Gastrointestinal Microbiota of Seabirds

Meagan Dewar

Bachelor of Science, (Marine Biology) (BSc) Bachelor of Applied Biology (Hons)

Submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Deakin University

28th September, 2012

“Earthworms are more important than humans. The fact is that earthworms are more important than people from an ecological perspective for the simple reason that earthworms can survive without people but people will not be able to survive without earthworms. People are dependent on earthworms; earthworms are not dependent upon humans.

Worms can live on the planet without people; people can’t live on the planet without worms. Honeybees are more important than people, bacteria are more important than people – any species that is actually a foundation species which allows for the other species to survive is more important than the species up top.

The fact is that the ecological law of diversity dictates that diversity of species is essential for the survival of each species within that diversity. Humans are equal to other species for the simple reason that if the other species were removed from the eco-systems that people inhabit, humans could not survive."

Captain Paul Watson, Sea Shepherd

ACKNOWLEDGEMENTS

Over the past 4.5 years a lot of blood, sweat and tears have gone into achieving one thing, this thesis, and now that journey must come to an end. Throughout this time there have been numerous people who have helped me achieve my goal. First and foremost, I would like to thank my supervisors Dr Stuart Smith and Associate Professor John Arnould for this opportunity. I wish to thank Stuart for his encouragement, guidance, expertise and support over the past 4.5 years. Your assistance has been instrumental in achieving my goal. I wish to thank John for his advice, expertise and guidance. Your support has also been instrumental in helping me achieve my goal. I would also like to thank John Reynolds for your statistical assistance and advice.

I would also like to thank my collaborators Dr Peter Dann (Phillip Island Nature Parks), Dr Phil Trathan (British Antarctic Survey) and Dr Rene Groscolas (Centre National de la Recherche Scientifique) for your logistical support, sample collection and editorial assistance. I would also like to thank the support crew from BAS; Stacey Adlard and Fabrice Le Bouard and CNRS; Vincent Viblanc, Nelly Mallosse, Goetz Eichhorn and René Groscolas for assisting with sample collection from Possession and Bird Island.

I would also like to acknowledge my funding partners Holsworth Wildlife Research Endowment, Molecular Nutrition Group Strategic Research Centre (Deakin University), Phillip Island Nature Parks, Snowtown Valley, the French Polar Institute (IPEV) (project N° 119) and the British Antarctic Survey. Without your financial assistance this project would not have been possible.

A special thank you to Dr Tiana Preston, Dr Louise Kelly, Joel Maloney, David, Kaye & Belinda Dewar for their assistance in the field at Phillip Island and to Nicole Schuman, Marcus Salton and Julie Baxter for their assistance in the field on Notch Island. Also to Tiana and Louise thank you for all the great penguin nights on the St Kilda breakwater and thank you for your support and advice throughout this time.

To the Hutchesson family, Damien, Vanessa, Max and Oliver, thank you for your support, endless trips to the zoo, photography outings and the endless laughter. To my dearest friends Kathryn Trevaskis, Sarah Troia, Dimi Paraskevas, Suree Morrison and Marty Durkan I want to thank you for your love and support over the years and iii thank you for the many nights of gossip and laughter, but most of all thank you for your friendship. To my beautiful angels Bevan, Remy, Luca and Dylan, you have brought so much joy and happiness to my life. You always know how to make your Aunty Megs laugh, so thank you for making the hard times so much easier. To my family (Aunties, Uncles and Cousins), thank you for your support and believing in me over the years it has meant the world to me.

To my sister, thank you believing in me, for your support, our wonderful holidays together and for putting up with me over the past 4.5 years. I know at times it hasn’t been easy. But thanks.

To the Deakin crew Amy, Aimee, Sheena, Marissa, Paul and Lisa, thank you for sharing this journey with me. Thank you for your support, endless gossip sessions and daily coffee breaks.

To my animals, Kitty, Claw, Pepper, Jessie, Sultan and Sebastian, you have been my source of comfort and joy over the past 23 years. I will never forget your endless love. Rest in Peace. To my new kitty, Nala, thank you for your love and cuddles these past few months.

Finally, to the two most important people, my parents David and Kaye Dewar, without you this dream would never have become a reality. Thank you so very much for all the sacrifices you have made throughout my life to help me achieve my dreams. Thank you for your enduring love, support and belief in me.

I dedicated this thesis to my parents: David and Kaye Dewar.

Thank you for everything, without you this would not have been possible.

PUBLICATIONS FROM Ph.D.

Dewar, M.L. Arnould, J.P.Y. Dann, P. Trathan, P. Groscolas, R and Smith, S.C. (2012). Inter-specific variations in the gastrointestinal microbiota of penguins. MicrobiologyOpen (Early View) http://onlinelibrary.wiley.com/doi/10.1002/mbo3.66/full

FUTURE PAPERS FROM THESIS

Dewar, M.L. Arnould, J.P.Y. Dann, P. Reynolds, J. Smith, S.C (2012) Variations in the Gastrointestinal Microbiota of Little Penguins (Eudyptula minor) During Development (In Preparation)

Dewar, M.L. Arnould, J.P.Y. Trathan, P. Dann, P. Smith, S.C (2012) Influence of Fasting During Moult on the Gastrointestinal Microbiota of Penguins. (In Preparation)

Dewar, M.L. Arnould, J.P.Y. Dann, P. Smith, S.C (2012) Inter-Specific Variations in the Gastrointestinal Microbiota of Procellariiformes (In Preparation)

Dewar, M.L. Arnould, J.P.Y. Dann, P. Reynolds, J. Smith, S.C (2012) Variations in the Gastrointestinal Microbiota of Short-Tailed Shearwaters (Puffinus tenuirostris) During Development. (In Preparation)

OTHER PUBLICATIONS

Dewar, M.L., and Scarpaci, C. (2011). Microbial implications associated with stomach flushing of Little Penguins. The Victorian Naturalist 128 (4), 2011, 128–131

Saunders, S. Smith, S.C. Oppedisano, F. Dewar, M.L. Tang, M. (2012) Alteration of the intestinal microbiota by Lactobacillus rhamnosus GG and Peanut Oral Immunotherapy (P-POIT) in children with peanut allergy. Gut Pathogens (In preparation)

Smith, S.C. Chalker, A. Dewar, M.L. Arnould, J.P.Y (2012). Age-related differences revealed in Australian fur seal Arctocephalus pusillus doriferus gut microbiota. FEMS Microbiology Ecology (In Review)

COMMUNICATIONS

Oral Communications of PhD results

Dewar, M.L., Arnould, J.P.Y., Dann, P., Trathan, P., Smith, S.C. (2012). Variations in the Gastrointestinal Microbiota of Moulting and Non-Moulting Penguins. International Polar Year: Knowledge to Action. Montreal Canada 22-27 April 2012

Dewar, M.L., Arnould, J.P.Y., Trathan, P., Smith, S. (2011). Microbial Ecology of Penguins in Extreme Polar Regions. CAREX Life in Extreme Environments. 18-20th October 2011

Dewar, M.L, Arnould, J.P.Y., Trathan, P., Groscolas, R., Smith, S.C. (2010). Gastrointestinal microbiota of King Penguins (Aptenodytes patagonicus) and the Influence of geographical separation. 7th International Penguin Conference Abstracts. Boston, Massachusetts. 30 August – 3rd September 2010

Dewar, M.L., Arnould, J., Dann, P., Trathan, P., Groscolas, R., Smith, S.C. Exploring the gastrointestinal microbiota of king and little penguins using high throughput sequencing. (2010) 7th Annual Exercise and Nutrition Sciences Higher Degrees by Research Student Symposium, Deakin University Melbourne Australia. (Awarded best presentation)

Poster Presentation of PhD results

Dewar, M.L., Arnould, J.P.Y., Dann, P., Trathan, P., Groscolas, R., Smith, S. (2010). Changes in the gastrointestinal microbiota during fasting in penguins. 1st World Seabird Conference. Victoria, British Columbia, 7th -11th of September 2010 (Poster)

Dewar, M.L., Arnould, J.P.Y., and Smith, S. (2009). Gastrointestinal Tract of Little penguins. Seabird Group 10th International Seabird Conference. Brugge, Belgium 27th -30th March 2009 (Poster).

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ABSTRACT

The gastrointestinal (GI) tract contains a highly diverse and complex microbial ecosystem that consists of residential and transient microbes. At birth, the GI tract in avian species is devoid of microorganisms. Immediately after birth however, the GI microbiota is rapidly colonised by microbes. The GI microbiota plays a significant role in gut development and maturation, host biochemistry, physiology, metabolism, synthesis and secretion of essential vitamins, minerals and nutrients, energy extraction, fat metabolism and storage. To date, studies on the avian GI microbiota have primarily been focused on poultry, with a limited number of studies conducted on avian species living under natural conditions. This thesis aimed to address this knowledge gap through a study on the gastrointestinal microbiota of seabirds using faecal samples collected via cloacal swabs. The study was designed to characterise the gastrointestinal microbiota of marine seabirds under natural conditions and to examine the variation in microbial composition during development and to examine the influence of fasting during moult on the GI microbiota. The study species in this thesis included; king (Aptenodytes patagonicus), gentoo (Pygoscelis papua), macaroni (Eudyptes chrysolophus) and little penguin (Eudyptula minor), short-tailed shearwater (Ardenna tenuirostris), fairy prion (Pachyptila turtur) and common diving petrel (Pelecanoides urinatrix). DNA extracted from faecal samples was analysed using quantitative Real Time PCR (qPCR) and 16S rRNA pyrosequencing. Quantitative data obtained from the qPCR analysis highlighted significant differences in the abundance of Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria between all penguins (Chapter 2), Procellariiformes (Chapter 5), during development of little penguins (4) and in fasting penguins (study 4). No significant differences were observed during development in short-tailed shearwaters. The pyrosequencing analysis identified that the penguin microbiota was dominated by Firmicutes, Bacteroidetes, Fusobacteria and Proteobacteria. However there were significant differences in the relative abundance of the major phyla. There was a relatively high level of similarity between gentoo and macaroni penguins. In common diving petrels (CDP) the microbiota was dominated by Firmicutes, Bacteroidetes and Proteobacteria, whereas the microbiota of short-tailed shearwaters (STS) and fairy prions (FP) was dominated by Firmicutes. There was a high level of similarity between the oil producing seabirds (STS & FP) and a low level of similarity between the oil producing (STS & FP) and non-oil producing (CDP) viii seabirds. Potential factors influencing the microbiota of penguins are most likely to be associated with diet, prey associated microbiota and host phylogeny, whereas the microbiota of Procellariiformes are most likely to be associated with digestive physiology (production of stomach oil, digestive anatomy) and host phylogeny. The development of the seabird microbiota, differs greatly between species (little penguin & short-tailed shearwater). In the little penguin, there were significant differences in the microbial composition throughout development and a significant upward trend in the abundance of Firmicutes and Bacteroidetes. In the short-tailed shearwater, however, there were no significant differences throughout development and appears relatively stable throughout development with a high level of similarity between each age class. In addition, fasting during moult significantly influenced the microbial composition of both king and little penguins, with the degree to which moult impacts the microbial composition differing between the two species. In little penguins, there is still a high level of similarity between early and late moult (60%), with little variation in the abundance of the major phyla observed with both the qPCR and pyrosequencing analysis. In contrast, there were significant differences between early and late moult with a 75% increase in the level of Fusobacteriaceae. In addition, there was a low level of similarity (20%) between early and late moult in king penguins. This research provides a baseline for future work regarding the microbial composition of seabirds. This thesis has identified that a complex community inhabits the GI tract of seabirds and provides insight into the successional changes that occur during development and how fasting influences the microbial composition.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS iv ABSTRACT ix LIST OF FIGURES xiv LIST OF TABLES xvi CHAPTER 1 1 Introduction 1 Gastrointestinal microbiota 1 Role of Gastrointestinal Microbiota 1 Gastrointestinal Physiology 1 Immune System 2 Metabolism & Adiposity 2 Establishment of Gastrointestinal Microbiota 3 Factors Influencing Gastrointestinal Microbiota 5 Gastrointestinal Microbiota & Fasting 6 Avian Gastrointestinal Microbiota 7 Spheniscidae Taxonomy and Distribution 8 King Penguin (Aptenodytes patagonicus) 9 Macaroni Penguin (Eudyptes chrysolophus) 10 Gentoo Penguin (Pygoscelis papua) 11 Little Penguin (Eudyptula minor) 12 Procellariiform Taxonomy and Distribution 14 Family Procellariidae 15 Short-tailed Shearwater (Ardenna tenuirostris) 15 Fairy Prion (Pachyptila turtur) 16 Family Pelecanoididae 17 Common Diving Petrel (Pelecanoides urinatrix) 17 Penguins as a model of Avian Gastrointestinal Microbiota 17 Procellariiformes as a model of Avian Gastrointestinal Microbiota 18 Research Objectives and Thesis Structure 26 Preface 27 CHAPTER 2 28 Inter-specific variations in the gastrointestinal microbiota of Penguins 28

Introduction 29 Methods 32 Results 35 Discussion 45 CHAPTER 3 49 Variations in the Gastrointestinal Microbiota of Little Penguins 49 (Eudyptula minor) During Development Introduction 50 Methods 51 Results 55 Discussion 64 CHAPTER 4 67 Influence of Fasting During Moult on the Gastrointestinal Microbiota 67 of Penguins Introduction 68 Methods 69 Results 71 Discussion 82 CHAPTER 5 85 Inter-Specific Variations in the Gastrointestinal Microbiota of 85 Procellariiformes Introduction 86 Methods 87 Results 89 Discussion 97 CHAPTER 6 101 Variations in the Gastrointestinal Microbiota of Short-Tailed 101 Shearwaters (Puffinus tenuirostris) During Development Introduction 102 Methods 103 Results 105 Discussion 116 CHAPTER 7 119 General Discussion 119

CHAPTER 8 126 Future Studies 128 Bibliography 134 Appendix 153 Appendix 1, Taxonomic breakdown of bacteria present in the GI 153 microbiota of penguins Appendix 2, Taxonomic Breakdown of bacteria present in little penguins 165 during development Appendix 3, Taxonomic Breakdown of bacteria present in moulting 173 penguins Appendix 4, Taxonomic Breakdown of bacteria present in Procellariiform 185 Seabirds. Appendix 5, Taxonomic Breakdown of bacteria present in short-tailed 196 shearwaters during development.

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LIST OF FIGURES

Figure 1.1, King Penguin 9 Figure 1.2, Macaroni Penguin 11 Figure 1.3, Gentoo Penguin 12 Figure 1.4, Little Penguin 13 Figure 1.5, Short-tailed Shearwater chick 16 Figure 2.1 Variation in the abundance of the major phyla; Firmicutes, 37 Bacteroidetes, Proteobacteria and Actinobacteria in king, gentoo, little and macaroni penguins Figure 2.2, Similarity of the major bacterial phyla of penguin species 38 using qPCR. Figure 2.3 Relative abundance of major phyla from the GI microbiota of 40 gentoo, king, little and macaroni penguins Figure 2.4, MDS Analysis of the gut microbiota of penguin species 41 Figure 2.5, Analysis of Similarity of the GI microbiota of gentoo, king, 42 little and macaroni penguins Figure 2.6, Relative abundance of major families from the GI microbiota 43 of gentoo, king, little and macaroni penguins. Figure 2.7, Variation in species diversity in king, gentoo, little and 44 macaroni penguins. Figure 3.1, Artificial nest box used by little penguin in this study 52 Figure 3.2, Adult little penguins and chicks in nesting box 53 Figure 3.3, Changes in the abundance of the major phyla; Actinobacteria 57 (a), Bacteroidetes (b), Firmicutes (c) and Proteobacteria (d) during development in little penguin chicks. Figure 3.4, Similarity of the major bacterial phyla in little penguins 58 during development using qPCR. Figure 3.5, Relative abundance of major phyla during development in 60 little penguin chicks. Figure 3.6, Similarity of the gut microbiota of little penguins during 61 development. Figure 3.7, Relative abundance of major Families during development in 62 little penguin chicks. Figure 3.8, Variation in species diversity during development and in 63 adult little penguins Figure 4.1, Variation in the abundance of the major phyla; Firmicutes, 72 Bacteroidetes, Proteobacteria and Actinobacteria during moult in little penguins. Figure 4.2, Variation in the abundance of the major phyla; Firmicutes, 73 Bacteroidetes, Proteobacteria and Actinobacteria during moult in king penguins. Figure 4.3, Similarity of the major bacterial phyla in little penguins 74 during moult using qPCR. Figure 4.4, Similarity of the major bacterial phyla in king penguins 75 during moult using qPCR. Figure 4.5, Relative abundance of major phyla in moulting little penguins 77 using 16S rRNA pyrosequencing Figure 4.6, Relative abundance of major families from the GI microbiota 78 of moulting little penguins

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Figure 4.7, Relative abundance of major phyla in moulting king penguins 80 using 16S rRNA pyrosequencing Figure 4.8, Relative abundance of major families from the GI microbiota 81 of moulting king penguins. Figure 5.1, Variation in the abundance of the major phyla; Firmicutes (a), 90 Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) in Common Diving Petrels, Fairy Prions and Short-tailed Shearwaters Figure 5.2, Similarity of the major bacterial phyla in Procellariiform 91 seabirds using qPCR. Figure 5.3, Relative abundance of major phyla in three Procellariiform 93 Seabirds; a) Common Diving Petrels, b) Fairy Prions, and c) Short-tailed Shearwaters Figure 5.4, Cluster Analysis of the GI microbiota Common Diving 94 Petrels (CDP), Fairy Prions (FP) and Short-tailed Shearwaters (STS). Figure 5.5, Relative abundance of major Families in common diving 95 petrels, fairy prions and short-tailed shearwaters. Figure 5.6, Species diversity of Procellariiformes Microbiota 96 Figure 6.1, Short-tailed shearwater breeding colony. Sticks represent nest 106 sites used in this study Figure 6.2, Natural burrow of short-tailed shearwater 107 Figure 6.3, Short-tailed shearwater chick 108 Figure 6.4, Changes in the abundance of the major phyla; Firmicutes (a), 109 Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) during development in short-tailed shearwater chicks. Figure 6.5, Similarity of the major bacterial phyla in short-tailed 110 shearwater chicks during development using qPCR Figure 6.6, Relative abundance of major the phyla in short-tailed 112 shearwater chicks during development Figure 6.7, Similarity of the gut microbiota of short-tailed shearwater 113 chicks during development Figure 6.8, Relative abundance of major Families during development in 114 short-tailed shearwater chicks. Figure 6.9, Species diversity at different age classes 115 Figure 7.1, MDS/Cluster analysis of seabirds 121 Figure 7.2, Relative abundance of the major phyla for all seabird species 122 using 16S rRNA pyrosequencing

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LIST OF TABLES

Table 1.1, Metabolites contributed by gut bacteria 4 Table 1.2 Microbial compositions of avian species from previous 16S 8 rRNA studies Table 1.3, Bacteria isolated from penguins from previous studies 19 Table 1.4, Microorganisms isolated from Procellariiform seabirds from 23 previous studies Table 2.1 Quantitative real time PCR primer sequences used in this 34 study to detect and quantify major phyla present in penguin faeces. Table A1, Taxonomic breakdown of bacteria present in the GI 153 microbiota of penguins Table A2, Taxonomic Breakdown of bacteria present in little penguins 165 during development Table A3.1, Taxonomic Breakdown of bacteria present in moulting 173 little penguins Table A3.2, Taxonomic Breakdown of bacteria present in moulting 178 king penguins Table A4, Taxonomic Breakdown of bacteria present in Procellariiform 185 Seabirds. Table A5 Taxonomic Breakdown of bacteria present in short-tailed 196 shearwaters during development.

CHAPTER 1 INTRODUCTION

Gastrointestinal Microbiota The gastrointestinal (GI) tract contains a highly diverse and complex microbial ecosystem that consists of residential and transient microbes [67, 143, 163, 257]. The GI microbiota was once considered to be a “collection of freeloading commensals” [162] that did not provide benefit to their host. However, recent studies have identified that a complex and mutually beneficial relationship exists between a host and its microbiota. This complex relationship is said to be the result of a long and complex coevolution [154]. The GI microbiota plays a considerable role in gut development and maturation, host biochemistry, physiology, metabolism, synthesis and secretion of essential vitamins, minerals and nutrients [168, 180, 299].

Role of Gastrointestinal Bacteria The use of germ free animals has highlighted the importance of the GI microbiota to its vertebrate host. These studies have shown that the GI microbiota has a profound influence on the nutritional, physiological, immunological and metabolic processes of the host [168, 299], playing a significant role in energy harvest, fat metabolism, secretion and synthesis of nutrients, vitamins, amino acids and the production of short chain fatty acids from the diet consumed [67, 94, 248, 257, 267]. In the absence of bacteria the GI tract does not fully mature and an animal’s ability to secrete and absorb essential vitamins and nutrients from their diet is significantly reduced [25, 199, 214, 215]. Germ free animals are not only more susceptible to disease, but also require a greater caloric intake to achieve and maintain a normal body weight [9]. This indicates that members of the GI microbiota modulate fat deposition [185].

Gastrointestinal Physiology The use of germ free models such as mice; chickens and zebrafish have revealed a range of functions that are influenced by the presence of a commensal microbiota. Rawls et al [215] used zebrafish, to examine the influence of the GI microbiota on the development of the GI tract. Zebrafish are a useful germ free model to examine the influence of the GI microbiota on gut development. Firstly, the structure of the GI tract is similar to that of human hosts and secondly because they are completely transparent during the fry and juvenile stages. When examining the GI tract of germ

1 free Zebrafish, Rawls et al [215] noted that in the absence of a commensal GI microbiota, the epithelium of the GI tract is arrested in aspects of differentiation. In addition, the absence of a GI microbiota reduces a host’s ability to secrete and absorb essential nutrients and extract energy from dietary sources [38].

Immune System The GI microbiota has also been shown to be responsible for the establishment and maintenance of the host’s immune system through its effect on gut morphology, nutrition, and pathogenesis of intestinal diseases [257]. The indigenous microbiota also suppresses the colonisation of invading bacteria which provides protection to the host against pathogenic species [143, 159, 240] and can prevent enteric infection by secreting defensins, while phagocytes and M cells continually survey the microbiota in the lumen for potential pathogens. In recent studies, microbial components of the indigenous microbiota have been shown to stimulate the immune system [215].

Metabolism & Adiposity Animal studies have demonstrated that the gastrointestinal microbiota has a profound influence on the modulation of host metabolism. The GI microbiota appears to be able to modify a number of lipids in serum, adipose tissue and in the liver, with drastic effects on triglycerides and phosphatidylcholine. The resident microbiota is also responsible for the production of metabolites that contribute to host fitness and survival [132]. Some of these metabolites are listed in table 1.1 below.

There have been multiple studies linking the presence of gut bacteria to adiposity, which have noted that changes to the microbial composition can significantly increase the level of adiposity experienced by the host causing obesity [9, 89, 126, 152, 156, 177, 180, 213, 260, 263]. When germ free animals are inoculated with the GI microbiota of conventionally raised hosts, the body fat level of the germ free host rapidly increases, despite decreased food intake [9]. Conventionalisation of germ free mice increases the production of SCFA’s, which are absorbed and metabolised by the body and supresses the expression of fasting induced adipose factor (Fiaf). The suppression of Fiaf, leads to increased enzymatic activity of lipoprotein lipase (LPL), increasing the uptake of lipids into adipocytes and other tissues [38]. In addition, both the GI microbiota and host genetics have been identified as risk factors in the development of obesity. Research has indicated that obesity is associated with a 2 higher proportion of Firmicutes and a decreased proportion of Bacteroidetes increasing energy production [9, 153, 154, 263]. Turnbaugh et al [264] documented that members from the Phylum Firmicutes were associated with the breakdown of complex carbohydrates, polysaccharides, and sugars into simple sugars and fatty acids which are then absorbed and utilised as an energy source by the host. In chickens Panda et al [196] documented that the supplementation of diet with butyrate increased their overall body mass. As butyrate is a SCFA derived from microbial fermentation, this observation indicates that butyrate producing microbes could contribute to host adiposity.

Establishment of Gastrointestinal Microbiota In nature, bacteria are found everywhere and with the availability of nutrients are able to colonise new habitats, including the GI tract [6]. At birth, the GI tract is considered to be devoid of microorganisms. In mammals, the microbial community is said to be inherited from the mother through intimate contact with faecal and vaginal microbes during birth and from breast milk [194]. In birds, however, new born chicks, acquire their microbiota from the surface of the egg, surrounding environment (i.e. nest) and their first meal [6, 99, 101, 159, 162, 217, 256, 291]. In addition, vertical transmission of microbes via regurgitation of an undigested or partially digested meal is also said to influence the microbiota of the chick [110, 142, 205]. Through ecological succession, the sterile GI tract, is rapidly colonised forming a dense and complex microbial community within 2 years in humans [164, 166, 168, 199, 240, 268] and 40 days in chickens [16, 17, 68, 90, 217].

Table 1.1, Metabolites contributed by Gut Bacteria (table modified from Nicholson et al [182]).

Metabolites Related Bacteria Potential Biological Ref er en ce Funct i o n Sh o r t - c h a i n f a t t y Clostridial clusters IV Decreased colonic pH, [39, 196, 231, acids: acet ate, and XIVa of inhibit growth of 288] propionate, butyrate, Firmicutes, including pat hogens; st imulat e isobutyrate, 2- species Eubacterium, water and sodium methylpropionate, Roseburia, absorption; cholesterol valerate, isovalerate, Faecalibacterium synthesis; provide energy hexonate to the colonic epithelium cells; implicated in obesity Bile acids Lactobacillus, Absorb diet ary fat s and [117, 223, Bifidobacteria, lipid soluble vitamins, 251] Enterobacteria, facilitate lipid absorption, Bacteroides, maintain intestinal barrier Cl o st r i u m function, regulate triglycerides, cholesterol, glucose and energy homeostasis Indole derivatives Clostridium Protect against stress- [20, 139, 292] sporogens, E.coli induced lesion in the GI tract, modulate expression of proinflammatory genes, increase expression of ant i-inflammatory genes, and strengthen epithelial cell barrier properties. Implicated in GI pathologies brain-gut axis, and a few neurological conditions Lipids: conjugated Campylobacter Impact intestinal [43, 235] fatty acids, LPS, jejuni, Clostridium permeability, active peptidoglycan, saccharolyticum, intestine-brain-liver acylglycerols, Bifidobacterium, neural axis to regulate cholest erol, Roseburia, Klebsiella, glucose homeostasis, LPS triglycerides Enterobacter, induces chronic syst emic Citrobacter, inflammation, enhance Cl o st r i d i u m the immune system, alter lipoprotein profiles

At birth, the neonate gut is an aerobic environment that is rapidly colonised by aerobic and facultative anaerobes such as Escherichia coli and Streptococci [5, 68, 168, 217, 268]. Within the first few days both E. coli and Streptococci are present in extremely high numbers of around 108 – 1010 CFU of faeces [168]. Research suggests that these pioneering bacteria reduced the amount of oxygen present in the GI tract through oxidation/reduction, creating an environment that is highly favourable to facultative and obligate anaerobes [168, 199] such as Bacteroides, Clostridia, and Bifidobacterium [168, 245].

Although the microbial succession in the developing vertebrate has been studied in many animals, including humans, chickens, and pigs [168] to date, little is known about the microbial succession in the developing seabird.

Factors Influencing Gastrointestinal Microbiota Throughout life, the GI tract is continuously exposed to microbes from the surrounding environment, diet and through colonial living [87, 262, 299]. In addition, other factors such as host phylogeny, physiology, gut morphology, nesting material, diet, prey associated microbiota, antibiotic use and weight influence the microbial composition [73, 87, 88, 131, 153, 154, 181].

In a revolutionary study on the microbial composition of 60 species of terrestrial mammals, Ley et al [154] discovered that host phylogeny, diet and gut physiology influenced the composition [154, 262]. In addition, Ochman et al [186] and Yildrim et al [290] also showed that evolutionary lineages determine the composition of the gut microbiota of a vertebrate species. However, more recent evidence points to diet being the main driving force behind differences in the gut microbiota of phylogenetically similar hosts [178, 181]. In accordance with Ley et al [154], Nelson [181] identified that both host phylogeny and diet influence the gut microbiota of Antarctic seals. In addition, Banks et al [12] documented that host phylogeny was a major driving force behind differences in the GI microbiota of adéliepenguins. These differrences however, need to be examined in seabirds in more detail.

During development, nesting environment has been identified as a major source of microbial colonisation avian species [162, 256]. Lucas and Heeb [162] discovered that when blue tit chicks were fostered in to the nest of great tits the blue tit chick

5 acquired the bacterial microbiota of their foster siblings. Whilst Torok [256], identified that the microbial composition of the litter (nest) material influenced the microbial composition of chickens during the first week of development.

Whilst host environment and phylogeny have been shown to influence the microbial composition of the infant, diet factor influencing the microbial composition throughout life [140]. Dietary changes modify the composition of the GI microbiota by altering the intestinal environment and metabolic activity of the GI tract. When examining the microbiota of 60 mammals Ley et al [153, 154] identified that individuals clustered together according to their dietary preference (i.e. herbivore, carnivores, omnivore). In addition, Blanco et al [24] identified significant changes in the microbial composition of red kites in response to changes in diet, within an increase in pathogenic species of bacteria Thus diet influences GI microbiota profoundly.

Gastrointestinal Microbiota and Fasting Previous studies examining the effect of fasting on the GI microbiota of vertebrate (hamsters, python, mice, termites), have shown that fasting not only alters the composition and diversity of the GI microbiota, and influence the host’s immune defences [73, 74, 241]. Research has also shown that complex interrelationships exist between a host, its GI microbiota and the hosts nutritional status, diet and physiological state [73, 74]. These studies however, have concentrated on animals that are relatively inactive during times of nutrient deprivation. Unlike other vertebrates, penguins do not hibernate during times of fasting. In penguins fasting occurs during the breeding season (incubation of eggs and chick brooding) and moult, where penguins replace their entire plumage whist fasting on land. The moulting fasts can last anywhere from 2 to 5 weeks. Therefore, penguins must survive long periods of starvation while also coping with increased metabolic demands for feather synthesis and thermoregulation [102, 281]. Therefore, penguins provide an attractive model for investigating the influence of fasting that is associated with increased metabolic demands. To date the influence of fasting on the microbiota of a seabird has not been examined.

Avian Gastrointestinal Microbiota In comparison to mammals, there is limited information available on the avian microbiota with the exception of poultry. Most avian based studies on microbial composition have used selective culture based techniques to investigate specific bacterial populations and/or to identify the presence of known human and veterinary pathogens [142, 158]. From the limited number of studies conducted on avian species using 16S rRNA sequencing to profile the GI microbiota, has identified similarities between mammals and birds especially at the higher taxonomic level (phylum). However, at lower taxonomic levels (genus and species) there are significant differences, with avian specific genera and species being identified [101, 142, 158, 160, 161]. From these 16S rRNA studies it has been identified that the avian GI microbiota is dominated by Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Tenericutes; however the relative abundances of these phyla differ from species to species as shown in table 1.2.

Table 1.2 Microbial compositions of avian species from previous 16S rRNA studies (table modified from Kohl [142])

Phyla Adélie Gull Canada Turkey Chicken Parrot Penguins Geese Firmicutes 39.2 54.6 71.6 32.3 70 72.9 Bacteroidetes 14.7 1.1 10.1 54.2 1.9 0.2 Actinobacteria 32.3 6.4 7.0 <0.1 4.9 12.0 Proteobacteria 9.8 23 10.4 3.4 21.5 14.9 Tenericutes 3.9 8.9 0.2 <0.1 1.6 14.9 Reference [12] [161] [160] [234] [297] [289]

Spheniscidae Taxonomy and Distribution

The family Spheniscidae are a group of flightless seabirds, containing 16-19 species [76, 247]. Penguins occupy an extensive range including Antarctica, the Galapagos Islands, South Africa, Australia, New Zealand and South America [27]. Rough estimates of penguin abundance suggest that there are 23.6 million breeding pairs of penguins worldwide [218, 286] with 10 of the 19 species, considered vulnerable to extinction (www.iucnredlist.org) [27, 218].

Penguins are long lived seabirds, spending most of their lives at sea, only returning to land to breed and moult. Penguins undergo prolonged periods of fasting during breeding and moult [31, 55, 119, 283]. Prior to undergoing their annual fasts, penguins build up large reserves of white adipose tissue and protein for long periods of fasting during incubation and moult [124]. During the breeding season, most of the fasting occurs during incubation and brooding of the chick. Throughout this time, penguins must rely on their endogenous fat stores for nourishment [49]. In addition, penguins are able to store copious amount of undigested food in their stomach for long periods of time, which is used to provision their chicks [124]. During moult penguins replace their entire plumage whilst fasting on land and cannot return to sea because of the consequences of reduced waterproofing and thermal insulation [105, 119]. Similarly to the breeding season, penguins must rely on endogenous fat and protein reserves for feather synthesis and nourishment for the duration of moult which can take between 2-5 weeks [50, 218].

This thesis will examine the GI microbiota of four members of the family Spheniscidae, the king (Aptenodytes patagonicus), macaroni (Eudyptes chrysolophus), gentoo (Pygoscelis papua) and little (Eudyptula minor).

King Penguin (Aptenodytes patagonicus)

Figure 1.1, King penguin (Photography by Meagan Dewar)

Of the 19 species of penguins, the king penguin (Aptenodytes patagonicus) is the second largest species, standing between 85-90cm tall and is one of two species comprising the genus Aptenodytes. King penguins have an extensive geographical range; breeding throughout the sub-Antarctic and Antarctic Islands between 45° and 65° south, including South Georgia Islands, Crozet Archipelago, Macquarie Island, Prince Edward Islands, and Kerguelen Islands [70, 84]. Current estimates on the global population size of king penguin’s ranges from 1.5 million breeding pairs [14]. At sea king penguins forage north of Antarctic pack ice near where the Antarctic/sub-Antarctic waters meet the Indian, Australasian and Atlantic Ocean regions. The diet of king penguins consists of Myctophid fish (90%), with crustaceans and cephalopods [52, 53, 192, 211, 224].

King penguins have a long and complex breeding cycle and are the only penguin species, whose breeding cycle lasts more the a year. After a period of courtship, female king penguins lay one large egg (November), with both parents taking turns to incubate the egg (54 days) [247]. During guard stage, one parent must remain with the chick, whilst the other resumes foraging. Once chicks are able to thermoregulate, 9 both parents return to sea to resume foraging, whilst chicks join crèches within the colony. From January to the end of the Austral summer (April), king penguin chicks will have built up large fat reserves [105, 118]. Due to sharp declines in food availability during the winter, adults must forage further away from the colony, leaving their chicks to fasting for extensive periods of time, which can last for up to 5 months [86, 218, 278]. In September, parents return to the colony and feeding resumes with both parents provisioning their chicks until fledging in Nov/Dec. In its entirety, the breeding season for a king penguin takes between 12-14 months, with king penguins breeding once every 2 years. After breeding king penguins return to sea to replenish their energy reserves to prepare for moult which can last up to 5 weeks [218].

Macaroni Penguin (Eudyptes chrysolophus)

Eudptid or Crested penguins are characterised by their crests of yellow or orange plumes and red or red-brown eyes. Of the eudyptids, macaroni penguins are the second largest [218]. Macaroni penguins are the most abundant penguin species [71, 82] with 9-11.8 million breeding pairs globally [286].

Macaroni penguins are the most southerly breeding of all the eudyptids. There are approximately 216 colonies of macaroni penguins, breeding at 50 sites throughout the Antarctic and Sub-Antarctic islands, including; South Georgia, Chile, South Sandwich Islands, Crozet, Kerugelen Islands, Heard and McDonalds Islands and locally on the Antarctic peninsula [15, 75, 285, 287]. During the breeding season, the diet of macaroni penguin consists mainly of the Euphausiid krill Euphausia superb (>90%), with small fish and cephalopods becoming more predominant at the end of the breeding season [32, 76, 82].

