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While there have been many investigatory studies on the systematic and .... Jane Hughes – Part of my interest in this project was that it had a molecular.
A Systematic and Taxonomic Review of Two Australo-Pacific Snake Genera (Elapidae: Oxyuranus And Pseudonaja)

Christopher James Gregory A.A., B.Sc., M.Sc.

Griffith School of Environment Science, Environment, Engineering and Technology Griffith University

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy

April 2010

© Christopher James Gregory

―The classification of snakes is a hard test of intellectual honesty...‖ First written by Underwood (1967), later adapted by Minton, Jr. and Salanitro (1972)

GENERAL ABSTRACT

This study investigated two protocols for use with taxonomic research and provides a significant step toward a comprehensive taxonomic revision of two Australo-Pacific snake genera: taipans (Elapidae: Oxyuranus) and brown snakes (Elapidae: Pseudonaja). These snakes are of interest to many people for reasons such as the proximity of their distribution to major urban centres, the potential lethality associated with their venom, and the morphological variation found in most sub-taxa. Distributed throughout Australia and parts of Indonesia and Papua New Guinea, taipans and brown snakes are noted both for their morphological variability and sub-taxa similarity. These two reasons are partially why, since the first species of brown snake was erected in 1854, 48 different (sub)species of Oxyuranus and Pseudonaja have been described as valid taxa. A third reason may be methodological: the use of inappropriate sampling techniques and laboratory methods. The present study tested and validated a novel systematic sampling methodology for taxonomic and systematic research, investigated the appropriateness for the inclusion of archival DNA in molecular research, and conducted phylogenetic and morphologic analyses of past and present data collected from these taxa.

A review of the taxonomic history of these snakes (presented in Chapter 1) shows that the evolutionary relationships of Oxyuranus and Pseudonaja sub-taxa have long been uncertain. Previous taxonomic and systematic inquiries have been descriptive in nature, inconsistent characters have been selected for analysis, specimen selection has been opportunistic, and molecular studies have been reliant on fresh tissue for genetic analyses. These issues may be, in part, why there is still taxonomic uncertainty regarding the number of valid taxa associated with these snakes. This study attempted to uncover the evolutionary relationships of these snakes using novel and improved techniques. These techniques were first tested for their adequacy, then employed in further experiments designed to resolve Oxyuranus and Pseudonaja taxonomy.

While there have been many investigatory studies on the systematic and nomenclatural taxonomy of Australo-Pacific elapids, most researchers have chosen their samples

General Abstract

v

opportunistically, and few have attempted to do so in a systematic manner. Chapter 2 investigates the efficacy of morphological characters from previous regional and national identification keys to Pseudonaja using a systematic sampling regime. Many identification keys were less useful than previously believed (accuracy rates as low as 62%) when using snakes selected from throughout their distributional range. Therefore, it was assumed that all past researchers employed an opportunistic sampling strategy (based also on personal comments from some of the original authors, as well as the presence of low sample sizes in the literature) which was not able to include the full range of morphological variation associated with these snakes. Several additional sources of morphological variation (some new, some previously described yet subsequently ignored) were tested and several characters which improved identification of Pseudonaja species were found. These characters were used to create a new, temporary identification guide with a 96% success rate. In short, a systematic sampling regime appears to be the most appropriate protocol for taxonomic and phylogenetic research.

With increasing frequency, analyses of molecular data are complementing or replacing traditional morphological analyses within taxonomic research. Karyomorphic analyses in the 1980s helped move Australian elapid systematics into the modern era of evolution research and subsequent phylogenetic analyses of mitochondrial data in the 2000s were largely confirmatory of earlier karyomorphic results. However, previous molecular work may have been hindered not only by the opportunistic selection of snakes (typically collected near large human population centres), but also by reliance on the use of fresh tissue. Fresh tissue from these taxa is not easily available throughout their distributions. Thus, to uncover the full genetic diversity of Oxyuranus and Pseudonaja, DNA extracted from archival, formalin-fixed tissue is required. Chapter 3 details a comparison of 47 methods of archival DNA extraction, along with further investigations on the efficacy of using selected polymerase chain reaction (PCR) additives, several Taq-replacements, different sizes of targeted gene fragments, different sizes of starting tissue, and how the time of storage since extraction affects PCR success. Although the use of archival material is not as fruitful as using fresh tissue, methods employing one of two main extraction strategies (exposing tissue to a high heating step [≥ 90° C] or magnetic attraction of DNA molecules) were shown to maximise PCR success, and reactions often benefited with the use of bovine serum albumin or dithiothreitol.

