This file is part of the following reference:

Gainsford, Ashton (2016) A multi-disciplinary evaluation

of the hybrid anemonefish Amphiprion leucokranos:

behaviour shaping evolutionary outcomes of hybridization.

PhD thesis, James Cook University.

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http://dx.doi.org/10.4225/28/58c62be6f0a34

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A MULTI-DISCIPLINARY EVALUATION OF THE

HYBRID ANEMONEFISH AMPHIPRION LEUCOKRANOS:

BEHAVIOUR SHAPING EVOLUTIONARY OUTCOMES

OF HYBRIDIZATION

Thesis submitted by

Ashton Gainsford (BSc Hons)

In November 2016

For the degree of Doctor of Philosophy

College of Science and Engineering

James Cook University

Townsville, Queensland, Australia

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STATEMENT OF CONTRIBUTION OF OTHERS

A number of collaborators and colleagues have contributed in various ways to this thesis and the subsequent manuscripts which are published, submitted or in preparation for submission. A summary of these contributions are listed below.

Nature of Assistance Contribution Co-contributors

Intellectual support Project design Dr. L. van Herwerden (JCU) & Prof. G.P. Jones (JCU, ARC CoE)

Proposal writing Dr. L. van Herwerden (JCU) & Prof. G.P. Jones (JCU, ARC CoE)

Data analysis support Chapter 2: S.R. Montanari (Aims@JCU) & Dr. M. van der Meer (JCU)

Statistical support Chapter 5: Prof Emer. R. Jones (JCU)

Editorial assistance Chapters 2-4: Dr. L. van Herwerden (JCU), Prof. G.P. Jones (JCU, ARC CoE); Chapter 4: Dr. J-P. Hobbs (CU); Chapter 4-5: H. Epstein (Aims@JCU, ARC CoE)

Data collection Fieldwork assistance Dr. M. Lal (JCU), K. Thomas Brown (USP), T. Millitz (JCU), Dr. J-P. Hobbs (CU), H. Campbell, Dr. L. Bostrom-Einarsson (JCU, ARC CoE), Dr. H.B. Harrison (JCU, ARC CoE), T. Bowling, & Dr. M.C. Bonin (ARC CoE)

Laboratory assistance F.M. Heindler (JCU) & H. Epstein (Aims@JCU, ARC CoE)

Diving & boating support

L. Choquette & Solomon Dive Adventures, Mahonia Na Dari, Kimbe Bay & (NIMRF, Kavieng)

Histology S. Reilly (JCU)

Otolith sectioning Dr. D. Lou (Tropical Fish Ageing)

Financial assistance Research funding Prof. G.P. Jones (JCU, ARC CoE), Dr. J-P. Hobbs (CU), & Dr. L. van Herwerden (JCU)

The research presented and reported in this thesis was conducted in compliance with the National Health and Medical Research Council (NHMRC) Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th Edition, 2004 and the Qld Animal Care and Protection Act, 2001. The proposed research study received animal ethics approval from the JCU Animal Ethics Committee Approval Number A1633 and A1791.

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ACKNOWLEDGEMENTS

“Solitude is a human presumption. Every quiet step is thunder to beetle life underfoot, a tug of impalpable thread on the web pulling mate to mate and predator to prey, a beginning or an end. Every choice is a world made new for the chosen.” – Barbara Kingsolver

The journey to completing my dissertation was not travelled alone and I will be forever

grateful to my family, friends and colleagues who have shared in this experience. Each small

kindness contributed to me finishing, which sometimes felt beyond reach. Many favours, words of

encouragement, insights, late night phone calls, blind faith and understanding were shared without

question. You’re all lovely and wonderful and I will never forget it.

A few special mentions – Carol and Brian Gainsford, your generosity inspires me and I am

proud beyond measure to be your daughter. Thank you for always encouraging curiosity and

adventure, even though it has put considerable distance between us. You have given me spirit and

strength, and a family with unconditional love. Thank you for your unwavering support every step

of the way.