Figure 1.2 Macaroni Penguin (photography by M. Dewar 2012)

In September/October every year macaroni penguins, return to the colony for the beginning of the breeding season. Macaroni penguins breed on steep terrain such as lava flows, rock falls, and side of cliffs, caves and hills. Their nests are made out of rocks, sticks and pebbles. Macaroni breeding sites are extremely crowded, which can contain anywhere from 1-2 million breeding pairs [285]. In contrast to other penguin species, the eggs of eudyptid penguins are not uniform in size. The first egg is significantly smaller than the second egg, and is abandoned before hatching, with up to 50% being abandoned prior to the second egg being laid [270, 281, 282, 284]. Following an incubation period of 5 weeks, one parent remains at the nest to brood the chick whilst the other returns to sea to forage. During the guard stage, male cares for the chick whilst the female does most of the foraging, returning to the colony every 2-3 days to provision the chick Post guard stage (25 days), and both parents return to sea. In macaroni penguins, chick rearing takes approximately 60-70 days [282]. Once the chicks fledge, adult macaroni penguins return to sea to build-up large reserves of protein and lipid in preparation for moult. In macaroni penguins the moulting can take up to 40 days [282, 284].

Gentoo Penguin (Pygoscelis papua) Gentoo penguins (Pygoscelis papua) are the largest of all Pygoscelis penguins [218, 283]. Gentoo penguins have a circumpolar distribution mainly located on Sub-

Antarctic Islands and along the Antarctic Peninsula [218], with 80% of the global population of gentoo penguins breed at the Falkland Islands [95], South Georgia [258], and the Antarctic peninsula [218]. Globally there is an estimated abundance of between 300,000 to 387,000 breeding pairs [165, 218]. Their diet consists mainly of rock cod, crustaceans and cephalopods, with females consuming more crustaceans than males and males consuming more fish than females [208].

Figure1.3, Gentoo Penguin (Photography by Meagan Dewar)

In mid to late October, Gentoo penguins return to their colonies to breed. Like other Pygoscelis penguins, gentoo penguins construct a pebble nest and lay two eggs, with both the male and female taking turns to incubate their eggs. Similar to other penguin species, chicks remain with their parents until approximately 3-4 weeks of life. Chicks then enter a crèche until fledging at around 14 weeks of age [218]. In January, adult gentoo penguins begin their annual moult which lasts for a period of 15-21 days [218].

Little Penguin (Eudyptula minor) Little penguins (Eudyptula minor) are the smallest of all living species at only 35- 45cm tall with an average body mass of 0.8-1.3kg and are the only species within the genera Eudyptula [7, 78, 218, 244].

Little penguins are distributed along the southern coast of Australia, New Zealand and the Chatham Islands [77, 218]. In Victoria, little penguins can be found along the entire coast from Portland to Gabo Island. To date, no global population size has been estimated for the little penguins. However, in Australia the current population is

12 estimated to be just under 1 million breeding pairs, with the largest colonies located on Gabo Island (35,000 birds) and at the Summerland Peninsula on Phillip Island (25,000) [81, 84]. At the Summerland Peninsula, Phillip Island little penguins are present in the colony throughout the year including the winter and non-breeding season [77]. On average the lifespan for little penguins is 6.5 years. However, these penguins have been recorded to live over 20 years of age at St Kilda (Zoe Hogg, Earth Care, Personal Communication) and Summerland Peninsula [79, 80, 219] The diet of little penguins predominantly consists of Clupeiformes with Engraulis australis and Sardinops neopilchardus dominating [56, 58, 170, 218], with the remainder of the diet including; barracouta, warehou, cod and cephalopods [56, 58].

Figure 1.4, Adult little penguin with chicks (Photography by Meagan Dewar)

The breeding season for little penguins occurs from August to February [172] Unlike most other penguins, egg laying is not synchronised within a little penguin colony, with egg laying occurring in some individuals as early and June and July. However, in most penguins the peak egg laying time usually occurs during September-October. Little penguins produce a clutch of two eggs with both parents alternating between incubation and foraging [218]. The incubation period lasts on average between 31-40 days [59, 220]. Similar to all penguin species, little penguin chicks are unable to self thermoregulate, and therefore parents must take turns guarding their chick whilst the other forages. The guard stage in little penguins occurs during the first 2-3 weeks [218, 244]. During the guard stage, chicks are fed on average every 1-2 days [60].

13 whereas during the post-guard stage adults alternate between short and long foraging trips [230]. Fledging occurs between 9-10 weeks of age [220].

Procellariiformes Taxonomy and Distribution The order Procellariiformes is made up of four families; Diomedeidae (Albatrosses), Procellariidae (Petrels and Shearwaters), Hydrobatidae (Storm petrels) and Pelecanoididae (Diving petrels), 23 genera and 128 species. Procellariiformes are long lived seabirds, that are characterised by their tubular nostrils, grooved, hooked beaks and their feet are well set back on their bodies and their ability to produce stomach oils (all except the Pelecanoididae) [76]. Procellariiformes are the most abundant seabirds in the Southern Ocean [172, 279] and are the most endangered bird taxa, with 36 species threatened with extinction (www.iucnredlist.org).

The ability to produce stomach oils is a unique trait restricted to all members of the Order Procellariiformes, except for diving petrels. Stomach oils are produced in the proventriculus through partial digestion of prey. The oil fractions of the prey are then concentrated and retained for long periods of time via delayed gastric emptying [225, 271]. This adaptation reduces the cost of travelling long distance to provide their chick with a highly concentrated food source that is high in energy and fats [76, 271, 273]. Stomach oils have also been shown to greatly influence the health and development of the Procellariiform chick [226, 271]. Stomach oils are neutral dietary lipids. The chemical composition of stomach oils differs from species to species, but almost always contains wax esters and triglycerides [271].

Procellariiformes lay a single egg with both parents sharing incubation duties (6-12 weeks). Similarly to penguins, Procellariiformes brood their chicks during the early stages of development due to their inability to thermoregulate, with both parents taking turns to guard the chick whilst the other forages for food [11, 255, 272]. After this initial guard period, Procellariiformes use a twofold breeding strategy which incorporates both short and long foraging trips [48, 275, 276]. Short foraging trips are used to provision the chick, whilst the long foraging trips are used by adults to replenish their dwindling fat reserves [275]. Procellariiform chicks accumulate large fat reserves as a result of over feeding by their parents during the early stages of development. As a result Procellariiform chicks are often obese and can accumulate a peak mass of 136% of their parents body mass [272]. Fat accumulation in chicks is 14 essential to enable them to survive prolonged periods of fasting in the later stages of development [125, 222]. Procellariiform chicks fledge at around 3 months for the smaller species and 9 months for the larger species [11, 255, 272].

Family Procellariidae The procellariids are the most diverse and numerous of the Order Procellariiformes [272]. The family Procellariidae consists of four distinct groups; the fulmarine petrels, the gadfly petrels, the prions and the shearwaters [272]. This thesis will examine the GI microbiota of two members of the family Procellariidae; the Short- tailed Shearwater (Ardenna tenuirostris) and the Fairy Prion (Pachyptila turtur).

Short-tailed Shearwater (Ardenna tenuirostris, formally Puffinus tenuirostris) The Short-tailed shearwater (Ardenna tenuirostris) is a medium sized (40-45cm), long lived, pelagic seabird that spend the bulk of its life at sea [23]. Short-tailed shearwaters are the most abundant of all Procellariiformes with an estimated global population size of 23 million individuals, with the majority breeding in Tasmania and Victoria [36].

All members of the family Procellariidae, including the Short-tailed shearwater nest in simple scrapes or burrows, lined with vegetation. Short-tailed shearwaters spend the winter months in the northern hemisphere foraging in the northern Pacific and Arctic Ocean during the winter months [123, 237]. In October, short-tailed shearwaters return to the southern hemisphere to begin the breeding season. Prior to egg laying, adult shearwaters embark on long foraging journeys to replenish their energy and fat reserves [236]. Egg laying is synchronised over a two to three week period, with all eggs laid by the last week in November. Males take the first incubation shift which lasts for up to 10-14 days [237], with the entire incubation period lasting 50-55 days Short-tailed shearwaters only lay one egg per season, therefore if breeding fails it is not replaced [44, 272]. In contrast to other seabirds, the guard stage in Procellariiformes is extremely short, lasting for between 2-3 days. This is because Procellariiformes are able to regulate body temperature and reduce brooding time. This ability is in part due to the subcutaneous fat deposits laid down during the first few days [272].

Figure 1.5, Short-tailed shearwater chick (Photography by M. Dewar 2010)

Like all Procellariiformes, short-tailed shearwaters use a twofold foraging strategy with birds alternating between two short foraging trips of 1-4 days, with one long foraging trip of approximately 8-19 days [141, 277]. Weimerskirch & Cherel [277] documented the dominance of Australian krill Nyctiphanes australis in the stomach contents of short-tailed shearwaters from Tasmania when returning from short foraging trips and Antarctic krill and myctophid fish dominating the stomach contents after long foraging trips. [277]. However, in Victorian shearwaters, there is little evidence to suggest that shearwaters forage on Antarctic species (Schumann et al, unpublished; [173]). Although chicks do not fledge until around 97days of age, adults begin their migration north when their chicks are around 85 days old at the beginning of April. For the next 2-3 weeks chicks must survive off their endogenous fat stores until fledging at 97 days of age [190].

Fairy Prion (Pachyptila turtur) Fairy prions are the smallest of six species within the genera Pachyptila [37]. Fairy prions found throughout the southern hemisphere, breeding on coastal islands including; South Georgia, the Chatham, Snares Island, Antipodes and Bass Strait Islands (Australia), Crozet Islands [84] with a current global population estimate of 5 million individuals [35]. Recent studies have identified the diet of Fairy Prions at Lady Julia Percy Island, in Victoria consists exclusively of the Australian krill

Nyctiphanes australis (Schumann et al, unpublished). Eggs are laid between October and November with adults sharing incubation duties with eggs hatching around 25 days later. Similarly to other Procellariidae’s, Fairy prions have a short guard stage of 1-5 days, with chicks fledging after 31-45 days [128]. Unlike many other petrel species, there is limited information on the biology of Fairy Prions.

Family Pelecanoididae In contrast to other members of the Order Procellariiformes, diving petrels (Pelecanoididae), do not produce stomach oils [272]. Pelecanoides is the only genus within the family Pelecanoididae, and contains four species including; P. urinatrix, P. garnotii, P. magellani and P. georgicus. In this thesis we will be examining the GI microbiota of the Common Diving Petrel (P. urinatrix).

Common Diving Petrel (Pelecanoides urinatrix) Common diving petrels are the only diving petrel known to breed in southern Australia, ranging from the from Seal Island group and nearby islands off Wilsons promontory, to west of Lady Julia Percy Island and Lawrence rocks [202]. Common diving petrels have a global population size of 16 million individuals [35]. The diet of Common Diving Petrels used in this study on Notch Island, consists entirely of Nyctiphanes australis which is a Euphausiid Krill [233]. In contrast to many other Procellariiformes, common diving petrels commence their breeding in mid-winter, as opposed to spring, with chicks fledging between November and December [172]. Similarly to other Procellariiformes, common diving petrels lay only one egg, with both parents sharing incubation. Guard stage on average lasts 6.7 days, with chicks fledging at 45-60 days old [172]. Similarly to the fairy prion, to date there is limited data available on the biology of this species.

Penguins as a Model of Avian Gastrointestinal Microbiota Although penguins are one of the most extensively studied species in the southern hemisphere, little is known about the resident microbiota. To date the composition and role of this ecosystem remains uncharacterised for many seabird species, including penguins, with earlier studies based upon the use of culture dependent techniques to provide description of the composition and abundance of members of the microbial community [28, 205, 254, 298, 299] or specific microbial pathogens 17 such as Salmonella [191, 195] Campylobacter [28, 34, 116, 133, 151, 210] and Pasturella multocida [85, 149, 150, 274]. However, cultivation techniques do not accurately reflect the actual microbial composition but only those microbes that can be cultured using selective media and as a result, provide an inaccurate account on the composition and abundance of microbes from complex ecological systems such as the gastrointestinal tract. Therefore, earlier studies on microbial composition of penguins are considered incomplete. For this reason many microbiologists have turned to molecular methods to characterise and explore the microbial composition of complex ecosystems such as the gastrointestinal tract, and marine ecosystems. In penguins, the use of molecular based methods to examine the microbial composition has been limited to just two studies on adélie penguins [12, 294]. Zdanowski et al [294] examined the microbial composition of freshly deposited adélie penguin guano while Banks et al [12] examined the influence of geographical separation and host phylogeny on the microbial composition of adélie penguins. Both studies, documented that adélie penguins are highly dominated by Firmicutes (41%), Actinobacteria (35%), Proteobacteria (12.5%). However, Banks et al [12] also noted the presence of Bacteroidetes (5%). Table 1.2 below lists microbes isolated from penguins.

Procellariiformes as a Model of an Avian Gastrointestinal Microbiota The order Procellariiformes is made up of four families; Diomedeidae (Albatrosses), Procellariidae (Petrels and Shearwaters), Hydrobatidae (Storm petrels) and Pelecanoididae (Diving petrels). These birds are characterised by the following traits; they are long lived, have external tubular nostrils, have the ability to produce stomach oils (all except the Pelecanoididae), grooved, hooked beaks and their feet are well set back on their bodies [76].

The production of stomach oils occurs during the digestion of food [271]. This adaptation reduces the cost of travelling long distances to provide their chick with a highly concentrated food source that is high in energy and fats [76]. Unlike penguins, there are no previous publications examining the microbial composition of Procellariiform seabirds, with all previous investigations isolating pathogenic microorganisms and identifying the cause of mortality. In table 1.3, below lists the microorganisms previously isolated from Procellariiform seabirds.

Table 1.3, Bacteria isolated from Penguin from previous studies

Penguin Common Name Bacteria Reference

Aptenodytes Emperor Chlamydia spp [41] forsteri Penguin Salmonella spp [191, 195]

Escherichia [135] intermedia

Bacillus spp [135]

Alcalgenes

aquamarines Pygoscelis papua Gentoo Penguin Achromobacter [28] spp Actinomyces

Campylobacter lari Salmonella spp [28] [29]

Yersinia spp

Campylobacter [28] jejuni [29] Edwardsiella [29] species Alcaligenes [242] faecalis Pseudomonas [242]

Escherichia coli [242]

Table 1.3, Continued Pygoscelis Adélie Penguin Campylobacter lari [28] adeliae Campylobacter jejuni Pasteurella [149] multocida Alcaligenes faecalis [242] Citrobacter freundii [242] Enterobacter spp [242] Escherichia coli [184, 242] Staphylococcus spp [184] Clostridium spp Salmonella spp [28, 176, 187] Yersinia spp [28] Mycoplasma spp [148] Chlamydia spp [174] Bacillus spp [212] Brachybacterium spp Streptomyces spp Arthrobacter spp Psychrobacter spp [217] Pseudomonas spp Moraxellaceae/ [294] Pseudomonadaceae Flavobacteriaceae [294] Micrococcaceae Actinobacteria [12] Firmicutes Bacteroidetes Proteobacteria Pygoscelis Chinstrap Penguin Bacillus spp [135] antarctica

Table 1.3 continued. Pygoscelis antarctica Alcalgenes [135] cont. aquamarines Micrococcus spp

Staphylococcus spp

Eubacterium spp

Sarcina ureae

Streptococcus spp

Escherichia coli

Corynebacterium spp

Salmonella spp [28]

Yersinia spp

Campylobacter lari

Campylobacter jejuni

Pasteurella multocida [72]

Escherichia coli [242]

Eudyptes chrysocome Southern Chlamydia spp [174] Rockhopper Escherichia coli [242]

Enterobacter spp [242]

Pasteurella multocida [85, 175]

Bacillus spp [40]

Eudyptes Macaroni Penguin Campylobacter jejuni [33] Chrysolophus Pasteurella multocida [72]

Eudyptes Schlegeli Royal Penguin Chlamydia spp [174]

Bacillus spp [40]

Table 1.3, Continued

Eudyptes Schlegeli Royal Penguin Escherichia coli [242]

Megadyptes antipodes Yellow-eyed Corynebacterium [2-4] Penguin amycolatum Spheniscus Magellanic Corynebacterium [113] magellanicus Penguin spheniscorum Corynebacterium spp [205]

Eudyptula minor Little Penguin Erysipelothrix [26] rhusiopathiae Escherichia coli [242]

Spheniscus demersus African Penguin Mycoplasma [96] sphenisci Salmonella [65] typhimurium Aptenodytes King Penguin Escherichia coli [242] patagonicus Citrobacter freundii

Table 1.4, Microorganisms isolated from Procellariiform seabirds from previous studies

Procellariiform Common Name Bacteria Reference

Thalassarche Black-Browed Salmonella [195] melanophrys Albatross

Thalassarche Grey-Headed Salmonella [195] chrysostoma Albatross

Pasteurella [274] multocida Thalassarche Atlantic Yellow- chlororhynchos nosed Albatross Erysipelothrix rhusiopathiae

Macronectes Southern Giant Pasteurella [150] giganteus Petrel multocida

Escherichia coli

Enterococcus [135] faecalis

Bacillus subtilis

Brevibacterium brunneum

Alcaligenes faecalis

Mycoplasma [148] gallisepticum

Mycoplasma synoviae

Salmonella

Table 1.4, continued.

Campylobacter lari [28]

Campylobacter jejuni

Yersinia

Catharacta Brown Skua Chlamydophila [130] lonnbergi abortus

Campylobacter lari [28]

Alcaligenes faecalis [135]

Micrococcus

Eubacterium alactolyticum

Staphylococcus epidermidis

Enterococcus faecalis

Proteus

Escherichia coli [13, 135]

Streptococcus

Table 1.4 Continued

Pseudomonas [13]

Acinetobacter

Micrococcus

Pasteurella [13, 149, 197] multocida

Salmonella [28]

Campylobacter lari [151]

Campylobacter [28] jejuni

Yersinia

Catharacta South Polar Skua Salmonella [28, 187] maccormicki Campylobacter lari [28]

Yersinia

Campylobacter jejuni

Research Objectives and Thesis Structure

The overall objective of this thesis was to characterise the microbial composition of the gastrointestinal microbiota of Spheniscidae and Procellariiformes. More specifically, the aims of this thesis were to:

Characterise the microbial composition of sub-Antarctic and temperate penguins;

Investigate the influence of fasting during moult on the faecal microbiota of king and little penguins;

Investigate the microbial colonisation of the faecal microbiota of little penguin chicks from birth to fledging (as a model for development);

Characterise the microbial composition of Procellariiform seabirds; and

Investigate the microbial colonisation of the faecal microbiota of short-tailed shearwater chicks from birth to fledging.

The following Chapters in this thesis are written as a series of five standalone manuscripts (Chapters 2-6) addressing particular aspects of the objectives and aims stated above. The collection of manuscripts means that some repetition and overlap occurs in the introduction and methods sections of some Chapters.

In Chapter 2, the microbial composition of king, gentoo, macaroni and little penguins was investigated using Quantitative Real Time PCR (qPCR) and 16S rRNA amplicon sequencing. Using qPCR and 16S rRNA amplicon sequencing the influence of fasting during moult was investigated in little and king penguins in Chapter 3. Chapter 4 investigated the variations in the gastrointestinal microbiota of little penguin chicks from hatching to fledging. In Chapter 5 the microbial composition of short-tailed shearwaters, fairy prions and common diving petrels was investigated using Quantitative Real Time PCR (qPCR) and 16S rRNA amplicon sequencing. Chapter 6 investigated the variations in the gastrointestinal microbiota of Short-tailed Shearwater chicks during development. The major findings from these Chapters are compiled and discussed in Chapter 7.

Preface

All opinions and concepts presented in this thesis are my own. Dr Stuart Smith and Associate Professor John Arnould provided structural and editorial suggestions in a supervisory capacity and have therefore been included as co-authors of all manuscripts. In Chapter 2, Dr Rene Groscolas (CNRS) coordinated and collected samples from king penguins, from Possession Island, Crozet Archipelago. In Chapters 2 and 3 Dr Phil Trathan (BAS) coordinated and collected samples from king, gentoo and macaroni penguins from Bird Island, South Georgia and has made some editorial suggestions. In Chapters 2-5 Dr Peter Dann (PINP) supervised field work, provided logistical support and organised ethics and permit for field work conducted at Phillip Island Nature Parks. As such, Dr Groscolas, Dr Trathan and Dr Dann have been included as co-authors of manuscripts resulting from the above Chapters.

All work was carried out under the approved Department of Sustainability and Environment Wildlife Research Permits (#10004713 and #10005156) and approval by the Phillip Island Nature Parks Animal Ethics Committee (#1.2008, #2.2009, #3.2009) and Deakin University Animal Welfare Committee (#A31/2008). Sub- Antarctic samples collected from Crozet Archipelago were obtained under Permit N°2008-69. All sub-Antarctic samples were imported under AQIS Permit (#IP090088774).

CHAPTER 2

Inter-specific variations in the gastrointestinal microbiota of penguins A version of this chapter has been accepted for publication in MicrobiologyOpen Dewar, M.L. Arnould, J.P.Y, Dann, P, Trathan, P, Groscolas, R and Smith, S.C. (2013). MicrobiologyOpen, Early View (http://onlinelibrary.wiley.com/doi/10.1002/mbo3.66/full)

Summary Despite the enormous amount of data available on the importance of the gastrointestinal microbiota in vertebrate (especially mammals), information on the GI microbiota of seabirds remains incomplete. As with many seabirds, penguins have a unique digestive physiology that enables them to store large reserves of adipose tissue, protein and lipids. This study used quantitative real time polymerase chain and 16S rRNA gene pyrosequencing to characterise the inter-specific variations of the gastrointestinal microbiota of four penguin species: the king, gentoo, macaroni and little penguin.

The qPCR results indicated that there were significant differences in the abundance for all of the major phyla. A total of 132,340, 18,336, 6,324 and 4,826 partial length 16S rRNA gene sequences were amplified from faecal samples collected from king, gentoo, macaroni and little penguins respectively. A total of 13 phyla were identified with Firmicutes, Bacteroidetes, Proteobacteria and Fusobacteria dominating the composition, however, there were major differences in the relative abundance of the phyla. In addition, this study documented the presence of known human pathogens such as Campylobacter, Helicobacter, Prevotella, Veillonella, Erysipelotrichaceae, Neisseria and Mycoplasma. However, their role in disease in penguins is unknown. To our knowledge, this is the first study to provide an in depth investigation of the gastrointestinal microbiota of penguins.

Introduction Penguins are a distinctive group of flightless seabirds found exclusively in the southern hemisphere, occupying an extensive geographic range extending from the Galapagos Islands to the Antarctic continent [247]. Penguins like all seabirds, spend most of their lives at sea, only coming to land to breed and moult [218, 227, 247]. Penguins have a unique digestive physiology that enables them to store large amounts of undigested food, build up large reserves of adipose tissue (fat) and store large amounts of protein and lipids for long periods of fasting during breeding and moult [124].

The diet of all penguin species is derived from the sea and can include; crustaceans, cephalopods, and fish, with composition varying from species to species and season. The diet of the king penguin predominantly consists of myctophid fish (family Myctophidae), For king penguins at Possession Island, (Crozet), two species of myctophid (Krefftichthy andersoni and Electrona carlsbergi) make up over 90% of the overall diet, whilst the remainder is composed of squid and other myctophid species [30, 49, 53, 54, 105, 211, 224]. At the beginning of the breeding season, the diet of macaroni penguin predominantly consists of the krill Euphausia superba (95%), but is then switched to a diet myctophid fish (Krefftichthy andersoni) [51, 104, 115, 224]. The diet of gentoo penguins is comprised of fish (nototheniids), cephalopods (Longio gahi) and Krill (Euphausia superba), with both temporal and spatial variation observed within the diet [208]. Whilst little penguins eat a variety of fish and small cephalopods, including anchovy (Engraulis spp), pilchard (Sardinops sagax), sandy sprat (Hyperlophus vittatus), juvenile barracouta (Thyrsites atun) and Krill (Nyctiphanes autralis) [56, 57, 103].

The fish and krill based diet of all four penguin species are a rich source of lipids, proteins, in n-3 and n-6 polyunsaturated fatty acids (PUFA) such as EPA, arachidonic acid and DHA. Krill are also a rich source of provitamin E, phospholipids, flavonoids, vitamin A, alpha-linolenic acid, astacin and other essential nutrients [137, 295] which are all essential for penguins during the pre-breeding and pre-moulting [53, 112, 192, 252] with penguins storing high levels of n-3 PUFA in their sub-cutaneous adipose tissue [211, 243]. In addition, diets high in n-3 and n-6 PUFA’s are essential for normal metabolism, growth, heart health and neurological development [243]. In avian species, arachidonic is a polyunsaturated omega-6 fatty 29 acid that is important for the development of tissues in the chick embryo, with roles in signal transduction and eicosanoid synthesis [243].

The gastrointestinal (GI) tract contains a diverse and complex microbial ecosystem made up of hundreds of different species of micro-organisms, that has coevolved with its host [67, 143, 163, 257]. The GI microbiota has a profound influence on the nutritional, physiological, immunological and metabolic processes of the host [168, 180, 298, 299], playing a significant role in energy harvest, fat metabolism, secretion and synthesis of nutrients, vitamins, amino acids and the production of short chain fatty acids from the diet consumed by the host [67, 94, 248, 257]. Rawls et al [214, 215]. identified that in the absence of microbial colonisation the GI tract results in an immature and arrested differentiation. Furthermore, research has shown that the absence of a functional GI microbiota reduces an animal’s ability to secrete and absorb essential vitamins and nutrients from their diet [25, 199].

To date, the vertebrate GI microbiota has been extensively studied and shown to be dominated by two main phyla; Firmicutes (usually 60-80% of total composition) and Bacteroidetes (usually 20-40% of total composition) in multiple vertebrate species including mammals (such as humans, rodents and pinnipeds), poultry, and livestock (i.e. cattle, pigs). [101, 108, 109, 152, 154-156]. The avian microbiota however, is comprised of approximately 640 species from 140 genera, with only 10% of the avian microbiota being cultured in the laboratory [257]. However, much of what we know about the avian microbiota is based from research on poultry [6, 83, 297].

In a study on the microbial composition of 60 species of terrestrial mammals, Ley et al [154] identified that host phylogeny, diet and gut morphology influenced the composition [154, 262] In addition, Ochman et al [186] and Yildrim et al [290] also showed that evolutionary lineages determine the composition of the gut microbiota of a vertebrate species. However, more recent evidence points to diet being the main driving force behind differences in the gut microbiota of phylogenetically similar hosts [178, 181]. In accordance with Ley et al [154], Banks et al [12] documented that host phylogeny is the main driving force behind differences in the GI microbiota of adélie penguins in the Ross Sea. In addition, to host phylogeny and diet, Nelson [181] identified that prey associated microbiota significantly dominated the predator microbiota. 30

Despite the enormous amount of data available on the importance of microbes in mammals, it is surprising that so little research has been carried out on the avian microbiota, with most studies focusing on the poultry microbiota (chicken, turkey) [6, 297] To date the composition and role of this ecosystem remains incomplete for seabirds, including penguin, with the exception of culture dependant techniques which have been used to describe the composition and abundance of specific bacteria within the microbial community [28, 205, 254, 298, 299], or specific microbial pathogens such as Salmonella [191, 195] Campylobacter [28, 34, 116, 133, 151, 210] and Pasturella multocida [85, 149, 150, 274]. However, traditional cultivation techniques do not accurately reflect the microbial composition of the GI ecosystem, and therefore, earlier studies on microbial composition of penguins are considered incomplete. For this reason many microbiologists have turned to molecular methods to characterise and explore the microbial composition of complex ecosystems such as the gastrointestinal tract, and marine ecosystems. In penguins, the use of molecular based methods to examine the microbial composition has been limited to just two studies on adélie penguins [12, 294]. Zdanowski et al [294] examined the microbial composition of freshly deposited adélie penguin guano while Banks et al [12] examined the influence of geographical separation and host phylogeny on the microbial composition of adélie penguins. Both studies, documented that adélie penguins are highly dominated by Firmicutes (41%), Actinobacteria (35%), Proteobacteria (12.5%). However, Banks et al [12] also noted the presence of Bacteroidetes (5%).

Given the ease in obtaining faecal samples from penguins, compared to the alternative which would require the euthanisation of penguins to obtain to observe the microbial composition of the entire GI tract, we chose to analyse the microbial compositon of the penguin GI tract via faeces collected via rectal swab as performed in previous studies [156, 194, 199, 262]. It has been shown that bacterial cells within a faecal material are representative of the microbiota in the colonic lumen, colonic mucosa and bacteria originating from other regions of the GI tract [91, 268]. Thus, although faeces do not fully represent the entire microbial community from the GI tract, a substantial proportion of bacterial species from this environment may be present [91, 268] and thus the analysis of faecal material should be considered adequate for investigating the microbial community of different species.

In order to understand the complex relationship between a host and its microbiota, one must first characterise the GI microbiota of healthy individuals at baseline before elucidating the role of microbiota, and the influence alterations of the GI microbiota will have on health and disease of an individual or population [157, 262]. Therefore, the goal of this study was to exanine variations in the microbial compostion of four penguin species; king, gentoo macaroni and little (Eudyptula minor). The aims of this study were; 1) Examine the variation in the abundance of four dominant phyla within the vertebrate GI microbiota using quantitative real time PCR and 2) to examine the community composition of the GI microbiota of four penguin species.

Materials and Methods Sample Collection

Faecal samples were collected from king (Aptenodytes patagonicus) (n = 12), gentoo (Pygoscelis papua) (n = 12), macaroni (Eudyptes chrysolophus) (n = 12) and little penguin (Eudyptula minor) (n = 12) upon returning to the colony from a recent foraging trip during the breeding season. Samples from gentoo and macaroni penguins were collected from Bird Island, South Georgia (54 00′S, 38 03′W). Samples from king penguins were collected from Baie du Marine, Possession Island Crozet Archipelago (46°25'S, 51°52 E). Samples from little penguins were collected from Phillip Island, Australia (38.4833° S, 145.2333° E). Upon return to the colony, birds were captured by hand, and a cloacal swab was collected (Copan Eswabs, Copan™ Italy) and stored in liquid nitrogen for transport.

Sample Analysis DNA Extraction and Real Time PCR DNA was extracted using the Qiagen™ QIAamp DNA Stool Mini Kit (Hilden, Germany) following the manufacturer’s instructions. The major phyla selected for analysis in this study were chosen from previous studies that had examined the predominant gastrointestinal microbiota of vertebrates (mammals, chickens, and adélie penguins) [91], which included Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria. Due to the absence of seabird specific bacterial primers, the primer sequences used in this study were obtained from previously published papers (see Table 2.1). The primer sequences and annealing temperature for the chosen bacterial groups are listed in table 2.1. The quantitative real time PCR was performed on the

Stratagene MX3000P. Each PCR reaction mixture comprised of 5µl of Brilliant II SYBR green (Stratagene™), 20 pmol/µL of forward and reverse primer, 2ng of template DNA and made up to a final volume of 20µl with nuclease free water. The cycling conditions were 95⁰C for 2 mins, followed by 40 cycles of 95⁰C for 5 secs, followed by annealing temperature (listed in table 2.1) for 30 secs with all samples were run in triplicate. Bacterial concentration was determined by comparing the threshold value (Ct. Values) with a standard curve. The standard curve was created by using a serial 10 fold dilution from DNA extracted from a pure culture of E.coli ranging from 102 – 1010 CFU.

PCR amplification of 16S ribosomal RNA gene sequences

Gentoo, Little & Macaroni Penguin Samples From the original samples, 4 individuals per species were chosen for 16S rRNA pyrosequencing. All samples/species were pooled together with the attachment of MID tag barcodes (i.e. Barcode 338R_BC0495 “TCACTGGCAGTA” was attached to all little penguin samples). Samples were then amplified using universal primers Roche adapter A (5’GCC TCC CTC GCG CCA TCA GT-3’) and reverse 338R (5’- CAT GCT GCC TCC CGT AGG AGT-3’) to amplify the V2-V3 region. Following amplification, samples were sequenced on the Roche/454 GS FLX Titanium Genome Sequencer by Engencore (USA) according to Fierer et al (2008). All sample preparation and sequencing was performed by Engencore (USA) according to the Roche 454 and Fierer et al (2008) protocol. Following sequencing, barcodes were removed using Roche SFF software (Roche Applied Science, Indianapolis, IN). King Penguin Samples Results from a previously unpublished study on the microbial composition of king penguins were included in this study. In the previous study, the V2-V3 region was amplified using primers 8F (5’ AGAGTTTGATCCTGG 3’) [12, 22, 91, 152-154, 221] and 519R (5’ TTACCGCGGCTGCT 3’) [146]. Following PCR amplification, samples were pooled and run on the Roche 454 FLX genome sequencer at CSIRO.

Phylogenetic Target Forward Reverse Reference

Firmicutes GGC AGC AGT RGG GAA TCT ACA CYT AGY ACT CAT CGT TT [92] TC

GGA RCA TGT GGT TTA ATT AGC TGA ACG ACA ACC ATG Bacteroidetes [179] CGA TGA T CAG

Actinobacteria ACG GGC GGT GTG TAC A TCC GAG TTR ACC CCG GC [121]

Alphaproteobacteria, Deltaproteobacteria AGT GTA GAG GTG AAA TT ACG GGC GGT GTG TAC A [22] Fusobacterium

Betaproteobacteria CTC GTG TAG CAG TGA ACG GGC GGT GTG TAC A [22]

CMA TGC CGC GTG TGT ACT CCC CAG GCG GTC DAC Gammaproteobacteria [22] GAA TTA

AGC GTT AYT CGG AAT CAC CCC CGT CTA TTC CTT TGA GTT Epsilonproteobacteria [179] TGG TT

Table 2.1 Quantitative real time PCR primer sequences used in this study to detect and quantify major phyla present in penguin faeces.

Bioinformatics Quality control, removal of chimera’s, sequence alignment, identification and Operational Taxonomic Units (OTU) classification were performed by Ribocon GmbH (Germany) as per the protocol of Prusse et al [206, 207]. The 16S rRNA sequences reported in this study have been submitted to EMBL under accession number ERP001504. Statistical Analysis To determine if there were significant differences between all penguin species for the major phyla for qPCR analysis, a one-way ANOVA with Tukeys HSD for pairwise comparison was performed in SPSS, and was considered to be significantly different if P = < 0.05. A multidimensional scaling plot (MDS), species diversity and cluster analysis was performed on both the qPCR and pyrosequencing data using Primer E v6 as per Clarke [64]. The samples that share the highest similarity will be represented on the plot by points plotted closest together, while samples that share limited similarity will be represented on the plot by points that are plotted farthest apart [221]. Diversity Indices Microbial diversity in the faecal samples of the four penguin species was estimated with the Shannon-Weiner diversity index (H’). This index was calculated by the following equation:

Shannon-Weiner index H’ = -∑i pi log(pi)

Where pi is the proportion of the total count arising from the ith species

Results Quantitative Real Time PCR (qPCR) The abundance of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were detected from DNA extracted from faecal samples of king, gentoo, macaroni and little penguins and using phyla specific primers. Proteobacteria dominates the GI microbiota of gentoo, macaroni and little penguins, whereas Bacteroidetes, dominated the GI microbiota of king penguins. Bacteroidetes was the second most abundant phyla in gentoo, macaroni and little penguins, followed by Firmicutes and Actinobacteria. In king penguins, Proteobacteria was the second most abundant phyla, followed by Firmicutes and Actinobacteria. The abundance of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria in little penguins were significantly 35 lower in comparison to other penguin species (P = 0.0001). Firmicutes was significantly higher in macaroni penguins in comparison to king (P = 0.002) and gentoo (P = 0.008) penguins. While, significant differences in the abundance Proteobacteria were observed between king and gentoo penguins (P = 0.041). For Actinobacteria there were no significant differences observed between macaroni, king and gentoo penguins (Figure 2.1).

The results from the MDS analysis, shows evidence that individual little penguins are clustering together, indicating limited variation within this species. Some individual gentoo, king and macaroni penguins appear to be clustered together, whilst other individuals appear to be separated, indicating similarities between the three penguin species and also the potential for individual variation, within each species (Figure 2.2).

