Evolutionary tales of maskrays (Neotrygon, Dasyatidae), flatheads (, ) and tuskfishes (Choerodon, Labridae).

by Melody Puckridge Institute for Marine and Antarctic Studies

Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy University of Tasmania October 2013

DECLARATION OF ORIGINALITY

This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright.

Melody Puckridge

Date: 27/10/2013

STATEMENT OF ETHICAL CONDUCT

The research associated with this thesis abides by the international and Australian codes on human and experimentation, the guidelines by the Australian Government's Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University.

Melody Puckridge

Date: 27/10/2013

AUTHORITY OF ACCESS

The publishers of the papers comprising Chapters 2 and 3 hold the copyright for that content, and access to the material should be sought from the respective journals. The remaining non published content of the thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968.

Melody Puckridge

Date: 27/10/2013 ii

PUBLICATIONS

Chapter 2 Puckridge M, Last PR, White WT, Andreakis N (2013) Phylogeography of the Indo- West Pacific maskrays (Dasyatidae, Neotrygon): a complex example of chondrichthyan radiation in the Cenozoic. Ecology and Evolution 3, 217-232.

Chapter 3 Puckridge M, Andreakis N, Appleyard SA, Ward RD (2013) Cryptic diversity in flathead (Scorpaeniformes: Platycephalidae) across the Indo-West Pacific uncovered by DNA barcoding. Molecular Ecology Resources 13, 32-42.

Chapter 4 Puckridge M, Last PR, Gledhill D, Andreakis N (2013) Revisiting latitudinal diversity gradients in the Indo-West Pacific: Phylogeography of the flathead fishes (Platycephalidae). Journal of Biogeography (submitted).

Supplementary publications Last P, White WT, Puckridge M (2010) Neotrygon ningalooensis n. sp. (Myliobatoidei: Dasyatidae), a new maskray from Australia. Aqua, International Journal of Ichthyology 16, 37-50.

White WT, Last PR, Dharmadi, Faizah R, Chodrijah U, Prisantoso BI, Pogonoski JJ, Puckridge M and Blaber SJM (2013) Market fishes of Indonesia (=Jenis-jenis ikan yang di Indonesia). ACIAR Monograph No. 155. Australian Centre for International Agricultural Research: Canberra.

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STATEMENT OF CO-AUTHORSHIP

The concept development, survey design, implementation, production, analysis and interpretation of data, writing and final editing of the manuscripts were the responsibility of the primary author and PhD candidate, M. Puckridge. Additional input from co-authors was as follows:

For Chapter 2: P.R. Last helped develop ideas, chose the focus group and provided editorial input to refine the manuscript. W.T. White provided the great majority of tissue samples as well as editorial input to refine the manuscript. N. Andreakis helped develop ideas, provided guidance with analyses, their interpretation and manuscript layout as well as editorial revisions.

For Chapter 3: N. Andreakis provided guidance with the analyses and editorial input to the manuscript. S.A. Appleyard offered general laboratory advice as well as editorial input to the manuscript. R.D. Ward helped develop ideas, provided guidance on interpretation of analyses and manuscript layout as well as editorial revisions.

For Chapter 4: P.R. Last helped develop ideas, chose the focus group and provided editorial input to the manuscript. D. Gledhill helped develop ideas and provided editorial input to the manuscript. N. Andreakis provided guidance with analyses and their interpretation and editorial input to the manuscript.

