The Genetic and Physiological Characteristics of the Symbiodinium spp. in the endemic anemone Anthopleura aureoradiata Jennifer N Howe A thesis submitted to Victoria University of Wellington in partial fulfilment of the requirements for the degree of Masters of Science in Marine Biology 2013 Anthopleura aureoradiata ii Abstract Photosynthetic dinoflagellates of the genus Symbiodinium form symbiotic relationships with many marine hosts, including cnidarian corals and sea anemones. This partnership is extremely successful in tropical waters leading to a great diversity of coral species and Symbiodinium types. Environmental condition in the tropics are stable, changes to which can lead to destabilization of the symbiotic interactions between the host and symbiont, which in turn can lead to total breakdown of the partnership and expulsion of the symbiont. Temperate symbiotic cnidarian species, especially sea anemones, are less common but locally abundant. Environmental conditions are highly variable with extreme differences in light and temperature. Adaptation to these conditions has led to the success of resilient partnerships, but also to less diversity of Symbiodinium types. This study looked at the relationship between the endemic New Zealand anemone, Anthopleura aureoradiata, and its symbiotic relationship with the Symbiodinium cells it harbours. The aim was to determine why and how this symbiotic relationship is so resilient to the temperate conditions by 1) determining the molecular identity of the Symbiodinium spp. within the anemone, throughout its latitudinal range and through the seasons, and whether any seasonal changes differed between two habitats, the rocky shore and mudflats; 2) comparing the identity of the Symbiodinium spp. in New Zealand with those from four species of anemones from Europe (Cereus pedunculatus, Anthopleura ballii and Anemonia viridis from the south-west of England and Aiptasia mutabilis from Brittany (France)) to establish any differences or similarities between the northern and southern hemispheres; 3) determining whether resilience to environmental conditions is attributed to the Symbiodinium photoprotective mechanisms. A. aureoradiata were collected in early autumn in five sites from the top (Parengarenga Harbour) to the bottom (Stewart Island) of New Zealand for the latitudinal study. Seasonal anemones were collected from a rocky shore in Wellington Harbour (Point Halswell, Kau Bay) and a mudflat at Pauatahanui Inlet. Symbiodinium types were identified to subcladal level using ITS2 sequencing. A low diversity of types was found, with all anemones harbouring algal cells identified as being similar, or identical to, Symbiodinium sp. Mediterranean clade A (Med clade A) and Symbiodinium sp. Amed iii (Amed). 96.55% of the anemones from the latitudinal study, all the winter anemones, 87.50% of the summer anemones and almost 78% of the autumn anemones harboured Symbiodinium cells most similar or identical to Med clade A. All Symbiodinium sequences from the European anemones also were identified as being similar or identical to Med clade A or Amed, suggesting that the Symbiodinium in A. aureoradiata are likely not endemic. It is not known whether anemones harbour both types simultaneously and whether a change in dominant symbiont type occurs with seasons within anemones by “shuffling”. The photophysiology of the Symbiodinium cells isolated from the anemones was studied using an Imaging-PAM fluorometer whilst being maintained in six light and temperature treatments. The photosynthetic rate of PSII, energy quenching by NPQ, and photosystem recovery were measured to determine whether the Symbiodinium cells had a strong capacity for photoprotection and were able to down-regulate quickly to reduce photodamage to the chloroplast. The main outcome of this study is that the Symbiodinium cells within A. aureoradiata are very effective in protecting themselves against photo-damage by activating an efficient NPQ system. Down-regulation of the quantum efficiency of PSII under high light conditions appeared to cease altogether. Whether this was a true measurement of down-regulation to stop photodamage, or whether these clade A types use an alternative electron transport that bypasses PSII, and can therefore not be measured with the I-PAM fluorometer technique used, needs to be addressed in future studies. iv Declaration STATEMENT OF ORIGINALITY The following work presented in this thesis is, to the best of my knowledge, original. It is a representation of my own work throughout my Masters of Science course, except as acknowledged throughout the text. All figures presented represent original work unless otherwise stated. The contained material has not been submitted for any degree at this or any