16S rRNA gene Pyrosequencing Microbial Composition of penguins A total of 132,340, 18,336, 6,324 and 4,826 partial 16S rRNA gene sequences were amplified from faecal samples collected from king, gentoo, macaroni and little penguins respectively. With 97% sequence similarity a total of 1331, 2195, 1362 and 561 Operational Taxonomic Units (OTU) were identified respectively.

From the phylogenetic analyses, there were 13 phyla represented in penguin gut microbiota; Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Fibrobacteres, Planctomycetes, Spirochaetes and Synergistetes and four recently classified candidates (BD1-5; OP10, SR1 and TM7) were also represented. The most abundant phyla present in all penguin populations included the Bacteroidetes, Firmicutes, Proteobacteria and Fusobacteria. Although all four species of penguin are dominated by similar phyla, there are differences in the abundance of each phylum between all four penguin species. Gentoo penguins were dominated by Fusobacteria (55%), Firmicutes (18%), Proteobacteria (18%) and Bacteroidetes (7%). In king penguins, Firmicutes (48%), Fusobacteria (24%) and Bacteroidetes (18%) dominate. The dominant phyla in little penguins were Proteobacteria (30%), Firmicutes (24%), Bacteroidetes (22%), Planctomycetes (11%) and Actinobacteria (7%). Whilst in

36 macaroni penguins, Firmicutes (45%), Proteobacteria (29%) and Bacteroidetes (19%) dominate (Figure 2.3).

*P = 0.0001, **P < 0.01, ***P < 0.05 Figure 2.1 Variation in the abundance of the major phyla; Firmicutes (a), Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) in king, gentoo, little and macaroni penguins. Proteobacteria dominates the GI microbiota of gentoo, macaroni and little penguins, followed by Firmicutes, Bacteroidetes and Actinobacteria. Whereas Bacteroidetes dominates the king penguin microbiota, followed by Proteobacteria, Firmicutes and Actinobacteria.

Figure 2.2, Similarity of the major bacterial phyla of penguin species using qPCR. Similarity of faecal microbiota of penguin species displayed in MDS plot performed in Primer E. There is a high level of similarity between individual little penguins and gentoo penguins (with some outliers), indicating limited individual variation. Although king and macaroni penguins do cluster together, it is not a tight as little and gentoo, indicating potential individual variation within the population.

MDS analysis is used to examine the similarity between samples, with highly similar samples clustering closely together on the plot, whereas samples of limited similarity are further apart on the plot. The MDS analysis of the penguin microbiota demonstrates a high level of similarity between gentoo and macaroni penguins. For king and little penguins, there is little similarity with other penguin species (Figure 2.4). The analysis of similarity results also indicates a high level of similarity between gentoo and macaroni penguins, sharing approximately 50% of the microbial species. Little penguins share approximately 40% of their microbiota with gentoo and macaroni penguins. Whereas king penguins have the lowest level of similarity, sharing less than 10% of their microbiota with the other penguin species (Figure 2.5).

At family level, gentoo penguins were highly dominated by Fusobacteriaceae (55%) with Fusobacterium the dominant genus. In king penguins, Leuconostocaceae, Campylobacteriaceae and Porphyromonadaceae were the most dominate families with Leuconostoc, Weissella, Helicobacter, Proteiniphilum and Petrimonas the predominant genera. In little penguins the dominant families were Enterobacteriaceae, Porphyromonadaceae and Phycisphaeraceae with Enterobacter, Escherichia and Klebsiella the dominant genera within the family Enterobacteriaceae. Barnesiella was the dominant genera of Porphyromonadaceae, while Phycisphaeraceae was dominated by an unclassified genus. In macaroni penguins Leuconostocaceae and Porphyromonadaceae were the dominant families with Leuconostoc and Petrimonas the predominant genera (Figure 2.6).

Diversity Diversity index was calculated for total microbial composition and for each major phylum for each penguin species using the Shannon-Weiner index in Primer E. For total microbial composition, little penguins had the highest microbial diversity with H = 3.543, followed by macaroni, king and gentoo penguins with a diversity index of H = 3.379, H = 2.889 and H = 2.363 respectively (Figure 2.7).

Figure 2.3, Relative abundance of major phyla from the GI microbiota of gentoo, king, little and macaroni penguins using 16S rRNA pyrosequencing. Bacteroidetes, Firmicutes and Proteobacteria dominate the microbial composition of king, little and macaroni penguins. Fusobacteria, Firmicutes and Proteobacteria dominate the GI microbiota of gentoo penguins.

Figure 2.4, Similarity of the gut microbiota of penguin species. There is a high level of similarity between gentoo and macaroni penguins, as indicated by the close clustering. For king and little penguins, there is a low level of similarity to other penguin species.

Figure 2.5, Analysis of Similarity of the GI microbiota of gentoo, king, little and macaroni penguins. The results from the analysis of similarity of bacterial profile indicate that the similarity between gentoo and macaroni penguins is approximately 50%. In little penguins there is a similarity of approximately 40% between little, gentoo and macaroni penguins. Whereas king penguins have a very low level of similarity to all other penguin species with less than 10% similarity.

Figure 2.6, Relative abundance of major families of gentoo, king, little and macaroni penguins using 16S rRNA pyrosequencing. Fusobacteriaceae dominates the GI microbiota of gentoo penguins. In king penguins, Leuconostocaceae, Campylobacteriaceae and Porphyromonadaceae dominate. Little penguins Enterobacteriaceae, Porphyromonadaceae and Phycisphaeraceae dominate the GI microbiota. Macaroni penguins Leuconostocaceae, Porphyromonadaceae and Moraxellaceae were the most dominant families.

Figure 2.7, Variation in species diversity in king, gentoo, little and macaroni penguins. Species diversity was calculated by Shannon-Weiner index using Primer E. Little penguins have the highest level of species diversity with H = 3.543, followed by macaroni (H = 3.379), king (H = 2.889) and gentoo (H =2.363).

Discussion Prior to this study, limited information was available on the microbial composition and diversity of the penguin microbiota and the variation that exists between different penguin species. In the current study, the microbial composition of king, gentoo, macaroni and little penguins were characterised using Quantitative Real Time PCR (qPCR) and 16S rRNA amplicon pyrosequencing. Quantitative Real Time PCR was used to quantify the abundance of specific phylogenetic groups and to examine the influence of individual variation, whilst the 16S rRNA amplicon sequencing was used to provide a more in-depth characterisation of the microbial diversity of four different penguin species.

Quantitative Real Time PCR The results from the qPCR demonstrated that there was significant variation between all penguin species, especially between little penguins and the three sub-Antarctic species for all major phyla. In gentoo, macaroni and little penguins, Proteobacteria appears to be the most dominant phyla with an abundance of 1.5 x 108, 8.0 x 107 and 1.82 x 106 CFU (colony forming units) respectively. Whilst in king, Bacteroidetes is the most abundant phyla present with an abundance of 6.6 x 107 CFU. Actinobacteria had the lowest abundance level for all penguin species ranging from 8.8 x 103 to 7.7 x 105 in king penguins (Figure 2.1).

The MDS results indicate that there is some clustering within the different species, with the majority of individuals for each species clustering together, indicating that there is some variation in the microbial composition of different penguin species (Figure 2.2). However, the clustering of individuals within each species does not appear to be tight and therefore could indicate individual variation within each species (Figure 2.2).

16S rRNA gene pyrosequencing Influence of “Primer Sequences” on Pyrosequencing results In this study, we included data from a previously unpublished study on the microbial composition of king penguins (Dewar et al, unpublished) and although the primer sequence used to amplify the V2-V3 region analysis of the sequence data produced has shown that the primer sets used in this study have a similar coverage of the bacterial taxa present in all penguin species as shown in table A1 (Appendix). Thus 45 we feel that the sequences used in the king penguin study do not influence the variation in microbial composition between king penguins and the other penguin species.

Microbial Composition of penguins Pyrosequencing provided a more detailed characterisation with 161,826 sequence reads a total of 5,449 OTUs were identified from all penguin species, from13 phyla; Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Fibrobacteres, Planctomycetes, Spirochaetes and Synergistetes and four recently classified candidates (BD1-5; OP10, SR1 and TM7). Firmicutes, Bacteroidetes, Proteobacteria and Fusobacteria were found to dominate the penguin microbiota. There were significant differences in the results from the qPCR and 16S rRNA pyrosequencing, with the qPCR results indicating that Bacteroidetes and Proteobacteria were the most dominant phyla in all four species of penguins, whilst the pyrosequencing has identified that the proportion of Firmicutes and Bacteroidetes are more dominant than Proteobacteria. These differences could be due to the qPCR primer sets being designed based upon data from terrestrial vertebrate microbiota, (such as humans, rodents and poultry) and that they do not cover the entire microbial composition of the penguin microbiota. As the 16S rRNA pyrosequencing amplifies all bacterial DNA within the V2-V3 bacterial region, it is likely to provide a more accurate and thorough coverage of the bacteria present.

The dominance of Bacteroidetes and Firmicutes at phylum level in penguins is in accordance with previous studies on other vertebrate species; including many mammalian species [91, 121, 145, 153, 249] . Members of the phylum Firmicutes are associated with the breakdown of complex carbohydrates, polysaccharides, sugars and fatty acids, which are then utilised by the host as an energy source with high abundances of Firmicutes being linked to increased adiposity [93, 253]. Members of the phylum Bacteroidetes in humans have been associated with vitamin synthesis, polysaccharide metabolism, and membrane transport [120]. Although the penguin microbiota in this study is similar to that of other vertebrate species (i.e. dominance of Firmicutes and Bacteroidetes), they do differ from other vertebrates in regards to the relatively high abundance of Fusobacteria (2-55%) and Proteobacteria (5-30%) and their functional roles in penguins is not known. 46

The composition of the faecal microbiota varies amongst all four penguin species, with differences observed at both the phylum and family level. These results indicate that there are significant variations. However, the cause of these variations is unknown. Possible factors such as diet, prey associated microbiota, phylogenetic differences, and external environment, have all been found to influence a hosts microbial composition [12, 120, 154, 181]. In adélie penguins, Banks et al [12] identified that host phylogeny had a greater influence over host microbial composition in comparison to geographical location. Similarly, Ley et al [154] noted that host phylogeny and diet appear to majorly influence the composition at a species and population level, observing distinct differences between herbivores, omnivores and carnivores. In Antarctic pinnipeds, Nelson [181] identified that not only did diet and host phylogeny influence the GI microbiota, but that prey associated microbiota dominated the GI microbiota of the higher predator.

The pyrosequencing data from this study have identified a significant proportion of “unclassified” bacteria, indicating penguins harbour a large novel group of resident bacteria in their digestive systems. Earlier studies in terrestrial vertebrates have discovered that previously ‘unclassified’ bacteria to be responsible for important metabolic, immunological or physiological functions [203, 296]. These bacteria could have the potential to be involved in significant functions relating to digestive physiology (synthesis and storage of proteins, lipids, PUFA’s), health and disease in penguins and need to be analysed further.

The 16S rRNA pyrosequencing analysis also highlighted the presence of potential pathogens such as Campylobacter, Helicobacter, Prevotella, Veillonella, Streptococcus, Erysipelotrichaceae, Neisseria, Ureaplasma and Mycoplasma. The identification of these pathogens is of significant concern due to their potential impact on wildlife health and survival. Although these are known pathogens in humans and other vertebrates there is limited data available linking these pathogens to disease except for a member of the family Erysipelotrichaceae, which was associated with the death of a little penguin in captivity [26]. Also, Campylobacter spp have previously been identified in many penguin species, however, there have been no studies linking the presence of this bacterium to disease in sub-Antarctic penguins [28, 34, 116, 151] In addition, Helicobacter spp are widely distributed amongst many vertebrate species including marine mammals (i.e. pinnipeds, 47 cetaceans) [111, 193] and although some species have been associated with disease in humans and some marine mammals [127, 193] there is no data linking the presence of Helicobacter to illness or disease in marine seabirds. The presence of these pathogens may also indicate transmission from humans, which needs further investigation. Because of their dense colonial living, penguins are more susceptible to rapid pathogen transfer, which could potentially lead to major disease outbreaks [116]. Therefore, the presence of any known pathogen warrants further investigation to ascertain if these organisms are in fact pathogenic to penguins and if so, what would the potential impact be on the population.

Conclusion

This study has identified, that there is large variation within the faecal microbiota of sub-Antarctic and temperate penguin species and that these microbiota appear to be dominated by five major phyla; Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria and Actinobacteria. Although, the cause of these differences is yet to be determined, host phylogeny, and diet could potentially play a major role in determining the final microbial composition of an individual [12, 154]. This study also identified the presence of known mammalian pathogens that could potentially cause illness or disease within a penguin population and these findings warrant further investigation as to the roles of these pathogenic bacteria in penguins.

CHAPTER 3

Variation in the Gastrointestinal Microbiota of Little Penguins (Eudyptula minor) During Development

Abstract At birth (emergence from egg), the gastrointestinal (GI) tract of all avian species is considered to be a completely sterile environment that is rapidly colonised. Factors such as microbial load on the eggshell, nesting environment and the first meal are all known to influence the microbial colonisation of the GI tract of the chick. Immediately after birth, the microbiota changes from a completely sterile aerobic environment to a complex anaerobic environment dominated by anaerobic bacteria. To date the development of the microbiota in a long lived seabird has not been examined. Therefore this study aimed to characterise the GI microbiota of little penguin chicks from birth to fledging (56-70 days), using Quantitative Real Time PCR (qPCR) and 16S rRNA pyrosequencing sequencing. Results from the qPCR analysis showed an upward trend for Firmicutes and Bacteroidetes during developing in the little penguin chick. No trend was observed for Proteobacteria or Actinobacteria. 16S rRNA pyrosequencing data identified that there are significant fluctuations in the microbial composition of little penguins from hatching to fledging. The similarity between all age classes is around 20%, with similarity slightly increasing to 40% between weeks 5, 7 and 9. The newly hatched chick is quickly colonised by aerobic and facultative anaerobic bacteria, with Enterobacteriaceae dominating the microbiota throughout the first three weeks of development. By week 5, the composition is dominated by Fusobacteriaceae and Clostridiaceae, while Fusobacteriaceae and Clostridiaceae continue to dominate throughout development until fledging. Similarly to other vertebrates, there is an initial increase in species diversity, followed by a steady decline by fledging. This study describes the first molecular examination of the microbial community of the digestive tract of little penguin chicks throughout development. Little penguin chicks display similar ecological successional changes as other vertebrates with aerobic and facultative anaerobes being the first microbes to colonise the GI tract and slowly transitioning to a microbiota dominated by strict anaerobes. However, unlike other vertebrate species, the little penguin microbiota at fledging is still relatively unstable and does not resemble that of the adult microbiota. 49

Introduction In nature, bacteria are found everywhere and with the availability of nutrients are able to colonise new habitats, including the GI tract [6]. At birth (emergence from egg), the GI tract is considered to be devoid of microorganisms. In mammals, the microbial community is said to be inherited from the mother through intimate contact with faecal and vaginal microbes during birth and from breast milk [194]. In birds, however, newborn chicks, acquire their microbiota from the surface of the egg, surrounding environment (i.e. nest) and their first meal [6, 99, 101, 159, 217, 256, 291]. In addition, vertical transmission of microbes via regurgitation of an undigested or partially digested meal is also said to influence the microbiota of the chick [110, 142, 205]. Through ecological succession, the sterile GI tract is rapidly colonised forming a dense and complex microbial community within 2 years in humans [164, 166, 168, 199, 240, 268] and 40 days in chickens [16, 17, 68, 90, 217].

At birth, the neonate gut is an aerobic environment that is rapidly colonised by aerobic and facultative anaerobic bacteria such as Escherichia coli and Streptococci immediately after birth [5, 68, 168, 217, 268]. After initial colonisation, aerobic bacteria modify the GI tract environment, by reducing the level oxygen through oxidation/reduction. This process creates an environment that is highly favourable to facultative and obligate anaerobes [168, 200], such as Bacteroides, Clostridia, and Bifidobacterium [168, 245] before stabilising as an anaerobic environment.

To examine the development of the GI microbiota in a long lived seabird, the little penguin was used as a model. Little penguins (Eudyptula minor) breed between August to February [172] with peak egg production occurring between September and October [220]. Little penguins nest in burrows, rocks, crevices and artificial nest boxes lined with vegetation. Little penguins produce two eggs which are incubated by both parents for 31-40 days [59, 220]. During the early stages post hatch (2-3 weeks), little penguin chicks are unable to self thermoregulate, and therefore one parent must remain with the chick during this period (guard stage) [218, 244], During the guard stage, chicks are fed on average every 1-2 days [60], whereas during the post-guard stage adults alternate between short and long foraging trips [230]. Fledging occurs between 9-10 weeks of age [218].

To date, there have been no previous publications exploring the microbial colonisation of the GI microbiota in long lived birds, especially birds that derives their entire nutrition from the marine environment. Therefore, the goal of this study was to characterise the colonisation of the GI microbiota of the little penguin hatching to fledging (7-10 weeks) using DNA derived from cloacal material. Quantitative Real Time PCR and 16S rRNA gene amplicon sequencing was used to identify bacteria at each stage during development and the samples were compared to examine changes in community structure over time.

Materials and Methods

Study site and Sample Collection To characterise the microbial composition of the little penguin chick during development, faecal samples were collected from little penguin chicks from the Phillip Island Nature Park. The study was conducted within the Summerland Peninsula, a previous housing estate, which had been reclaimed for penguin habitat. Due to severe compaction of the ground, burrows in the area consist of artificial wooden burrows. Burrows are lined with vegetation and excrement from previous breeding seasons (Figures 3.1 and 3.2). At the beginning of the breeding season, 10 nest boxes were randomly selected with the restriction that the nest contained 2 eggs at the beginning of the breeding season. Of the 20 eggs, 16 successfully hatched, with only 9 successfully fledging at the end of the breeding season. Following hatching, cloacal swabs were collected from each chick directly from the cloacae using Eswabs (Copan, Italy). On the same day every week, penguin chicks were removed from their nest, weighed, their stomach was inked (red or green food dye) for identification purposes and swabbed and then placed back into the nest within a 5-10 minutes. Samples were placed into an amine solution for preservation of the DNA and stored in liquid nitrogen (-196°C) for shipment and then stored at -20 until DNA extraction.

Figure 3.1, artificial nest box used by little penguins in this study (Photography by M. Dewar 2008).

Figure 3.2, inside of an artificial nesting box, with adult little penguin and chicks

Quantitative Real Time PCR DNA extractions and quantitative Real Time PCR analysis were conducted as per the protocol described in chapter 2. The qPCR primers used to analyse the abundance of major bacterial phyla and classes are outlined in Table 2.1 in chapter 2.

16S rRNA Pyrosequencing, Samples from 4 little penguin chicks at weeks 1, 3, 5, 7 and 9 were chosen for further analysis by 16S rRNA pyrosequencing. All samples/species were pooled together with the attachment of MID tag barcodes (i.e. Barcode 338R_BC0500 “CAGCTCAACTA” was attached to all week 1 samples). Samples were amplified and sequenced as per the protocol described in chapter 2. Results from Adult little penguins from chapter 2 were included for comparison.

Bioinformatics, Statistical Analysis and Diversity Quality control, removal of chimera’s, sequence alignment, identification and Operational Taxonomic Units (OTU) classification was performed by Ribocon GmbH (Germany) as per the protocol of Prusse et al [206, 207]. The 16S rRNA sequences reported in this study have been submitted to EMBL under accession number ERP001967. In order to explore the variation in bacterial counts, linear mixed models with individual birds as a random effect and common assessment times (weeks) as a fixed effect were fitted to the log-transformed data. Bacterial counts were log transformed (base 10), to uncouple apparent variance-mean relationships, after inspection of diagnostic plots of Studentized residuals versus predicted values [238].

The linear mixed models were restricted to six types corresponding to the six combinations of the following: Random effects (penguin chicks):

No autocorrelation in the repeated counts from the penguin chicks and homogeneous variances at each assessment (scaled identity model). No autocorrelation in the repeated counts from the penguin chicks and heterogeneous variances at each assessment (diagonal model). First order autoregressive process for the co-variation in the repeated counts (AR (1) model) and homogeneous variances.

Fixed effects (weeks): Week as a categorical factor. Week as a linear trend.

For each abundance measure, the fitted model with the smallest value of the Akaike Information Criterion (AIC) [144] was selected and the significance of the association of the common assessment times with variation in abundance was assessed using the F-test. If the selected model included week as a categorical factor, weekly means and their standard errors were reported. Otherwise, if the selected model included week as a linear trend, the estimated intercept and slope coefficients, and their standard errors, were reported. All analyses used the mixed model procedures in the IBM SPSS Statistics package (Version 20).

Principal Coordinates Analysis, diversity and cluster analysis was performed on qPCR, whilst a Multidimensional scaling plot (MDS), was performed on the pyrosequencing data. The samples that share the highest similarity will be represented on the plot by points plotted closest together, while samples that share limited similarity will be represented on the plot by points that are plotted farthest apart

Microbial diversity in the faecal samples of the four penguin species was estimated with the Shannon-Weiner diversity index (H’). This index was calculated by the following equation: Shannon-Wiener index H’ = -∑i pi log (pi) Where pi is the proportion of the total count arising from the ith

Results Quantitative Real Time PCR The abundance of Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria were detected from DNA extracted from faecal samples collected from little penguin chicks during development using phyla specific primers. Bacteroidetes and Firmicutes dominate the microbial composition throughout development, followed by Proteobacteria. Actinobacteria appeared to have the lowest abundance with less than 103 CFU. During weeks 1-3, 5, 7-9 Firmicutes dominated the microbial composition. Whilst during weeks 4, 6 and 10 Bacteroidetes dominated with 55 statistical differences observed throughout development in both Firmicutes and Bacteroidetes. When analysing the data using a linear mixed model with time as a covariate, a significant increasing linear trend was observed for both Firmicutes and Bacteroidetes (Figure 3.3), indicating that there was an underlying linear increase common to the observed abundance profiles for the 9 chicks/birds from hatching to fledging. The MDS results show a strong clustering of all individuals throughout development indicates that there is little variation according to qPCR results (Figure 3.4).

16S rRNA gene pyrosequencing A total of 17,106, 19,386, 9,705, 11,792 and 31,418 partial 16S rRNA gene sequences were amplified from faecal samples collected from little penguin chicks on weeks 1, 3, 5, 7 and 9 during development respectively. With 97% sequence similarity a total of 1,127, 1,099, 1,190, 1,998, and 1,694 Operational Taxonomic Units (OTU) were identified from little penguin chicks respectively. A total of 4,826 partial 16S rRNA gene sequences and 561 OTU’s were amplified from adult little penguins during the breeding season.

From the phylogenetic analyses, there were 10 phyla represented in developing little penguin gut microbiota Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Planctomycetes, Chlorobi, Cyanobacteria, Tenericutes, Verrucomicrobia and five recently classified candidates (Candidate division BRC1, OP10, TM7, SM2F11 and TM6) were also represented (Figure 3.5). Fusobacteria, Firmicutes and Proteobacteria dominate the developing microbiota. Proteobacteria (87%) dominated the microbiota of little penguin chicks during early development (week 1-3), with Fusobacteria increased in abundance by week 3. After week three, Fusobacteria, Firmicutes and Proteobacteria dominate the microbiota until fledging. In the adult penguin, the predominant phyla were Proteobacteria (30%), Firmicutes (24%), Bacteroidetes (22%), Planctomycetes (11%) and Actinobacteria (7%). Although Bacteroidetes is a dominant member of the adult microbiota, its presence in chicks is negligible.

Figure 3.3, Changes in the abundance of the major phyla; Actinobacteria (a), Bacteroidetes (b), Firmicutes (c) and Proteobacteria (d) during development in little penguin chicks. A significant upward trend was detected in both Firmicutes and Bacteroidetes.

Figure 3.4, Similarity of the major bacterial phyla in little penguins during development using qPCR. The PCoA cluster analysis was performed using Calypso and shows no clear clustering, indicating no significant differences between chicks during development.

At Family level, the microbiota of the week old chick was dominated by Gram negative aerobic and facultative anaerobic bacteria such as Enterobacteriaceae (69%) and Moraxellaceae (11%) with Enterobacter, and Psychrobacter the predominant genera. Similarly at week three, the microbiota was dominated by Enterobacteriaceae (44%), with the level of Fusobacteriaceae (33%) and Unclassified (14%) bacteria increasing. Escherichia again was the dominant genera. In weeks 5 and 9, the microbiota was dominated by Fusobacteriaceae (56%) and Clostridiaceae (17%), with Fusobacteria the predominant genera. In week 7, there is a sharp increase in the abundance of Vibrionaceae (34%), with Vibrio the dominant genera. In the adult penguin the predominant families were Enterobacteriaceae (16%), Porphyromonadaceae (13%), and Phycisphaeraceae (11%) (Figure 3.7).

The MDS/Cluster analysis indicates that level of similarity between the different age classes increases with age. During the early stages of development, the level of similarity between age classes is approximately 20%. In the later stages of development, there is an increase in similarity, with weeks 5, 7 and 9 sharing 40% of their microbiota. However, by fledging, the there is still a low level of similarity between the adult and chick microbiota (Figure 3.6).

Diversity The bacterial diversity in the little penguin chick gradually increases from week one (H = 1.797) to week 7 (H = 2.556), but then declines by week 9 (H = 1.21) and significantly increases in the adult penguin (H = 3.543) (Figure 3.8).

Figure 3.5, Relative abundance of major phyla during development in little penguin chicks. In weeks 1 and 3, Proteobacteria highly dominates the microbial composition. In weeks 5 and 8 Fusobacteria dominates the microbial composition. In week 7 Firmicutes and Proteobacteria dominate the microbial composition.

Figure 3.6, Similarity of the gut microbiota of little penguins during development using 16S rRNA Pyrosequencing. The results indicates that there is limited similarity between the different stages of development in little penguin chicks and also between little penguin chicks and adult penguins.

Figure 3.7, Relative abundance of major Families during development in little penguin chicks. Microbial composition of the developing GI tract changes from a dominance of Enterobacteriaceae and Fusobacteriaceae to a domination of Fusobacteriaceae over time. The adult microbiota is dominated by Enterobacteriaceae, Porphyromonadaceae and Phycisphaeraceae.

Figure 3.8 Variation in species diversity during development and in adult little penguins. Species diversity was calculated by Shannon-Wiener Index using Primer E. Species diversity increases with age until week 7. After week 7 we see a steep decline in species diversity. Adult little penguins have the highest level of species diversity in comparison to little penguin chicks.

Discussion Establishment and early colonisation of the GI tract has been recognised as a crucial stage in neonatal development with pioneering species responsible for influencing the GI pH, oxygen levels, mucosal structure and immunity and therefore, these pioneering species influence a hosts overall health and disease status throughout life [136, 138, 198]. In mammals the establishment and colonisation of the neonate GI tract has been extensively studied [168, 199, 268]. In avian species, however, data is restricted to the microbiota of poultry [17, 68, 134, 171, 256, 266]. To date there is no data available on the GI microbiota of long lived seabirds, nor the microbial succession that occurs during development. Therefore this study aimed to examine the establishment and microbial succession that occurs in little penguin chicks during development.

Quantitative data obtained by qPCR for Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria respectively demonstrated that the microbiota establishment occurs rapidly. However, unlike humans and chickens, the microbiota did not remain stable over time, indicating that the microbiota of little penguins stabilises after fledging. From overall abundance of the four major phyla examined in this study, that the total microbial population in a week old chick is over 1.3 x 107 CFU. By fledging the total microbial population is estimated at over 3.0 x 106. These estimates are consistent with other neonate vertebrates, a total population estimate of 108- 1010 CFU 7 days post birth [136, 168], with a final population size of around 1014 by the end of development. Throughout development an upward trend was observed for Firmicutes and Bacteroidetes, but no trend was observed for Proteobacteria and Actinobacteria. The qPCR analysis indicates that an abundant population of microbes have colonised the GI microbiota of little penguin chicks by the first week of life.

Results obtained from pyrosequencing identified that the microbiota of little penguins is dominated by aerobic and facultative anaerobic bacteria from the phylum Proteobacteria, with Escherichia and Psychrobacter the major pioneering populations, followed by members of the phylum Firmicutes, with members of the phyla Bacteroidetes and Planctomycetes present in lower numbers. Although the presence of anaerobic bacterial populations begins to flourish in week 3, Enterobacteriaceae still dominates the microbiota. From week 3 to 5, there is a significant transition from an environment dominated by aerobic and facultative 64 anaerobic bacteria to an environment dominated by strict anaerobes with Fusobacteria and Clostridium dominating the microbiota for the remainder of development, except in week 7, when Vibrionaceae dominates.

The microbial composition of the little penguin during early development is dominated by microbes that are known to colonise the infant microbiota of many other vertebrates. Members of the family Enterobacteriaceae such as Escherichia, Enterobacter, Klebsiella, and Citrobacter are responsible for the absorption of dietary fats, lipids, and soluble vitamins, maintain intestinal barrier function, regulate triglycerides, impact intestinal permeability, activate intestine-brain-liver neural axis, enhance the immune system, alteration of lipoprotein profiles, protection against stress-induced lesions, modulation of proinflammatory and anti-inflammatory gene expression, and strengthen epithelial cell barrier properties. However, these microbes have also been implicated in GI pathologies, brain-gut axis, and a few neurological conditions [43, 117, 139, 223, 235, 251, 292]. While Psychrobacter, which has previously not been isolated from the infant microbiota, has been shown to have probiotic properties boosting the immune system and weight gain of Groupers [250]. By week 3, butyrate producing bacteria begin to flourish and by week 5 dominate the microbiota [204].

Butyrate is an essential short-chain fatty acid produced in the colon. The main effects butyrate has on the intestinal tract in humans include, influencing ion absorption, cell proliferation and differentiation, immune regulation, and is an important anti- inflammatory agent and is an essential energy source for colonocytes and has been associated with colonic health [42]. In chickens, butyrate supplementation can lead to a significant increase in host defence peptide gene expression, enhance antibacterial properties of monocytes against pathogenic bacteria, boost host immunity and increase host adiposity [196].

Conclusion Similar to other infant vertebrates, the little penguin microbiota is dominated by aerobic and facultative anaerobic bacteria, with the total population, estimated at over 107 CFU by 7 days, with a significant upward trend observed throughout development in Firmicutes and Bacteroidetes populations [134, 166]. During the early stages of development the microbiota is dominated by members of the family 65

Enterobacteriaceae which have been identified as pioneering populations associated with gut maturation and development, absorption of dietary fats and lipids, and soluble vitamins, enhance the immune system, alteration of lipoprotein profiles, modulation of proinflammatory and anti-inflammatory gene expression, and shave also been implicated in GI pathologies [20, 43, 117, 139, 223, 235, 251, 292]. These pioneering microbes are said to alter the GI environment towards an environment that favours the growth of anaerobic bacteria. Similarly to other studies, the microbiota of the little penguin began to transition from an aerobic/facultative anaerobic microbial population, to a strict anaerobic population by week 3, and continued throughout development, with the anaerobic population being dominated by butyrate producing microbes. In poultry, butyrate has been shown to not only boost the immune system and protect against the colonisation of pathogenic bacteria, but has also been shown to influence host adiposity. In little penguins, there is a strong link between host body mass and fledging survival. Therefore, the high abundance of butyrate producing microbes in little penguin chicks could influence chick body mass and therefore fledging survival. However, this would require further analysis.

CHAPTER 4

Influence of Fasting During Moult on the Gastrointestinal Microbiota of Penguins

Summary Many seabirds including penguins are adapted to long periods of fasting, due to reproduction, prey availability and moult, which are all a normal part of their lifecycle. In the past many studies have focused their attention on understanding the physiology of fasting; with limited research being done to understand the impact fasting has on the composition of the gastrointestinal (GI) microbiota. Previous studies examining the influence of fasting in other vertebrates (Syrian hamsters, mice and pythons) identified that fasting not only influences the microbial composition and diversity, but can also influence the immune response. However, the influence of fasting on the GI microbiota has not been investigated in seabirds. Therefore, the present study aimed to examine the microbial composition and diversity of the GI microbiota of fasting penguins during early and late moult. Faecal samples were collected from little (Eudyptula minor) and king penguins (Aptenodytes patagonicus) during moult and analysed using Quantitative Real Time PCR (qPCR) and 16S rRNA gene pyrosequencing.

The results from this study indicated that there was little change in the abundance of the major phyla during moult, except for a significant increase in the level of Proteobacteria in king penguins when using qPCR. In accordance with these results, the 16S rRNA gene pyrosequencing did not identify any significant changes in the relative abundance of the major phyla in moulting little penguins. However, there were significant differences between early and late moulting little penguins at lower taxonomic levels (i.e. Family). In king penguins, however, significant variation in the microbial composition was observed at all taxonomic levels (i.e. phylum, family) with a significant increase of Fusobacteria from 1 - 75% of the total composition. Similarly to mammals and reptiles, our result indicated that fasting during moult, alters the microbial composition of both little and king penguins and that the microbial composition during early and late moult differs for each penguin species, indicating that responses to moulting could be species specific.

Previous studies examining the effect of fasting on the GI microbiota of vertebrate (hamsters, python, mice), have shown that fasting not only alters the composition and diversity of the GI microbiota, but it also influences the host’s immune defences [73, 74, 241] and that interrelationships exist between a host, its microbiota and the host’s nutritional status, diet and physiology [73, 74] These studies however, have concentrated on animals that are relatively inactive during times of nutrient deprivation. Unlike other vertebrates, penguins have to survive long periods of starvation while also coping with increased metabolic demands for feather synthesis and thermoregulation [102, 281]. Therefore, penguins provide an attractive model for investigating the influence of fasting that is associated with the increased metabolic demands of moult.

Many seabirds, including penguins, are adapted to long periods of fasting due to periodic fluctuations in nutrient availability, breeding and moult [31, 55, 119]. In general, moult occurs post breeding in most penguin species and can last anywhere from 2-5 weeks depending on the species. During moult penguins replace their entire plumage whilst fasting on land and cannot return to sea because of the consequences of reduced waterproofing and thermal insulation [105, 119]. Throughout moult penguins must rely on endogenous fat and protein reserves for feather synthesis and 68 nourishment and is considered to be the most stressful and energetically costly period within the penguin life cycle due to increased metabolic demands for feather synthesis and thermoregulation [50]. Many penguin species experience high rates of mortality during moult as a consequence of inadequate storage of fat and protein reserves [50, 77, 114]. Because of its role in host adiposity, immune function and regulation and metabolism, the GI microbiota could potentially influence host health and survival during this stressful period and therefore needs to be investigated. The king and little penguin experience different periods of moult, with moult lasting 2-3 weeks in little penguins, and 5 weeks in king [218, 247]. Therefore, these two species provide an opportunity to examine the influence of moult and the length of moult in the microbiota. The goal of this study was to gain an accurate understanding of how fasting during moult influences the microbial composition of king (Aptenodytes patagonicus) and little (Eudyptula minor) penguins.

Materials and Methods Sample Collection Faecal samples were collected from king (Aptenodytes patagonicus) (n = 12) and little penguins (Eudyptula minor) (n = 9) during early and late moult. King penguins were located at Bird Island, South Georgia (54 00′S, 38 03′W), while little penguins were located at the Summerland Peninsula, Phillip Island, Australia (38.4833° S, 145.2333° E). A cloacal swab was collected from all birds during the early and late moult using Copan E-swabs (Copan™, Italy), and placed into an amine solution for preservation of the DNA and frozen for storage.

DNA Extraction and Real Time PCR DNA was extracted using the Qiagen™ QIAamp DNA Stool Mini Kit (Hilden, Germany) following the manufacturer’s instructions.

DNA was extracted using the Qiagen™ QIAamp DNA Stool Mini Kit (Hilden, Germany) following the manufacturer’s instructions. The major phyla selected for analysis in this study were chosen from previous studies that had examined the predominant gastrointestinal microbiota of vertebrates (mammals, chickens, and adélie penguins) [91], which included Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria. Due to the absence of seabird specific bacterial primers, the primer sequences used in this study were obtained from previously published papers (see 69

Table 2.1). The primer sequences and annealing temperature for the chosen bacterial groups are listed in table 2.1. The quantitative real time PCR was performed on the Stratagene MX3000P. Each PCR reaction mixture comprised of 5µl of Brilliant II SYBR green (Stratagene™), 20 pmol/µL of forward and reverse primer, 2ng of template DNA and made up to a final volume of 20µl with nuclease free water. The cycling conditions were 95⁰C for 2 mins, followed by 40 cycles of 95⁰C for 5 secs, followed by annealing temperature (listed in table 2.1) for 30 secs with all samples were run in triplicate. Bacterial concentration was determined by comparing the threshold value (Ct. Values) with a standard curve. The standard curve was created by using a serial 10 fold dilution from DNA extracted from a pure culture of E.coli ranging from 102 – 1010 CFU.

