The addition of farmed bivalves increases habitat complexity, potentially enhancing species diversity. Additionally, biodeposits generated by farmed bivalves may increase the organic load, attracting concomitant detritivores . The physical modification of the habitat and the alteration of fluxes of organic matter towards the bottom make bivalves ecosystem engineers . Although responses of trait-based functional diversity in macrofauna to bivalve farming are well documented, there is little information on how these functional traits affect the trophic structure within the assemblage. Functional traits provide a mechanistic link between food web composition and energy fluxes . The role of functional traits of co-occurring species reflects the trophic structure in a food web , suggesting that the trait-based functional structure of an assemblage is strongly correlated with its trophic structure . Thus, understanding the ecological effects on the macrofauna requires responses of functional traits, trophic structure, and food web of the assemblage, in order to obtain comprehensive knowledge about improving farming practices and management. Although many studies regarding trophic ecology have suggested that identifying trophic structure and determining feeding relationships is crucial to mechanistically understand assemblage functioning , the exploration of ecological effects of bivalve farms by combining functional and trophic structures in macrofaunal assemblages is unknown. Stable isotopes , particularly carbon and nitrogen , have been widely used for quantifying trophic structure within assemblages such as quantifying trophic niche and the diet of consumers . δ13C can reflect the potential source of dietary carbon source of the organisms, while δ15N can be used to quantify the trophic level in the food chain . This technique has been widely used to describe the trophic structure of macrofaunal assemblage, grow bucket facilitating our understanding of biological processes relevant to disturbance.

In this study, the macrofaunal assemblage was characterized before and after the seeding of Manila clam in a bottom-based farm. For that purpose, the variation of trait-based functional structure and SI-based trophic structure between both periods were analyzed. The aims of the present study are: 1) to identify the functional and trophic responses of the macrofaunal assemblage to bottom-based Manila clam farming; and 2) to identify the feeding relationships of the macrofaunal assemblage in both periods. By achieving these aims, this study will provide insights on the ecological effects of clam farming and explore how the effects could be used to improve farming practices and management. Field investigations were carried out in two periods. The farming area was firstly sampled in July 2019, approximately 1 month before seeding , which defined the natural assemblage of the farming area. The second sampling was carried out in July 2020 , after approximately one year of farming operations. Ten sampling sites, evenly distributed within the farm, were set to collect macrofauna samples in both periods . Macrofauna was collected using a 0.1 m2 Van Veen grab with three replicates per site and sieved through a 1 mm mesh. The collected macrofauna was sorted and identified at the lowest possible taxonomic level. Potential food sources were also sampled at each sampling site in both periods to analyze δ13C and δ15N signature. As no seagrass or macroalgae was observed in the study area, four major food sources, sediment organic matter , particulate organic matter , zooplankton, and phytoplankton were used as the primary food sources for the assemblage. The detailed procedure used for sampling food sources is found in Supplementary Materials . Trophic structure was described by the isotopic spaces which were built using the average values of an iso-space C-N biplot for each species . Standard ellipse area was used to represent the isotopic richness rather than total area because it is comparable to the standard deviation for univariate data and is more robust to differences in sample size and less sensitive to extreme values than the TA . The trophic niche was calculated using the R package “SIBER” . Additionally, to better understand the variations in trophic structure between the two assemblages, the macrofauna species were partitioned into 3 functional feeding groups based on their feeding mode: filter feeder, bottom deposit feeder, and carnivore. The trophic structures of each functional feeding group were also described using SEAc based on the same approach. Functional structures were described by the multivariate functional space generated from 12 functional traits in 4 categories including mobility, vertical position, normal adult size, and longevity . The selected traits reflect the trophic position of animals in the macrofaunal assemblage .

To better virtualize the variation of specific traits, a multidimensional functional space containing the selected traits was built through a principal coordinate analysis conducted on a Bray-Curtis distance matrix . The first two axes were used to plot the resulting functional space and traits for the two assemblages. Additional PCoA was also applied to the 3 functional feeding groups to better reflect the trait-based functional variations of the two assemblages. PCoA approach was calculated using the R package “vegan” . Additionally, to describe the trophic and functional structure, three indices were calculated for both the SI-based trophic indices and trait-based functional indices. The detailed procedure used for calculating trophic and functional indices is found in Supplementary Materials . Both SI-based trophic indices and trait-based functional indices were calculated for each feeding group. To better visualize the variation between each feeding group of the two assemblages, each index was illustrated using AFTER – BEFORE differences . In the present study, the number of filter-feeder species decreased from nine to four after one year of farming, indicating a strong negative impact on this group due to clam farming. Because the abundance of the farmed clam was highly above natural levels, other filter feeders were strongly impacted likely due to the competition for food and habitat. As functional richness is often strongly correlated to species number , the decrease in the number of species resulted in reduced functional richness in the filter feeder group. Additionally, an increase of clam predators, attracted by the enhanced population of farmed bivalves, may raise the predation pressure on other filter-feeders, amplifying the negative effects on their populations . Under this predation pressure, filter feeders with larger body sizes may have more chances to survive , which could explain the observed shift towards a larger body size in the filter feeder group. For the detritus feeder group, the niche width represented by SEAc illustrated a large shift, with a narrower δ13C range after clam farming. Given that farmed bivalves increase sedimentation rates through biodeposits and, accordingly, change the organic content of the sediment , it is expected that SOM would be influenced by these abundant biodeposits and become more easily consumed by some detritivores . This is also supported by a higher δ15N values after clam farming in the present study. Accordingly, the biodeposit-enhanced SOM could contribute to a large proportion of detritus feeders’ diet and be further transferred into higher trophic levels through cascading effects .

