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    The power of metabarcoding for investigating the tropic interactions of mesopredators through dietary studies

    Hughes, Thomas David (2023) The power of metabarcoding for investigating the tropic interactions of mesopredators through dietary studies. Doctoral thesis (PhD), Manchester Metropolitan University.

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    Abstract

    Diet studies can contribute knowledge about the ecology of a species, its interactions with other species and information about the wider environment. They are therefore important tools in effective conservation planning. Studying the diet of mesopredators is of increasing importance due to the widespread loss of apex predators, resulting in the dominance of the smaller predators. Despite the increasing importance of mesopredators in ecosystems, they remain understudied relative to larger more charismatic apex species. Recent advances in the field of metabarcoding has led to a renaissance in the number of diet studies being undertaken due to the increased ability to identify prey items, especially small items or those that little or no identifiable remains for more traditional morphological methods to assess. Metabarcoding is capable of processing hundreds or even thousands of samples simultaneously, presenting opportunities to conduct largescale studies with far more powerful conclusions than the smaller scale studies commonly conducted in the past. Although metabarcoding has become a powerful tool, it suffers from a wide variety of biases that need to be accounted for, and doing so has increased the size, complexity and cost of metabarcoding studies. One way of conducting large scale studies cost-effectively is to use a tagged barcode approach to create metabarcoding libraries. While this approach is cost effective, it needs a wide variety of specialist equipment to prepare the libraries for sequencing, making it difficult for some smaller laboratories to execute such studies. In this thesis, I developed a library preparation protocol that could be used in the absence of some of the quality control equipment commonly recommended in all tag-based library preparation techniques, thereby increasing the accessibility of metabarcoding work to a range of laboratories. I validated this method showing it produced precise mock communities, with a good level of accuracy. This methodology was then validated using a novel hierarchical marker strategy to assess the full breadth of the diet of two mesocarnivores, Neotropical and Eurasian Otters, more effectively than previous studies. I found that invertebrates represented a significant portion of otter diets, something previously assessed. Metabarcoding diet also revealed wider changes in the environment, including the spread of invasive species outside of their reported range. The methodology also revealed the previously unknown importance of plants and scavenging behaviour to the critically endangered Bermuda skink, informing both in-situ and ex-situ conservation plans. Overall, this work demonstrated the role that cost-effective metabarcoding methodology can contribute to ecological and conservation understanding.

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