16S ribosomal RNA gene pyrosequencing Individual samples were amplified using universal primers Roche adapter A (5’GCC TCC CTC GCG CCA TCA GT-3’) and reverse 338R (5’-CAT GCT GCC TCC CGT AGG AGT-3’) to amplify the V2-V3 region. Samples were pooled following PCR amplification with the attachment of MID tag barcodes to ensure that bacterial DNA from all members of each species was equally represented. Samples from the same species were pooled together for sequencing on the Roche/454 FLX Genome Sequencer by Engencore (USA). All sample preparation and sequencing was performed by Engencore (USA) according to the Roche 454 protocol.

Bioinformatics, Statistical Analysis & Diversity Quality control, removal of chimera’s, sequence alignment, identification and Operational Taxonomic Units (OTU) classification was performed by Ribocon GmbH (Germany) as per the protocol of Prusse et al [206, 207]. The 16S rRNA sequences reported in this study have been submitted to EMBL under accession number ERP0001595

To determine if there was significant differences between all penguin species for the major phyla for qPCR analysis, paired samples T-test were performed in SPSS, and were considered to be significantly different if P<0.05. A Principal Coordinates Analysis (PCoA), diversity and cluster analysis was performed on the qPCR, whilst a Multidimensional scaling plot was performed on the pyrosequencing data. The samples that share the highest similarity will be represented on the plot by points 70 plotted closest together, while samples that share limited similarity will be represented on the plot by points that are plotted farthest apart [12, 22, 91, 152-154].

Microbial diversity in the faecal samples of the two penguin species was estimated with the Shannon-Weiner diversity index (H’). This index was calculated by the following equation: Shannon-Wiener index H’ = -∑i pi log (pi) Where pi is the proportion of the total count arising from the ith species

Results Quantitative Real Time PCR The abundance of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria from DNA obtained from faecal samples collected from king and little penguins during early and late moult were examined using qPCR. In little penguins a significant decrease in the abundance of Bacteroidetes was observed during moult (Figure 4.1). In king penguins a significant increase in the abundance of Proteobacteria was observed during moult (Figure 4.2). In both king and little penguins, Bacteroidetes is the most abundant phyla followed by Firmicutes, Proteobacteria and Actinobacteria in little penguins and Proteobacteria, Firmicutes and Actinobacteria in king penguins.

The PCoA results indicate that during early moult, all individuals cluster together indicating little variation in the microbial composition in little penguins, with 1 outlier. However, by late moult, individuals appear to cluster into two main groups and two outliers, indicating the presence of individual variation in response to moult (Figure 4.3). The cluster analysis also indicates a low level of similarity between early and late moulting little penguins. For king penguins, the PCoA results shows that most individuals cluster together during both of moult, but especially during early moult, with most individuals tightly clustered together indicating that there is little variation between individuals during the early moult. This clustering indicates that the microbial compositions between early and late moult in general are different (Figure 4.4).

*P = 0.02 Figure 4.1, Variation in the abundance of the major phyla; Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria during moult in little penguins. The abundance of the major bacterial phyla was determined by comparing the threshold value (Ct values) with a standard curve.

*P = 0.005 Figure 4.2, Variation in the abundance of the major phyla; Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria during moult in king penguins. Proteobacteria significantly increases during moult.

Figure 4.3, Similarity of the major bacterial phyla in little penguins during moult using qPCR. The PCoA results indicate that there is a low level of similarity between early and late moulting little penguins. There is also a high level of individual variability.

Figure 4.4, Similarity of the major bacterial phyla in king penguins during moult using qPCR. There is distinct clustering of early and late moult. This clustering indicates that there is a high level of variation between individuals during the late moult state, including two outliers

16S rRNA gene, pyrosequencing

Variations in the microbial composition of little penguins during moult A total of 4,986 and 5,856 partial 16S rRNA gene sequences were amplified from faecal samples collected from little penguins during the early and late moult respectively. With 97% sequence similarity a total of 954 and 1,003 Operational Taxonomic Units (OTU) were identified in little penguins during the early and late moult respectively. A total of 10 phyla were represented in the GI microbiota of both early and late moulting little penguins. These included Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Fusobacteria, Planctomycetes, Proteobacteria, Spirochaetes, Tenericutes and 1 recently classified candidate (division TM7). In addition, members from the phyla Cyanobacteria and Deferribacteres were also present during early moult, while Synergistetes and two additional classified candidates (Candidate division BRC1 and Candidate division SR1) were present during the late moult.

The microbiota of little penguins during early moult was dominated by Firmicutes (33%), Proteobacteria (19%), Fusobacteria (17%), Bacteroidetes (10%) and Actinobacteria (8%). Similarly, little penguins during late moult have a similar composition to individual little penguins in early moult, with Firmicutes (32%), Proteobacteria (21%), Bacteroidetes (20%) and Actinobacteria (10%), except, for the relative abundance of Fusobacteria which had significantly declined from 17% to 1% (Figure 4.5). The similarity in the microbial composition between early and late moulting little penguins was approximately 55%, indicating that microbial composition varies during moult.

The predominant families dominating the microbial composition of little penguins during the early moult were Fusobacteriaceae, Bacilliaceae and Porphyromonadaceae with Fusobacterium, Oceanobacillus and Petrimonas the dominant genera. During late moult, Porphyromonadaceae, Neisseriaceae and Lachnospiraceae dominate the population (Figure 4.6). The dominant genera included Proteiniphilum, Petrimonas, uncultured Neisseriaceae and Lachnospiraceae.

Figure 4.5, Relative abundance of major phyla in moulting little penguins using 16S rRNA pyrosequencing. Firmicutes, Proteobacteria Bacteroidetes and Fusobacteria dominate the microbial composition during early moult in little penguins. By late moult, Fusobacteria has completely disappeared.

Figure 4.6, Relative abundance of major families from the GI microbiota of moulting little penguins using 16S rRNA pyrosequencing. Fusobacteriaceae, Bacilliaceae and Porphyromonadaceae dominate the GI microbiota during early moult, while, Porphyromonadaceae, Neisseriaceae and Lachnospiraceae dominate during late moult.

Variations in the microbial composition of king penguins during moult A total of 15,151 and 16,749 partial 16S rRNA gene sequences were amplified from faecal samples collected from king penguins during the early and late moult respectively. With 97% sequence similarity a total of 2196 and 1551 Operational Taxonomic Units (OTU) were identified in king penguins during the early and late moult respectively. A total of 6 phyla were represented in gut microbiota of both early and late moulting king penguins; Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, Tenericutes, and four recently classified candidates (BD1-5, Candidate division OD1, Candidate division SR1, Candidate division TM7). In addition, members from the phyla Verrucomicrobia, Acidobacteria, Chloroflexi, Cyanobacteria, Planctomycetes, Synergistetes were represented in early moulting king penguins, while members from the phyla Chlorobi and Lentisphaerae were represented in late moulting king penguins.

There is a significant difference between early and late moult in king penguins. In early moult the microbiota is dominated by Firmicutes (37%), Proteobacteria (34%) and Bacteroidetes (20%) dominated the microbiota of king penguins during early moult, with Leuconostocaceae, Campylobacteriaceae and Porphyromonadaceae (11%), the dominant families. By late moult, however, Fusobacteria (75%) dominates (Figure 4.7), with Fusobacteriaceae the dominant family (Figure 4.9). The level of similarity between early and late was less than 30%. A significant decline in the level of species diversity was also observed (3.41 to 1.1).

Diversity The overall diversity in the microbial composition of little penguins declines slightly from 3.635 to 3.231 in early to late moult. However, in king penguins, the level of diversity significantly decline from 3.409 to 1.157 in early and late moult respectively.

Figure 4.7, Relative abundance of major phyla in moulting king penguins using 16S rRNA pyrosequencing. During early moult, Proteobacteria, Firmicutes and Bacteroidetes dominate the microbial composition. By late moult, Fusobacteria dominates the microbial composition, comprising over 70% of the total composition.

Figure 4.8, Relative abundance of major families from the GI microbiota of moulting king penguins. During early moult Leuconostocaceae, Campylobacteriaceae and Porphyromonadaceae are the dominant families, whilst Fusobacteriaceae dominates the GI microbiota during late moult.

Discussion Previous studies examining the effect of fasting on the GI microbiota of vertebrate (hamsters, python, mice, termites), have shown that fasting not only alters the composition and diversity of the GI microbiota, and influence the host’s immune defences [73, 74, 241]. Research has also shown that complex interrelationships exist between a host, its GI microbiota and the hosts nutritional status, diet and physiological state [73, 74]. These studies, however, have concentrated on animals that are relatively inactive during times of nutrient deprivation. Unlike other vertebrates, penguins do not hibernate during times of fasting. In penguins fasting occurs during the breeding season (incubation of eggs and chick brooding) and moult, where penguins replace their entire plumage whist fasting on land. The moulting fasts can last anywhere from 2 to 5 weeks. Therefore, penguins must survive long periods of starvation while also coping with increased metabolic demands for feather synthesis and thermoregulation [102, 281]. Penguins provide an attractive model for investigating the influence of fasting that is associated with increased metabolic demands. To date the influence of fasting on the microbiota of a seabird has not been examined. Therefore, this study aimed to examine the influence of fasting during moult in king and little penguins using qPCR and 16S rRNA pyrosequencing.

Quantitative Real Time PCR The quantitative results from the qPCR analysis showed that moult significantly decreased that abundance of Bacteroidetes in little penguins and significantly increased the abundance of Proteobacteria in king penguins. In accordance with other fasting vertebrates, there is a low level of similarity between early and late moult in both penguin species, indicating that moult does alter the microbial composition of king and little penguins [73, 74, 241].

16S rRNA gene Pyrosequencing Results obtained from pyrosequencing from the 16S rRNA gene sequencing show significant differences to that of the qPCR, with the 16S rRNA sequencing identifying significant differences in the microbial composition and diversity between early and late moulting penguins. Similar to other vertebrate species, the most predominant phyla in early moulting penguins were Firmicutes (33-37%), Bacteroidetes (10-20%) and Proteobacteria (19-34%) [73, 91, 147, 154]. However, 82 unlike other vertebrates, king penguins were dominated by families Leuconostocaceae, Campylobacteriaceae, Porphyromonadaceae, and Helicobacteriaceae, while, little penguins were dominated by Fusobacteriaceae, Bacilliaceae, Porphyromonadaceae and Neisseriaceae.

Increased levels of members from the phyla Firmicutes are associated with increased adiposity, by enhancing energy extraction and through modulation of the genes that regulate fat storage [9, 152, 156, 259, 260, 263] . Although Firmicutes dominates the microbial composition during early moult in both king and little penguins, it does not constitute a large proportion of the total composition, as in other vertebrates that have large fat stores (i.e. Australian sea lions, polar bears) [108, 147]. This is quite surprising, considering that penguins build up large reserves of fat prior to moult. Therefore, one would expect the microbiota of penguins during early moult to have a microbial profile similar to that of other vertebrates that are able to store large fat deposits (i.e. pinnipeds). In accordance with previous studies, a significant shift in the microbial composition was observed in king and little penguins. In Syrian hamsters Sonoyama et al [241] documented that the microbial composition during fed, fasted and hibernating hamsters were all highly dominated by the Phylum Firmicutes. However, the abundance of the class Clostridia was lower in fasted hamsters, while Akkermansia mucinphila a mucin degrader were significantly increased in the fasted state. In Burmese pythons, Costello et al [73] documented that the microbial composition during feeding was dominated by Clostridium, Lactobacillus, and Peptostreptococcus, whilst during fasting, the microbial community was dominated by Bacteroidetes, Rikenella, Synergistes and Akkermansia. Whereas Gupta et al [122] documented a 35-fold and 12-fold increases in the levels of Campylobacteriaceae and Helicobacteriaceae in malnourished children when comparing them to healthy children. In this study we saw the complete disappearance of Campylobacteriaceae and Neisseriaceae by late moult and a 52% decline in the abundance of Helicobacteriaceae in king penguins. Whilst in little penguins we see a 60% increase in the level of Neisseriaceae and a 58% decline in the level of Enterobacteriaceae from early to late moult.

Due to the absence of data on the functional role of microbes in penguins we can only infer what impact these changes to the microbiota will have on penguins. Throughout moult the little penguin microbiota was associated with potentially 83

Butyrate producing microbes (Fusobacteria and Clostridia) [8, 204], known gastrointestinal commensals and known human and veterinary pathogens [169, 216] such as Campylobacteriaceae which has also been associated with disease in penguins [113]. Whilst the king penguin microbiota is dominated by microbes that are associated with butyrate production, chitin degradation, a novel probiotic (Psychrobacter) and known gut commensals [8, 204, 250]. Known pathogens such as Campylobacter, Escherichia coli, and Helicobacter also dominate the microbiota during early moult [204]. By late moult, the microbiota is dominated by Fusobacteria, which is a known butyrate producer. In mammals Butyrate is an essential short-chain fatty acid produced in the colon. The main effects butyrate has on the intestinal tract in humans include, influencing ion absorption, cell proliferation and differentiation, immune regulation, and is an important anti-inflammatory agent [42]. In chickens, butyrate supplementation leads to a significant increase in host defence peptide gene expression, enhance antibacterial properties of monocytes against pathogenic bacteria, boost host immunity and increase host adiposity [196]. Therefore the presence of butyrate producing microbes could influence host adiposity levels prior to moult.

Conclusion The results from the qPCR and pyrosequencing both indicate that fasting does alter the microbial composition of both king and little penguins during moult. However, it appears that the microbial composition of king penguins is more affected by fasting than little penguins with the length of fast the most probable cause for this difference. The reduction in the level of ‘potential’ pathogenic bacteria could be in part due to the anti-microbial properties of butyrate. However, further investigation of more species is required to examine the influence of moult on GI microbiota.

CHAPTER 5 Inter-Specific Variations in the Gastrointestinal Microbiota of Procellariiform Seabirds

Summary Despite the enormous amount of data available on the importance of the gastrointestinal microbiota in vertebrate (especially mammals), to date there is currently no information available on the microbiota of seabirds, with the unique ability to produce and store stomach oils. All Procellariiformes, except diving petrels have the unique ability to produce and store ‘stomach oils’ in their proventriculus. Stomach oils are produced in the proventriculus through partial digestion of prey. The oil fractions of the prey are then concentrated and retained for long periods of time via delayed gastric emptying. Therefore, short-tailed shearwaters, fairy prions and common diving petrels offer the unique opportunity to examine the microbiota of seabirds that consume the same diet, but differ in digestive physiology. Therefore this study compared the microbiota short-tailed shearwaters, fairy prions and common diving petrels using quantitative real time PCR and 16S rRNA gene pyrosequencing. The qPCR results indicated that overall there were significant differences between all Procellariiform seabirds for Firmicutes and Bacteroidetes, but no significant difference in Proteobacteria and Actinobacteria. From the phylogenetic analyses, a total of 16 classified phyla represented in the Procellariiform microbiota, with Firmicutes and Proteobacteria dominating the microbial composition of all three Procellariiform seabirds, and Bacteroidetes also dominating the microbial composition of common diving petrel. The microbial composition of short-tailed shearwater and fairy prion are highly similar sharing 50- 60% of the same operational taxonomic units OTUs, whereas the similarity between short-tailed shearwaters, fairy prions and common diving petrels is approximately 20%. As all three species share a similar diet, it can be assumed that diet is not the major influence for the difference in microbial composition. Therefore, other possible causes for these differences are likely to be host phylogeny, digestive physiology or retention time.

Introduction An intricate and complex relationship exists between a host and its microbiota. The GI microbiota plays a significant role in energy extraction, fat metabolism and storage, production of short chain fatty acids and host adiposity [9, 259, 293, 298] and has a profound influence on the modulation of host metabolism. The GI microbiota has the ability to modify a number of lipids in serum, adipose tissue and in the liver, with drastic effects on triglycerides and phosphatidylcholine. The resident microbiota is also responsible for the production of metabolites that contribute to host fitness and survival [132]. The use of germ free animals has highlighted the importance of the GI microbiota on its vertebrate host. Germ free animals are not only more susceptible to disease, but also require a greater caloric intake to achieve and maintain a normal body weight [9]. However, when germ free animals are inoculated with the microbiota of conventionally raised hosts, the body fat level of the germ free host rapidly increases, despite decreased food intake [9], indicating that members of the GI microbiota may modulate fat deposition [185]. Many metabolites that are essential host nutrition are the product of microbial fermentation. These metabolites are responsible for the absorption of dietary fats lipids, and soluble vitamins, maintain intestinal barrier function, regulate triglycerides, impact intestinal permeability, activate intestine-brain-liver neural axis, enhance the immune system, alteration of lipoprotein profiles, protection against stress-induced lesions, modulation of pro-inflammatory and anti-inflammatory gene expression, and strengthen epithelial cell barrier properties and have been associated with influencing host adiposity [43, 117, 139, 223, 235, 251, 292]. However, to date there is currently no information available on the microbiota of seabirds, with the unique ability to produce and store stomach oils. Therefore, this study offers the unique opportunity to examine the influence of stomach oils on the microbiota.

Procellariiformes are long lived seabirds, that consume a diet high in dietary lipids and are characterised by their ability to produce stomach oils (all except the Pelecanoididae), [42]. Stomach oils are produced in the proventriculus through partial digestion of prey. The oil fractions of the prey are then concentrated and retained for long periods of time via delayed gastric emptying. [76]. This adaptation reduces the cost of travelling long distances to provide their chick with a highly concentrated food source that is high in energy and fats [225, 271]. The absence of stomach oils in diving petrels is explained by differences in their digestive 86 morphology in comparison to other Procellariiformes. Procellariiformes, have a highly extensible proventriculus, small gizzard and a unique arrangement of their duodenal loop. The proventriculus acts as a separating funnel, separating the lighter lipid layer from the denser aqueous layer. The dense aqueous layer is the first to pass through the digestive tract, while the lipid layer is retained longer. In diving petrels, however, the dorsal positioning of the pylorus and gizzard reduces the retention time of the digesta, with the lighter lipid layers emerging first [243]. In diving petrels the retention time of digesta is said to preclude the formation of stomach oils [225]. The diet of short-tailed shearwaters, fairy prions and common diving petrels is dominated by Australian krill Nyctohpanes australis. Their krill based diet is a rich source of n- 3 polyunsaturated fatty acids (PUFA), proteins and minerals [271]. Krill are also a rich source of provitamin E, phospholipids, flavonoids, vitamin A, Alpha-linolenic acid, astacin and other essential nutrients [69, 183, 269]. In avian species diets high in n-3 PUFA’s are essential for normal metabolism, growth, heart health and neurological development [137, 295].

In order to understand the complex relationship between a host and its microbiota, one must first characterise the GI microbiota of healthy individuals at baseline before elucidating the role of microbiota, and the influence alterations of the GI microbiota will have on health and disease of an individual or population [157, 262]. The goal of this study was to examine variations in the microbial compostion of three members of the Order Procellariiformes; short-tailed shearwater, fairy prion and common diving petrel using quantitative real time PCR and 16S rRNA pyrosequencing.

Methods Sample Collection Faecal samples were collected from Short-tailed Shearwaters (Puffinus tenuirostris) from Phillip Island, (Australia), Common Diving Petrels (Pelecanoides urinatrix) from Notch Island, (Bass Strait, Australia) and Fairy Prions (Pachyptila turtur) from Lady Julia Percy, (Australia) when returning to their colonies at night. Short-tailed shearwaters were captured by hand, whereas Common Diving Petrels and Fairy Prions were captured by mist netting.

To obtain faecal samples a sterile Copan E-swab (Copan™, Italy) was inserted in to the cloaca. The samples was then placed into an amine solution for preservation of the DNA and frozen for storage and stored at -80 degrees until analysis.

Sample Analysis Quantitative Real Time PCR DNA extractions and quantitative Real Time PCR analysis were conducted as per the protocol described in chapter 2. The qPCR primers used to analyse the abundance of major bacterial phyla and classes are outlined in Table 2.1 in chapter 2.

16S rRNA Pyrosequencing Faecal samples were analysed using the 16S rRNA pyrosequencing protocols described in chapter 2.

Bioinformatics, Statistical Analysis and Diversity Index Quality control, removal of chimera’s, sequence alignment, identification and Operational Taxonomic Units (OTU) classification was performed by Ribocon GmbH (Germany) as per the protocol of Prusse et al [206, 207], as per Chapter 2.

As the data was considered to be non-parametric all data was log transformed in SPSS. To determine if there was significant differences between Procellariiform seabirds for the major phyla for qPCR analysis, the data was log transformed and a one-way ANOVA, was performed in SPSS, and was considered to be significantly different if P<0.05. Multidimensional scaling plot (MDS), diversity and cluster analysis was performed on both the qPCR and pyrosequencing data.

Microbial diversity in the faecal samples of the three Procellariiformes was estimated with the Shannon-Weiner diversity index (H’). This index was calculated by the following equation: Shannon-Wiener index H’ = -∑i pi log (pi) Where pi is the proportion of the total count arising from the ith species

Results

Quantitative Real Time PCR (qPCR) The abundance of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were detected from DNA extracted from faecal samples of common diving petrels, fairy prions and short-tailed shearwaters using phyla specific primers (Table 2.1). Overall there were significant differences between all Procellariiform seabirds for Firmicutes and Bacteroidetes, but no significant difference in Proteobacteria and Actinobacteria. Short-tailed shearwaters had a significantly higher abundance of Firmicutes in comparison to fairy prions (P = 0.0001) and a significantly high abundance of Bacteroidetes in comparison to fairy prions (P = 0.021) and common diving petrels (P = 0.023). In addition, fairy prions had a significantly higher abundance of Firmicutes in comparison to common diving petrels (P = 0.016) but no significant difference between Bacteroidetes, Proteobacteria and Actinobacteria (Figure 5.1).

The results from the MDS analysis indicates a strong clustering between all fairy prion samples indicating little variation amongst individual fairy prions. For short- tailed shearwaters and common diving petrels there is loose clustering of all individuals indicating that there is more individual variation within these two species (Figure 5.2).

16S rRNA gene pyrosequencing A total of 10,993, 9,877 and 23,967 partial 16S rRNA gene sequences were amplified from faecal samples collected from common diving petrel, fairy prion and short-tailed shearwater respectively. With 97% sequences similarity a total of 2,684, 809 and 1,800 Operational Taxonomic units (OTU) were identified for common diving petrel, fairy prion and short-tailed shearwater respectively.

Figure 5.1, Variation in the abundance of the major phyla; Firmicutes (a), Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) in Common Diving Petrels, Fairy Prions and Short-tailed Shearwaters. There were significant differences between all seabirds for Firmicutes and Bacteroidetes but no significant difference between Actinobacteria and Proteobacteria.

Figure 5.2, Similarity of the major bacterial phyla in Procellariiform seabirds using qPCR. MDS analysis shows tight clustering individual fairy prions indicting little individual variation of the main phyla. While most individuals are clustering together for short-tailed shearwaters and common diving petrels, at least one third do not cluster, indicating potential individual variation.

From the phylogenetic analyses, a total of 16 phyla represented in the Procellariiform microbiota; Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria, Planctomycetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Spirochaetes, Synergistetes, Tenericutes, and Verrucomicrobia and four recently classified candidates (BD1-5, OP10, SR1, and TM7). Firmicutes and Proteobacteria dominated the microbial composition of all three Procellariiform seabirds, with Bacteroidetes also dominating the microbial composition of common diving petrel (Figure 5.3). The level of similarity between short-tailed shearwater and fairy prion is relatively high (50-60%). In comparison, the level of similarity between common diving petrels and short-tailed shearwaters and fairy prions is relatively low (20%) (Figure 5.4).

At family level Ruminococcaceae, Lachnospiraceae and Porphyromonadaceae dominate the microbial composition of common diving petrels with Faecalibacterium, Uncultured Lachnospiraceae, Petrimonas and Barnesiella the dominant genera. In fairy prions and short-tailed shearwaters Leuconostocaceae and Streptococcaceae are the dominant families, with Leuconostoc, Weissella, and Lactococcus the dominant genera in fairy prions and Leuconostoc and Lactococcus the dominant genera in short-tailed shearwaters (Figure 5.5).

Diversity Common diving petrels have a high level of diversity in comparison to the short- tailed shearwater and fairy prion (Figure 5.6).

Figure 5.3, Relative abundance of major phyla in three Procellariiform Seabirds; a) Common Diving Petrels, b) Fairy Prions, and c) Short-tailed Shearwaters. Firmicutes, Bacteroidetes and Proteobacteria dominate the common diving petrel microbiota, whereas, Firmicutes dominates the GI microbiota of short-tailed shearwaters and fairy prions.

Figure 5.4, Cluster Analysis of the GI microbiota Common Diving Petrels (CDP), Fairy Prions (FP) and Short-tailed Shearwaters (STS). The results from the cluster analysis indicate that the similarity of the GI microbiota between fairy prions and short-tailed shearwaters is approximately 50%, whilst the similarity between common diving petrels and fairy prions and common diving petrels and short-tailed shearwaters is relatively low with only 20% similarity.

Figure 5.5, Relative abundance of major Families in common diving petrels, fairy prions and short-tailed shearwaters. Common diving petrels are dominated by Ruminococcaceae, Lachnospiraceae and Porphyromonadaceae. Fairy prions and short-tailed shearwaters are dominated by Leuconostocaceae and Streptococcaceae.

Figure 5.6, Species diversity of Procellariiformes microbiota. Common diving petrels have significantly higher species diversity compared to short-tailed shearwaters and fairy prions.

Discussion An intricate and complex relationship exists between a host and its microbiota. The GI microbiota plays a significant role in energy extraction, fat metabolism and storage, production of short chain fatty acids and host adiposity [9, 259, 293, 298] and has a profound influence on the modulation of host metabolism. The GI microbiota has the ability to modify a number of lipids in serum, adipose tissue and in the liver, with drastic effects on triglycerides and phosphatidylcholine. The resident microbiota is also responsible for the production of metabolites that contribute to host fitness and survival [132]. However, to date there is currently no information available on the microbiota of seabirds, with the unique ability to produce and store stomach oils. Therefore, this study offers the unique opportunity to examine the influence of stomach oils on the microbiota. In the current study, the microbial composition of short-tailed shearwaters, fairy prions and common diving petrels were characterised using Quantitative Real Time PCR (qPCR) and 16S rRNA pyrosequencing.

The quantitative results from the quantitative qPCR analysis demonstrated that the microbial composition of all Procellariiformes was significantly different. Specifically, there was a significant difference in the abundance of Firmicutes and Bacteroidetes. In all three Procellariiformes, Proteobacteria was the most abundant phyla with an abundance of 2.66 x 108, 2.54 x 106 and 1.20 x 108 CFU (colony forming units) for short-tailed shearwater, fairy prion and common diving petrel. Actinobacteria had the lowest abundance level for all Procellariiformes ranging from 1.74 x 101 to 4.93 x 103 (Figure 5.1). The MDS results show strong clustering of individual fairy prions. Most individuals of common diving petrels and short-tailed shearwaters loosely cluster together with almost one third of individuals not clustering together (Figure 5.2).

Results obtained from pyrosequencing identified that the microbiota of that the microbiota of all three Procellariiformes were dominated by Firmicutes and Proteobacteria, with Bacteroidetes also dominating the microbiota composition of common diving petrels, which is in contrast to the qPCR results. These differences could be due to the qPCR primer sets being designed based upon data from terrestrial vertebrate microbiota, (such as humans, rodents and poultry) and that they do not cover the entire microbial composition of the microbiota of Procellariiformes. 97

In accordance with previous studies on other marine vertebrates such as polar bears and Australian sea lions the GI microbiota of short-tailed shearwaters and fairy prions is highly dominated by the phylum Firmicutes [108, 147]. [91]. Whereas the domination of Firmicutes and Bacteroidetes in common diving petrels is similar to that of other vertebrate species; including penguins (Chapter 2), terrestrial mammals, Arctic and Antarctic pinnipeds and avian species [73, 109, 142, 145, 154, 181] Members of the phylum Firmicutes are associated with the breakdown of complex carbohydrates, polysaccharides, sugars and fatty acids, which are then utilised by the host as an energy source [93, 253] with high abundances of Firmicutes being linked to adiposity. Members of the phylum Bacteroidetes in humans have been associated with vitamin synthesis, polysaccharide metabolism, and membrane transport [120]. Although the common diving petrel microbiota in this study is similar to that of other vertebrate species they do differ from other vertebrates in regards to the relatively high abundance of Proteobacteria (5-30%).

In humans and mice the ratio of Firmicutes to Bacteroidetes in obese and lean individuals differs, with obese individuals having a higher abundance of Firmicutes and relatively lower abundance of Bacteroidetes in comparison to their lean counterparts [152, 156, 263]. In oil producing Procellariiformes the relative proportion of Firmicutes (77%) to Bacteroidetes (1-2%) is similar to the relative proportions found in obese humans and mice. [152]. This ratio of Firmicutes to Bacteroidetes has also been found in another marine vertebrate that also has a predisposition to obesity the Australian Sea Lion (80% Firmicutes, 2% Bacteroidetes). The high level of body fat in Procellariiformes therefore may be a combination of a rich lipid based diet and a microbiome that is high in Firmicutes. In the common diving petrels however, the proportion of Firmicutes to Bacteroidetes is almost 1:1, as opposed to 77:1in oil producing Procellariiformes. Unlike most Procellariiformes, common diving petrels do not migrate over large distances and are thought to be fairly sedentary remaining close to coastal waters all year round and therefore may not require large fat reserves [84].

At family level short-tailed shearwaters and fairy prions are dominated by Leuconostocaceae and Streptococcaceae, with Leuconostoc, Weissella, and Lactococcus the dominant genera of short-tailed shearwaters and Leuconostoc and Lactococcus are the dominant genera of fairy prions. There is little similarity 98 between common diving petrels and the oil producers (20%). Due to the absence of data on the functional role of microbes in seabirds we can only infer what impact these changes to the microbiota will have on Procellariiformes. The microbiota of short-tailed shearwaters and fairy prions are dominated by species that are not commonly found in the vertebrate gastrointestinal tract (Leuconostoc & Lactococcus), are commonly used as starter cultures for dairy products and are associated with the skin microbiota of animals [21, 46, 188]. Weissella is associated with the vertebrate GI tract and is also used as a probiotic [21] The microbiota of common diving petrels is associated with butyrate producing microbes (Fusobacteria and Ruminococcus) [8, 204] and microbes associated with anti-inflammatory properties. Butyrate is an essential short-chain fatty acid produced in the colon. The main effects butyrate has on the intestinal tract in humans included, influence of ion absorption, cell proliferation and differentiation, immune regulation, and is an important anti-inflammatory agent [42]. In chickens, butyrate supplementation leads to a significant increase in host defence peptide gene expression, enhance antibacterial properties of monocytes against pathogenic bacteria, boost host immunity and increase host adiposity [196].

As all three species share a similar diet, it can be assumed that diet is not the reason for the difference in microbial composition. Therefore, the cause of these differences is likely to be due to differences in host phylogeny, digestive physiology or retention time. Ley et al [154] identified that host phylogeny and gut physiology are significant predictors of host microbial composition. In addition, however retention time of digesta can also influence the microbial composition of the GI tract [246]. It is well known that the digestive physiology in diving petrels differs to that of other Procellariiformes, but retention time of digesta is also significantly shorter than other Procellariiformes [225]. For example in the diving petrel Pelecanoides georgicus the retention time for lipids was 2.3hrs, whereas in Shy Albatross (Macronectes giganteus) and other large Procellariiformes the retention time is 12.5h and in smaller oil producing Procellariiform Antarctic Prion (Pachyptila desolata) the average retention time was 15hrs [225].

Penguins Vs. Procellariiformes The penguin microbiota is dominated by Firmicutes, Bacteroidetes, Fusobacteria and Proteobacteria (Chapter 2), while the GI microbiota of Procellariiformes is 99 dominated by Firmicutes, Proteobacteria and in common diving Bacteroidetes is also dominant (Chapter 5) (Figure 7.2). In penguins, diet, and diet associated microbiota could potentially be a major factors influencing the microbiota of seabirds, with the krill eating penguins clustering closely together on the MDS plot, in comparison to the king and little penguins (Figure 2.4) [12, 153]. Whereas, in the Procellariiformes, digestive physiology and retention time of digesta appear to be the major factors influencing the microbial composition (Figure 5.4), with a high level of similarity between the two oil producing seabirds and a low level of similarity between the oil and non-oil producing seabirds. In contrast to the sequencing results of penguins (Chapter 2), the Procellariiformes did not have a large proportion of “unclassified” bacteria present and most sequences were able to be identified down to genus level. In addition, although opportunistic pathogens such as members from the family Enterobacteriaceae, Campylobacteriaceae, Helicobacteriaceae and Neisseriaceae are present in the microbiota of all Procellariiformes, they are not present in significant numbers <1%.

Conclusion This study has identified that there is large variation within the faecal microbiota of Procellariiformes which can be divvided into oil producing and non-oil producing such as common diving petrels. Firmicutes and Proteobacteria dominate the GI microbiota of oil producing Procellariiformes, whilst Firmicutes, Proteobacteria and Bacteroidetes dominate the GI microbiota of common diving petrels. Although, the cause of these differences is yet to be determined, host phylogeny, digestive physiology and retention time could potentially play major roles in determining the final microbial composition of individual seabird species [12, 153, 246].

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CHAPTER 6 Variations in the Gastrointestinal Microbiota of Short-Tailed Shearwaters (Puffinus tenuirostris) During Development

Summary At birth (emergence from egg) the gastrointestinal (GI) tract of all avian species is considered to be completely sterile and is colonised immediately after emerging from the egg. Factors such as microbial load on the eggshell, nesting environment and their first meal are all said to influence the microbial colonisation of the GI microbiota of the chick. Immediately after birth, successional changes occur within the microbiota until a climax community is achieved. To date, the development of the microbiota in a long lived seabird has not been examined. Therefore, this study aimed to characterise the microbiota of short-tailed shearwater chicks from hatching until fledging (98 days), using Quantitative Real Time PCR (qPCR) and 16S rRNA pyrosequencing sequencing. Results from the qPCR and 16S rRNA pyrosequencing analysis identified no significant differences or trends in the microbiota of short- tailed shearwaters from hatching to fledging. The pyrosequencing analysis identified that the GI microbiota of short-tailed shearwater chicks was dominated by gram positive facultative anaerobes and obligate anaerobes from the phylum Firmicutes (73-85%) throughout development. The ratio of Firmicutes to Bacteroidetes in short- tailed shearwater chicks (77:1) were similar to that of obese humans and mice (100:1), indicating that the GI microbiota may play a role in fat deposition during development. This study describes the first molecular examination of the microbial community of the digestive tract of short-tailed shearwater chicks throughout development. Short-tailed shearwater chicks display similar ecological successional changes as other vertebrates with aerobic and facultative anaerobes being the first microbes to colonise the GI tract and slowly transitioning to a microbiota dominated by strict anaerobes.

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Introduction In nature, bacteria are found everywhere and are able to colonise new habitats with the availability of nutrients. As bacteria are dispersed by water, soil and wind there are very few places that are unable to be colonised by bacteria, including the GI tract [6]. At birth the GI tract is considered to be devoid of microorganisms. In mammals, the microbial community is said to be inherited from the mother through intimate contact with their mothers faecal and vaginal microbes and from breast milk [194]. In birds however, the new born chick, acquires its microbiota from the surface of the egg, surrounding environment (i.e. nest) and their first meal [6, 99, 101, 159, 162, 217, 256, 291]. In addition, vertical transmission of microbes via regurgitation of an undigested or partially digested meal is also said to influence microbial colonisation [110, 205]. Through ecological succession, the sterile GI tract, is rapidly colonised overtime forming a dense and complex microbial community. In humans this process usually takes around 2-3 years [164, 166, 168, 240], whilst in chickens, stabilisation of the composition of the GI microbiota occurs within 40 day [16, 17, 68, 90, 217].