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Melody Puckridge Dr. Patti Virtue Candidate Supervisor Institute for Marine & Antarctic Studies Institute for Marine & Antarctic Studies University of Tasmania University of Tasmania

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ACKNOWLEDGEMENTS

Nikos Andreakis and his fabulous mind coached me through the daunting world of phylogenetics (not to mention the entirety of PhD land itself) – sharing his vast understanding of science through high level, philosophical discussions that have changed the way I look at the world down to the minutiae (and frustration!!!) of software troubleshooting and back again – all delivered with great integrity, humour and patience. Being stationed ‘remotely’ (let’s face it, Townsville is the middle of nowhere) meant helping me often spilled into his evenings and weekends. To that end, I am also very grateful to his beautiful family, Gabriella, Christina and Iannis, for sharing his focus. Peter Last and his exceptional knowledge of life in the oceans is the reason I’ve had the privilege of working with such fascinating fishes. If I have managed to make any inroads into telling their wondrous stories it is because he seeded my thinking with their possibilities. Bob Ward has been a constant guide in my scientific endeavours and if it weren’t for his initial support, I may not have been able to take on this particular endeavour. His pragmatic approach and scientific clarity of thinking are something to aspire to. Sharon Appleyard graciously stepped in to mentor me through the laboratory process and schooled me in how to methodically work through the black magic of genetics for which I am incredibly grateful, as this is by no means an art that comes naturally to me. There were times I wondered if waving my pipette as a wand would’ve been a more useful next step in coaxing dormant samples to show themselves and hand over their transcripts. I am also very grateful to Guy Abel and Peter Grewe who, at the start, took time out to help me find my feet in the lab. Patti Virtue, keeper of the eternally optimistic spirit, has been invaluable. Her presence, guidance, intuition and nous form the ultimate cheering squad and an essential force in the challenging times of a PhD. William White has been a fantastic early mentor. Not only did he coach my interests in the world of fishes, but also introduced me to the wonders of fieldwork in Eastern Indonesia. Although ‘pleasant’ is light years away from describing the rotting markets and cramped, makeshift hotel bathroom ‘laboratories’ we got to process in for long, arduous days upon days, I always loved the adventure and learning of those trips. And armed with a better understanding of fish IDs (thanks to Will), I was equipped to walk into those fish markets and be simultaneously horrified and delighted at the incredible diversity of life (or rather – death) that lay in buckets, baskets, boxes, across tables or strewn on the ground. The conservationist and the collector in me have certainly had their battles on those Indonesian shores. The steadfast fish tax crew at large have been awesome. Specific thanks to Al Graham, John Pogonoski and Dan Gledhill for allowing me to pester at regular intervals for samples, reference materials or even just the odd bit of stationary that looked useful... And of course, thank you to the many people who collected material used in the following thesis.

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Without their independent interest and meticulous efforts to sample and store material, I would’ve had a snowballs chance in hell of targeting work of this scope. Specifically, material has come through the hands of: William White, Peter Last, Daniel Gledhill, Alastair Graham, John Pogonoski, Gordon Yearsley, Gary Fry, Ross Daley, Allan Connell, Jenny Giles, Ian Jacobsen, Ashlee Jones, Peter Kyne, Alec Moore, Gavin Naylor, Simon Pierce, Lucy Scott, Bernard Séret, Mike Sugden, Jeremy Vaudo, Kohji Mabuchi, Ken Graham and David Fairclough. William White, Allan Connell and Gary Fry made effort to collect samples specifically for this project with greatly appreciated enthusiasm. Permission to use other material and sequences originally donated to the Barcode of Life Database was kindly granted by Martin Gomon, Mark McGrouther, Seinen Chow, Junbin Zhang, Allan Connell, Amy Driscoll, Les Knapp, Chris Meyer, Serge Planes, Jeff Williams, Phil Heemstra, Elaine Heemstra and Monica Mwale. And thank you very much to Dirk Steinke for sub-sampling and orchestrating the transfer of all of this material from the Biodiversity Institute of Ontario in Guelph. Neil Aschliman and Gavin Naylor kindly provided advice on RAG1 primers for the stingrays. Shannon Corrigan, John Pogonoski, Peter Smith, Alan Butler and Nic Bax all provided fantastic and insightful feedback that greatly helped to improve various chapters of this thesis. I would not have lasted without the mentoring and encouragement of Alan Williams. His insights, integrity, warmth and enthusiasm have been formative influences over these past years and his friendship brings to mind the words of Charles Darwin that ‘A [person’s] friendships are one of the best measures of [their] worth’. Among the jewels of my friendship treasure trove are also my fellow Block 4 roomies, particularly Jess Ford, whose companionship and student room giggles have been the daily staple. And of course, in there is Lev Bodrossy, my true blue mate, confidant and coordination doppelgänger, whose company is synonymous with almost every event of the last few years. My family has been fabulous. In addition to their eye rolling about my PhD, Dad has taken time to tutor me in the world of Adobe software. If a picture truly does speak a thousand words, my illustrator skills alone will’ve saved me years of writing, given the snails pace it moves at. Of course, no chapter of my life would be complete without Mum’s moral support down the phone to help me find humour in the less humorous. And Matti – who will likely never read this, because he’ll always have ‘better things to do’ but whose big brother support is largely why I’ve learnt to reach for the challenging fruit higher in the tree of life. And last, but in no way least, Toby Jarvis, my partner and buddy, thank you for supporting me - in all ways! - and keeping me company throughout the journey. For this, and for you, I am eternally grateful.

