Understanding the network of interactions that support biological communities are vital for management and conservation of ecosystems. Characterising species interactions are vital for predicting ecosystem response to perturbation, however quantifying such interactions remains a challenge. Traditionally, behavioural observational data and morphological analysis of stomach contents have been used to identify prey-predator dynamics, but these methods are time consuming and introduce biases. Stable isotope analysis (SIA) of carbon and nitrogen have been extensively applied to trophic web studies to identify sources of carbon input and offer trophic level discrimination as well as provide long-term assimilation data. However, species level prey composition data is usually not attainable. DNA-based diet determination methods have been applied as an alternative to SIA as it produces data with high taxonomic resolution revealing species specific prey composition. Although, DNA-based methods also have limitations such as PCR and quantification biases. We propose that the complementary use of SIA and DNA-based techniques is necessary to obtain accurate representation of interactions within an ecosystem.

In this study we used a combination of carbon and nitrogen SIA techniques with dietary metabarcoding of intermediate trophic level fish from mangrove ecosystems. These fish are important conduits of energy transfer between basal organisms and top predators but have been rarely studied as they are not commercially important species. The SIA analysis reflected distinct carbon input between coastal and lagoon sites, where coastal fish were more enriched in 13C. The results also unexpectedly reflected enriched 15N signatures for species Anchoa mitchili (glass minnow/bay anchovy), a pelagic fish that usually occupies lower trophic levels. Due to the lack of prey SIA ratios, we were not able to confirm causes for elevated 15N values observed in glass minnows, but dietary metabarcoding data revealed possibilities of ichthyoplankton ingestion. The mitochondrial cytochrome c oxidase subunit I (COI) and V4 region of the 18S ribosomal DNA-encoding gene were used to characterise prey diversity from the stomach contents.

Subsequently, the metabarcoding data was used to construct ecological networks which reflected that the intermediate trophic level fish had more specialised feeding preferences during wet season and generalised feeding patterns in mangrove habitats due to the greater availability of resources. In addition, network models suggest that coastal networks are more resistant to potential extinction events.

Using similar metabarcoding techniques, we explored the role of diet and trophic niche divergence in the benthic and littoral ecomorphs of Astatotilapia calliptera. The stomach contents of benthic individuals consisted of nematodes, Bacillariophyta (diatoms) and copepods while the diet of littoral individuals comprised molluscs, annelids and fungi, supporting previous SIA data analysis. The dietary analysis reflected that the ecomorphs were likely generalist consumers but when resources are scarce they have the ability to expand their range and explore specialist prey.

Our findings demonstrate that the complementary use of SIA and dietary metabarcoding techniques can provide insights into interactions not offered when either technique is applied independently. The combination of these methods offer strong potential to develop a deeper understanding of feeding ecologies, that can be integrated into effective conservation and management strategies.