At birth, the infant gut is an aerobic environment that is rapidly colonised by aerobic and facultative anaerobic bacteria, such as Escherichia coli and Streptococci [5, 68, 168, 217, 268]. Within the first few days both E.coli and Streptococci are present in extremely high numbers of around 108 – 1010 CFU of faeces [168]. Research suggests that these pioneering bacteria reduce the amount of oxygen present in the GI tract through oxidation/reduction, creating an environment that is highly favourable to facultative and obligate anaerobes [168, 199], such as Bacteroides, Clostridia, and Bifidobacterium [168, 245], which flourish in a low oxygen environment.

To date, the development of the GI microbiota in a long lived seabird has not been examined. Therefore, the short-tailed shearwater was used as a model to examine the development of the GI microbiota in a long lived seabird. Short-tailed shearwaters breed between September and April with egg laying occurring in the last week of November [237]. Prior to egg laying, adult shearwaters embark on long foraging journeys to replenish their energy and fat reserves [236]. Short-tailed shearwater nest in simple scrapes or burrows, lined with vegetation. Short-tailed shearwaters lay one egg, which is incubated for a period of 50-55 days [44, 272]. In contrast to other seabirds, Procellariiformes are able to regulate body temperature much sooner than other seabirds, reducing the guard stage to 2 - 3 days. This ability to regulate body temperature is in part due to the subcutaneous fat deposits laid down during the first 102 few days [272]. Like all Procellariiformes, short-tailed shearwaters (Ardenna tenuirostris)use a twofold foraging strategy during the breeding season, with adults alternating between two short foraging trips of 1 - 4 days, with one long foraging trips of approximately 8 - 19 days [141, 277]. Previous studies have documented that the diet of short-tailed shearwaters is dominated by Australian krill Nyctiphanes australis [44, 141, 173]. However, in contrast to previous studies, Weimerskirch & Cherel [277], discovered that during long foraging trips, Antarctic krill and Myctophid fish dominated the diet of short-tailed shearwaters. In Victorian shearwaters, however, there is little evidence to suggest that shearwaters forage on Antarctic species with only Nyctiphanes australis present in dietary samples (Schumann et al, unpublished [173]). Although chicks do not fledge until around 97days of age, adults begin their migration north when their chicks are around 85 days old at the beginning of April. For the next 2-3 weeks chicks must survive off their endogenous fat stores until fledging at 97 days of age [190].

The goal of this study was to characterise the microbial community of the short- tailed shearwaters digestive tract during development from newly hatched chicks to fledging juveniles (7-10 weeks) using DNA derived from faecal material. Quantitative Real Time PCR and 16S rRNA gene amplicon sequencing was used to identify bacteria at each stage during development and the samples were compared to examine changes in community structure over time. To our knowledge, this is the first study to investigate the microbial composition of short-tailed shearwater chicks. This

Methods Sample Collection To characterise the microbial composition of the short-tailed shearwater chick during development faecal samples were collected from short-tailed shearwater chicks from the Phillip Island Nature Park. On Phillip Island, short-tailed shearwaters burrow among the sand dunes and line their nests with surrounding vegetation (Figure 6.1). At the beginning of the breeding season, 20 burrows were randomly selected with the restriction that the nest contained an egg at the beginning of the breeding season. Of the 20 eggs, 17 successfully hatched, with only 10 chicks successfully fledging at the end of the breeding season. Following hatching, cloacal swabs were collected from each chick directly from the cloacae using Eswabs (Copan, Italy). On the same day

103 every week, short-tailed shearwater chicks would be removed from their nest, weighed, inked (red or green food dye) for identification purposes and swabbed and then placed back into the nest within 5-10 minutes to avoid stress. Samples were placed into an amine solution for preservation of the DNA and stored in liquid nitrogen (-196°C) for shipment and then stored at -20°C until DNA extraction.

16S rRNA Pyrosequencing Samples from 4 short-tailed shearwater chicks for week 2, 5, 8, 11 and 14 were analysed 16S rRNA pyrosequencing as per protocols described in chapter 2. Results from adult short-tailed shearwaters in Chapter 5 were included for comparison.

Bioinformatics, Statistical and Diversity analysis Quality control, removal of chimera’s, sequence alignment, identification and Operational Taxonomic Units (OTU) classification was performed by Ribocon GmbH (Germany) as per the protocol of Prusse et al [206, 207], as per Chapter 2 methodology.

In order to explore the variation in bacterial counts, linear mixed models with individual birds as a random effect and common assessment times (weeks) as a fixed effect were fitted to the log-transformed data. Bacterial counts were log transformed (base 10), to uncouple apparent variance-mean relationships, after inspection of diagnostic plots of Studentized residuals versus predicted values [238].

The linear mixed models were restricted to six types corresponding to the six combinations of the following: Random effects (short-tailed shearwater chicks): No autocorrelation in the repeated counts from the short-tailed shearwater chicks and homogeneous variances at each assessment (scaled identity model).

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No autocorrelation in the repeated counts from the short-tailed shearwater chicks and heterogeneous variances at each assessment (diagonal model). First order autoregressive process for the co-variation in the repeated counts (AR (1) model) and homogeneous variances. Fixed effects (weeks): Week as a categorical factor. Week as a linear trend.

For each abundance measure, the fitted model with the smallest value of the Akaike Information Criterion (AIC) [239] was selected and the significance of the association of the common assessment times with variation in abundance was assessed using the F-test. If the selected model included week as a categorical factor, weekly means and their standard errors were reported otherwise, if the selected model included week as a linear trend, the estimated intercept and slope coefficients, and their standard errors, were reported. All analyses used the mixed model procedures in the IBM SPSS Statistics package (Version 20). A Principal Coordinates Analysis (PCoA) diversity and cluster analysis was performed on the qPCR whilst a Multidimensional scaling plot (MDS), was performed on the pyrosequencing data.

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PCoA results, also did not detect any significant differences between age classes with no distinct clustering of age classes or individuals (Figure 6.5).

Figure 6.1, Short-tailed shearwater breeding colony. Sticks represent nest sites used in this study (Photography by Meagan Dewar).

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Figure 6.2, Natural burrow of short-tailed shearwater (Photography by Meagan Dewar)

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Figure 6.3, Short-tailed shearwater chick (Photography by Meagan Dewar).

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Figure 6.4, Changes in the abundance of the major phyla; Firmicutes (a), Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) during development in short-tailed shearwater chicks. There were no significant differences or trend detected during development.

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Figure 6.5, Similarity of the major bacterial phyla in short-tailed shearwater chicks during development using qPCR. No clustering of age classes or individuals was observed.

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16S rRNA gene pyrosequencing A total of 12,522, 5,089, 24,222, 20312, and 16,131 partial 16S rRNA gene sequences were amplified from faecal samples collected from short-tailed shearwater chicks on weeks 2, 5, 8, 11 and 14 respectively. With 97% sequence similarity a total of 1,040, 655, 1,786, 1,469 and 1,124 Operational Taxonomic Units (OTU) were identified from short-tailed shearwater chicks respectively. A total of 23,967 partial 16S rRNA gene sequences and 1,800 OTU’s were amplified from adult short-tailed shearwaters at the beginning of the breeding season

From the phylogenetic analyses, there were 13 phyla represented in developing short-tailed shearwater gut microbiota; Firmicutes, Fusobacteria, Proteobacteria, Planctomycetes, Bacteroidetes, Acidobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, Gemmatimonadetes, Synergistetes, Tenericutes and seven recently classified candidates (Candidate division BRC1, OD1, OP10, OP11, TM7, BD1-5 and SM2F11). Throughout development Firmicutes dominated the microbiota, comprising over 80% of the total composition except for weeks 8 and 11 where Firmicutes comprises 62 and 73% (Figure 6.6). There was a high level of similarity in the microbial composition of short-tailed shearwater chicks throughout development with 60% similarity between all weeks except for week 5, where the level of similarity declined to 40%. (Figure 6.7). In the adult short-tailed shearwater, Firmicutes and Proteobacteria were the dominant phyla (Figure 6.6). The level of similarity between the adult short-tailed shearwaters and its offspring was 40% (Figure 6.6).

At family level, Leuconostocaceae and Planococcaceae dominated throughout development, with Leuconostoc and Lysinibacillus the dominant genera. During weeks 2 and 5 Leuconostocaceae is the most dominant family, followed by Planococcaceae. However by week 8, Planococcaceae dominated the microbiota until fledging in week 14 (Figure 6.8). In adult shearwaters, Leuconostocaceae and Streptococcaceae (Figure 6.6) are the dominant families, with Leuconostoc, Weissella, Lactococcus and Streptococcus the dominant genera (Figure 6.8).

Species Diversity The species diversity in the short-tailed shearwater chick fluctuates throughout development. The adult short-tailed shearwater has a high level of diversity (Figure 6.9). 111

Figure 6.6, Relative abundance of the major phyla in short-tailed shearwater chicks during development. Firmicutes dominates the microbiota of short-tailed shearwater chicks throughout development and in the adult microbiota.

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Figure 6.7, Similarity of the gut microbiota of short-tailed shearwater chicks during development. The result indicates that there is a high level of similarity between weeks 2, 8, 11 and 14 with 60%, with limited similarity between week 5 and the other weeks with 40%. In addition, there is around 40% similarity between adult short-tailed shearwaters and their offspring.

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Figure 6.8, Relative abundance of major Families during development in short-tailed shearwater chicks. Planococcaceae and Leuconostocaceae dominate the microbial composition of short-tailed shearwater chicks throughout development, whereas, Leuconostocaceae and Streptococcaceae dominate the adult microbiota.

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Figure 6.9, Species diversity at different age classes. Species diversity fluctuates in the short-tailed shearwater chick, throughout development.

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Discussion Establishment and early colonisation of the GI tract has been recognised as a crucial stage in neonatal development with pioneering species responsible for influencing the GI pH, oxygen levels, mucosal structure and immunity and therefore, these pioneering species influence a hosts overall health and disease status throughout life [136, 138, 198]. In mammals the establishment and colonisation of the neonate GI tract has been extensively studied [168, 199, 268]. In avian species, however data is restricted to the microbiota of poultry [17, 68, 134, 171, 256, 266]. To date there is no data available on the GI microbiota of long lived seabirds, nor the microbial succession that occurs during development. Therefore this study aimed to examine the establishment and microbial succession that occurs in little penguin chicks during development. This study aimed to analyse the microbial composition of the developing short-tailed shearwater from hatching to fledging using Quantitative Real Time PCR and 16S rRNA pyrosequencing.

Quantitative data obtained by qPCR analysis for Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria demonstrated that the microbiota in short-tailed shearwater chicks occurs rapidly after birth, with over 4.1 x 106CFU present at 2 weeks of age (estimation calculated from abundance of Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria), which is similar to little penguin chicks (Chapter 3) and other vertebrates with the microbial population estimated at around 2 x 106 in little penguins and 108 -1010 in humans [136, 168]. In contrast to other vertebrates however, the short-tailed shearwater microbiota appears to stabilise quite rapidly with the absence of any significant changes in bacterial abundance.

In accordance with the qPCR results, the pyrosequencing analysis, demonstrated that there was little change in the microbiota of short-tailed shearwater chicks during development. In previously examined neonates including penguins (Chapter 3), the GI microbiota is initially colonised by gram negative aerobic and facultative anaerobes, followed by facultative and strict anaerobes [5, 68, 168, 217, 268]. In the short-tailed shearwater however, the microbiota at 2 weeks of age is colonised by gram positive facultative and obligate anaerobes. However, as samples were not analysed during the first week post hatch, there is a possibility that these early pioneering species could have been present. In comparison to other marine vertebrates, the microbial composition of short tailed shearwater chicks appears to be 116 more similar to that of Australian fur seal pups than other seabirds including the little penguin (Chapter 3). In Australian fur seal pups the microbiota at 2, 6, and 9 months is heavily dominated by gram positive bacteria from the phylum Firmicutes (>80%) including; members from the Order Clostridia and Bacilli (Smith et al, unpublished). In addition, little variation was observed in the microbial composition of both the short-tail shearwater and Australian fur seal (>60%), with high similarity throughout development (60%) except for week 5 (Smith et al unpublished). Even during the final stages of development when adults abandon their chicks to return to the northern hemisphere, leaving chicks to survive off their endogenous fat reserves [190] a high level of similarity is maintained. There is low level of similarity between the adult and chick indicating that the adult has negligible influence of the GI microbiota of the chick.

Throughout development the microbiota of short-tailed shearwater chicks is dominated by Firmicutes. In humans and mice, the ratio of Firmicutes to Bacteroidetes has been shown to influence a host’s adiposity level by enhancing energy extraction and modulating genes that regulate fat storage [152, 156, 259, 260, 263] and is currently considered to play a crucial role in obesity. In obese individuals the ratio of Firmicutes to Bacteroidetes is 100:1, whereas in their lean counterparts the ratio is 10:1 [152, 156, 263]. Similarly to adult short-tailed shearwater (Chapter 5), the ratio of Firmicutes to Bacteroidetes in short-tailed shearwater chicks ranges from 73:1 to 85:1 and is similar to that of obese humans, mice and Australian sea lions, except for week 5, where the ratio is 62:18 [152, 156, 263]. Following birth, the body mass of short-tailed shearwater chicks rapidly increases and by week 7, the body mass of chicks exceeds that of the adult shearwater by 15-50% [189]. While many factors, such as a high lipid diet and gut physiology, may influence body mass, the high abundance of Firmicutes of the short-tailed shearwater chick may confer a predisposition towards developing excess body fat stores. A chick’s ability to rapidly accumulate large fat reserves enables adults to feed them less regularly and enables chicks to survive the long periods between feeds and is essential for survival post fledging [201].

Due to the absence of data available on the role of microbes in seabirds, the potential role of the microbiota in short-tailed shearwaters can only be elucidated from previous research. At genus level the microbiota of short-tailed shearwater chicks is 117 dominated by Lysinibacillus, a known soil bacterium. Other genera known to dominate the microbiota of short-tailed shearwater chicks are Leuconostoc and Lactococcus, which are lactic acid bacteria commonly used in the food industry as starter cultures for dairy products and have been associated with fermentation [1, 21, 188]. However, to date their role in the GI microbiota is still unknown and requires further investigation.

Conclusion The microbial composition of short-tailed shearwater chicks is highly dominated by the phylum Firmicutes. In contrast to other infants, the short-tailed shearwater microbiota is dominated by gram positive facultative and obligate anaerobic bacteria and is stable throughout development. The ratio of Firmicutes to Bacteroidetes in the short-tailed shearwater chick strongly resembles that of obese humans, mice and Australian sea lions and fur seals. However, due to the absence of information on the functional role of the dominant microbiota in short-tailed shearwaters and other vertebrates, it is extremely difficult to elucidate the role of the microbiota short-tailed shearwater development.

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CHAPTER 7 General Discussion

Introduction In order to understand the complex relationship between a host and its microbiota, one must first characterise the GI microbiota of healthy individuals at baseline before elucidating the role of microbiota, and the influence alterations of the GI microbiota will have on health and disease of an individual or population [157, 262]. Therefore, the main goal of this project was to characterise the assemblage and community composition of the seabird microbiota in its natural environment. Seabirds are a group of birds that obtain their entire nutrition from the marine environment [100, 105, 232]. The diet of seabirds is high in lipids, proteins and polyunsaturated fatty acids [211]. This research to my knowledge is the first to use both Quantitative Real Time PCR and 16S rRNA pyrosequencing to characterise the GI microbiota from members of the Orders Sphenisciformes and Procellariiformes.

In recent years, research has been carried out on the mammalian [108, 109, 147, 154, 181, 290], avian [12, 162, 289, 297], human [155, 156, 168], reptilian [73, 167] and fish [18, 214, 215, 228] gut microbiota. These studies have increased our knowledge and understanding on the complex relationship that exists between a host and its microbiota and that the GI microbiota has a profound influence on the nutritional, physiological, immunological and metabolic processes of the host. The GI microbiota is essential for the development maturation of the immune system [214, 215], developmental regulation of the intestinal physiology, energy harvest, fat metabolism and production of short-chain fatty acids [66, 167, 168, 180, 215, 298, 299]. The colonisation process during infancy has also been shown to affect a hosts predisposition to disease later in life [66].

Over the past decade, a large number of internal and external factors have been found to influence the colonisation and stabilisation of the GI microbiota. These factors include, diet, host phylogeny, intestinal physiology, age, external environment (i.e. nesting material), antibiotics and stress [73, 87, 88, 131, 153, 154, 181]. However, the major limitation in being able to link the composition of the GI microbiota to host function is an absence of knowledge concerning the indigenous community that is present under natural conditions.

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Therefore, characterisation of the vastly diverse ecosystem of the GI tract is the first step in exploring its role in digestive physiology, gut development and maturation, immune function and disease [91]. Seabirds live in an unpredictable environment and have had to adapt to fluctuations in availability of prey, oceanic currents and periods of fasting during breeding and moult. Therefore, knowledge on the composition and role of the GI microbitoa is essential to further understand the ecology of seabirds.

This thesis provides greater understanding of the composition of the GI microbiota of seabirds under natural conditions during different life stages (i.e. chicks, adults, moult). This discussion outlines the general conclusions of the research that has been covered in this thesis.

Gastrointestinal Microbiota of Seabirds The results from this thesis identified that the gastrointestinal microbiota of seabrids differ not only between avian orders (Spheniscidae & Procellariiformes) but also between species, with a relatively low level of similarity between all seabirds examined in this study (Figure 7.1). The penguin microbiota is dominated by Firmicutes, Bacteroidetes, Fusobacteria and Proteobacteria (Chapter 2), while the GI microbiota of Procellariiformes is dominated by Firmicutes, Proteobacteria and in common diving Bacterodetes is also dominant (Chapter 5) (Figure 7.2). In penguins, diet, and diet associated microbiota could potentially be a major factors influencing the microbiota of seabirds, with the krill eating penguins clustering closely together on the MDS plot, in comparison to the king and little penguins (Figure 2.4) [12, 154]. In contrast, the Procellariifomres, digestive physiology and rention time digesta appear to be the major factors influencing the microbial composition (Figure 5.4), with a high level of similarity between the two oil producing seabirds and a low level of similarity between the oil and non-oil producing seabirds. Although diet is not a major factor influencing the differences in Procellariiform seabird microbiota, it appears to be a major influential factor in seabirds with the krill eating seabirds (short-tailed shearwaters, fairy prions, macaroni & gentoo penguins and common diving petrels) clustering closesly together (Figure 7.1) [12, 153, 228, 246].

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Figure 7.1, Level of similarity between seabirds. Short-tailed shearwaters and fairy prions are highly similar in their microbial composition. In addition, gentoo and macaroni penguins are highly similar to common diving petrels. Little and king penguins are highly disimilar to other seabird species.

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Figure 7.2, Relative abundance of the major phyla for all seabird species using 16S rRNA pyrosequncing. Little, king and macaroni penguins and common diving petrels are dominated by Fimicutes, Bacteroidetes and Proteobacteria. Gentoo penguins are dominated by Fusobacteria. Short-tailed shearwaters and fairy prions are dominated by Firmicutes.

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The dominance of Bacteroidetes and Firmicutes in penguins and common diving petrels, is in accordance with previous studies on other vertebrate species; including Antarctic seals [91, 121, 145, 153, 249]. However, the relatively high abundance of Fusobacteria (2-55%) and Proteobacteria (5-30%) differ from other vertebrates. The domination of Firmicutes in oil producing Procellariiformes is very similar to that of other marine vertebrates including; polar bears and Australian sea lion [108, 147]. To date, there is no knowledge available in the literature on the functional role of the GI microbiota in seabirds and therefore, information on the role of these microbes must be elucidated from previous research in other vertebrates. The penguin and common diving petrel microbiota appears to be associated with microbes that are responsible for responsible for the absorption of dietary fats, lipids, and soluble vitamins, maintain intestinal barrier function, regulate triglycerides, impact intestinal permeability, activate intestine-brain-liver neural axis, enhance the immune system, alteration of lipoprotein profiles, protection against stress-induced lesions, modulation of proinflammatory and anti-inflammatory gene expression, and strengthen epithelial cell barrier properties. However, these microbes have also been implicated in GI pathologies, brain-gut axis, and a few neurological conditions [43, 117, 139, 223, 235, 251, 292], butyrate production [204] and microbes with probiotic properties [250]. In contrast, the Procellariidae microbiota appears to be dominated by lactic acid bacteria that are commonly used in the food industry and the skin microbiota of some vertebrates [22, 47, 188].

Development of the Gastrointestinal Microbiota in Seabirds Establishment and early colonisation of the GI tract has been recognised as a crucial stage in infant development with pioneering species responsible for influencing the GI tract pH, oxygen levels, mucosal structure and immunity [136, 138, 198]. Immediately after birth, the GI microbiota is rapidly colonised and undergoes successional changes until a dense and stable community is achieved. In most vertebrates this process can take anywhere from 40 days [16, 17, 68, 90, 217] to 2 years [164, 166, 168, 240]. The results from this thesis identified that the development of the seabird microbiota, differs greatly between species. In the little penguin, there were significant differences throughout development and significant upward trends in the abundance of Firmicutes and Bacteroidetes, whereas, in the short-tailed shearwater there were no significant differences throughout development and appears relatively stable throughout development with a high level of similarity 123 between each age class. There is low similarity between adults and chicks for both the short-tailed shearwaters and little penguin, indicating that the adult microbiota has a negligible influence over the chick’s microbiota.

The microbial composition of the little penguin is dominated by microbes that are responsible for the absorption of dietary fats, lipids, and soluble vitamins, maintain intestinal barrier function, regulate triglycerides, impact intestinal permeability, activate intestine-brain-liver neural axis, enhance the immune system, alteration of lipoprotein profiles, protection against stress-induced lesions, modulation of proinflammatory and anti-inflammatory gene expression, and strengthen epithelial cell barrier properties. However, these microbes have also been implicated in GI pathologies, brain-gut axis, and a few neurological conditions [43, 117, 139, 223, 235, 251, 292], butyrate production [204] and microbes with probiotic properties [250]. In the short-tailed shearwater chick, however the GI microbiota appears to be associated with soil and lactic acid bacteria that are not commonly associated with the GI tract.

In seabirds, pre-fledging body mass is associated with juvenile survival and therefore, the accumulation of fat reserves is important for post fledging survival. In little penguin chicks the relatively high abundance of butyrate producing microbes could influence body mass, as previously observed in poultry [196]. Although the ratio of Firmicutes to Bacteroidetes strongly resembles that of obese humans, mice and Australian pinnipeds, the microbes within the phylum Firmicutes (Lysinibacillus, Leuconostoc and Lactococcus) have previously not been associated with energy extraction, lipid accumulation or obesity.

The differences between little penguins and short-tailed shearwaters could be in part due to differences diet composition, digestive physiology, nesting environment and host phylogeny, all of which have been previously shown to strongly influence the microbial composition of vertebrates [12, 154, 246, 256]. Due to the absence of information on the functional role of the dominant microbiota, we are unable to elucidate the role of the GI microbiota in fledging survival of little penguins and short-tailed shearwaters.

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Influence of Fasting on the Gastrointestinal Microbiota of Penguins Once a year all penguins must undergo moult to replace their old warn plumage with new feathers. Prior to moult penguins spend large amounts of time at sea building up their protein and lipid reserves necessary for the upcoming moult [218]. During moult penguins replace their entire plumage whilst fasting on land and cannot return to sea because of the consequences of reduced waterproofing and thermal insulation [105, 119]. Similarly to the breeding season, penguins must rely on endogenous fat and protein reserves for feather synthesis and nourishment for the duration of moult which can take between 2-5 weeks [50, 218].

This study aimed to examine the influence of fasting during moult on the GI microbiota of two penguins; the king (Aptenodytes patagonicus) and little penguin (Eudyptula minor). The results from this thesis identified significant changes in the microbial composition of both king and little penguins. This study also documented that the degrees to which moult impacts the microbial composition differs between the two penguin species. In little penguins, there is still a high level of similarity between early and late moult (60%), with little variation in the abundance of the major phyla observed with both the qPCR and pyrosequencing analysis. In contrast, there were significant differences between early and late moult with a 75% increase in the level of Fusobacteriaceae. In addition, there was a low level of similarity (20%) between early and late moult in king penguins. Due to the absence of knowledge in the literature regarding the role of microbes in a seabird host, we are unable to elucidate the consequences of fasting as a result of microbial changes to the microbial composition.

Quantitative Real Time PCR Vs. 16S rRNA Pyrosequencing. The primer sets used in this study appear to be limited in their ability to provide an accurate estimate of the abundance of major phyla within the faecal samples of seabirds. The limitation of the primers could be in part due to the fact that they have been designed from bacterial sequences obtained from human and chickens and therefore, do not cover all members of the bacterial phyla found in the penguin microbiota. The use of the pyrosequencing data from this project to design more accurate primer sets could be useful in providing more accurate abundance estimates of the major phyla present in seabirds.

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CHAPTER 8 Conclusions and Future Directions

Conclusions

This research provides a baseline for future work regarding the microbial composition of seabirds. This thesis has identified that a complex community inhabits the GI tract of seabirds and provides insight in to the succession changes that occur during development and how fasting influences the microbial composition. The results obtained in this thesis allow us to conclude;

The results from this thesis identified that the gastrointestinal microbiota of seabrids differs not only between avian orders (Spheniscidae & Procellariiformes) but also between species, and at different life stages

The microbiota of penguins and common diving petrels, is dominated by Firmicutes, Bacteroidetes and Proteobacteria, with Fusobacteria

The GI microbiota of oil producing Procellariiformes (short-tailed shearwaters and Fairy Prions) is highly dominated by Firmicutes and Proteobacteria to a less extent.

Diet and prey associateed microbiota appear to be major contributing factors influencing the GI microbiota of seabird species with strong clustering of krill eating seabirds. However, within the Order Procellariiformes, digestive physiology appears to be the major factor influencing microbial composition, with high similarity between the two oil-producing Procellariiformes and a low level of similarity between oil and non-oil producing Procellariiformes. As diet between these three seabirds, is highly similar, diet and prey associated microbiota do not appear to explain these differences.

Signficant differences in the colonisation of the GI microbiota during development in different seabird species, with significant changes observed in the little penguin microbiota throughout development, whereas, no signifcant changes were observed in the short-tailed shearwater microbiota throughout development. Therefore, is no ‘core’ microbiota colonising the GI microbiota of seabirds during development.

Fasting during moult influences the microbial composition of both king and little penguins, but to differing degrees. In little penguins these changes appear to be subtle, with only a 40% difference in the microbial composition. In king penguins 126 however, not only is there a significant increase in the level of Fusobacterium but there is little similarity between the early and late moult (20%). These differences, could in part be due to the differences in moult length (i.e. 2 weeks in little penguins vs. 5 weeks in king penguins).

Limitations

Quantitative Real Time PCR Primer Sets

Due to the absence of ‘seabird’ specific oligonucleotide primers, the primers used in this study were from previous research on the dominant microbiota of other vertebrate hosts including humans and chickens. Although, Zdanowski et al [294] did use ‘penguin specific’ bacterial primers in their study, upon further testing of these primers in our laboratory, these primers were not non-selective towards the particular primer as stated in the paper (i.e. Firmicutes primer was able to detect Bifidobacteria). Therefore, for this thesis we used bacterial primers that were specific to a given phylum. However, the results from the 16S rRNA pyrosequencing of this study, which was conducted after the qPCR analysis, identified that the primer sets used in this thesis, are unable to detect all members of the major phyla in the study animals of this thesis and therefore, are limited in their ability to provide an accurate estimate of the abundance of major phyla within the faecal samples of seabirds. If more time and funds were available, the data from the 16S pyrosequencing could have been used to design primer sets that were more specific to seabirds.

16S rRNA gene pyrosequencing

Due to the availability of limited funds and the high costs associated with pyrosequencing, samples were pooled together following amplification and prior to sequencing on the Roche GS FLX 454 sequencer. By pooling samples, after amplification, ensured that bacterial DNA from all members of each species was equally represented and enable us to look at changes in the microbiota at a community level. However, by pooling samples we were unable to examine individual differences and also prevented us from removing potential outliers that may influence the overall result.

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Future Studies

This thesis has provided knowledge of the resident GI microbiota of two seabird Orders, the Sphenisciformes and Procellariiformes. To further our knowledge of the GI microbiota of seabirds and its role in health, digestive physiology, survival and disease the following studies are recommended.

Microbiome of penguins

Microbiome of fasting penguins

Introduction of alien species by humans in Antarctic penguins

Microbiome of Procellariiformes

The developing microbiota and immune system in Seabirds

Study 1: Microbiome of Penguins

Summary: In the past, only two studies have focused on examining the “residential” microbiota of penguins using molecular based technologies [12, 294], by examining the microbial ecology of adélie penguins. Whereas other research has focused on identifying the presence of known human and veterinary pathogens, or the microbial composition using traditional culture techniques. Whereas for temperate penguins, there have been no investigations into the microbial ecology utilising molecular based techniques. To date there is no data available on; 1) the stability of the penguin microbiome over time and 2) role of microbes in penguins; including the role of potential pathogens, except when death has been caused by an infectious agent (i.e. Avian Cholera) [26, 45, 62, 63, 85, 106, 113, 149, 151].

The recent advances of metagenomic techniques allows for a more comprehensive and unbiased assessments of the microbial diversity by allowing the examination of microbes that are not easily cultured in a laboratory [264]. Metagenomics allows for the characterisation of the microbial community within the gut [107] and can elucidate important processes for the gut microbes and the host. In addition, metagenomics can provide insight into the insight into the relationships between the host and its gut microbiota [10, 209, 264, 280]. By coupling metagenomics with metabolomics (study of metabolites) will shed light on how microbial communities function in the gastrointestinal tract of penguins [261].

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Methods: Samples from penguins will be collected from Antarctic (i.e. emperor, royal, gentoo, and adélie), Sub-Antarctic (i.e. macaroni, king), Temperate (i.e. little, rockhopper, snares, yellow-eyed) and Tropical (i.e. Humboldt, Galapagos) penguin species. For statistical significance, a population size between 10-20 individuals/species will be required. Metagenomic analysis of samples will be completed on the Roche 454 GS FLX Titanium. Metagenomic analysis will elucidate information on the microbial population and potential functionality of the microbial population. Metabolites present in samples are identified by GC mass spectroscopy. Results are then run through genomic and metabolite databases to identify microbes and metabolites present in the sample.

Significance: This research is significant because it starts to elucidate the role of microbes in penguin health, metabolism and digestive physiology which has not previously been done before. In addition, this study would also provide valuable information on forces influencing the GI microbiota of penguins (i.e. host phylogeny, diet, location).

Study2: Microbiome of fasting penguins

Summary: Like many seabirds, penguins have had to adapt to periodic fluctuations in nutrient availability and to prolonged periods of fasting [31, 55, 119] especially during moult and breeding. Previous studies examining the effect of fasting on the GI microbiota of vertebrate (hamsters, python, mice), have shown that fasting not only alters the composition and diversity of the GI microbiota, but it also influences the host’s immune defences [73, 74, 241] and that interrelationships exist between a host, its microbiota and the hosts nutritional status, diet and physiology [73, 74].

Unlike many other vertebrates, penguins undergo two distinct of fasting. During the breeding season, adult penguins undergo long periods of fasting during egg incubation and during guard and post-guard stage of chick rearing, alternating foraging trips between short and long foraging trips. In little penguins, long foraging trips are often associated with adult penguins replenishing their energy reserves [230]. In contrast, during moult penguins replace their entire plumage whilst fasting on land and cannot return to sea because of the consequences of reduced waterproofing and thermal insulation [105, 119]. During both the breeding and moulting fast penguins must rely on endogenous fat and protein reserves built up

129 prior to fast [50]. Inadequate storage of fat and protein reserves during moult can result in high rates of mortality [50, 77, 114] whereas inadequate reserves during the breeding season, can result in abandonment of chicks. To date no studies have examined the changes in microbial functionality during fasting vertebrates. By coupling metagenomics with metabolomics, this study will shed light on how fasting during moult affects the composition of the microbiota and identify changes in the function and presence of metabolites in the gastrointestinal tract of penguins [261].

Methods: Because of their availability throughout the year, the fact that little penguins are present in the colony all year round, and the ease of following the same individual over time, make the little penguin the perfect penguin species to examine the influence of fasting during breeding and moult. Metagenomic analysis of samples will be completed on the Roche 454 GS FLX Titanium. Metagenomic analysis will elucidate information on the microbial population and potential functionality of the microbial population. Metabolites present in samples are identified by GC mass spectroscopy. Results are then run through genomic and metabolite databases to identify microbes and metabolites present in the sample.

Significance: This study will enable scientists to examine if the GI microbiota influences the penguins ability to store large reserves of proteins and lipids prior to the fast and how the microbial composition changes during the fasting process.

Study 3: Impact of Humans on the microbial composition of Antarctic penguins

Summary: A recent article highlighted the introduction of alien species to the so called ‘pristine’ environment of the Antarctic, since the arrival of man. Chown et al [61] discovered that both scientists and tourists inadvertently brought alien species with them on their clothing to the Antarctic. In addition, the study identified that scientists were the main vector introducing alien species such as seeds, plant structures, and propagules. But what about microbes? Frenot et al [98] identified that alien microbes, fungi, plants and animals have managed to invade sub-Antarctic islands and some parts of the Antarctic continent, with the main routes of introduction being associated with human activity in the region. However, when Bonnedahl et al [28] examined the faeces of penguins at six tourist locations on the Antarctic peninsula, for the presence of human associated pathogenic bacteria, failed to isolate any human-associated pathogenic bacteria from penguins. However, this study only concentrated on Campylobacter, Salmonella and Yersinia, therefore, 130 excluding other possible human-associated pathogens. Greikspoor et al [116] not only identified the presence of Campylobacter jejuni in macaroni penguins, but that the C. jejuni isolates were of a genotype common among humans, indicating the bacterium was possibly of human origin. The use of molecular based methods such as 16S rRNA in this thesis has helped identify the presence of known human and veterinarian pathogens in seabirds. However, this thesis was unable to identify whether or not these microbes posed any threat to penguin health. Therefore, by incorporating methods such as metagenomics and metatranscriptomics scientists will be able to examine the potential influence of human presence on the GI microbiota of penguins.

Methods: To examine the impact of humans on the GI microbiota of penguins, we will examine the GI microbiota of penguin species frequently visited by tourists (king, gentoo, adélie). Samples will be collected from three populations. These populations will be selected based on 1) frequency of tourisms, 2) presence of scientists and 3) absence of human contact. To examine the microbial composition of penguins, genomic DNA will be amplified using V2-3 region bacterial primers and then run on the Roche 454 GS FLX Titanium.

Significance: This study will provide the first in depth investigation to see if proximity to humans and civilisation negatively impacts penguin microbiota and if scientists and tourists are capable of introducing alien microbes to penguins and other Antarctic wildlife and the impacts these alien species can have on penguin health and survival.

Study 4: Microbiome of Procellariiformes

Summary: Unlike penguins, there are no previous publications examining the microbial composition of Procellariiform seabirds, with all previous investigations isolating pathogenic microorganisms and identifying the cause of mortality.

The recent advancement of metagenomic techniques allows for a more comprehensive and unbiased assessment of the microbial diversity by allowing the examination of microbes that is not easily cultured in a laboratory [264]. Metagenomics allows for the characterisation of the microbial community within the gut [107] and can elucidate important processes for the gut microbes and the host. In addition metagenomics can provide insight into the insight into the relationships

131 between the host and its gut microbiota [10, 209, 264, 280]. By coupling metagenomics with metabolomics’ (study of metabolites) will shed light on how microbial communities function in the gastrointestinal tract of penguins [261].

Methods: Samples are run on the Roche 454 GS FLX Titanium to sequence the microbial population, whilst metabolites present in samples are identified by GC mass spectroscopy. Results are then run through genomic and metabolite databases to identify microbes and metabolites present in the sample.

Significance: This research is significant because it starts to elucidate the role of microbes in seabird health, metabolism and digestive physiology which has not previously been done before. In addition, this research could elucidate the influence of stomach oils on microbial community composition.