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ABSTRACT

The tropical Indo-West Pacific (IWP) is the most biologically diverse marine region on earth with a number of competing hypotheses proposed to explain the evolutionary events responsible for this area’s biotic complexity. These hypotheses are based on varying interpretations of species distributions radiating from the Indo-Australian Archipelago (IAA) at the centre of the region. The IAA represents a geologically dynamic area formed by colliding tectonics; mechanisms relating to tectonic activity are therefore commonly considered responsible for the biodiversity here. In the present study, a) maskrays of the Neotrygon (Dasyatidae), b) flatheads of the family Platycephalidae (Scorpaeniformes) and c) tuskfishes of the genus Choerodon (Labridae) were studied with respect to their , phylogeny and phylogeography. These groups are characterized by having demersal life histories and a high proportion of narrow-ranging endemics. Furthermore, some of the widely distributed species across the region are suggestive of either recent jump-dispersal events or fragmentation of once pan- tropical populations. The sampling effort in this study focused on exploring diversity among species, and within selected taxa, by obtaining multiple individuals across species’ distribution ranges, where possible. Molecular phylogenies, coupled with molecular clock approximations, were employed to a) assess nominal species validity b) DNA barcode Evolutionarily Significant Units (ESUs) and c) explore the geological and evolutionary processes responsible for observed trends in diversity and species distribution in maskrays, flatheads and tuskfishes. Both mitochondrial and nuclear molecular markers were used in this study. The chosen markers amplified consistently across species, showed high content of phylogenetic signal and were able to resolve closely related species, thus allowing the inference of robust phylogenies and molecular clock dating. Parallel genealogical trajectories have been recovered within rays of the genus Neotrygon and platycephalin flatheads (the most comprehensively sampled subfamily of Platycephalidae). These congruent spatial and temporal patterns suggest that species differentiation predominantly occurred across the tropical IAA throughout the mid to late Miocene. The most widely distributed and derived forms in these groups – Neotyrgon kuhlii and Platycephalus indicus – were found to consist of cryptic species complexes with considerable lineage diversity across their IWP range. Phylogeographic comparisons with other, less well sampled platycephalid species confirm that diversification patterns are consistent across multiple species, suggesting species diversity in shallow water marine fauna may be grossly underestimated. In contrast, tuskfishes of the genus Choerodon showed unclear phylogeographic structuring. However, mixed ancestral and derived lineages, endemic to Australian waters, suggest the Australian region has acted as both a refuge for lineage survival and source of radiation in this group since the mid-Miocene. Patterns are

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consistent with centrifugal speciation, a process that may be common across marine groups here. In conclusion, the tectonic suture zones across the Australian and Eurasian Plates have played an essential role in triggering the area’s mega-diversity and biotic complexity. Tectonic rafting, the establishment of new habitats in the form of shallow seas, tropical coastlines and island arcs, together with glacio-eustatic sea level oscillations, have likely favoured rapid species diversification through vicariance and allopatric speciation. The imprint of these large-scale geological and climatic events has been retained within the evolutionary history of maskrays, flatheads and tuskfishes. These uncovered patterns may represent a common trend for many other marine taxa with similar ranges and geographical distributions.

