The development and application of DNA metabarcoding to non-invasively assess seabird diets, using albatrosses as a model by Julie McInnes Institute for Marine and Antarctic Studies Submitted in fulfilment of the requirements for the Doctor of Philosophy University of Tasmania June, 2017 i Abstract The diets of marine organisms provide valuable insights into their behaviour, ecology, population vulnerabilities and the role they play in marine foodwebs. Many seabird populations are threatened by interactions with commercial fisheries resulting in incidental mortality or competition for resources, and global environmental changes are affecting the abundance and availability of prey. Understanding their prey requirements and dietary flexibility in this context is valuable for effective conservation and management. Conventional studies using stomach contents analysis can be invasive and suffer considerable drawbacks such as overestimation of prey represented by hard parts and underestimation of soft- bodied prey. DNA metabarcoding of scats provides a non-invasive dietary analysis method that identifies prey DNA and overcomes some of the drawbacks of conventional methods. However, this method has rarely been used on seabirds. It is unknown whether DNA is viable in scats that have been exposed to the harsh weather typical of seabird colonies, if dietary information can be collected during all breeding stages, or whether DNA metabarcoding offers improvements over other dietary assessment methods for evaluating marine ecosystem changes and interactions with fisheries. Albatrosses provide an ideal model to develop and apply DNA metabarcoding to seabirds. They are one of the most threatened seabird groups due primarily to human activities impacting upon breeding populations, and are used as keystone monitoring species for identifying changes in marine ecosystems. In this thesis, I describe the development and assessment of DNA dietary analysis as a non-invasive tool to evaluate and monitor threats to seabird populations posed by changing environmental conditions and interactions with commercial fisheries. To achieve this I have used albatrosses as a model to: 1) Examine the current methods used to assess diets and identify gaps in our knowledge and propose a framework for future diet monitoring studies; 2) Develop optimised scat collection protocols to ensure high quality dietary data is obtained; 3) Determine the importance of gelatinous prey in the diets of a seabird indicator species used for ecosystem monitoring; and 4) Assess the application of DNA metabarcoding to detect fishery discards in the diets of threatened seabirds across broad geographic ranges and determine implications for conservation and management. ii The first component of this thesis was a systematic review of the literature to identify the methods used to assess albatross diets. I investigated the spatial and temporal application of these methods and species studied to identify knowledge gaps. Most albatross studies have focused on the chick- rearing period, and diet during other breeding phases is comparatively poorly known. There was a pronounced shift over time in the preferred method of characterising diets, from the morphological examination of prey remains to stable isotope analysis of tissue. This shift has reduced the volume of detailed taxonomic information available from morphological studies. Additionally, there are few long-term dietary datasets available. This reduction in recent prey information and paucity of long- term studies impacts our ability to monitor broader changes in marine ecosystems and has implications for management of threatened albatrosses. DNA-based dietary analysis provides a potential method to fill some of these information gaps and provide high taxonomic resolution of prey. Shy albatross (Thalassarche cauta) were used as a case study to investigate how DNA amplification success and the proportion of food DNA detected are influenced by both environmental and physiological parameters. Albatross colonies are often remote and exposed; therefore it is unknown if dietary DNA can easily be obtained from scats in these conditions or during all breeding stages. A broad ranging universal PCR primer set enables identification of all major prey groups; however, this method also amplifies non-food DNA. Both the amount and type of non-target DNA varies with sample freshness, the collection substrate, fasting period and developmental stage of the consumer. I developed optimised scat collection protocols to enable high quality dietary DNA to be collected during all breeding stages. These will also minimise contamination issues from non-target DNA and provide standardised field methods in this rapidly expanding area of research. I was able to apply DNA metabarcoding to assess the diets of black-browed albatross at seven colonies across their species range through collaboration with a global network of researchers. This circumpolar species has suffered population declines due primarily to incidental mortality from commercial fisheries. They are used as an indicator species to identify changes in the overall species composition of an ecosystem by the Commission for the Conservation of Antarctic and Marine Living Resources (CCAMLR) Ecosystem Monitoring Program. As such, they provide an ideal model species to evaluate the use of DNA metabarcoding to monitor marine based threats. Albatross diets were examined at a low level of taxonomic resolution using universal primers to assess the importance of gelatinous prey. Diets are conventionally assessed from stomach content iii analyses which cannot easily detect soft-bodied prey. Such biases may impact our detection of important ecosystem regime shifts. Fish was the main dietary item at most sites, however scyphozoan jellyfish DNA was present in 37% of samples and up to 80% of samples at some sites. Warmer oceans and overfishing of finfish are predicted to favour jellyfish populations, therefore there is a need to review dietary assessment methods used for ecosystem monitoring. Future seabird monitoring programs should be designed to detect diet changes across the full prey spectrum, including jellyfish, so any potential impact on seabird breeding success and survival can be evaluated. Group-specific primers for bony fish were used to identify the diversity of fish prey consumed by black-browed albatross at five colonies and identify any overlaps with commercial fishery species (either target, bycatch or bait species). Across all sites, 51 fish species from 33 families were identified. There was extensive geographic variation but little inter-annual variability in fish species consumed. The prevalence of commercial fishery species detected in the diets of the albatross during the breeding season highlights that interactions and/or competition with fisheries are still ongoing for this species, particularly at the Falkland and Kerguelen Islands. This study highlights the potential value of DNA metabarcoding as a fishery resource management tool. This body of work has shown that DNA metabarcoding of seabird scats provides a non-invasive dietary method for identifying and monitoring marine based threats, which can be applied during all stages of the breeding season. The ability to detect gelatinous prey and the high taxonomic resolution delivered means it provides a valuable alternate or complementary dietary method for ecosystem monitoring and fishery resource management. The development of DNA dietary analysis techniques described in this thesis will enable researchers and conservation efforts around the world to obtain further information on ecosystem linkages. This will enable ongoing monitoring and evaluation of marine threats to seabird populations. iv Declaration of originality: This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief contains no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Julie McInnes 07/06/2017 Statement of authority of access: The publishers of the papers comprising Chapters 1 and 2 hold the copyright for that content. Access to the material should be sought from the respective journals. The remaining non-published content of the thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. Julie McInnes 07/06/2017 v Acknowledgements This PhD has been an incredible journey and many people have made this trip enjoyable and possible. Firstly, to my four amazing supervisors Simon Jarman, Rachael Alderman, Mary-Anne Lea and Ben Raymond; your support, knowledge and encouragement has been astounding, as has your tolerance of my puns. Thanks to Simon for his patience while I learned the ways of the geneticist and for always keeping his door open for me to discuss the project and whatever else was on my mind. I’m sorry that moving across the globe did not allow you to escape this. Thanks to Rachael for sharing her knowledge and passion for albatross and their conservation, and the weeks spent on Albatross Island sharing my ever-increasing excitement at the sound of an albatross producing data. Thanks to Mary-Anne for always providing opportunities to develop as a researcher and encouraging me to stand tall and