Study 5: The developing microbiota and immune system in Seabirds

Summary: Studies investigating the developing microbiota and immune system has been limited to poultry. To date there are no publications examining the development of the microbiota and its influence on the developing immune system in seabirds. The composition of the microbial community within the gastrointestinal tract of vertebrates is fundamental for the development and function of an effective and competent immune system [19, 47, 229]. Research utilising gnotobiotic mice have shown that in the absence of a diverse gastrointestinal microbiota, the mice had a compromised immune system and a higher susceptibility to disease [97, 214, 215, 229, 265]. In addition, research has shown that any alteration of the host’s microbiota during post natal development can have profound effects on the establishment of the immune system [129, 229]. This thesis identified that the development of the GI microbiota varies from one seabird species to another, with little penguins experiencing relatively high variation throughout development, whereas short-tailed shearwaters experience very little variation. Further research into the factors influencing the colonisation process of the GI microbiota and the role the developing GI microbiota has on the immune system will provide valuable information on the influence of early microbial colonisation on health and disease state of adults.

Methods: Samples will be collected from little penguins and short-tailed shearwaters from hatch and until fledging. To investigate the development of the immune system in seabirds, we must first identify the lymphocyte and inflammatory markers such as

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Tcell, Bcell and MHC II CD4, CD8, and cytokines IFNg, IL4, IL10, IL12, IL2, and IL6. This will be achieved by taking blood and tissue samples from captive Adult penguin species, using flow cytometric methods. Once the markers have been identified, blood samples will be collected at weekly time points and analysed by quantification of gene expression using qRT-PCR with primers that would be developed in this study.

To investigate the developing microbiota, faecal samples would initially collected on a daily basis from birth for the first week (to examine the microbial composition immediately after birth), then weekly thereafter. Samples are run on the Roche 454 GS FLX Titanium to sequence the microbial population, whilst metabolites present in samples are identified by GC mass spectroscopy. Results are then run through genomic and metabolite databases to identify microbes and metabolites present in the sample.

Significance: This study will help identify the role the developing GI microbiota has on the development of the innate and adaptive immune system of seabirds. This knowledge will be invaluable in providing information on factors that influence survival and health of penguin chicks.

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Appendix 1 Table A1, Taxonomic Breakdown of bacteria present in the GI microbiota of penguins. KP = King, GP = Gentoo, LP = Little, MP = Macaroni

Taxonomic group (SILVA tax, release 106) KP GP LP MP Bacteria;Acidobacteria;Acidobacteria;11-24; 0 0 0 2 Bacteria;Acidobacteria;Holophagae;iii1-8; 0 6 0 0 Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales; 0 1 0 0 Acidimicrobineae;Acidimicrobiaceae Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales; 0 9 0 0 Acidimicrobineae;Iamiaceae Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales; 1 0 0 0 Acidimicrobineae;Iamiaceae;Iamia; Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales; 0 18 113 81 Acidimicrobineae;Sva0996 marine group Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales 0 32 46 27 ;Actinomycineae;Actinomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 74 0 0 0 Actinomycineae;Actinomycetaceae;Actinomyces; Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 2 0 0 0 Actinomycineae;Actinomycetaceae;uncultured; Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales 2 0 0 0 ;Actinomycineae;Actinomycetaceae;Varibaculum; Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 0 31 0 Corynebacterineae;Corynebacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 13 0 0 0 Corynebacterineae;Corynebacteriaceae;Corynebacterium; Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 10 0 3 Corynebacterineae;Dietziaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 0 0 1 Corynebacterineae;Family Incertae Sedis Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 7 41 5 Corynebacterineae;Mycobacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 0 17 0 Frankineae;Frankiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 3 26 0 Frankineae;Nakamurellaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 0 0 1 Frankineae;Sporichthyaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 0 0 4 Micrococcineae;AKAU3644 Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 16 0 1 Micrococcineae;Dermacoccaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 11 0 4 Micrococcineae;Dermatophilaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 3 0 0 0 Micrococcineae;Dermatophilaceae;Dermatophilus Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 12 0 9 Micrococcineae;Intrasporangiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 1 0 13 Micrococcineae;Microbacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 2 0 0 Micrococcineae;Micrococcaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 0 0 2 Micromonosporineae;Micromonosporaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 18 0 1 PeM15; Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 38 61 21 Propionibacterineae;Nocardioidaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 1 0 0 0 Propionibacterineae;Propionibacteriaceae;Tessaracoccus; Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 1 0 0 0 Propionibacterineae;Propionibacteriaceae;uncultured; Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 0 4 0 Streptosporangineae;Thermomonosporaceae 153

Bacteria;Actinobacteria;Actinobacteria;OPB41; 0 0 0 2 Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae;AKIW543; 0 2 0 0 Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae;Solirubrobacterales; 0 0 0 1 480-2; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae; 15 0 0 2 Bacteroides; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;gir-aah93h0; 87 0 0 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae; 2 0 0 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae 0 0 480 2 ;uncultured; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;p-2534-18B5 gut group; 99 0 0 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae; 5 0 0 0 Dysgonomonas; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae; 0 4 0 4 Odoribacter; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae; 712 0 0 18 Paludibacter; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae; 0 237 101 723 Parabacteroides; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae; 18262 87 18 13 Petrimonas; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae; 1550 243 22 147 Porphyromonas; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae; 1387 2 0 2 Proteiniphilum; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae; 0 13 88 5 uncultured; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella; 6 2 0 6 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;RF16; 0 0 0 42 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;RC9 gut 0 0 97 127 group; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7; 0 0 0 14 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;vadinHA21; 182 0 0 1 Bacteria;Bacteroidetes;BD2-2; 0 4 202 10 Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae; 0 0 4 0 Adhaeribacter; Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Fibrella; 0 0 0 8 Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae; 0 0 0 2 Hymenobacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae; 0 3 0 0 Brumimicrobium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae; 1 0 0 0 Brumimimicrobium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae; 1 0 0 0 Crocinitomix; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae; 3 0 0 0 Cryomorpha; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae; 12 1 0 0 Fluviicola; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae; 0 1 0 0 Owenweeksia; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 29 29 0 2 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 8 0 0 0 Aequorivita; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 2 0 0 0 Arenibacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 26 0 0 0 Bergeyella; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 0 0 0 1 Bizionia; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 0 204 0 25 Capnocytophaga; 154

Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 681 0 0 15 Chryseobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 0 90 0 4 Cloacibacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 26 169 35 2 Coenonia; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 528 79 0 5 Flavobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 9 0 0 0 Gelidibacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 2 0 0 0 Lacinutrix; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 0 31 0 0 Myroides; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 20 0 0 0 Ornithobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 0 0 0 1 Psychroserpens; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 0 1 0 0 Salinimicrobium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 2 0 0 0 uncultured; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 0 2 0 1 Wautersiella; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 62 0 0 0 Weeksella; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;NS9 marine group; 0 1 0 0 Bacteria;Bacteroidetes;SB-1; 0 1 0 0 Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;hitinophagaceae; 0 3 3 19 Sediminibacterium; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;hitinophagaceae; 32 12 0 0 uncultured; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 1 0 0 0 Cyclobacteriaceae;Aquiflexum; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 2 0 0 0 Cytophagaceae;Leadbetterella; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;E6aC02; 5 0 0 0 Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 5 0 0 Saprospiraceae;Lewinella; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 2 0 0 0 Saprospiraceae;uncultured; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 1 0 0 Sphingobacteriaceae;Parapedobacter; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 37 0 0 0 Sphingobacteriaceae;Pedobacter; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 6 0 0 0 Sphingobacteriaceae;Sphingobacterium; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 41 0 0 Sphingobacteriaceae;uncultured; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;WCHB1-69; 1 0 0 0 Bacteria;Bacteroidetes;VC2.1 Bac22; 362 0 0 0 Bacteria;BD1-5; 53 0 0 0 Bacteria;Candidate division BRC1; 0 3 18 0 Bacteria;Candidate division OP10; 1 0 0 0 Bacteria;Candidate division OP11; 0 10 2 0 Bacteria;Candidate division SR1; 937 8 3 4 Bacteria;Candidate division TG-1;Lineage IIb; 1 0 0 0 Bacteria;Candidate division TM7; 1278 7 0 0 Bacteria;Candidate division WS6; 0 0 0 1 Bacteria;Chlorobi;Chlorobia;Chlorobiales;SJA-28; 0 1 0 0 Bacteria;Chlorobi;Ignavibacteria;Ignavibacteriales;uncultured; 0 19 0 3 155

Bacteria;Chloroflexi;Anaerolineae;Anaerolineales;Anaerolineaceae; 37 0 0 5 uncultured Bacteria;Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae;Caldilinea; 0 0 0 5 Bacteria;Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae;uncultured; 0 0 0 1 Bacteria;Chloroflexi;Thermomicrobia;JG30-KF-CM45; 0 4 12 13 Bacteria;Cyanobacteria;Chloroplast; 5 14 0 0 Bacteria;Cyanobacteria;SubsectionIV;Anabaena; 0 1 0 0 Bacteria;Deinococcus-Thermus;Deinococci;Deinococcales;Deinococcaceae; 0 3 0 0 Deinococcus; Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Amphibacillus; 0 0 0 3 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Anoxybacillus; 0 8 0 7 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Filobacillus; 0 0 0 2 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Geobacillus; 1 0 0 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Natronobacillus; 0 0 0 2 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Sediminibacillus; 0 0 16 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Virgibacillus; 0 5 0 0 Bacteria;Firmicutes;Bacilli;Bacillales;Family XI Incertae Sedis;Gemella; 0 3 0 6 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Incertae Sedis; 18 0 0 0 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Planomicrobium; 0 0 0 12 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Sporosarcina; 12 0 0 0 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;uncultured; 2 0 0 0 Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Nosocomiicoccus; 0 11 0 0 Bacteria;Firmicutes;Bacilli;Bacillales;Thermoactinomycetaceae; 0 1 0 0 Thermoactinomyces; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Allofustis; 3 0 0 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Atopostipes; 0 3 0 3 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 1 0 0 0 Carnobacterium Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Trichococcus; 2 4 0 1 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;uncultured; 17 2 0 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Catellicoccus; 424 22 0 29 Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Enterococcus; 2 0 0 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Vagococcus; 3 8 316 82 Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus; 17 0 0 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Fructobacillus 0 1067 68 1100 Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Leuconostoc; 380 91 57 785 Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Weissella; 753 0 0 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;MOB164; 1 0 0 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;P5D1-392; 0 0 0 1 Bacteria;Firmicutes;Bacilli;Lactobacillales;PeH08; 0 3 0 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Rs-D42; 0 524 63 254 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Lactococcus; 121 131 95 32 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus; 0 2 0 4 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;uncultured; 0 6 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Alkaliphilus; 3217 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 0 0 41 0 Caloranaerobacter; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Caminicella; 7 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 0 8 27 5 Candidatus Arthromitus; 156

Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 36 123 49 42 Clostridiisalibacter; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium; 2256 12 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium; 4 0 0 0 Candidatus Arthromitus; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Proteiniclasticum 0 0 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Sarcina; 0 0 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;uncultured; 0 1 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Dethiosulfatibacter; 3 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Eubacteriaceae;Acetobacterium; 0 0 16 25 Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae 0 5 0 9 Sedis;Proteiniborus; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis; 617 33 29 42 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 15759 1 0 0 Sedis;Finegoldia; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 5758 0 0 5 Sedis;Gallicola; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 63 2 0 3 Sedis;Helcococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 188 0 0 0 Sedis;Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 66 17 0 22 Sedis;Parvimonas; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 2062 1 0 1 Sedis;Peptoniphilus; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 384 2 18 2 Sedis;Soehngenia; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 16 3 5 3 Sedis;Sporanaerobacter; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 7 55 0 14 Sedis;Tepidimicrobium; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 287 109 0 44 Sedis;Tissierella; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 78 1 0 0 Sedis;uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae 12 3 0 37 Sedis;Fusibacter; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae 1291 3 0 0 Sedis;Guggenheimella; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae Sedis; 8 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae 18 0 0 0 Sedis;Anaerovorax; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae 8 0 0 0 Sedis;Eubacterium; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae 0 1 0 2 Sedis;Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae 52 0 0 1 Sedis;Mogibacterium; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae 11 0 0 3 Sedis;uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 1 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Anaerostipes; 0 6 38 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Blautia; 5 9 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Catabacter; 0 2 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Coprococcus; 0 1 41 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Epulopiscium; 662 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Howardella; 0 709 14 10 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Incertae Sedis; 1604 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Moryella; 0 0 1 2

157

Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Oribacterium; 0 7 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 0 0 0 2 Pseudobutyrivibrio; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Roseburia; 1 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Shuttleworthia; 0 18 32 188 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;uncultured; 553 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;livecontrolB21; 5 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;Peptococcus; 8 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;uncultured; 0 0 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Filifactor 500 191 109 3 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae; 3876 0 0 0 Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae; 0 0 0 1 Proteocatella; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae; 1781 0 0 0 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Proteiniborus; 995 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 28 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Anaerofilum; 0 0 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 815 0 0 0 Anaerotruncus; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 0 1 Anaerotruncus; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 16 19 20 Faecalibacterium; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Fastidiosipila 222 2 49 9 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Incertae 2 0 0 0 Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Papillibacter; 0 1 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 0 5 Saccharofermentans; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 4 6 12 20 Subdoligranulum; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;uncultured; 1923 1 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Syntrophomonadaceae; 1 0 0 0 Pelospora; Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae; 0 0 0 3 Phascolarctobacterium; Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Selenomonas; 0 21 41 13 Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;uncultured; 407 0 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella; 8 0 0 1 Bacteria;Firmicutes;Clostridia;D8A-2; 0 0 1 2 Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae; 0 2 0 3 Coprobacillus; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae; 1 0 0 0 Incertae Sedis; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae; 0 1 0 2 Solobacterium; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae; 0 1 0 0 Turicibacter; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae 0 1 0 0 ;uncultured; Bacteria;Firmicutes;Mollicutes;Acholeplasmatales;Acholeplasmataceae; 13 0 0 0 Acholeplasma; Bacteria;Firmicutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae; 656 0 0 0 Mycoplasma; Bacteria;Firmicutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae; 15212 0 0 0 Ureaplasma; Bacteria;Firmicutes;Mollicutes;RF9; 605 0 0 0 158

Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;ASCC02; 0 2 0 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;boneC3G7; 0 8 0 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7; 2825 45 0 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae; 1 9956 82 1 Cetobacterium; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae; 28570 0 0 0 Fusobacterium; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae; 0 4 0 0 Psychrilyobacter; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae; 45 3 0 0 Streptobacillus; Bacteria;Gemmatimonadetes;Gemmatimonadetes;AT425-EubC11 terrestrial 0 0 0 1 group; Bacteria;Gemmatimonadetes;Gemmatimonadetes;Gemmatimonadales; 0 1 0 0 Gemmatimonadaceae;Gemmatimonas; Bacteria;Lentisphaerae;Lentisphaeria;BS5; 0 0 0 6 Bacteria;Nitrospirae;Nitrospira;Nitrospirales;OPB95; 0 0 0 3 Bacteria;Planctomycetes;OM190; 0 63 541 11 Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales; 0 8 0 0 Caulobacteraceae;Phenylobacterium; Bacteria;Proteobacteria;Alphaproteobacteria;DB1-14; 3 0 0 0 Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;alphaI cluster; 0 0 0 2 Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Beijerinckiaceae; 0 5 0 0 uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;DUNssu371; 0 0 0 2 Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Nordella; 1 0 0 0 Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Phyllobacteriaceae 0 0 0 2 ;Mesorhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae; 0 13 0 1 Ensifer; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae; 0 2 0 0 Rhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Xanthobacteraceae 0 0 0 5 ;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 1 0 0 Rhodobacteraceae; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 7 Rhodobacteraceae;Maritimibacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 57 0 0 0 Rhodobacteraceae;Paracoccus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales 0 5 0 0 ;Rhodobacteraceae;Phaeobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 65 0 3 Rhodobacteraceae;Pontibaca; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 19 0 0 2 Rhodobacteraceae;Pseudorhodobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 2 Rhodobacteraceae;Roseibacterium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 9 0 0 Rhodobacteraceae;Roseobacter clade OCT lineage; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 3 0 0 0 Rhodobacteraceae;Roseobacter clade;Roseovarius; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 6 0 0 Rhodobacteraceae;Roseovarius; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 4 0 0 Rhodobacteraceae;Seohaeicola; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 2 0 0 Rhodobacteraceae;Sulfitobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 15 0 0 Rhodobacteraceae;Tateyamaria; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 9 0 0 Rhodobacteraceae;Thalassococcus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 1 0 0 3 159

Rhodobacteraceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales; 0 0 2 0 Acetobacteraceae;Roseomonas; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 0 13 0 0 Erythrobacteraceae;Altererythrobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 1 0 0 0 Erythrobacteraceae;Erythrobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 2 0 0 1 Sphingomonadaceae;Blastomonas; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 1 0 0 0 Sphingomonadaceae;Sandarakinorhabdus; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 0 29 0 0 Sphingomonadaceae;Sphingobium; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 1 0 0 0 Sphingomonadaceae;Sphingopyxis; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae; 0 0 0 1 Alcaligenes; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae; 0 23 0 0 Castellaniella; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae; 1 14 0 0 Derxia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae; 0 5 0 1 Parasutterella; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae; 0 3 0 0 Pusillimonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae; 7 0 0 0 Sutterella; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae; 1 208 0 0 uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 6 0 0 Burkholderiaceae;Cupriavidus; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 1 0 0 Burkholderiaceae;Limnobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 1 5 1 Burkholderiaceae;Polynucleobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 24 0 2 Burkholderiaceae;Ralstonia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 7 39 0 51 Comamonadaceae; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 36 0 4 Comamonadaceae;Acidovorax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 1 Comamonadaceae;Albidiferax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 10 0 Comamonadaceae;Aquabacterium; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales 0 0 112 2 ;Comamonadaceae;Chlorochromatium; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 3 0 4 Comamonadaceae;Delftia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 21 0 1 Comamonadaceae;Diaphorobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 2 0 0 Comamonadaceae;Hydrogenophaga; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 5 1 Comamonadaceae;Paucibacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 2 Comamonadaceae;Piscinibacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 3 0 0 2 Comamonadaceae;Polaromonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 10 0 3 Comamonadaceae;Rhodoferax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales 0 8 0 2 ;Comamonadaceae;Simplicispira; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 0 0 0 Comamonadaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 4 0 4 Comamonadaceae;Variovorax; 160

Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 1 0 0 Oxalobacteraceae; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 7 0 7 Oxalobacteraceae;Herminiimonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales 0 0 0 1 ;Oxalobacteraceae;Massilia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 7 0 4 Oxalobacteraceae;Naxibacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 1 0 0 Oxalobacteraceae;Undibacterium; Bacteria;Proteobacteria;Betaproteobacteria;H2SRC138; 0 0 0 2 Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales; 0 55 0 5 Hydrogenophilaceae;Petrobacter; Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales; 0 0 0 2 Methylophilaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 3 0 0 0 Aquitalea; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 0 7 0 0 Conchiformibius; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 0 2 1 0 Deefgea; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 0 19 0 20 Eikenella; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 171 0 0 0 Laribacter; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 4 29 65 139 Microvirgula; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 33 0 0 0 Neisseria; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 111 0 0 0 uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 0 6 0 5 Uruburuella; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 0 0 0 3 Vitreoscilla; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae; 0 22 62 199 Vogesella; Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomonadales; 0 3 0 0 Gallionellaceae;Sideroxydans; Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomonadales; 0 0 0 2 Nitrosomonadaceae;Nitrosomonas; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 2 0 7 Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 2 0 2 Azoarcus; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 3 0 0 0 Azospira; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 1 0 0 Dechloromonas; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 2 0 4 Methyloversatilis; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 37 0 35 Sterolibacterium; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 1 0 1 Uliginosibacterium; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 1 0 0 uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 0 0 1 Zoogloea; Bacteria;Proteobacteria;Betaproteobacteria;SC-I-84; 0 2 0 0 Bacteria;Proteobacteria;Betaproteobacteria;UCT N117; 0 2 0 1 Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales; 0 7 0 0 Bdellovibrionaceae;Bdellovibrio; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales; 0 14 55 28 Desulfobacteraceae;Desulfobacterium; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales; 91 2 0 0 Desulfobulbaceae;Desulfobulbus; 161

Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales; 0 8 0 11 Desulfobulbaceae;Desulfotalea; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales; 0 1 0 11 Desulfobulbaceae;uncultured; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales; 1 0 0 0 Desulfohalobiaceae;uncultured; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales; 0 1 0 0 Desulfovibrionaceae;Desulfovibrio; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfuromonadales; 3 0 0 0 GR-WP33-58; Bacteria;Proteobacteria;Deltaproteobacteria;GR-WP33-30; 0 3 0 5 Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;Cystobacterineae 0 1 0 0 ;uncultured; Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;JG37-AG-15; 0 0 0 1 Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;Nannocystineae; 0 0 0 3 Haliangiaceae;Haliangium Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;Nannocystineae; 0 0 0 2 Nannocystaceae;Nannocystis Bacteria;Proteobacteria;Deltaproteobacteria;Order IncertaeSedis; 0 1 0 5 Syntrophorhabdaceae;Syntrophorhabdus; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 42 17 16 31 Campylobacteraceae;Arcobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 4651 3 0 0 Campylobacteraceae;Campylobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 2 66 0 0 Campylobacteraceae;Sulfurospirillum; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 638 2 0 0 Helicobacteraceae;Helicobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 0 49 23 21 Helicobacteraceae;Sulfurimonas; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 1 0 0 0 Helicobacteraceae;Sulfurovum; Bacteria;Proteobacteria;Gammaproteobacteria;Acidithiobacillales; 0 4 0 2 KCM-B-112; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales; 0 0 1 0 Alteromonadaceae;Glaciecola; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales; 0 88 0 0 Alteromonadaceae;Haliea; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales; 0 0 0 3 Alteromonadaceae;Marinobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;NB-1d; 0 0 0 1 Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales 0 93 0 0 ;Psychromonadaceae;Psychromonas; Bacteria;Proteobacteria;Gammaproteobacteria;B38; 0 4 22 14 Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales; 0 4 1 5 Cardiobacteriaceae;Cardiobacterium; Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales; 9 0 0 0 Cardiobacteriaceae;Suttonella; Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales; 4 0 0 1 Cardiobacteriaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales; 0 1 0 0 Chromatiaceae;Thiocystis; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales; 2 2 0 1 Granulosicoccaceae;Granulosicoccus; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 96 0 106 Enterobacteriaceae;Cedecea; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 4 0 Enterobacteriaceae;Citrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 104 516 57 Enterobacteriaceae;Edwardsiella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 183 104 31 Enterobacteriaceae;Erwinia; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 143 2 Enterobacteriaceae;Hafnia-Obesumbacterium; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 5 12 0 Enterobacteriaceae;Klebsiella; 162

Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 8 0 Enterobacteriaceae;Morganella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 0 1 Enterobacteriaceae;Rahnella; Bacteria;Proteobacteria;Gammaproteobacteria;Legionellales;Coxiellaceae; 7 0 7 0 Aquicella; Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales; 0 3 4 2 Crenotrichaceae;Crenothrix; Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;Hyd24-01; 0 5 18 56 Bacteria;Proteobacteria;Gammaproteobacteria;NKB5; 34 1 0 0 Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales; 0 2 0 0 Alcanivoracaceae;Alcanivorax; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;SAR86 0 6 0 4 clade; Bacteria;Proteobacteria;Gammaproteobacteria;Order Incertae Sedis;Family 0 1 0 0 Incertae Sedis;Marinicella; Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae 0 0 36 0 ;Aggregatibacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae 0 33 0 2 ;Haemophilus; Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae 0 9 3 0 ;Pasteurella; Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae 0 79 153 133 ;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales; 0 0 0 4 Moraxellaceae;Acinetobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales; 0 17 2 11 Moraxellaceae;Alkanindiges; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales; 0 1364 0 635 Moraxellaceae;Enhydrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales 8 0 0 0 ;Moraxellaceae;Psychrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales; 0 234 63 29 Pseudomonadaceae;Cellvibrio; Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Francisellaceae; 27 0 0 0 Francisella; Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Piscirickettsiace 20 0 0 0 ae;Methylophaga; Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Thiotrichaceae; 0 1 0 0 Leucothrix; Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae; 0 0 0 3 Photobacterium; Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae; 0 0 0 1 Vibrio; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 3 0 0 Sinobacteraceae;Steroidobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 3 0 0 Xanthomonadaceae; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 13 0 4 Xanthomonadaceae;Arenimonas; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 3 0 0 Xanthomonadaceae;Ignatzschineria; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 17 0 1 Xanthomonadaceae;Luteimonas; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 3 4 0 23 Xanthomonadaceae;Lysobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 1 0 0 Xanthomonadaceae;Rhodanobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 1 6 0 2 Xanthomonadaceae;Stenotrophomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales 0 0 0 1 Xanthomonadaceae;Thermomonas; Bacteria;Spirochaetes;Spirochaetes; 0 3 0 96 Bacteria;Spirochaetes;Spirochaetes;Spirochaetales;Spirochaetaceae; 45 0 0 0 Treponema; Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Jonquetella; 10 2 0 0 163

Bacteria;Tenericutes;Mollicutes;Anaeroplasmatales;Anaeroplasmataceae; 0 1 0 0 Anaeroplasma; Bacteria;Tenericutes;Mollicutes;Entomoplasmatales;Candidatus 0 36 109 4 Hepatoplasma; Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae; 0 2 0 0 Mycoplasma; Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae; 0 4 0 2 Ureaplasma; Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales; 0 0 0 7 Verrucomicrobiaceae;Akkermansia; Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales; 0 51 57 102 Verrucomicrobiaceae;uncultured; Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales; 0 0 0 2 Verrucomicrobiaceae;Verrucomicrobium; Unclassified; 4434 0 0 0

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Appendix 2 Table A2, Taxonomic Breakdown of bacteria present in little penguins during development (Chapter 3). A = Adult, 1 = week 1, 3 = Week 3, 5 = week 5, 7 = Week 7, 9 = Week 9. Taxonomic group (SILVA tax, release 106) A 1 3 5 7 9 Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 113 0 1 11 12 1 Actinomycetales;Actinomycineae;Actinomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 46 0 15 8 5 5 Actinomycetales;Corynebacterineae;Corynebacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 31 0 0 1 8 3 Actinomycetales;Corynebacterineae;Dietziaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 2 1 0 Actinomycetales;Corynebacterineae;Family Incertae Sedis Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 9 0 Actinomycetales;Corynebacterineae;Mycobacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 41 0 0 0 1 0 Actinomycetales;Corynebacterineae;Nocardiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 2 0 0 0 0 Actinomycetales;Frankineae;Fodinicola Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 17 0 0 0 0 0 Actinomycetales;Frankineae;Geodermatophilaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 1 0 Actinomycetales;Frankineae;Nakamurellaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 26 0 0 0 0 0 Actinomycetales;Frankineae;Sporichthyaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 1 0 Actinomycetales;Frankineae;uncultured Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 3 1 0 Actinomycetales;Micrococcineae;Bogoriellaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 8 21 23 2 Actinomycetales;Micrococcineae;Brevibacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 1 1 Actinomycetales;Micrococcineae;Cellulomonadaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 2 4 6 12 1 Actinomycetales;Micrococcineae;Dermabacteraceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 2 1 12 4 8 Actinomycetales;Micrococcineae;Microbacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 3 8 19 6 Actinomycetales;Micrococcineae;Micrococcaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 0 1 Actinomycetales;Propionibacterineae;Nocardioidaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 61 4 0 1 2 0 Actinomycetales;Propionibacterineae;Propionibacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 1 0 0 Actinomycetales;Streptomycineae;Streptomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 4 0 0 0 0 1 Bifidobacteriales;Bifidobacteriaceae;Bifidobacterium Bacteria;Actinobacteria;Actinobacteria;Coriobacteridae; 0 0 0 0 4 28 Coriobacteriales;Coriobacterineae;Coriobacteriaceae Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae; 0 5 1 0 2 0 Rubrobacterales;Rubrobacterineae;Rubrobacteriaceae Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae; 202 5 1 5 2 13 Bacteroides; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 480 0 0 0 0 0 Porphyromonadaceae;Barnesiella; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 101 0 6 498 109 12 Porphyromonadaceae;Petrimonas; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 18 0 0 0 6 0 Porphyromonadaceae;Porphyromonas; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 22 0 0 50 31 5 Porphyromonadaceae;Proteiniphilum; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 0 0 0 2 0 0 Porphyromonadaceae;uncultured; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae; 88 0 0 1 0 0 Prevotella; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae; 0 1 0 0 3 2 uncultured; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae; 0 5 0 0 5 0 Alistipes; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7; 97 10 1 6 1 2 Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;Echini 0 0 0 0 0 1 cola; 165

Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;uncult 0 0 0 0 1 0 ured; Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae; 4 0 0 0 0 0 Arcicella; Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae; 0 0 0 5 0 0 Hymenobacter; Bacteria;Bacteroidetes;Cytophagia;Order III Incertae Sedis;Family 0 0 0 0 1 0 Incertae Sedis;Balneola; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 0 1 2 0 Cryomorphaceae;Brumimicrobium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales 0 0 0 0 2 0 ;Flavobacteriaceae;Aequorivita; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 9 30 5 8 3 Flavobacteriaceae;Chryseobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 3 0 0 1 0 Flavobacteriaceae;Cloacibacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales 35 0 0 0 3 0 ;Flavobacteriaceae;Flavobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 0 0 4 0 Flavobacteriaceae;Gelidibacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales 0 246 0 0 0 0 ;Flavobacteriaceae;Myroides; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 0 1 36 0 Flavobacteriaceae;Ulvibacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 1 0 0 0 Flavobacteriaceae;uncultured; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 3 0 0 0 17 1 Chitinophagaceae;uncultured; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 0 0 0 4 1 Saprospiraceae;uncultured; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 1 0 0 0 0 Sphingobacteriaceae;Olivibacter; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 3 0 1 4 0 Sphingobacteriaceae;Pedobacter; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 86 51 0 0 0 Sphingobacteriaceae;Sphingobacterium; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 0 0 2 5 0 Sphingobacteriaceae;uncultured; Bacteria;Candidate division BRC1; 0 0 0 1 0 0 Bacteria;Candidate division OD1; 18 0 0 0 0 0 Bacteria;Candidate division OP10; 0 1 0 0 0 0 Bacteria;Candidate division SR1; 2 0 0 0 0 0 Bacteria;Candidate division TM7; 3 2 0 3 2 0 Bacteria;Chlorobi;Chlorobia;Chlorobiales;OPB56; 0 0 0 0 1 1 Bacteria;Cyanobacteria;Chloroplast; 12 0 3 0 1 2 Bacteria;Cyanobacteria;SubsectionI;Synechococcus; 0 0 0 2 3 0 Bacteria;Firmicutes;Bacilli;Bacillales;Alicyclobacillaceae; 0 0 0 1 0 0 Alicyclobacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae; 0 0 0 0 2 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Alkalibacillus; 0 0 0 0 3 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Amphibacillus; 0 0 0 0 2 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus; 0 0 1 0 32 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Filobacillus; 0 0 0 0 1 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Geobacillus; 0 0 0 0 28 8 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Gracilibacillus; 0 0 0 0 1 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Halobacillus; 0 0 0 0 8 1 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Lentibacillus; 0 0 0 5 14 1 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Natronobacillus; 0 0 0 0 1 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Oceanobacillus; 0 0 0 0 7 4 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Paucisalibacillus; 0 0 0 1 44 2 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Sediminibacillus; 0 0 1 0 1 1 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;uncultured; 0 0 4 5 220 43

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Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Virgibacillus; 16 0 0 3 50 10 Bacteria;Firmicutes;Bacilli;Bacillales;Family XI Incertae Sedis;Gemella; 0 0 2 7 20 21 Bacteria;Firmicutes;Bacilli;Bacillales;Family XII Incertae 0 0 0 0 16 0 Sedis;Exiguobacterium; Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae; 0 0 0 0 0 4 Ammoniphilus; Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Brevibacillus 0 0 0 1 0 0 Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Paenibacillu; 0 8 0 2 1 0 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;IncertaeSedis; 0 0 0 0 8 0 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 0 0 0 0 20 0 Jeotgalibacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 0 0 0 4 14 0 Lysinibacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 0 0 0 2 0 0 Planomicrobium; Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Sporosarcina; 0 0 7 3 407 4 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;uncultured; 0 0 0 0 4 0 Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae; 0 0 0 0 1 0 Jeotgalicoccus; Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae; 0 4 0 0 0 0 Macrococcus; Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae; 0 126 173 9 9 0 Staphylococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae; 0 0 0 1 13 8 Dolosicoccus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 0 0 1 0 121 8 Atopococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 0 0 0 0 134 8 Atopostipes; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 0 5 0 0 2 0 Carnobacterium; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 0 2 0 3 1 0 Dolosigranulum; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 0 0 1 13 361 64 uncultured; Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae; 0 310 25 8 6 7 Enterococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae; 0 0 14 1 0 0 Vagococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae; 316 44 13 5 26 48 Lactobacillus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae; 68 332 63 279 229 42 Leuconostoc; Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae; 57 270 57 76 73 20 Weissella; Bacteria;Firmicutes;Bacilli;Lactobacillales;Rs-D42; 0 0 0 0 58 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 63 64 31 35 61 14 Lactococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 95 17 2 10 8 51 Streptococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 0 0 0 0 1 0 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 1 0 0 0 14 0 Alkaliphilus; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 41 0 0 0 8 166 Candidatus Arthromitus; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 27 0 0 0 80 0 Clostridiisalibacter; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 49 262 587 139 284 4795 Clostridium; 8 5 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Sarcina; 1 16 0 0 2 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae 16 0 0 4 7 1 Sedis;Proteiniborus; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 29 0 0 0 18 0 Sedis;Finegoldia; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 0 0 0 1 0 Sedis;Gallicola; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 0 0 0 93 0 Sedis;Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 0 0 0 0 1 167

Sedis;Parvimonas; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 0 0 1 0 0 Sedis;Peptoniphilus; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 0 0 1 0 0 Sedis;Soehngenia; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 18 0 0 0 0 0 Sedis;Sporanaerobacter; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 5 0 0 0 8 1 Sedis;Tepidimicrobium; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 0 0 1 155 7 Sedis;Tissierella; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 0 0 0 9 0 Sedis;uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 38 0 0 0 1 4 Blautia; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae 0 0 0 0 4 0 ;Caldicoprobacter; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 0 0 0 0 0 1 Coprococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 41 0 0 0 0 0 Dorea; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 14 6 59 463 145 2319 Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 1 0 0 0 0 0 Oribacterium; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 0 0 0 0 0 2 Pseudobutyrivibrio; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 32 14 2 20 57 3 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae; 0 0 0 0 2 0 Desulfonispora; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae; 0 0 0 1 1 0 Peptococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae; 1 0 0 0 0 0 Filifactor; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae; 109 37 28 398 136 322 Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae; 0 0 5 0 0 2 Peptostreptococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae; 0 0 0 0 0 2 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 0 15 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 0 1 0 0 Anaerotruncus; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 1 2 0 5 Faecalibacterium; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 19 0 0 3 0 0 Fastidiosipila; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 49 0 0 0 0 1 Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 0 0 0 3 Ruminococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 0 0 0 9 Subdoligranulum; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 12 1 1 11 9 87 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae; 41 23 2 7 2 3 Veillonella; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 1 0 1 1 0 0 Erysipelotrichaceae;Allobaculum; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 0 0 0 0 15 2 Erysipelotrichaceae;Erysipelothrix; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 0 0 0 0 0 1 Erysipelotrichaceae;Turicibacter; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 0 0 0 0 0 2 Erysipelotrichaceae;uncultured; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7; 0 0 4 67 2 37 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae; 0 0 15 25 1 183 Cetobacterium; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 82 55 6441 548 992 2095 Fusobacteriaceae;Fusobacterium; 0 4 Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales; 541 105 34 470 83 23 Phycisphaeraceae;CL500-3;