Yvette, Dean and Chiara-Lee – you are the craziest, kindest and most spirited bunch of

bloody drongos anyone could ever hope to call family. Thank you for reminding me to see humour

in each and every moment and for sharing in my trials and triumphs. You are my best friends.

Mary – you taught me how to catch a fish and a hell of a lot about research in the field for

which I will be forever grateful; thanks for all the epic adventures and laughs along the way. Hannah

– your enthusiasm for my research, challenging conversations, tireless assistance in the lab and

editing in the final weeks of this long journey are appreciated beyond words. Lisa, Gigi, Emma and

Margaux – you are one solid group of fearless, strong ladies. Your friendship has picked me up and

empowered me to keep moving forward. Thank you for always celebrating each other’s successes,

big and small – your joy is powerful and contagious.

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To my supervisors, Lynne and Geoff, I cannot thank you enough for the time and effort

you have invested in developing me as a researcher, whilst also juggling your own demanding lives

and many responsibilities. Lynne, you took me under your wing with blind faith, you have

challenged my thinking and ability, and offered me many opportunities to increase my skills and

responsibilities in academia. Thank you for your time, guidance and friendship.

Formally, I would also like to acknowledge and thank the following institutions and

funding bodies for financial support of my research and professional development: Sea World

Research and Rescue Foundation Inc., the College of Marine and Environmental Sciences and

Australian Research Council Centre of Excellence for Coral Reef Studies, and the Comparative

Genomics Centre at James Cook University, and the Australian Coral Reef Society. Finally, I was

generously supported by an Australian Postgraduate Award for three and a half years with full

tuition waiver while completing my dissertation, as well as a living stipend through the Graduate

Research Scheme (JCU) to complete my dissertation writing, for which I am beyond grateful.

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GENERAL ABSTRACT

Hybridization is an evolutionarily significant and common occurrence in nature.

Interspecific breeding between closely related taxa challenges established phylogenies and

questions what constitutes a species; where hybridization may drive rapid evolution of taxa and

provide a source of evolutionary novelty to ecosystems. Despite the enhanced study of

hybridization in recent time, a clear understanding of the mechanisms which initiate and maintain

hybridization remains limited. Hybrid zones are ideal for testing theoretical questions regarding

speciation and elucidating patterns of variation among species. For many taxa, reproductive success

is skewed towards large, socially dominant individuals within groups, and reproductive traits and

mating systems are expected to influence the outcomes of hybridization and gene exchange between

hybridizing species. In this thesis, I addressed how ecology and behaviour contribute to

maintenance and persistence of the Amphiprion leucokranos hybrid zone. This research employed

a combination of ecological, phylogenetic and population genetic assays, as well as an observational

study and behavioural experiment to test key concepts of the importance of hybridization to

evolution of species.

A suite of 42 novel and published microsatellite markers were developed and tested in

Chapter 2 to facilitate investigation into the relatedness of taxa within the hybrid zone. The

influence of ecology and behaviour on the outcomes of hybridization were tested in Chapter 3,

addressing how habitat use and relative size differences of parent species and hybrids drive patterns

of gene exchange. Findings confirmed A. leucokranos to be the hybrid of closely related

A. chrysopterus and A. sandaracinos, and subsequently verified that behavioural isolation, habitat

use and species-specific size differences dictates the direction and degree of back-crossing and

subsequent introgression. Hybridization took place exclusively with A. chrysopterus in the

dominant female rank and the smaller A. sandaracinos in the sub-dominant male rank, based on

mtDNA cytochrome b and multiple nDNA microsatellite loci. Overlap in habitat, depth and host

anemone use was found, with hybrids intermediate to parents and co-habitation in over 25% of

anemones sampled. Hybrids, intermediate in body size, colour and pattern, were classified 55% of

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the time as morphologically first generation hybrids relative to parents, whereas 45% of hybrids

were more like A. sandaracinos, suggesting back-crossing. Unidirectional introgression of

A. chrysopterus mtDNA into A. sandaracinos via hybrid back-crosses was found, wi