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ABBREVIATIONS

16S Ribosomal RNA (mitochondrial marker) ANFC Australian National Fish Collection Aus Australia BBM Bayesian Binary MCMC analysis BI Bayesian inference BOLD Barcode of Life Database COI Cytochrome oxidase subunit 1 (mitochondrial marker) DNA Deoxyribonucleic acid EAC East Australian Current ENC1 A single-copy ectodermal-neural cortex I-like protein (nuclear marker) GTR General time reversible model of evolution HKY Hasegawa, Kishino and Yano model of evolution hLRTs Hierarchical likelihood ratio tests HPD High posterior density IAA Indo-Australian archipelago ILD Incongruence length difference test Indo Indonesia IWP Indo-West Pacific K2P Kimura-2 Parameter model of evolution Ma Million years ago MCMC Markov chain Monte Carlo ML Maximum likelihood MP Maximum parsimony MRCA Most recent common ancestor mtDNA Mitochondrial DNA My Million years nDNA Nuclear DNA NGS Next Generation Sequencing NSW New South Wales, Australia PCR Polymerase Chain Reaction Qld Queensland, Australia RAG 1 Recombination-activating gene 1 (nuclear marker) RAG 2 Recombination-activating gene 2 (nuclear marker) TBR Tree bisection reconnection (a branch swapping algorithm) TIMef Transition model of evolution with equal base frequencies Tmo-4c4 Titin-like protein (nuclear marker) TrN Tamura Nei model of evolution TrNef TrN model of evolution with equal base frequencies TVM Transversion model of evolution WA Western Australia, Australia

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TABLE OF CONTENTS

DECLARATION ii STATEMENT OF ETHICAL CONDUCT ii AUTHORITY OF ACCESS ii PUBLICATIONS iii STATEMENT OF CO-AUTHORSHIP iv ACKNOWLEDGEMENTS v ABSTRACT viii ABBREVIATIONS ix TABLE OF CONTENTS x LIST OF FIGURES xiii LIST OF TABLES xvii

CHAPTER 1 1 1.1 INTRODUCTION 2 1.1.1 Description of the study area 2 1.1.2 Current theories 3 1.1.3 Evolutionary patterns & their interpretation 4 1.1.2 Geological history 4 1.1.5 The model systems 8 1.1.5.1 The maskrays 9 1.1.5.2 The flathead fishes 9 1.1.5.3 The tuskfishes 10 1.1.6 Phylogenetics & phylogeography 10 1.1.7 Thesis summary 11

CHAPTER 2 Phylogeography of the Indo-West Pacific maskrays (Dasyatidae, Neotrygon): a complex example of chondrichthyan radiation in the Cenozoic 12 2.1 ABSTRACT 14 2.2 INTRODUCTION 15 2.3 METHODS 17 2.3.1 Specimen collection & geographical locations 17 2.3.2 DNA extraction, PCR amplification & sequencing 17 2.3.3 Sequence alignments, data exploration, model & outgroup selection 18 2.3.4 Phylogenetic analyses, clade delineation & network reconstruction 18 2.3.5 Partition of genetic diversity & barriers to gene-flow 19 2.3.6 Past demographic history 20 2.3.7 Divergence time estimates 20 2.4 RESULTS 22 2.4.1 Phylogenetic reconstructions 22 2.4.2 Species level phylogenies & clade delineations in Neotrygon 24 2.4.3 Network analysis 27 2.4.4 Barriers to gene-flow within N. kuhlii 28 2.4.5 Past population demographic history & divergence time estimates 28