168

Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales; 0 0 0 4 0 0 Caulobacteraceae;Asticcacaulis; Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales; 0 4 1 1 0 0 Caulobacteraceae;Brevundimonas; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 0 1 0 Beijerinckiaceae;Chelatococcus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 2 0 0 0 Bradyrhizobiaceae;Bradyrhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 3 0 0 0 0 Brucellaceae;Pseudochrobactrum; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 1 0 0 Hyphomicrobiaceae;Devosia; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 1 0 0 Methylobacteriaceae;Methylobacterium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 0 2 0 Phyllobacteriaceae;Mesorhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 4 0 0 Rhizobiaceae;Rhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 4 5 3 Rhodobacteraceae;Paracoccus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 0 7 1 Rhodobacteraceae;Pontibaca; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 0 1 0 Rhodobacteraceae;Rhodobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 0 6 0 Rhodobacteraceae;Roseovarius; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 0 5 0 Rhodobacteraceae;Rubellimicrobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 0 12 5 Rhodobacteraceae;Thalassococcus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 1 5 0 Rhodobacteraceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales; 2 0 0 0 0 0 KCM-B-15; Bacteria;Proteobacteria;Alphaproteobacteria;SAR11 clade; 0 0 2 0 0 0 Chesapeake-Delaware Bay; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 0 3 0 0 0 0 GOBB3-C201; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 0 0 0 0 6 0 Sphingomonadaceae;Blastomonas; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 0 3 0 0 0 0 Sphingomonadaceae;Novosphingobium; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 0 0 0 5 0 0 Sphingomonadaceae;Sphingobium; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales; 0 0 0 0 3 1 Sphingomonadaceae;Sphingomonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 0 1 0 Alcaligenaceae;Achromobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 0 34 0 Alcaligenaceae;Alcaligenes; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 3 8 0 Alcaligenaceae;Castellaniella; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 2 0 0 Alcaligenaceae;Parasutterella; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 3 3 0 Alcaligenaceae;Pusillimonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 0 25 2 Alcaligenaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 2 0 0 0 Burkholderiaceae;Cupriavidus; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 5 5 2 1 3 0 Burkholderiaceae;Ralstonia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 7 1 1 2 1 Comamonadaceae;Acidovorax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 3 0 0 0 0 Comamonadaceae;Aquabacterium; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 10 0 0 0 0 0 Comamonadaceae;Brachymonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 112 3 0 2 0 1 Comamonadaceae;Comamonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 0 5 0 Comamonadaceae;Ottowia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 5 1 0 0 0 0 Comamonadaceae;Pelomonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 1 0 0 0 Comamonadaceae;uncultured; 169

Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 5 1 7 Oxalobacteraceae; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 3 0 0 Oxalobacteraceae;Herbaspirillum; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 5 1 0 Oxalobacteraceae;Janthinobacterium; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 250 0 0 3 0 Oxalobacteraceae;Massilia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 6 0 0 0 0 Oxalobacteraceae;Undibacterium; Bacteria;Proteobacteria;Betaproteobacteria;H2SRC138; 0 0 0 4 0 0 Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales; 0 2 0 0 0 0 Methylophilaceae;LD28 freshwater group; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 1 0 0 0 0 0 Neisseriaceae;Eikenella; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 0 9 2 0 4 0 Neisseriaceae;Microvirgula; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 65 0 0 0 0 0 Neisseriaceae;Neisseria; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 62 0 0 0 0 0 Neisseriaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 0 0 1 0 1 0 Neisseriaceae;Vitreoscilla; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 0 1 0 0 0 0 Rhodocyclaceae;Azoarcus; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 0 2 0 0 0 0 Rhodocyclaceae;Propionivibrio; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 0 0 0 0 2 0 Rhodocyclaceae;uncultured; Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales; 0 0 3 0 0 0 Bdellovibrionaceae;Bdellovibrio; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales; 55 0 0 0 0 0 Desulfobulbaceae;Desulfobulbus; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfuromonadales; 0 0 0 0 0 1 GR-WP33-58; Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;0319-6G20; 0 5 2 0 0 0 Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 0 4 3 0 2 1 Campylobacteraceae;Arcobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 16 0 0 11 5 0 Campylobacteraceae;Campylobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales; 23 0 0 0 0 0 Helicobacteraceae;Sulfurovum; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales; 0 7 0 1 1 0 Aeromonadaceae;Aeromonas; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales; 0 0 0 0 1 0 Aeromonadaceae;Oceanimonas; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales; 0 0 0 0 2 1 Aeromonadaceae;Oceanisphaera; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales; 0 0 1 1 142 4 Aeromonadaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales; 0 0 0 0 1 0 Aeromonadaceae;Zobellella; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales; 0 0 0 0 2 0 Alteromonadaceae;Gilvimarinus; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales; 1 0 0 0 0 0 Alteromonadaceae;Haliea; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales; 0 2 0 0 15 1 Alteromonadaceae;Marinobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales; 0 0 0 0 1 0 NB-1d; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales; 0 8 0 0 0 0 Psychromonadaceae;Psychromonas; Bacteria;Proteobacteria;Gammaproteobacteria;B38; 0 71 2584 0 0 0 Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales; 22 0 0 2 1 0 Cardiobacteriaceae;Cardiobacterium; Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales; 1 0 0 0 0 0 Cardiobacteriaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales; 0 0 0 0 1 0 Chromatiaceae;Nitrosococcus; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales; 0 1 0 0 0 0 Chromatiaceae;Rheinheimera; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales; 0 0 0 0 1 0 Ectothiorhodospiraceae;Thioalkalispira;

170

Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 0 0 0 1 Enterobacteriaceae;Biostraticola; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales 0 16 0 1 0 8 ;Enterobacteriaceae;Cedecea; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 245 59 9 19 2 Enterobacteriaceae;Citrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 4 0 21 0 0 0 Enterobacteriaceae;Cronobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 2 0 0 1 Enterobacteriaceae;Dickeya; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 15 2 0 1858 Enterobacteriaceae;Edwardsiella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 516 10510 25 6 8 6 Enterobacteriaceae;Enterobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 14 0 1 0 Enterobacteriaceae;Erwinia; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 104 640 8506 1 2 1 Enterobacteriaceae;Escherichia-Shigella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 143 65 0 0 0 0 Enterobacteriaceae;Klebsiella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales 12 26 0 0 0 0 ;Enterobacteriaceae;Kluyvera; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 8 0 0 0 0 0 Enterobacteriaceae;Pantoea; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 354 18 0 0 0 Enterobacteriaceae;Raoultella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 0 0 0 1 Enterobacteriaceae;Salmonella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 2 0 0 0 Enterobacteriaceae;Serratia; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 48 0 0 0 Enterobacteriaceae;Tatumella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales; 0 0 0 0 0 1 Enterobacteriaceae;Yersinia; Bacteria;Proteobacteria;Gammaproteobacteria;Legionellales; 7 0 0 0 0 0 Legionellaceae;Legionella; Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales; 4 0 0 1 0 0 Hyd24-01; Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales; 18 0 0 0 1 0 Methylococcaceae;Methylomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales; 0 0 0 0 3 0 Alcanivoracaceae;Alcanivorax; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales; 0 0 0 0 1 0 Halomonadaceae;Cobetia; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales; 0 0 0 0 5 0 Halomonadaceae;Halomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales; 0 0 0 0 1 0 Oceanospirillaceae;Pseudospirillum; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales; 0 0 0 0 4 0 Oceanospirillaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales; 36 0 0 0 0 0 Pasteurellaceae;Haemophilus; Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales; 3 12 0 0 0 0 Pasteurellaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales; 153 602 8 19 16 4 Moraxellaceae;Acinetobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales 0 3 0 0 0 0 ;Moraxellaceae;Alkanindiges; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales; 2 13 2 4 0 0 Moraxellaceae;Enhydrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales; 0 1309 101 32 47 9 Moraxellaceae;Psychrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales; 63 656 0 2 138 1 Pseudomonadaceae;Pseudomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Salinisphaerales; 0 0 0 0 1 0 Salinisphaeraceae;Salinisphaera; Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales; 0 5 0 0 0 0 Vibrionaceae;Photobacterium; Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales; 0 0 2 23 400 68 Vibrionaceae;Vibrio; 2 Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 0 0 0 0 1 Sinobacteraceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 0 0 1 0 0 Xanthomonadaceae; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 0 0 0 32 0 Xanthomonadaceae;Ignatzschineria; 171

Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 0 15 0 0 0 Xanthomonadaceae;Luteibacter; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 0 0 0 3 0 Xanthomonadaceae;Lysobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 0 0 0 2 0 Xanthomonadaceae;Rhodanobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 91 1 0 2 0 Xanthomonadaceae;Stenotrophomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 0 0 0 1 0 Xanthomonadaceae;Thermomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales; 0 0 0 0 6 0 Xanthomonadaceae;uncultured; Bacteria;SM2F11; 0 1 0 0 0 0 Bacteria;Tenericutes;Mollicutes;Mycoplasmatales; 109 0 0 0 0 0 Mycoplasmataceae;Mycoplasma; Bacteria;Tenericutes;Mollicutes;Mycoplasmatales; 0 0 1 23 0 0 Mycoplasmataceae;Ureaplasma; Bacteria;TM6; 0 1 0 0 0 0 Bacteria;Verrucomicrobia;OPB35 soil group; 0 0 0 2 0 0 Unclassified; (this class will be removed with SILVA 108) 57 69 238 14 120 22

172

Appendix 3

Table A3.1, Taxonomic Breakdown of bacteria present in moulting little penguins

Taxonomic group (SILVA tax, release 106) Early Late moult Moult Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 34 189 Actinomycineae;Actinomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 244 358 Corynebacterineae;Corynebacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 7 0 Corynebacterineae;Dietziaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 16 0 Corynebacterineae;Family Incertae Sedis Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 2 0 Corynebacterineae;Mycobacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 5 0 Corynebacterineae;Nocardiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 0 1 Frankineae;Sporichthyaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 21 0 Micrococcineae;Brevibacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 23 0 Micrococcineae;Dermabacteraceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 9 1 Micrococcineae;Microbacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 17 9 Micrococcineae;Micrococcaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 9 29 Propionibacterineae;Propionibacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Bifidobacteriales; 3 1 Bifidobacteriaceae;Bifidobacterium Bacteria;Actinobacteria;Actinobacteria;Coriobacteridae;Coriobacteriales; 2 0 Coriobacterineae;Coriobacteriaceae Bacteria;Actinobacteria;Actinobacteria;MB-A2-108; 2 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Bacteroides; 54 25 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Paludibacter; 0 8 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Petrimonas; 230 414 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas; 45 18 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Proteiniphilum; 122 608 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;uncultured; 1 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella; 9 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;Blvii28 1 0 wastewater-sludge group; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7; 2 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;vadinHA21; 8 58 Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;uncultured; 1 0 Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Persicitalea; 1 0 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 6 9 Chryseobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Flavobacterium 0 1 ; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 0 1 Ornithobacterium; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Chitinophagaceae; 2 0 Ferruginibacter; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Chitinophagaceae;uncultured 7 0 ; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Sphingobacteriaceae; 3 0 Sphingobacterium; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;ST-12K33; 0 2 173

Bacteria;Bacteroidetes;VC2.1 Bac22; 0 2 Bacteria;Candidate division BRC1; 0 1 Bacteria;Candidate division SR1; 0 2 Bacteria;Candidate division TM7; 19 27 Bacteria;Chloroflexi;Anaerolineae;Anaerolineales;Anaerolineaceae;uncultured; 2 39 Bacteria;Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae;Caldilinea; 1 0 Bacteria;Cyanobacteria;Chloroplast; 5 0 Bacteria;Deferribacteres;Deferribacteres;Deferribacterales;SAR406 clade(Marine 1 0 group A); Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae; 2 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus; 10 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Halolactibacillus; 2 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Lentibacillus; 58 32 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Oceanobacillus; 92 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Paucisalibacillus; 17 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;uncultured; 62 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Virgibacillus; 162 3 Bacteria;Firmicutes;Bacilli;Bacillales;Family XI Incertae Sedis;Gemella; 83 2 Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Ammoniphilus; 0 1 Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Paenibacillus; 3 0 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Incertae Sedis; 0 1 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Sporosarcina; 11 0 Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Jeotgalicoccus; 0 3 Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Nosocomiicoccus; 0 12 Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus; 2 55 Bacteria;Firmicutes;Bacilli;Bacillales;Thermoactinomycetaceae;Thermoactinomyces; 5 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;Dolosicoccus; 26 1 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Atopococcus; 39 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Atopostipes; 7 3 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;uncultured; 105 2 Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Enterococcus; 20 9 Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus; 3 48 Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Leuconostoc; 162 221 Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Weissella; 117 89 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Lactococcus; 40 62 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus; 52 27 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;uncultured; 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Alkaliphilus; 0 6 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridiisalibacter; 0 28 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium; 177 24 Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae Sedis;Proteiniborus; 36 44 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Finegoldia; 96 172 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Gallicola; 0 27 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Helcococcus; 1 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Parvimonas; 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Peptoniphilus; 9 27 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Soehngenia; 1 8 174

Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Sporanaerobacter; 4 41 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Tepidimicrobium; 0 17 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Tissierella; 0 6 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;uncultured; 7 15 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae Sedis;Guggenheimella; 5 24 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae Sedis;Mogibacterium; 1 3 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Anaerostipes; 3 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Blautia; 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Incertae Sedis; 9 3 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;uncultured; 99 722 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;Peptococcus; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Filifactor; 11 16 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Incertae Sedis; 83 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Faecalibacterium; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Fastidiosipila; 16 79 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcus; 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Saccharofermentans; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;uncultured; 2 40 Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella; 6 6 Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Allobaculum; 0 1 Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Turicibacter; 1 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7; 7 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Cetobacterium; 2 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium; 817 1 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Streptobacillus; 1 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;uncultured; 0 4 Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales;Phycisphaeraceae;CL500-3; 43 29 Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Bradyrhizobiaceae; 2 0 Bradyrhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Family Incertae 0 1 Sedis;Nordella; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Hyphomicrobiaceae;Devosia; 7 3 Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 7 0 Paracoccus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 0 1 Pontibaca; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 1 0 Roseibacterium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 2 0 Rubellimicrobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 12 0 Thalassococcus Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 3 0 uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;Rhodospirillaceae; 12 0 Thalassospira; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;GOBB3-C201; 0 2 Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae; 0 3 Sphingomonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;uncultured; 1 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae; 1 8 Burkholderia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae; 0 3 Polynucleobacter; 175

Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Ralstonia; 0 2 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 2 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 1 6 Acidovorax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 13 0 Comamonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 4 0 uncultured Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae; 2 0 Herbaspirillum; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae; 11 0 Janthinobacterium Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;Massilia; 0 1 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae; 0 6 Undibacterium; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Eikenella; 2 3 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Microvirgula; 0 3 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria; 42 142 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Stenoxybacter; 1 0 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;uncultured; 298 805 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Vogesella; 2 0 Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Azoarcus; 0 1 Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Azospira; 9 0 Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 1 Denitratisoma Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;uncultured; 0 2 Bacteria;Proteobacteria;Betaproteobacteria;SC-I-84; 0 9 Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobulbaceae; 26 7 Desulfobulbus; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulfohalobiaceae; 5 2 uncultured; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae; 8 21 Campylobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae; 12 3 Sulfurovum Bacteria;Proteobacteria;Gammaproteobacteria;Acidithiobacillales;Acidithiobacillaceae; 1 0 Acidithiobacillus; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;Aeromonadaceae; 0 1 Aeromonas; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;Aeromonadaceae; 4 1 uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Alteromonadaceae; 1 0 Marinobacter Bacteria;Proteobacteria;Gammaproteobacteria;B38; 2 0 Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;Cardiobacteriaceae; 6 12 Cardiobacterium; Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;Cardiobacteriaceae 0 2 ;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;Chromatiaceae; 1 0 Rheinheimera; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 1 0 Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 22 22 Citrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 1 0 Cronobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 100 34 Enterobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 135 54 Escherichia-Shigella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 25 9 Klebsiella; 176

Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 3 0 Kluyvera; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 2 0 Pantoea; Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;Hyd24-01; 0 1 Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;Methylococcaceae; 4 17 Methylomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;Halomonadaceae; 3 0 Cobetia; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;Halomonadaceae; 13 0 Halomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Pasteurella; 6 0 Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;uncultured; 1 0 Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae; 43 39 Acinetobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae; 9 0 Psychrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae; 0 3 uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae; 4 14 Pseudomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Thiotrichaceae;Thiothrix; 0 1 Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;Vibrio; 49 7 Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae; 7 0 uncultured; Bacteria;Spirochaetes;Spirochaetes;Spirochaetales;Spirochaetaceae;Treponema; 27 83 Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Jonquetella; 0 15 Bacteria;Tenericutes;Mollicutes;Acholeplasmatales;Acholeplasmataceae;Acholeplasma; 2 0 Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;Mycoplasma; 273 287 Bacteria;Tenericutes;Mollicutes;RF9; 6 30 Unclassified; 315 467

177

Appendix 3 Table A3.2, Taxonomic Breakdown of bacteria presents in moulting king penguins (Chapter 4).

Taxonomic group (SILVA tax, release 106) Early Late Moult Moult Bacteria;Acidobacteria;Acidobacteria;SJA-149; 6 0 Bacteria;Acidobacteria;Holophagae;iii1-8; 2 0 Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales; 3 2 Acidimicrobineae;Acidimicrobiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 115 0 Actinomycineae;Actinomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 225 0 Corynebacterineae;Corynebacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 4 0 Corynebacterineae;Dietziaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 6 0 Corynebacterineae;Nocardiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Frankineae; 5 0 Frankiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Frankineae; 1 0 uncultured Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae 5 3 ;Dermatophilaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae 3 0 ;Intrasporangiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae 11 1 ;Microbacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae 6 0 ;Micrococcaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae 3 0 ;Promicromonosporaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales; 29 0 Propionibacterineae;Propionibacteriaceae Bacteria;Actinobacteria;Actinobacteria;Coriobacteridae;Coriobacteriales;Coriobacterineae; 19 0 Coriobacteriaceae Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Bacteroides; 1 2 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae;Marinifilum; 4 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae;uncultured; 0 1 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Paludibacter; 0 2 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Petrimonas; 1367 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas; 72 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Proteiniphilum; 296 230 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;uncultured; 4 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella; 0 3 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;uncultured; 2 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7; 1 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;vadinHA21; 3 0 Bacteria;Bacteroidetes;BD2-2; 8 0 Bacteria;Bacteroidetes;Class Incertae Sedis;Order Incertae Sedis;Family Incertae 0 1 Sedis;Prolixibacter; Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;Algoriphagus; 6 2 Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Flexibacter; 2 0 Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Leadbetterella; 5 0 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;Brumimicrobium; 0 1 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;Cryomorpha; 0 2 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;Owenweeksia; 2 0 178

Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 3 0 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Aequorivita; 0 6 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae; 123 79 Chryseobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Coenonia; 985 0 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Flavobacterium; 90 40 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Gelidibacter; 1 23 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Mariniflexile; 4 0 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Ornithobacterium 6 1 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Psychroserpens; 1 0 Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Sufflavibacter; 2 0 Bacteria;Bacteroidetes;SB-1; 4 3 Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Chitinophagaceae; 3 1 Ferruginibacter; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Chitinophagaceae;uncultured; 15 4 Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;env.OPS 17; 7 0 Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;KD1-131; 11 15 Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Sphingobacteriaceae; 9 0 Pedobacter Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;WCHB1-69; 16 25 Bacteria;Bacteroidetes;vadinHA17; 0 1 Bacteria;Bacteroidetes;VC2.1 Bac22; 2 0 Bacteria;Bacteroidetes;WCHB1-32; 0 1 Bacteria;BD1-5; 18 1 Bacteria;Candidate division OD1; 9 2 Bacteria;Candidate division SR1; 29 5 Bacteria;Candidate division TM7; 38 1 Bacteria;Chlorobi;Chlorobia;Chlorobiales;SJA-28; 0 2 Bacteria;Chloroflexi;Anaerolineae;Anaerolineales;Anaerolineaceae;uncultured; 1 0 Bacteria;Cyanobacteria;Chloroplast; 16 0 Bacteria;Cyanobacteria;SubsectionIII; 13 0 Bacteria;Cyanobacteria;SubsectionIII;Geitlerinema; 9 0 Bacteria;Cyanobacteria;SubsectionIII;Leptolyngbya; 26 0 Bacteria;Cyanobacteria;SubsectionIII;Phormidium; 9 0 Bacteria;Cyanobacteria;SubsectionIV;Anabaena; 11 0 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus; 10 0 Bacteria;Firmicutes;Bacilli;Bacillales;Family XII Incertae Sedis;Exiguobacterium; 0 1 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Planomicrobium; 1 0 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Sporosarcina; 16 1 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Trichococcus; 5 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Enterococcus; 15 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus; 310 2 Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Pediococcus; 4 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Leuconostoc; 1966 1 Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Weissella; 932 1 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Lactococcus; 842 2 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus; 187 7 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;uncultured; 6 0

179

Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Alkaliphilus; 5 3 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Caloranaerobacter; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridiisalibacter; 120 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium; 117 1718 Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Proteiniclasticum; 6 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae Sedis;Proteiniborus; 16 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis; 0 16 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Finegoldia; 358 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Gallicola; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Parvimonas; 5 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Peptoniphilus; 32 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Soehngenia; 35 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Sporanaerobacter; 15 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Tepidimicrobium; 5 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Tissierella; 83 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;uncultured; 14 47 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae Sedis;Fusibacter; 2 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae Sedis;Guggenheimella; 6 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae Sedis;Anaerovorax; 0 1 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae Sedis;Incertae Sedis; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIV Incertae Sedis;Anaerobranca; 19 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Blautia; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Epulopiscium; 0 15 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;IncertaeSedis; 152 275 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;uncultured; 147 40 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;Peptococcus; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;IncertaeSedis; 9 122 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Proteocatella; 4 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 2 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Anaerotruncus; 0 4 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Fastidiosipila; 31 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Incertae Sedis; 8 63 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcus; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Saccharofermentans; 1 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;uncultured; 46 77 Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella; 38 0 Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Erysipelothrix; 0 1 Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Incertae Sedis; 0 1 Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Turicibacter; 0 1 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;boneC3G7; 0 1 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7; 0 19 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Cetobacterium; 1 32 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium; 137 12546 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Leptotrichia; 1 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Streptobacillus; 1 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;uncultured; 6 0

180

Bacteria;Lentisphaerae;Lentisphaeria;BS5; 0 1 Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales;Phycisphaeraceae;CL500-3; 101 0 Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;Caulobacteraceae; 6 0 Brevundimonas; Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;Caulobacteraceae;uncultured 3 0 Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Methylobacteriaceae; 1 0 Methylobacterium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Methylocystaceae; 3 0 Pleomorphomonas; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae;Rhizobium; 17 0 Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 5 0 Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 9 0 Paracoccus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 0 2 Pseudorhodobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 7 0 Rhodobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae; 10 1 uncultured Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;Acetobacteraceae; 2 0 Acetobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;AKIW852; 8 0 Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae; 16 0 Sandarakinorhabdus; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae; 7 1 Sphingomonas; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae; 2 0 Sphingopyxis; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;uncultured; 4 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae; 78 0 Burkholderia Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Ralstonia; 2 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 1 4 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Acidovorax 30 38 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Albidiferax 14 58 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 13 0 Chlorochromatium; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 14 0 Comamonas Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 18 0 Diaphorobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 34 11 Hydrogenophaga; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Malikia; 0 4 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Paucibacter 18 1 ; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Pelomonas; 1 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 13 7 Polaromonas Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 0 1 Rhizobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Rhodoferax 1 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; 2 2 Simplicispira; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;uncultured; 83 3 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Variovorax 3 6 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae; 1 0 Herminiimonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae; 5 0 Janthinobacterium; 181

Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;Massilia; 1 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;Naxibacter; 1 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;uncultured; 4 0 Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae; 4 0 Undibacterium; Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales;Hydrogenophilaceae; 0 2 Thiobacillus; Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales;Hydrogenophilaceae; 0 2 uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales;Methylophilaceae;uncultured; 0 2 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Microvirgula; 27 0 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria; 30 0 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;uncultured; 64 0 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Vitreoscilla; 13 0 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Vogesella; 3 0 Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomonadales;Gallionellaceae; 1 0 Sideroxydans Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 1 0 Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Azospira; 5 1 Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 0 3 Dechloromonas Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Propionivibrio 0 1 Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 5 0 Sterolibacterium; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Thauera; 0 2 Thauera Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae; 1 0 Uliginosibacterium; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;uncultured; 1 1 Bacteria;Proteobacteria;Betaproteobacteria;UCT N117; 0 1 Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;Bacteriovoraceae; 0 1 Bacteriovorax; Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;Bdellovibrionaceae; 6 0 Bdellovibrio; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobacteraceae; 0 4 Desulfobacterium; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobulbaceae; 84 1 Desulfobulbus; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobulbaceae; 0 1 uncultured Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulfohalobiaceae; 7 0 uncultured; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulfovibrionaceae; 9 0 Desulfovibrio; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfuromonadales;GR-WP33-58; 2 0 Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;Nannocystineae;Haliangiaceae; 11 0 Haliangium Bacteria;Proteobacteria;Deltaproteobacteria;Order IncertaeSedis;Syntrophorhabdaceae; 0 1 Syntrophorhabdus; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae; 7 0 Arcobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae; 1725 0 Campylobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae; 9 0 Sulfurospirillum; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae; 1226 600 Helicobacter; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae; 0 1 Sulfurimonas; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae; 47 0 Sulfurovum; 182

Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae; 0 1 Wolinella; Bacteria;Proteobacteria;Gammaproteobacteria;Acidithiobacillales;Acidithiobacillaceae; 5 0 Acidithiobacillus; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;Aeromonadaceae; 4 0 Aeromonas; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;Aeromonadaceae; 0 1 Oceanimonas; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Pseudoalteromonadaceae; 0 7 Pseudoalteromonas; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Psychromonadaceae; 10 0 Psychromonas; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;Chromatiaceae;Thiocystis; 0 1 Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 1 0 Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 103 0 Citrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 96 0 Enterobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 15 51 Escherichia-Shigella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 20 0 Klebsiella; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 62 0 Kluyvera; Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae; 9 0 Serratia; Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;Methylococcaceae; 26 0 Methylomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;SAR86 clade; 5 0 Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Pasteurella; 10 0 Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae; 263 0 Acinetobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae; 1 0 Alkanindiges; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae; 34 0 Enhydrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae; 778 140 Psychrobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae; 2 0 uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae; 19 202 Pseudomonas; Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;Vibrio; 0 4 Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Sinobacteraceae;JTB255 1 0 marine benthic group; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae; 8 0 Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae; 8 0 Arenimonas; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae; 4 3 Dokdonella; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae; 0 1 Lysobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae; 5 4 Rhodanobacter; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae; 0 2 Silanimonas; Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae; 0 3 Thermomonas; Bacteria;Spirochaetes;Spirochaetes; 0 1 Bacteria;Spirochaetes;Spirochaetes;Spirochaetales;Spirochaetaceae;Treponema; 41 0 Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Jonquetella; 3 0 Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;Mycoplasma; 24 6 Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;Ureaplasma; 18 10 183

Bacteria;Tenericutes;Mollicutes;RF9; 7 1 Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;Verrucomicrobiaceae; 8 0 uncultured; Unclassified; 338 20

184

Appendix 4 Table A4, Taxonomic Breakdown of bacteria present in Procellariiform Seabirds. CDP = Co m m o n d i v i n g Pe t r e l , FP = Fa i r y Pr i o n , STS = Sh o r t - t a i l e d Sh e a r w a t e r Taxonomic group (SILVA tax, release 106) CDP FP STS

Bacteria;Acidobacteria;Acidobacteria;11-24; 4 0 0 Bacteria;Acidobacteria;Acidobacteria;Candidatus Chloracidobacterium; 3 0 0 Bacteria;Acidobacteria;Acidobacteria;Candidatus Solibacter; 1 0 0 Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae; 0 0 7 Acidimicrobiales;Acidimicrobineae;OCS155 marine group Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae; 1 0 0 Acidimicrobiales;Acidimicrobineae;Sva0996 marine group Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 208 13 55 Actinomycetales;Actinomycineae;Actinomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 342 24 44 Actinomycetales;Corynebacterineae;Corynebacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 13 0 0 Actinomycetales;Corynebacterineae;Mycobact eriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 5 0 0 Actinomycetales;Corynebacterineae;Nocardiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 7 0 1 Actinomycetales;Corynebacterineae;uncultured Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 19 7 Actinomycetales;Frankineae;Geodermatophilaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 5 0 0 Actinomycetales;Frankineae;Sporichthyaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 8 0 Actinomycetales;Frankineae;uncultured Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 1 0 13 Actinomycetales;Kineosporiineae;Kineosporiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 4 0 0 Actinomycetales;Micrococcineae;AKAU3644 Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 6 0 0 Act inomycet ales;Micrococcineae;Brevibact eriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 1 0 0 Actinomycetales;Micrococcineae;Cellulomonadaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 3 0 0 Actinomycetales;Micrococcineae;Dermabacteraceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 2 0 0 Act inomycet ales;Micrococcineae;Dermacoccaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 5 0 0 Actinomycetales;Micrococcineae;Intrasporangiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 7 0 26 Act inomycet ales;Micrococcineae;Microbact eriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 1 34 21 Actinomycetales;Micrococcineae;Micrococcaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 1 0 0 Actinomycetales;Micrococcineae;Sanguibact eraceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 2 0 0 Actinomycetales;Micromonosporineae;Micromonosporaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 3 0 0 Actinomycetales;Propionibacterineae;Nocardioidaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 39 36 60 Actinomycetales;Propionibacterineae;Propionibacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 1 0 Actinomycetales;Pseudonocardineae;Pseudonocardiaceae 185

Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 2 0 0 Actinomycetales;Streptomycineae;Streptomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 2 0 0 Actinomycetales;Streptosporangineae;Thermomonosporaceae Bacteria;Actinobacteria;Actinobacteria;Coriobacteridae; 8 24 0 Coriobacteriales;Coriobacterineae;Coriobacteriaceae Bacteria;Actinobacteria;Actinobacteria;OPB41; 1 0 0 Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae; 1 10 0 Rubrobacterales;Rubrobacterineae;Rubrobacteriaceae Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Bacte 265 10 19 roides; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 488 0 0 Porphyromonadaceae;Barnesiella; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 4 0 0 Porphyromonadaceae;Odoribacter; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 570 0 0 Porphyromonadaceae;Petrimonas; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 4 0 18 Porphyromonadaceae;Porphyromonas; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 315 0 0 Porphyromonadaceae;Proteiniphilum; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 1 0 0 Porphyromonadaceae;uncultured; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 409 0 13 Prevot ellaceae;Prevot ella; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 1 0 0 Prevot ellaceae;uncultured; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;RF16; 96 0 0 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;Alistipe 368 4 12 s; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;RC9 gut 2 0 0 group; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7; 17 74 74 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;vadinHA21; 30 0 0 Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Adhae 0 17 0 ribacter; Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Arcice 1 0 0 lla; Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Flexib 1 0 0 act er; Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Hyme 1 0 0 nobacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 1 0 Cryomorphaceae;Brumimicrobium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 2 0 0 Cryomorphaceae;Fluviicola; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 20 0 Cryomorphaceae;Owenweeksia; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 16 51 139 Flavobacteriaceae;Chryseobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 3 Flavobacteriaceae;Cloacibacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 589 0 0 Flavobacteriaceae;Coenonia; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales 7 6 13 ;Flavobact eriaceae;Flavobact erium;

186

Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 2 0 0 Flavobacteriaceae;Gelidibacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 3 0 0 Flavobacteriaceae;Krokinobacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 3 0 1 Flavobacteriaceae;Ornithobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 1 0 0 Flavobact eriaceae;Persicivirga; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 1 0 2 Flavobacteriaceae;uncultured; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 3 0 0 Flavobacteriaceae;Wautersiella; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;NS9 marine 2 0 0 group; Bacteria;Bacteroidetes;SB-1; 0 0 1 Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 11 0 0 Chitinophagaceae;uncultured; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 2 0 0 env.OPS 17; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 1 0 0 Saprospiraceae;Lewinella; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 2 0 0 ST-12K33; Bacteria;Bacteroidetes;VC2.1 Bac22; 17 0 12 Bacteria;BD1-5; 2 0 0 Bacteria;Candidate division OP10; 1 0 0 Bacteria;Candidate division SR1; 3 0 0 Bacteria;Candidate division TM7; 37 0 3 Bacteria;Chlorobi;Chlorobia;Chlorobiales;OPB56; 4 0 0 Bacteria;Chloroflexi;Anaerolineae;Anaerolineales; 6 0 0 Anaerolineaceae;uncultured; Bacteria;Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae; 0 7 0 Caldilinea; Bacteria;Chloroflexi;Thermomicrobia;JG30-KF-CM45; 2 0 0 Bacteria;Cyanobacteria;Chloroplast; 8 0 17 Bacteria;Cyanobacteria;SubsectionI; 1 0 0 Bacteria;Cyanobacteria;SubsectionI;Prochlorococcus; 0 8 0 Bacteria;Cyanobacteria;SubsectionI;Synechococcus; 0 7 10 Bacteria;Cyanobacteria;SubsectionII;SubgroupII;Pleurocapsa; 2 0 0 Bacteria;Deferribacteres;Deferribacteres;Deferribacterales; 0 0 1 Deferribacteraceae;Mucispirillum; Bacteria;Deinococcus- 4 0 0 Thermus;Deinococci;Deinococcales;Deinococcaceae; Deinococcus; Bact eria;Deinococcus-Thermus;Deinococci;Deinococcales; 1 0 0 Trueperaceae;Truepera; Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus; 0 0 40 Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae 0 0 1 ;Paenibacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae; 0 0 20 Saccharibacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Bhargavaea; 1 0 0 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 6 0 0 Lysinibacillus; 187

Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 0 0 19 Planomicrobium; Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 4 0 0 Sporosarcina; Bacteria;Firmicutes;Bacilli;Bacillales;Sporolactobacillaceae; 0 26 0 Sporolactobacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae; 0 19 0 Macrococcus; Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae; 2 0 36 Staphylococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales; 0 0 11 Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae; 0 0 2 Aerococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 2 0 0 Atopococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 0 0 5 Atopostipes; Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae; 0 72 0 Catellicoccus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae; 4 13 320 En t er o co ccu s; Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae; 17 51 117 Lact obacillus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae; 92 5337 7278 Leuconostoc; Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae; 88 47 6277 Weissella; Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 23 1069 3249 Lactococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 6 227 644 Streptococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 0 0 37 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 1 0 0 Alkaliphilus; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 18 0 0 Clostridiisalibacter; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 70 478 31 Clostridium; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 1 0 0 Sa r c i n a ; Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae 32 0 0 Sedis;Proteiniborus; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 64 0 0 Sedis;Finegoldia; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 1 0 0 Sedis;Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 5 0 0 Sedis;Peptoniphilus; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 8 0 0 Se d i s; Sp o r a n a e r o b a c t e r ; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 1 0 0 Sedis;Tepidimicrobium; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 1 8 0 Sedis;uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae 11 0 0 Sedis;Guggenheimella;

188

Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae 4 0 0 Sedis;Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae 1 0 0 Sedis;Mogibacterium; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae 10 0 0 Sedis;uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIV Incertae 1 0 0 Sedis;Anaerobranca; Bacteria;Firmicutes;Clostridia;Clostridiales;Gracilibacteraceae; 3 0 0 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 22 0 0 Anaerost ipes; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 183 0 7 Bl aut i a; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 0 0 6 But yr i vi br i o; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 1 0 0 Cat ab act er ; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 21 0 0 Coprococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 40 0 0 Dorea; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 425 24 6 Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 17 0 0 Lachnospira; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 15 0 6 Marvinbryantia; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 1 0 0 Oribact erium; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 176 0 1 Pseudobutyrivibrio; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 63 0 0 Roseburia; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 547 4 40 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae; 2 0 0 Peptococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae; 4 0 0 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales; 9 0 0 Peptostreptococcaceae;Filifactor; Bacteria;Firmicutes;Clostridia;Clostridiales; 48 171 25 Peptostreptococcaceae;Incert ae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales; 13 0 0 Peptostreptococcaceae;Peptostreptococcus; Bacteria;Firmicutes;Clostridia;Clostridiales; 1 0 7 Peptostreptococcaceae;uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 58 0 0 Anaerot runcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 962 0 0 Faecalibacterium; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 7 0 4 Fastidiosipila; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 72 0 2 Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 57 0 9