other institution. ---------------------------------------- ------------------------------------------- Candidate’s Signature Date ---------------------------------------- ------------------------------------------- Supervisor’s Signature Date v Acknowledgements There are many people I would like to thank who helped me with my work. First on the long list are my friends and colleagues from the Davy Lab. For taking me on and helping greatly with the production of this thesis, I thank Dr Simon Davy. For getting me started on the molecular work, I‟m extremely grateful to Dr Paul Fisher. Thanks to Steffi Pontasch, for giving me so much of her time to answer so many of my questions, and always with a smile. Thanks to Dr Suzanne Becker, Thomas Krueger and Shaun Wilkinson for clarifying the mysteries of the I-PAM, „trees‟ and statistics. Thanks to all my anemone collecting buddies from the lab, especially Emma Gibbin, who travelled up the North Island with me, and Steffi, who spent many hours crawling over the rocks at Point Halswell. Special thanks though to the most intrepid of the anemone hunters, Anne Wietheger, who travelled south with me to Stewart Island, and who also could be relied upon to face the dawn on my many, early morning collection trips. Fun times! Thanks to Dr Ross Hill, The University of New South Wales, who gave me advice on how to use DCMU on my anemones and to Dr Xavier Pochon, Cawthron Institute, for looking through my sequences and advising me on the structure of the genetic trees to use. Thanks to the technicians in the School of Biological Sciences, especially Neville Higgison, who rewired a room and helped in supplying much of the electrical equipment needed. I would not have been able to make a comparison of the New Zealand Symbiodinium types with the European ones without the assistance of Dr Keith Hiscock from the Marine Biological Association in Plymouth, England, who collected all the English anemones, and Laurent Leveque and Sylvie Tanguy from the Station Biologique de Roscoff, Brittany. Financial assistance was given to me by Victoria University with the Graduate Scholarship and the Masters (by Thesis) Scholarship, for which I am exceedingly grateful. Also thanks to the grandparents of Alison Morton, for the scholarship they have set-up in her memory, and to the Wellington Branch, New Zealand Federation of Graduate Women for the Masters by Thesis Scholarship awarded to me. vi My biggest thanks has to go to my family, Chris, Liam and Adam. My boys have put up with having an absent mother for too long. Without Chris this long slog would have been tremendously difficult. Life was made a lot easier with his support. Thank you; it‟s time to show the three of you my appreciation. vii Table of Contents ABSTRACT III DECLARATION V ACKNOWLEDGEMENTS VI Table of Contents ............................................................................................................................... viii List of Figures...................................................................................................................................... xiii List of Tables ....................................................................................................................................... xvi Abbreviations ....................................................................................................................................xviii CHAPTER 1 – INTRODUCTION 1 THE CNIDARIAN-DINOFLAGELLATE SYMBIOSIS 1 1.1 Symbiosis ......................................................................................................................................... 1 1.1.1 Definition and types of symbiosis 1 1.1.2 Cnidarian-dinoflagellate symbiosis 3 1.2 Cellular basis of cnidarian-dinoflagellate symbiosis ......................................................................... 6 1.2.1 Establishment of the symbiosis 6 1.2.2 Maintenance of the symbiosis 7 1.2.3 Breakdown of the symbiotic partnership 8 1.3 Genetic diversity of Symbiodinium................................................................................................. 10 1.3.1 Molecular identification within the genus Symbiodinium 10 1.3.2 Biogeographic and ecological variability in diversity 13 1.4 Photophysiology of Symbiodinium ................................................................................................. 15 1.5 Tolerance and acclimation to environmental stress by change of symbiont .................................. 21 1.6 Temperate vs tropical .................................................................................................................... 23 1.7 The study organism –Anthopleura aureoradiata ........................................................................... 25 viii 1.8 Aims and