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2.5 DISCUSSION 30 2.5.1 Taxonomic considerations 30 2.5.2 Species diversity in Neotrygon 31 2.5.3 Phylogeography & cryptic speciation in the Pleistocene 32 2.5.4 Population expansion-contraction & secondary contact in Pleistocene 34 2.5.5 Phylogeography in Neotrygon ningalooensis & N. annotata 34 2.5.6 Conclusions 35

CHAPTER 3 Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo-West Pacific uncovered by DNA barcoding 36 3.1 ABSTRACT 38 3.2 INTRODUCTION 39 3.3 METHODS 41 3.4 RESULTS 43 3.4.1. Cymbacephalus staigeri 46 3.4.2. Platycephalus endrachtensis 46 3.4.3. Cymbacephalus nematophthalmus 47 3.4.4. Inegocia japonica 47 3.4.5. Thysanophrys chiltonae 47 3.4.6. Thysanophrys celebica 47 3.4.7. Sunagocia arenicola 47 3.4.8. Platycephalus indicus 48 3.5 DISCUSSION 50

CHAPTER 4 Revisiting latitudinal diversity gradients in the Indo-West Pacific: Phylogeography of the flathead fishes (Platycephalidae) 54 4.1 ABSTRACT 56 4.2 INTRODUCTION 57 4.3 METHODS 59 4.3.1 Sample collection 59 4.3.2 DNA extraction, PCR amplification, sequencing & alignments 59 4.3.3 Outgroup comparisons 60 4.3.4 Data exploration & model selection 60 4.3.5 Phylogenetic analyses & network reconstruction 60 4.3.6 Molecular clock computations 61 4.4 RESULTS 63 4.4.1 Onigociinae 65 4.4.2 Platycephalinae 66 4.4.3 Genealogical network 69 4.4.4 Molecular clock approximations 69 4.5 DISCUSSION 73 4.5.1 Latitudinal diversity gradients in temperate & tropic regions 73 4.5.2 Diversification in the tropics 74 4.5.3 Migration direction since the Eocene 74 4.5.4 Diversification in the mid-Miocene 76 4.5.5 The Platycephalus indicus complex 76 4.5.6 Systematic considerations 77 4.5.7 Conclusions 78

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CHAPTER 5 Centrifugal speciation promotes species diversity in the Indo-West Pacific tuskfishes (Labridae: Choerodon) 80 5.1 ABSTRACT 82 5.2 INTRODUCTION 83 5.3 METHODS 85 5.3.1 Sample collection 85 5.3.2 Sampling coverage 85 5.3.3 Outgroup selection 85 5.3.4 Marker selection 86 5.3.5 DNA extraction, PCR amplification & sequencing 86 5.3.6 Phylogenetic reconstructions 86 5.3.7 Divergence time dating 87 5.3.8 Phylogeographic relationships 87 5.3.9 Ecological partitioning 88 5.4 RESULTS 89 5.4.1 Phylogenetic reconstruction 89 5.4.2 Divergence times & phylogeography 91 5.4.2.1 Clade 1 93 5.4.2.2 Clade 2 93 5.4.2.3 Clade 3 94 5.4.2.4 Clade 4 94 5.4.3 Inferring ancestral biogeography, habitat preference & body size 96 5.5 DISCUSSION 98 5.5.1 Taxonomic & phylogenetic implications 98 5.5.2 Evolutionary origins 98 5.5.3 The geography of speciation 99 5.5.4 Evolutionary drivers 99 5.5.5 Habitat association 100 5.5.6 Conclusions 101

CHAPTER 6 102 6.1 DISCUSSION 103 6.2 CONCLUSIONS 106

REFERENCES 107

APPENDICES APPENDIX 1 Supplementary information for Chapter 2 125 APPENDIX 2 Supplementary information for Chapter 3 135 APPENDIX 3 Supplementary information for Chapter 4 153 APPENDIX 4 Supplementary information for Chapter 5 163 APPENDIX 5 Supplementary publication 1 Neotrygon ningalooensis 167 APPENDIX 6 Supplementary publication 2 Market fishes of Indonesia 181

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