189

Ruminococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 243 0 0 Subdoligranulum; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 497 2 27 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae; 0 0 1 Anaerospora; Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae; 43 0 0 Dialister; Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae; 4 44 115 Veillonella; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 2 0 0 Erysipelotrichaceae;Holdemania; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 5 0 0 Erysipelotrichaceae;Incertae Sedis; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 0 13 0 Erysipelotrichaceae;Turicibact er; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 46 0 3 Erysipelotrichaceae;uncultured; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7; 2 0 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 0 8 3 Fusobacteriaceae;Cetobacterium; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 518 97 60 Fusobacteriaceae;Fusobacterium; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 1 0 0 Fusobacteriaceae;Propionigenium; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 3 0 0 Fusobacteriaceae;Psychrilyobacter; Bacteria;Fusobacteria;Fusobacteria;SHA-35; 0 6 0 Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales; 38 268 525 Phycisphaeraceae;CL500-3; Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales; 1 0 0 Phycisphaeraceae;SM1A02; Bacteria;Prot eobacteria;Alphaproteobacteria;Caulobacterales; 6 10 17 Caulobacteraceae;Brevundimonas; Bacteria;Proteobacteria;Alphaproteobacteria;DB1-14; 1 0 0 Bacteria;Proteobacteria;Alphaproteobacteria; 1 0 0 Rhizobiales;alphaI cluster; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 8 0 Bradyrhizobiaceae;Nitrobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 2 0 Bradyrhizobiaceae;Rhodopseudomonas; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 47 0 0 DUNssu371; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 1 0 0 Family Incertae Sedis;Nordella; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 1 0 0 Hyphomicrobiaceae;Devosia; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 9 13 120 Methylobacteriaceae;Methylobacterium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 18 Phyllobacteriaceae;Aquamicrobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 2 0 14 Rhizobiaceae;Rhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 1 0 0 Rhodobacteraceae;Loktanella;

190

Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 1 0 0 Rhodobacteraceae;Maritimibacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 15 0 0 Rhodobacteraceae;Paracoccus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 14 0 Rhodobacteraceae;Pseudorhodobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 2 0 0 Rhodobacteraceae;Roseovarius; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 1 0 0 Rhodobacteraceae;Seohaeicola; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 1 0 0 Rhodobacteraceae;Tateyamaria; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 3 0 0 Rhodobacteraceae;Thalassococcus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 9 0 5 Rhodobacteraceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales; 1 0 0 Acetobacteraceae;Acidiphilium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales; 1 0 0 Acetobacteraceae;Roseomonas; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales; 0 0 8 Rhodospirillaceae;Thalassospira; Bacteria;Proteobacteria;Alphaproteobacteria;Rickettsiales; 1 0 0 SM 2 D1 2 ; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Erythr 7 0 0 obacteraceae;Altererythrobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Erythr 2 0 0 obacteraceae;Porphyrobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;GOBB 2 0 0 3-C201; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphin 4 0 0 gomonadaceae;Novosphingobium; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphin 2 0 0 gomonadaceae;Sphingobium; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphin 2 0 43 gomonadaceae;Sphingomonas; Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphin 0 0 42 gomonadaceae;Sphingopyxis; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 3 0 0 Alcaligenaceae;Achromobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 0 0 Alcaligenaceae;Parasutterella; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 177 0 0 Alcaligenaceae;Sutterella; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 2 0 0 Alcaligenaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 161 Burkholderiaceae;Burkholderia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales 0 0 5 ;Burkholderiaceae;Cupriavidus; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 2 0 0 Burkholderiaceae;Polynucleobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 0 147 Comamonadaceae;Acidovorax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 29 0 1 Comamonadaceae;Comamonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 1 0

191

Comamonadaceae;Delftia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 8 21 Comamonadaceae;Diaphorobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 9 0 Comamonadaceae;Hydrogenophaga; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 1 Comamonadaceae;Paucibacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 0 0 Comamonadaceae;Pelomonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 3 0 0 Comamonadaceae;Piscinibacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 2 0 0 Comamonadaceae;Rhizobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 13 6 1 Comamonadaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 2 18 0 Comamonadaceae;Variovorax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 17 0 Oxalobacteraceae; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 6 Oxalobacteraceae;Herminiimonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 12 12 15 Oxalobacteraceae;Janthinobacterium; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 36 62 Oxalobacteraceae;Massilia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 18 0 Oxalobacteraceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 61 16 Oxalobacteraceae;Undibacterium; Bacteria;Prot eobacteria;Betaproteobacteria;Hydrogenophilales;Hydrog 3 0 0 enophilaceae;Thiobacillus; Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales;Hydrog 2 0 0 enophilaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales; 12 0 0 Methylophilaceae;Methylophilus; Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales; 4 0 0 Methylophilaceae;Methylotenera; Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales; 28 0 0 Methylophilaceae;uncultured; Bacteria;Prot eobacteria;Betaproteobacteria;Neisseriales; 1 0 0 Neisseriaceae;Conchiformibius; Bacteria;Prot eobacteria;Betaproteobacteria;Neisseriales; 1 22 75 Neisseriaceae;Microvirgula; Bacteria;Prot eobacteria;Betaproteobacteria;Neisseriales; 345 0 0 Neisseriaceae;Neisseria; Bacteria;Prot eobacteria;Betaproteobacteria;Neisseriales; 160 0 0 Neisseriaceae;uncultured; Bacteria;Prot eobacteria;Betaproteobacteria;Neisseriales; 0 20 17 Neisseriaceae;Vitreoscilla; Bacteria;Prot eobacteria;Betaproteobacteria;Neisseriales; 3 9 7 Neisseriaceae;Vogesella; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 2 0 0 Rhodocyclaceae;Azoarcus; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 1 0 0 Rhodocyclaceae;Azospira; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 1 0 0 Rhodocyclaceae;Denitratisoma;

192

Bacteria;Prot eobacteria;Betaproteobacteria;Rhodocyclales; 1 0 0 Rhodocyclaceae;Methyloversatilis; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 1 0 0 Rhodocyclaceae;uncultured; Bacteria;Prot eobacteria;Betaproteobacteria;UCT N117; 0 2 0 Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales; 0 0 4 Bacteriovoraceae;Bact eriovorax; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfo 22 0 0 bulbaceae;Desulfobulbus; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfo 2 0 0 bulbaceae;Desulfotalea; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulf 9 0 0 ohalobiaceae;uncultured; Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulf 12 0 0 ovibrionaceae;Bilophila; Bacteria;Prot eobacteria;Deltaproteobacteria; 0 0 5 Desulfuromonadales;M113; Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales; 2 0 0 0319-6G20; Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales; 3 0 0 Cystobacterineae;uncultured; Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Cam 1 0 38 pylobacteraceae;Arcobacter; Bacteria;Proteobacteria;Epsilonproteobacteria; 23 616 2 Campylobacterales;Campylobacteraceae;Campylobacter; Bacteria;Proteobacteria;Epsilonproteobacteria; 2 0 22 Campylobacterales;Campylobacteraceae;Sulfurospirillum; Bacteria;Proteobacteria;Epsilonproteobacteria; 0 0 79 Campylobacterales;Helicobacteraceae;Helicobacter; Bacteria;Proteobacteria;Epsilonproteobacteria; 17 0 0 Campylobacterales;Helicobacteraceae;Sulfurovum; Bacteria;Proteobacteria;Gammaproteobacteria; 12 0 0 Acidithiobacillales;Acidithiobacillaceae;Acidithiobacillus; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 Acidithiobacillales;KCM-B-112; Bacteria;Proteobacteria;Gammaproteobacteria; 2 9 16 Aeromonadales;Aeromonadaceae;Aeromonas; Bacteria;Proteobacteria;Gammaproteobacteria; 6 0 0 Alteromonadales;Alteromonadaceae;Alteromonas; Bacteria;Proteobacteria;Gammaproteobacteria; 2 0 0 Alteromonadales;Alteromonadaceae;BD1-7 clade; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 Alteromonadales;Alteromonadaceae;Glaciecola; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 Alteromonadales;Alteromonadaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Pseu 1 0 0 doalteromonadaceae;Pseudoalteromonas; Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Psych 2 0 0 romonadaceae;Psychromonas; Bacteria;Proteobacteria;Gammaproteobacteria;B38; 0 0 1 Bacteria;Proteobacteria;Gammaproteobacteria; 107 0 0 Cardiobacteriales;Cardiobacteriaceae;Cardiobacterium; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales; 1 0 0 Chromatiaceae;Rheinheimera; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 1 Enterobacteriales;Enterobacteriaceae; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 1 193

Enterobacteriales;Enterobact eriaceae;Cedecea; Bacteria;Proteobacteria;Gammaproteobacteria; 25 139 749 Enterobacteriales;Enterobacteriaceae;Citrobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 18 0 0 Enterobacteriales;Enterobacteriaceae;Edwardsiella; Bacteria;Proteobacteria;Gammaproteobacteria; 8 81 388 Enterobacteriales;Enterobacteriaceae;Enterobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 71 0 79 Enterobacteriales;Enterobacteriaceae;Escherichia-Shigella; Bacteria;Proteobacteria;Gammaproteobacteria; 0 4 29 Enterobacteriales;Enterobacteriaceae;Klebsiella; Bacteria;Proteobacteria;Gammaproteobacteria; 0 25 80 Enterobacteriales;Enterobacteriaceae;Kluyvera; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 1 Enterobacteriales;Enterobacteriaceae;Morganella; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 49 Enterobacteriales;Enterobacteriaceae;Pantoea; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 66 Enterobacteriales;Enterobacteriaceae;Pectobacterium; Bacteria;Proteobacteria;Gammaproteobacteria; 4 0 0 Enterobacteriales;Enterobacteriaceae;Plesiomonas; Bacteria;Proteobacteria;Gammaproteobacteria; 0 14 0 Enterobacteriales;Enterobacteriaceae;Rahnella; Bacteria;Proteobacteria;Gammaproteobacteria; 0 8 21 Enterobacteriales;Enterobacteriaceae;Raoultella; Bacteria;Proteobacteria;Gammaproteobacteria; 11 0 0 Methylococcales;Hyd24-01; Bacteria;Proteobacteria;Gammaproteobacteria; 6 0 12 Methylococcales;Methylococcaceae;Methylomonas; Bacteria;Proteobacteria;Gammaproteobacteria;NKB5; 1 0 0 Bacteria;Proteobacteria;Gammaproteobacteria; 4 0 0 Oceanospirillales;Halomonadaceae;Cobetia; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 Oceanospirillales;Halomonadaceae;Halomonas; Bacteria;Proteobacteria;Gammaproteobacteria; 8 0 0 Oceanospirillales;Halomonadaceae;Kushneria; Bacteria;Proteobacteria;Gammaproteobacteria; 7 0 0 Oceanospirillales;Oceanospirillaceae;Marinomonas; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 Pasteurellales;Past eurellaceae;Haemophilus; Bacteria;Proteobacteria;Gammaproteobacteria; 381 0 0 Pasteurellales;Pasteurellaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria; 56 218 1367 Pseudomonadales;Moraxellaceae;Acinetobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 5 11 175 Pseudomonadales;Moraxellaceae;Enhydrobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 173 0 23 Pseudomonadales;Moraxellaceae;Psychrobacter; Bacteria;Proteobacteria;Gammaproteobacteria 2 0 0 Pseudomonadales;Moraxellaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria 1 0 0 ;Pseudomonadales;Pseudomonadaceae;Cellvibrio; Bacteria;Proteobacteria;Gammaproteobacteria; 20 56 130 Pseudomonadales;Pseudomonadaceae;Pseudomonas; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 Salinisphaerales;Salinisphaeraceae;Salinisphaera; Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales; 6 0 0 194

Piscirickettsiaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales; 2 0 0 Thiotrichaceae;Leucothrix; Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales; 0 15 0 Vibrionaceae;Photobacterium; Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales; 8 0 0 Vibrionaceae;Vibrio; Bacteria;Proteobacteria;Gammaproteobacteria; 6 0 0 Xanthomonadales;Sinobacteraceae;JTB255 marine benthic group; Bacteria;Proteobacteria;Gammaproteobacteria; 2 0 0 Xanthomonadales;Xanthomonadaceae;Dokdonella; Bacteria;Proteobacteria;Gammaproteobacteria; 2 0 2 Xanthomonadales;Xanthomonadaceae;Dyella; Bacteria;Proteobacteria;Gammaproteobacteria; 3 0 0 Xanthomonadales;Xanthomonadaceae;Frateuria; Bacteria;Proteobacteria;Gammaproteobacteria; 21 0 10 Xanthomonadales;Xanthomonadaceae;Rhodanobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 158 0 24 Xanthomonadales;Xanthomonadaceae;Stenotrophomonas; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 Xanthomonadales;Xanthomonadaceae;Thermomonas; Bacteria;Spirochaetes;Spirochaetes;Spirochaetales; 36 0 0 Spirochaetaceae;Treponema; Bacteria;Synergistetes;Synergistia;Synergistales; 1 0 0 Synergistaceae;uncultured; Bacteria;Tenericutes;Mollicut es;Acholeplasmatales; 1 0 0 Acholeplasmataceae;Acholeplasma; Bacteria;Tenericutes;Mollicutes;Mycoplasmatales; 22 0 0 Mycoplasmataceae;Mycoplasma; Bacteria;Tenericutes;Mollicut es;RF9; 6 0 0 Bacteria;Verrucomicrobia;OPB35 soil group; 1 0 0 Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;Verruc 0 0 25 omicrobiaceae;Prosthecobact er; Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;Verruc 1 0 0 omicrobiaceae;Verrucomicrobium; Unclassified 135 37 260

195

Appendix 5 Table A5, Taxonomic Breakdown of bacteria present in short-tailed shearwaters during development. A = Adult, 2 = Week 2, 5 = Week 5, 8 = Week 8, 11 = Week 11 and 14 = Week 4. Taxonomic group (SILVA tax, release 106) A 2 5 8 11 14 Bacteria;Acidobacteria;Acidobacteria;Acidobacteriales; 0 0 2 0 0 0 Acidobacteriaceae;Edaphobacter; Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae; 0 0 0 19 0 0 Acidimicrobiales;Acidimicrobineae;Iamiaceae Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae; 7 0 0 0 0 0 Acidimicrobiales;Acidimicrobineae;OCS155 marine group Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 55 8 21 5 85 44 Actinomycetales;Actinomycineae;Actinomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 44 43 107 141 661 87 Actinomycetales;Corynebacterineae;Corynebacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 7 0 8 Actinomycetales;Corynebacterineae;Dietziaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 16 0 Actinomycetales;Corynebacterineae;Mycobacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae 0 3 6 15 12 6 ;Actinomycetales;Corynebacterineae;Nocardiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 1 0 1 11 13 0 Actinomycetales;Corynebacterineae;uncultured Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 0 1 Actinomycetales;Frankineae;Frankiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 7 0 0 0 0 0 Actinomycetales;Frankineae;Geodermatophilaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 3 0 0 7 Actinomycetales;Frankineae;Sporichthyaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 1 0 1 Actinomycetales;Frankineae;uncultured Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 13 0 0 0 0 0 Actinomycetales;Kineosporiineae;Kineosporiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 1 0 Actinomycetales;Micrococcineae;Bogoriellaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 24 10 74 95 16 Actinomycetales;Micrococcineae;Brevibacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 14 7 50 0 6 Actinomycetales;Micrococcineae;Dermabacteraceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 13 0 0 Actinomycetales;Micrococcineae;Dermacoccaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 13 4 17 21 0 Actinomycetales;Micrococcineae;Intrasporangiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 26 17 6 11 17 0 Actinomycetales;Micrococcineae;Microbacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 21 0 2 33 0 2 Actinomycetales;Micrococcineae;Micrococcaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 7 0 Actinomycetales;Micrococcineae;Promicromonosporaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 0 0 0 1 0 Actinomycetales;Micrococcineae;Ruaniaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae 0 0 9 39 0 3 ;Actinomycetales;Propionibacterineae;Nocardioidaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 60 18 6 5 5 15 Actinomycetales;Propionibacterineae;Propionibacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 7 2 0 0 0 Actinomycetales;Pseudonocardineae;Pseudonocardiaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae; 0 1 4 0 0 0 Actinomycetales;Streptomycineae;Streptomycetaceae Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae 0 0 0 7 0 0 ;Bifidobacteriales;Bifidobacteriaceae;Bifidobacterium Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae; 0 0 0 0 38 0 Solirubrobacterales;480-2; Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae; 0 0 0 3 0 0 Solirubrobacterales;Solirubrobacteriaceae;Solirubrobacter Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 19 0 2 0 0 7 Bacteroidaceae;Bacteroides; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 0 0 0 0 1 0 Marinilabiaceae;Marinifilum; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 0 0 0 0 0 5 Porphyromonadaceae;Paludibacter; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales 0 13 4 0 17 25 Porphyromonadaceae;Petrimonas; 196

Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 18 6 0 0 2 0 Porphyromonadaceae;Porphyromonas; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 0 1 1 11 6 7 Porphyromonadaceae;Proteiniphilum; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 13 0 4 0 25 12 Prevotellaceae;Prevotella; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 0 15 0 0 3 6 Prevotellaceae;uncultured; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales; 12 1 0 4 0 4 Rikenellaceae;Alistipes; Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7; 74 28 0 5 41 0 Bacteria;Bacteroidetes;Cytophagia;Cytophagales; 0 0 0 0 0 3 Cytophagaceae;Dyadobacter; Bacteria;Bacteroidetes;Cytophagia;Cytophagales; 0 0 0 1 0 0 Cytophagaceae;Fibrella; Bacteria;Bacteroidetes;Cytophagia;Cytophagales 0 0 0 0 0 1 ;Cytophagaceae;Pontibacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 2 0 0 0 Cryomorphaceae;Fluviicola; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 3 8 0 0 Cryomorphaceae;Owenweeksia; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 0 40 19 8 Flavobacteriaceae;Aequorivita; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 0 0 1 0 Flavobacteriaceae;Bergeyella; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 0 14 0 0 Flavobacteriaceae;Bizionia; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 139 67 12 68 68 46 Flavobacteriaceae;Chryseobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 3 0 0 0 0 0 Flavobacteriaceae;Cloacibacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 16 841 237 1218 17 Flavobacteriaceae;Coenonia; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 13 0 16 9 10 2 Flavobacteriaceae;Flavobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 11 70 0 0 Flavobacteriaceae;Gelidibacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 0 2 0 0 Flavobacteriaceae;Kriegella; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 1 7 4 7 16 0 Flavobacteriaceae;Ornithobacterium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 0 36 0 0 Flavobacteriaceae;Salinimicrobium; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 2 0 3 3 0 0 Flavobacteriaceae;uncultured; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 6 19 0 0 Flavobacteriaceae;Vitellibacter; Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales; 0 0 3 0 0 0 Flavobacteriaceae;Wautersiella; Bacteria;Bacteroidetes;SB-1; 1 0 0 0 0 0 Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 22 2 12 27 0 Chitinophagaceae;Sediminibacterium; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 0 0 0 35 0 KD1-131; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 0 0 7 0 0 Sphingobacteriaceae;Parapedobacter; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 0 9 19 0 13 Sphingobacteriaceae;Pedobacter; Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales; 0 0 0 4 0 0 Sphingobacteriaceae;Sphingobacterium; Bacteria;Bacteroidetes;VC2.1 Bac22; 12 0 0 0 0 0 Bacteria;BD1-5; 0 0 5 0 0 0 Bacteria;Candidate division BRC1; 0 0 0 2 0 0 Bacteria;Candidate division OD1; 0 0 3 4 3 0 Bacteria;Candidate division OP10; 0 0 0 3 0 0 Bacteria;Candidate division OP11; 0 0 0 0 0 2 Bacteria;Candidate division TM7; 3 31 0 23 0 22 Bacteria;Chloroflexi;KD4-96; 0 1 0 0 0 0 Bacteria;Cyanobacteria;Chloroplast; 17 42 16 0 20 0

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Bacteria;Cyanobacteria;SM2F09; 0 0 0 2 0 0 Bacteria;Cyanobacteria;SubsectionI;Prochlorococcus; 0 3 0 0 0 0 Bacteria;Cyanobacteria;SubsectionI;Synechococcus; 10 0 0 0 0 0 Bacteria;Deferribacteres;Deferribacteres;Deferribacterales; 1 0 0 0 0 0 Deferribacteraceae;Mucispirillum; Bacteria;Firmicutes;Bacilli;Bacillales;Alicyclobacillaceae; 0 0 0 1 0 0 Alicyclobacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus; 40 21 5 9 1 3 Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;uncultured; 0 0 2 11 6 0 Bacteria;Firmicutes;Bacilli;Bacillales;Family XII Incertae 0 0 1 0 0 0 Sedis;Exiguobacterium; Bacteria;Firmicutes;Bacilli;Bacillales;Family XII Incertae Sedis;Incertae 0 0 0 0 11 1 Sedis; Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae; 1 0 0 0 0 0 Paenibacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae; 20 0 0 0 0 0 Saccharibacillus; Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 0 377 1271 1370 6457 726 Lysinibacillus; 4 3 6 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 19 0 0 1 1 0 Planomicrobium; Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 0 0 15 64 0 14 Sporosarcina; Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae; 0 0 0 6 0 0 uncultured; Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae; 0 9 2 0 0 0 Macrococcus; Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae; 36 105 12 78 26 36 Staphylococcus Bacteria;Firmicutes;Bacilli;Lactobacillales; 11 0 0 0 0 0 Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae; 2 0 0 0 0 0 Aerococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 5 0 0 0 0 0 Atopostipes; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 0 0 0 0 1 0 Carnobacterium; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae; 0 0 0 0 0 4 Trichococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae 0 0 1 0 0 0 uncultured; Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae; 320 34 43 85 162 34 Enterococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae; 117 57 0 90 53 25 Lactobacillus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae; 0 2 0 0 0 0 Fructobacillus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae; 7278 371 1328 4936 5145 480 Leuconostoc; 2 9 Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae; 6277 116 34 110 186 145 Weissella; Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 3249 898 287 909 1974 998 Lactococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 644 259 57 173 375 300 Streptococcus; Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae; 37 0 2 0 0 0 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae; 31 46 13 65 67 41 Clostridium; Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae 0 0 0 0 3 0 Sedis;Proteiniborus; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 15 0 3 33 1 Sedis;Finegoldia; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 10 2 11 3 0 Sedis;Tissierella; Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae 0 3 9 0 0 18 Sedis;uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 7 3 0 0 0 0 Blautia; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 6 0 0 2 0 0 Butyrivibrio; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 0 0 5 0 0 0 Epulopiscium; 198

Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 6 110 9 23 14 27 Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 6 0 0 0 0 0 Marvinbryantia; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 1 0 0 0 0 0 Pseudobutyrivibrio; Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae; 40 7 0 21 27 0 uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales; 25 110 13 16 40 11 Peptostreptococcaceae;Incertae Sedis; 1 Bacteria;Firmicutes;Clostridia;Clostridiales; 7 13 0 16 0 7 Peptostreptococcaceae;uncultured; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 0 2 0 0 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 0 0 1 0 0 11 Anaerotruncus; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 4 2 0 0 0 0 Fastidiosipila; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 2 0 0 8 18 0 Incertae Sedis; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 9 0 0 0 0 0 Ruminococcus; Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae; 27 1 0 0 33 0 uncultured Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae; 1 0 0 0 0 0 Anaerospora; Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae; 0 0 0 24 0 0 Megamonas; Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae; 115 42 36 98 257 39 Veillonella; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 0 0 1 0 0 0 Erysipelotrichaceae;Allobaculum; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 0 0 0 1 0 0 Erysipelotrichaceae;Turicibacter; Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales; 3 0 0 0 0 0 Erysipelotrichaceae;uncultured; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7 0 0 0 109 0 0 Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 3 2 0 5 0 6 Fusobacteriaceae;Cetobacterium; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 60 41 87 133 79 399 Fusobacteriaceae;Fusobacterium; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 0 3 0 0 0 0 Leptotrichiaceae;Sneathia; Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales; 0 0 1 0 0 0 Leptotrichiaceae;uncultured; Bacteria;Gemmatimonadetes;Gemmatimonadetes; 0 0 0 6 0 0 AT425-EubC11 terrestrial group; Bacteria;Gemmatimonadetes;Gemmatimonadetes;S0134 0 0 0 5 0 0 terrestrial group; Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales; 525 236 40 345 152 136 Phycisphaeraceae;CL500-3; Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales; 17 23 3 15 10 8 Caulobacteraceae;Brevundimonas; Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales; 0 0 4 0 0 0 Caulobacteraceae;Caulobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales; 0 13 0 0 0 0 Caulobacteraceae;Phenylobacterium; Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales; 0 8 0 19 0 7 Caulobacteraceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 0 0 6 Bartonellaceae;Bartonella; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 0 0 21 Beijerinckiaceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 21 0 1 Bradyrhizobiaceae;Bradyrhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 2 0 0 0 0 Bradyrhizobiaceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 3 0 0 0 Brucellaceae;Pseudochrobactrum; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 25 0 0 Hyphomicrobiaceae;Devosia; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales 0 0 0 3 0 0 ;Hyphomicrobiaceae;Pedomicrobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 120 6 0 0 0 0 Methylobacteriaceae;Methylobacterium;

199

Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 0 0 6 P-102; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 18 0 0 0 0 0 Phyllobacteriaceae;Aquamicrobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 13 0 0 0 0 Phyllobacteriaceae;Mesorhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 14 0 0 8 0 7 Rhizobiaceae;Rhizobium; Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; 0 0 0 0 0 6 Xanthobacteraceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 6 17 0 0 Rhodobacteraceae;Paracoccus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 11 0 0 Rhodobacteraceae;Phaeobacter; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 27 0 0 Rhodobacteraceae;Pontibaca; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 1 0 0 Rhodobacteraceae;Roseovarius; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 0 0 0 21 0 0 Rhodobacteraceae;Thalassococcus; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales; 5 0 0 22 0 0 Rhodobacteraceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales; 0 0 0 0 0 1 MSB-1E8; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales; 8 0 0 0 0 0 Rhodospirillaceae;Thalassospira; Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales; 0 0 5 0 0 0 Rhodospirillaceae;uncultured; Bacteria;Proteobacteria;Alphaproteobacteria; 0 19 0 0 0 0 Sphingomonadales;Sphingomonadaceae;Sphingobium; Bacteria;Proteobacteria;Alphaproteobacteria; 43 0 0 1 2 0 Sphingomonadales;Sphingomonadaceae;Sphingomonas; Bacteria;Proteobacteria;Alphaproteobacteria; 42 0 0 0 0 0 Sphingomonadales;Sphingomonadaceae;Sphingopyxis; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 9 0 0 Alcaligenaceae;Achromobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 20 0 0 Alcaligenaceae;Pusillimonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 161 554 151 527 1168 388 Burkholderiaceae;Burkholderia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 5 0 0 0 1 3 Burkholderiaceae;Cupriavidus; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 1 0 0 Burkholderiaceae;Limnobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 23 4 23 80 33 Burkholderiaceae;Ralstonia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 0 9 0 Comamonadaceae; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 147 0 22 0 0 10 Comamonadaceae;Acidovorax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 11 0 24 15 7 Comamonadaceae;Aquabacterium; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 9 0 0 3 7 Comamonadaceae;Comamonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 3 0 0 Comamonadaceae;Delftia; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 21 1 3 0 20 2 Comamonadaceae;Diaphorobacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 0 0 0 0 0 Comamonadaceae;Paucibacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 14 1 0 0 0 Comamonadaceae;Pelomonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales 0 8 0 0 0 0 ;Comamonadaceae;Piscinibacter; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 14 0 0 Comamonadaceae;Polaromonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 1 0 0 0 0 7 Comamonadaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 0 0 1 0 Comamonadaceae;Variovorax; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 6 0 0 0 0 0 Oxalobacteraceae;Herminiimonas; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 15 87 33 47 30 42 Oxalobacteraceae;Janthinobacterium; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 62 0 0 8 0 0 Oxalobacteraceae;Massilia; 200

Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 0 0 2 0 0 0 Oxalobacteraceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales; 16 0 0 0 0 20 Oxalobacteraceae;Undibacterium; Bacteria;Proteobacteria;Betaproteobacteria;DR-16; 0 1 0 0 0 0 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 0 2 2 0 0 10 Neisseriaceae;Aquitalea; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 75 7 10 53 15 15 Neisseriaceae;Microvirgula; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 0 0 0 0 1 0 Neisseriaceae;Neisseria; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 0 0 1 0 10 0 Neisseriaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 0 0 0 0 0 10 Neisseriaceae;Uruburuella; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 17 6 2 9 10 10 Neisseriaceae;Vitreoscilla; Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; 7 0 0 0 0 0 Neisseriaceae;Vogesella; Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomonadales; 0 0 5 0 0 0 Nitrosomonadaceae;Nitrosospira; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 0 18 0 0 0 0 Rhodocyclaceae;Thauera;Thauera Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 0 0 0 5 9 0 Rhodocyclaceae;Uliginosibacterium; Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales; 0 0 0 3 0 0 Rhodocyclaceae;uncultured; Bacteria;Proteobacteria;Betaproteobacteria;TRA3-20; 0 0 1 0 0 0 Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales; 4 0 0 0 0 0 Bacteriovoraceae;Bacteriovorax; Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales; 0 0 0 1 0 0 Bdellovibrionaceae;Bdellovibrio; Bacteria;Proteobacteria;Deltaproteobacteria; 0 0 3 2 0 0 Desulfuromonadales;GR-WP33-58; Bacteria;Proteobacteria;Deltaproteobacteria; 5 0 0 0 0 0 Desulfuromonadales;M113; Bacteria;Proteobacteria;Deltaproteobacteria;GR-WP33-30; 0 0 0 16 0 0 Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales; 0 0 2 14 0 0 0319-6G20; Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales; 0 0 0 0 0 1 Sorangiineae;uncultured; Bacteria;Proteobacteria;Epsilonproteobacteria; 38 0 0 15 16 3 Campylobacterales;Campylobacteraceae;Arcobacter; Bacteria;Proteobacteria;Epsilonproteobacteria; 2 4 4 0 18 34 Campylobacterales;Campylobacteraceae;Campylobacter; Bacteria;Proteobacteria;Epsilonproteobacteria; 22 9 0 0 0 0 Campylobacterales;Campylobacteraceae;Sulfurospirillum; Bacteria;Proteobacteria;Epsilonproteobactea; 79 8 0 64 0 0 Campylobacterales;Helicobacteraceae;Helicobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 0 22 28 20 35 1 Acidithiobacillales;Acidithiobacillaceae;Acidithiobacillus; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales; 16 11 2 27 0 0 Aeromonadaceae;Aeromonas; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales; 0 7 8 110 6 0 Aeromonadaceae;Oceanimonas; Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales; 0 0 0 12 2 0 Aeromonadaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 3 34 9 0 Alteromonadales;Alteromonadaceae;Marinobacter; Bacteria;Proteobacteria;Gammaproteobacteria;B38; 1 0 0 0 0 0 Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 0 1 0 0 Cardiobacteriales;Cardiobacteriaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales; 0 0 0 9 0 0 Chromatiaceae;Nitrosococcus; Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales; 0 0 0 0 0 10 Granulosicoccaceae;Granulosicoccus; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 0 0 0 Enterobacteriales;Enterobacteriaceae; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 0 0 0 1 Enterobacteriales;Enterobacteriaceae;Cedecea; Bacteria;Proteobacteria;Gammaproteobacteria; 749 152 38 197 250 70 Enterobacteriales;Enterobacteriaceae;Citrobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 388 105 25 62 198 150 201

Enterobacteriales;Enterobacteriaceae;Enterobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 79 2 9 5 48 86 Enterobacteriales;Enterobacteriaceae;Escherichia-Shigella; Bacteria;Proteobacteria;Gammaproteobacteria; 29 7 0 0 0 0 Enterobacteriales;Enterobacteriaceae;Klebsiella; Bacteria;Proteobacteria;Gammaproteobacteria; 80 2 3 4 28 119 Enterobacteriales;Enterobacteriaceae;Kluyvera; Bacteria;Proteobacteria;Gammaproteobacteria; 1 0 2 11 0 0 Enterobacteriales;Enterobacteriaceae;Morganella; Bacteria;Proteobacteria;Gammaproteobacteria; 49 0 0 0 0 0 Enterobacteriales;Enterobacteriaceae;Pantoea; Bacteria;Proteobacteria;Gammaproteobacteria; 66 0 0 0 0 0 Enterobacteriales;Enterobacteriaceae;Pectobacterium; Bacteria;Proteobacteria;Gammaproteobacteria; 21 0 0 0 0 13 Enterobacteriales;Enterobacteriaceae;Raoultella; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 0 8 0 0 Legionellales;Coxiellaceae;Aquicella; Bacteria;Proteobacteria;Gammaproteobacteria; 0 3 0 0 0 0 Methylococcales;Crenotrichaceae;Crenothrix; Bacteria;Proteobacteria;Gammaproteobacteria; 12 4 0 0 2 1 Methylococcales;Methylococcaceae;Methylomonas; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 1 0 0 0 Methylococcales;Methylococcaceae;Methylosoma; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 4 0 0 0 Oceanospirillales;Halomonadaceae;Chromohalobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 0 0 1 0 Oceanospirillales;Oceanospirillaceae;Pseudospirillum; Bacteria;Proteobacteria;Gammaproteobacteria; 0 26 50 0 0 0 Pasteurellales;Pasteurellaceae;Pasteurella; Bacteria;Proteobacteria;Gammaproteobacteria; 1367 113 61 229 422 174 Pseudomonadales;Moraxellaceae;Acinetobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 175 19 9 16 8 15 Pseudomonadales;Moraxellaceae;Enhydrobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 23 6 90 17 19 0 Pseudomonadales;Moraxellaceae;Psychrobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 130 28 7 86 43 26 Pseudomonadales;Pseudomonadaceae;Pseudomonas; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 0 0 12 0 Thiotrichales;Piscirickettsiaceae;Cycloclasticus; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 0 0 1 0 Thiotrichales;Piscirickettsiaceae;endosymbionts; Bacteria;Proteobacteria;Gammaproteobacteria; 0 2 0 0 0 0 Thiotrichales;Thiotrichaceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria; 0 9 0 65 46 0 Vibrionales;Vibrionaceae;Vibrio; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 1 0 6 8 Xanthomonadales;Sinobacteraceae;JTB255 marine benthic group; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 0 0 0 13 Xanthomonadales;Sinobacteraceae;Steroidobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 0 6 0 0 Xanthomonadales;Sinobacteraceae;uncultured; Bacteria;Proteobacteria;Gammaproteobacteria; 0 6 0 0 0 0 Xanthomonadales;Xanthomonadaceae;Arenimonas; Bacteria;Proteobacteria;Gammaproteobacteria; 2 0 0 0 0 0 Xanthomonadales;Xanthomonadaceae;Dyella; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 2 9 0 0 Xanthomonadales;Xanthomonadaceae;Luteimonas; Bacteria;Proteobacteria;Gammaproteobacteria; 0 0 8 9 0 0 Xanthomonadales;Xanthomonadaceae;Lysobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 10 0 4 11 0 2 Xanthomonadales;Xanthomonadaceae;Rhodanobacter; Bacteria;Proteobacteria;Gammaproteobacteria; 24 7 1 11 0 3 Xanthomonadales;Xanthomonadaceae;Stenotrophomonas; Bacteria;SM2F11; 0 0 0 0 3 0 Bacteria;Synergistetes;Synergistia;Synergistales; 0 0 1 0 0 0 Synergistaceae;Jonquetella; Bacteria;Tenericutes;Mollicutes;Anaeroplasmatales; 0 0 0 8 0 0 Anaeroplasmataceae;Anaeroplasma; Bacteria;Tenericutes;Mollicutes;Mycoplasmatales; 0 0 0 0 0 1 Mycoplasmataceae;Mycoplasma; Bacteria;Verrucomicrobia;Verrucomicrobiae; 25 0 0 0 0 0 Verrucomicrobiales;Verrucomicrobiaceae;Prosthecobacter; Unclassified; 260 122 51 76 147 66

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