Increased trophic richness of the detritus feeder group was observed after farming, despite the width of SEAc was clearly reduced. This could be explained by two processes. Firstly, biodeposit-enhanced SOM expanded the trophic niche that was absent before clam farming so that animals that used the new food source would accordingly favor a higher trophic richness. Second, it should be noted that some deposit feeders, with a broad range of niche breadth, are able to switch their diets to the biodeposits from farmed clam ; although other species, such as nematodes, do not necessarily modify their diet in the presence of new biodeposits . Accordingly, detritus feeders could increase their reliance on the biodeposit-enhanced SOM, resulting in a smaller SEAc. These species could occupy the new trophic position generated from the biodeposit-enhanced SOM, while other detritus feeders could remain in their trophic position, contributing to the increase of trophic richness for this group. Similarly, the increased reliance on the biodeposit-enhanced SOM also resulted in a less balanced distribution in food sources, causing a reduced trophic evenness after farming. A positive relationship between SI-based trophic indices and traitbased functional indices was observed for detritivores. This has been commonly observed in previous studies as the trophic niche of a whole assemblage is defined by the complexity of trophic interactions, and consequently, the higher the diversity of functional traits, the wider the trophic niche . The functional trait composition of detritus feeders shifted from infauna to epifauna, with increased presence of swimmers and crawlers after clam farming. This could be caused by the physical presence of biodeposits likely on the surface of marine sediment, potentially attracting epibenthic detritivores featuring surface mobility . Moreover, the increased presence of swimmers and crawlers, and their associated traits, favored a higher functional richness after clam farming. It is widely accepted that aquaculture can cause changes in flows of matter and energy in coastal ecosystems, potentially affecting the benthic environment and food web, and accordingly influencing the macrofaunal assemblage . Our study illustrated that although the functional and trophic structure of macrofaunal assemblage largely changed after starting clam farming,dutch bucket for tomatoes the effect may be positive at the local scale. Manila clam farming enhanced the secondary productivity and provided biodeposits as additional food sources for the macrofaunal assemblage.

Due to the high density of farmed bivalves, the biodeposit may significantly promote lower-level consumers such as detritivores, which can then further influence carnivores through benthic food chain and higher trophic levels due to cascading effects. Given the common knowledge that higher functional richness and diversity represent a better status for the ecosystem , the increased functional richness in this study suggests positive ecological effects. Further, it is worth mentioning that the attracted carnivores included high-level predators that were absent before clam farming. Their presence increased the food chain length and enhanced the ecosystem functions and trophic interactions of the assemblage. Both functional and trophic richness of macrofaunal assemblage were observed to increase after one year clam farming, suggesting a more diversified biotic interaction and a more complex food web. The diversity of preys and consumers has a positive effect on the secondary production of aquatic ecosystems as prey diversity may enhance energy transfer within an ecosystem. This is partly due to niche complementarity as ecological differences between species lead to an optimal utilization of resources . Petchey and Gaston predicted that numerous species coexisting in biologically established communities employ a diverse array of functional traits that facilitate their coexistence and the increased partitioning of the trophic niche. Authors hypothesized that the trophic niche of a whole community is defined by the complexity of trophic interactions and that a higher diversity of functional modes results in a wider trophic niche. Therefore, the farming of Manila clam enhanced the range of functional traits to be represented in the ecosystem, providing opportunities to maximize resources use. An interest finding in the present study is that the diet of carnivores in the clam farm relied mainly on detritus feeders, which differs from the common knowledge that the carnivores in farms consume mainly on the farmed bivalves . This is a positive phenomenon in terms of clam production, suggesting that predation pressure from the carnivores concentrated on these detritivores that were attracted by the biodeposit from the farmed clam, rather than the clam themselves. Therefore, together with the enhancement of ecosystem functions and trophic interactions, mostly via biodeposit-enhanced sediments, clam farming seems to exert positive disturbance for the ecosystem. However, it is important to note that the functional and trophic variations in macrofaunal assemblage in the present study arose in a short period of time and at a small spatial scale . The functional and trophic structures of macrofaunal assemblage could be subsequently shaped by the cumulative effects of farmed clams over time. Therefore, a more comprehensive conclusion of ecological effects of bottom-based bivalve farming would be drawn by considering greater temporal and spatial effects on the macrofaunal assemblage. Iran is a semi-arid country with small remnants of natural forest . This circumstance has presented challenges due to the impacts of deforestation on natural environments.