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New Zealand seagrass (Zostera muelleri) response to acute sedimentation: Linking non-structural carbohydrate reserves to resilience A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biological Sciences at The University of Waikato by Stine Tang Sørensen 2020 Abstract Seagrass meadows form healthy near-coastal marine environments that provide a wide range of ecosystem services in New Zealand and Australia. However, seagrass habitats are declining regionally and globally. It is therefore essential that research fills the current knowledge gaps associated with seagrass disturbance-response regimes and develops standardised methods to measure seagrass health and resilience. Sedimentation associated with increased human activity is a major environmental stressor to seagrass; yet little is known about how sedimentation affects New Zealand’s only seagrass species, Zostera muelleri. This thesis, therefore explores how Z. muelleri responds to catastrophic burial (acute) events in Tauranga Harbour, New Zealand. Non-structural carbohydrates (NSC) are synthesised and stored in seagrass tissues when photosynthetic outputs exceed metabolic demands and, in turn, are mobilised when photosynthesis is unable to meet metabolic demands. Hence, NSC reserves can be used to measure the impact of a disturbance and/or the resilience of seagrass. A literature review in Chapter 2 illustrates that multiple methods are used to estimate NSC reserves. Experimental comparisons of a selection of these methods demonstrate that NSC estimates by different analytical methods cannot be compared. The outcome of Chapter 2 is the development of a 5-step standard analytical protocol for the quantitation of NSC reserves in seagrass (specifically Z. muelleri). The implementation of a standardised protocol will enable researchers to compare and synthesise results and, thus, increase the application of NSC as a seagrass health measure. Chapter 3 investigates the effects of acute sedimentation (i.e. burial) on Z. muelleri. In situ manipulative experiments across three sites in Tauranga Harbour, New Zealand were used to assess the spatial variation in resilience (i.e. resistance and recovery) to burial. Resistance and recovery following burial varied significantly between the three distinct morphotypes that existed in the three sites; a large, a small and an intermediate morphotype that overlapped with the small and the large morphotypes. These morphometric differences were believed to be phenotypic responses to site-specific environmental conditions. Having a large morphotype maximises the potential to capture light and store reserves and presumably an acclimation of Z. muelleri existing in a chronically impacted environment (high i sediment mud/organic matter (SOM) and low sediment grain size). The largest seagrass displayed high resistance to both single and repeated burial events of 2 cm (no significant effects). In contrast, the smaller seagrass had low resistance and the slowest ability to recover following burial (up to 251 days), whereas, the intermediate morphotype displayed a faster ability to recover (up to 168 days). Results, therefore, suggest that the resilience to burial events increases in populations that have acclimated to a chronically impacted environment. It is, however, noted that a degraded environment will eventually reach a limiting threshold from which recovery is not plausible and results should therefore be interpreted with some caution. While rhizome NSC reserves were not affected by burial treatments, these varied significantly throughout a full growing year across the three locations. NSC reserves were at the lowest levels in winter and spring, consistent with seasonal trends. Sucrose levels at the peak of the growing season (February) were significantly related to relative shoot cover the following winter (r2 = 0.54, P < .001) and spring (r2 = 0.66, P < .001). As such, chapter 3 also documented a link between seagrass cover and sucrose reserves. Estimates of NSC reserves from the literature vary greatly, presumably due to the wide variety of analytical methods used. Chapter 4 explores the variability of Z. muelleri NSC reserves and the specific partitioning of carbohydrate groups (sucrose and starch) across different spatial scales (within meadow, between sites, between regions), including temperate (Port Phillip Bay) and tropical populations (Townville and Magnetic Island) in Australia. When measured in spring, the total NSC reserves were similar across all spatial scales (193.59 ± 10.88 mg g-1 DW); however, the proportional contents of sucrose and starch varied significantly at different spatial scales. Sucrose contents varied at site level and were best explained by a combination of sediment pH and aboveground to belowground biomass ratio (r2 = 0.49, P <0.001). Starch contents varied between regions, with seagrass in the tropical Townsville containing significantly higher starch levels and as such lower sucrose to starch ratios. The best model for starch included only one variable; sediment surface temperature (r2 = 0.33, P < 0.001) and results therefore suggest that starch is influenced by large-scale processes linked to climatic processes. The adaptive strategies of seagrass to moderate NSC allocation appear to be of particular importance to seagrass’ ability to acclimate to a diverse range of environments. ii Chapter 5 synthesises the results of the three research chapters and provides recommendations for best practices. Of particular importance, is the method that was developed to ensure accurate measurements of NSC reserves in seagrasses, allowing this measure to be used as a monitoring tool of potential resilience and health of seagrass meadows. This thesis clearly illustrates that the partitioning of NSC groups (sucrose/starch) vary spatially and temporally and that these are influenced by distinct processes. Furthermore, it demonstrates that the phenotypic plasticity of Z. muelleri in New Zealand enhances their resilience to acute sedimentation events (i.e. burial). It is therefore suggested that the timing and location of human-induced disturbances (i.e. catchment developments, dredging of harbours and ports) should match optimum seagrass resilience (e.g., high sucrose reserves and consideration of phenotypic expression), as this is likely to increase the survival rates of the impacted seagrass meadows. The new insights gained from this research provides crucial information to ensure sustainable management of Z. muelleri into the future. iii Acknowledgements As the end of my PhD journey approaches, and I sit down to write my acknowledgements, I feel a great sense of gratitude and appreciation for the many valuable experiences I have made along the way, and for all the people who made it possible. First, a huge thanks to my two main supervisors, Professor Marnie Campbell and Professor Merilyn Manley-Harris, for your support and guidance throughout this entire project. Your abilities to share your knowledge, past experiences and expertise were crucial to my success. I am truly grateful for the many opportunities you provided me to allow me to grow both personally and professionally. Marnie, thank you for encouraging me and believing in my capabilities to convert this project from an MSc project to a PhD project. To my co-supervisors Dr Craig Sherman, Dr Steve Miller and Professor Chris Battershill, thank you ever so much for your kindness, technical support and guidance at different stages of my journey. Chris your encouragement and support were of specific importance in the very early and the final stages of this project, thank you. Particular gratitude also goes out to Craig and his beautiful family, who hosted me while working at Deakin University, Geelong, Australia. Your hospitality allowed me to complete the field work and sample analysis for Chapter 4 and made my experience unforgettable. I would also like to thank the organisations and companies that funded this project and essentially made it possible. Thanks to the Waikato Regional Council, the Port of Tauranga, the Waikato Graduate Women Educational Trust and the University of Waikato for the generous research grants and scholarships. At the University of Waikato’s coastal marine field station (CMFS) in Tauranga, I would like to thank all the amazing people who volunteered their time to assist me, which allowed me to execute a fairly optimistic amount of sampling. These people included Staci King, David Sailer, Amy Platt, Fenna Beets, Helen Cadwallader, Nicole Hodgson, Te Puea Dempsey, Arwyn Claydon, Diana Miechler, Sam McCormak, Ryan Koverman, Jane Cope and Ben Stewart. Special thanks go out to Staci for your never-failing enthusiasm to accompany me in the field, you have