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Detecting anthropogenic impacts on estuarine benthic communities A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biological Sciences at The University of Waikato by DANA CLARK 2021 “There’s no limit to how much you’ll know, depending on how far from zebra you go” – Dr Seuss ii Abstract Our estuaries, and the benefits that we derive from them, are threatened by the cumulative effects of interacting stressors. Separating the impacts of anthropogenic stressors from natural variability in the marine environment is extremely difficult. This is particularly true for estuaries, due to their inherent complexity and the prevalence of difficult-to- manage diffuse stressors. Successful management and protection of these valuable ecosystems requires innovative monitoring approaches that can reliably detect anthropogenic stressor impacts. In this thesis, I examined approaches for detecting the effects of three diffuse land-derived stressors (sedimentation, nutrient loading, and heavy metal contamination) on estuarine benthic communities. Using Gradient Forest analysis, I explored the relative importance of environmental factors, operating across multiple spatio-temporal scales, in influencing patterns of compositional turnover in estuarine benthic macroinvertebrate communities across New Zealand. Both land-derived stressors (sediment mud content and total sediment nitrogen and phosphorus content) and natural environmental variables (sea surface temperature, Southern Oscillation Index, and wind-wave exposure) were important predictors of compositional turnover, reflecting a matrix of processes interacting across space and time. Generalized linear models were used to link these turnover values to measures of benthic macroinvertebrate diversity, which are commonly used as indicators of ecological health. Based on compositional turnover, I could disentangle the negative effects of land-derived stressors from natural environmental variability. Critical stressor levels associated with high rates of compositional turnover were identified, providing a useful contribution to the current knowledge on land-derived stressor effects. Once I had determined that anthropogenic impacts could be disentangled from natural variability, I developed indicators (Benthic Health Models; BHMs) to assess estuary health in response to two dominant coastal stressors (sedimentation and heavy metal contamination). Benthic macroinvertebrate community data were used in separate canonical analyses of principal coordinates to create multivariate models of community responses to these stressors. Both models performed well (R2 = 0.81, 0.71), and were unaffected by regional and estuarine typology differences. They offer a sensitive and standardised approach to assessing estuarine health that allows separation of the two stressors. iii I also examined the potential for emerging molecular approaches to inform estuary health assessment. Recent advances in environmental genomics allow characterization of less visible forms of benthic biodiversity, offering a more holistic view of the ecosystem and potentially providing early warning signals of disturbance. A manipulative nutrient enrichment experiment was conducted in two estuaries and environmental DNA (eDNA) metabarcoding was used to examine the response of eukaryotic (18S rRNA), diatom only (rbcL) and bacterial (16S rRNA) communities. Multivariate analyses demonstrated differential changes in examined communities between sites, suggesting a context dependent response to nutrient enrichment. These patterns aligned with changes in morphologically identified macroinvertebrate communities, confirming concordance between eDNA-based and current monitoring approaches. This work represents a first step towards the development of molecular estuary monitoring tools, which could transform current approaches to ecosystem health assessment. This thesis demonstrates that the detection of anthropogenic impacts on estuarine benthic communities requires an understanding of the response of communities to stressors and how this response is modified by natural environmental processes operating at different spatio-temporal scales. My research contributes to the management and protection of estuaries by improving knowledge on the processes generating broad scale patterns in benthic macroinvertebrate communities, developing indicators that can be used to assess estuary health and demonstrating the potential of eDNA metabarcoding as a new tool for estuary health assessment. iv Preface This thesis comprises three research chapters (Chapters 2-4), which have been published in peer-reviewed international scientific journals. I was responsible for all field and laboratory work, data analysis and writing for this thesis. Unless referenced, the ideas in this thesis are my own and this work was carried out under the supervision of Professor Conrad Pilditch (University of Waikato), Dr Joanne Ellis (University of Waikato) and Dr Anastasija Zaiko (Cawthron Institute and the University of Auckland). Chapter 2 has been published in Marine Ecology Progress Series under the title, “The influence of land-derived stressors and environmental variability on the compositional turnover and diversity of estuarine benthic communities” by DE Clark, F Stephenson, JE Hewitt, JI Ellis, A Zaiko, A Berthelsen, RH Bulmer, and CA Pilditch. DOI:10.3354/meps13714. Chapter 3 has been published in Marine Pollution Bulletin Volume 150 (2020) under the title, “The development of a national approach to monitoring estuarine health based on multivariate analysis” by DE Clark, JE Hewitt, CA Pilditch, and JI Ellis. DOI: 10.1016/j.marpolbul.2019.110602. Chapter 4 has been published in Environmental Pollution Volume 267 (2020) under the title, “Environmental DNA metabarcoding reveals estuarine benthic community response to nutrient enrichment – Evidence from an in-situ experiment” by DE Clark, CA Pilditch, JK Pearman, JI Ellis, and A Zaiko. DOI: 10.1016/j.envpol.2020.115472. v Acknowledgements I have enjoyed undertaking this thesis – it is rare to have the opportunity to indulge your own research interests, supported by a team of established academics. I could not have hoped for a better supervisory panel. Many thanks to my primary supervisor Conrad Pilditch for your support and guidance over these last four years. Through your networks I have established my own research connections and made new friends. I admire the welcoming research environment you have created, your ability to think big and the time you invest in each of your students. Joanne Ellis, you were a great mentor before I embarked on my PhD, and I am so glad you could be part of my supervisory team. Thanks for sparking my interest in benthic ecology. I continue to be inspired by your ability to develop relationships, your openness to collaboration and your academic knowledge. Anastasija Zaiko, thank you for introducing me to the world of eDNA and providing valuable input to many aspects of my research. It has been enjoyable getting to know you and I hope we continue to work together in the future. I would also like to say a special thanks to Judi Hewitt who, although not officially a supervisor, has provided me with invaluable wisdom over the last several years. I have learned a lot and I appreciate you making time for me. Many thanks to Fabrice Stephenson for his patience with Gradient Forest and John Pearman for his bioinformatics expertise. Rebecca Gladstone-Gallagher, Anna Berthelsen, Shaun Bryant, Chris Cornelisen, Lisa Floerl, Simon Madill, Emma Newcombe, Holly Bennett, Sorrel O’Connell-Milne, Yuriy Malakhov, Jamie McAuley and Lais Miura helped in the field. Laura Biessy’s guidance was invaluable in the lab, Paula Casanovas provided helpful statistics advice, and Aneika Young helped me navigate iwi consultations. Thank you to all of the Waikato students for making me feel welcome, despite my remote location. I was lucky to remain working at the Cawthron Institute while I undertook my PhD. I am extremely grateful for Cawthron’s support, particularly that of Chris Cornelisen who championed funding for this project and gave me the flexibility I needed to complete my thesis. I also appreciate the guidance I have received from Jim Sinner, including his support to obtain funding for my research through the Oranga Taiao Oranga Tangata (OTOT) research programme (MBIE contract MAUX1502, led by Murray Patterson from Massey University). Anna Berthelsen, Javier Atalah and Eric Goodwin helped to compile vi the large environmental dataset upon which much of my research was based. Discussions with Anna were also influential in developing my research ideas and I appreciate the interest she took in my project. In addition to the funding