt & Kallstrom, 2007). Apart from that, studies relating genetic diversity to some (though particular) measure of ecosystem functioning have mainly focused on tolerance to toxic substances, indicating that genetically diverse populations resist to or recover better from contamination (Pease

The few studies relating genetic (genotypic) diversity to ecosystem functioning show that genetic diversity can be important for the stability and productivity of coastal ecosystems. Generally, increased genetic diversity enhances the continuity and reliability of ecosystems. The advantage of clonally reproducing organisms such as plants or algae is that they provide the relatively easy and thus straightforward possibility of setting up experimental groups of contrasting genotypic diversity. However, this method is not applicable to sexually reproducing organisms. For those, it is necessary to produce different levels of genetic diversity by controlled mating prior to the experiment. For example Gamfeldt & Kallstrom (2007) sampled natural populations of *G. duebeni* and then separated pairs of mating individuals to form different "families". With these families, they then formed inbred strains (lowest genetic diversity level) and strains of two to four different families (increasing diversity). This procedure provides reliable levels of contrasting genetic diversities, but it is time consuming and requires considerable effort and material. Nevertheless, it is probably the only reliable method of obtaining contrasting diversity levels for sexually reproducing organisms if natural inbred (representing low diversity) and outbred populations (high diversity) are unavailable. This is probably the main reason, why studies investigating the role of genetic diversity on the system functioning are still scarce.

Seagrass *Zostera marina*, pondweed or cordgrass *Spartina* ecosystems have been used widely to investigate the effect of species richness, species composition as well as food chain length and trophic cascades on ecosystem processes. For example all possible species combinations of four functionally and morphologically different submerged aquatic macrophytes (pondweed) were planted in artificial wading pools (Engelhardt & Ritchie, 2001). Although local wetlands are usually dominated by single vascular species, there was a clear effect of increased plant diversity leading to higher plant and algal biomass as well as lower loss of

**3.2 Effects of species diversity on benthic ecosystem functioning** 

with various proxies for ecosystem functioning across a range of deep-sea regions suggests that species diversity profoundly affects marine benthic ecosystem functioning (Danovaro et al., 2008). Therefore, mesocosm studies with benthic communities and systems provide useful options to take BEF studies to the next level.