General Abstract

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The two previously described novel and optimised techniques were used with phylogenetic analyses of all Oxyuranus and Pseudonaja sub-taxa (Chapter 4). A total of 388 full-length DNA sequences (773 base pairs of the ND4 and associated Histidine-, Serine-, and Leucine-tRNA mitochondrial genes) were extracted from fresh and archival tissue or were obtained from an online genetic database. Approximately 40% of all archival tissue attempted for use was successful. Full success was not possible, not only because of the presence of failed PCRs, but also because conservative methods were implemented to guard against the inclusion of any successfully amplified segment which may have contained base pair substitutions (one of several by-products of the fixation process inherent to most archival specimens). Phylogenetic analyses confirmed several previously-recognised relationships, while new relationships were also recovered. In particular, Pseudonaja modesta was most often recovered as the sister group to all other Oxyuranus and Pseudonaja. Final recovered relationships were retested numerous times and consistently reproduced with support values normally above 90%, while tests of several previously published results could not be easily replicated as originally described.

Chapter 5 presents the results of morphological analyses undertaken of over 1,400 specimens selected systematically from throughout their range, with no observer bias toward size, age, or gender. Analyses were conducted based on the molecular results obtained in the previous chapter, as well as based on original morphological descriptions taken from all previously published taxonomic hypotheses. As expected, most hypotheses presented with limited or no data were shown to have no support. Morphological delineations were consistent with genetic delineations, but new morphological variants were also discovered. As with genetic results, morphological results sometimes led to new hypotheses without strong support, which were assumed to be affected by rapid and recent evolutionary divergences. Finally, all type specimens were examined, with most assigned to their assumed appropriate groups. However, several type specimens failed to be ‗accurately‘ identified by discriminate function analyses. ‗Failed‘ type specimens could be consistently categorised by the time spent in storage (very old), their geographic origin (islands), or their authorship (incorrect labels by an author who had not examined any specimens).

General Abstract

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The present study has been able to address previously-developed hypotheses about the evolutionary relationships between these taxa, often with strong support for the findings presented here. Based on the weight of molecular, morphological, and geographic distribution evidence compiled during the present study, the following snakes and relationships1 were recognised as being valid: 1) Pseudonaja modesta is the sister group to all other ingroup taxa examined, should be elevated to its own genus, and contains four species (one of which is new); 2) Oxyuranus is the sister group to the remaining Pseudonaja, contains three species (pending further examination of O. temporalis), and one additional subspecies; 3) Pseudonaja contains eight species and an additional five subspecies (one of which is new). However, not all results obtained were clear, presumably due to rapid and recent evolutionary divergences. It is recommended that a further analysis of additional molecular data (such as that provided by nuclear genes) be undertaken before final relationships can be assumed. These analyses have begun, and additional ecological data (diet, habitat, and parasites) have been collected to help delineate and describe final taxonomic results. The results from this thesis, in addition to the extra data collected, provide the first comprehensive evaluation of the ecology and evolution of these snakes. Not only will this satisfy our curiosity and lead to new ecological investigations, but also will have conservation implications for the future management of taipans and brown snakes.

1

As per the rules of the International Code of Zoological Nomenclature (International

Code of Zoological Nomenclature 1999), any relationships presented within this thesis do not constitute an official taxonomic revision for nomenclatural purposes and are used for illustrative purposes only.

Acknowledgments

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STATEMENT OF ORIGINALITY AND PROOF OF RESEARCH PERMISSION

This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the dissertation contains no material previously published or written by another person except where due reference is made in the thesis itself.

All work was conducted under Griffith University Animal Ethics Committee protocol EAS/04/05/AEC. All material was handled under the following permits: Queensland – Environmental Protection Agency Scientific Purposes Permit WISP03547406 and Northern Territory – Permit to Take Wildlife for Commercial Purposes 24350.

Christopher James Gregory

ACKNOWLEDGMENTS

I fear the mental fatigue associated with the end of my candidature will cause me to overlook some of the people who have—directly or indirectly—helped me with my thesis. Should that be the case, please know that you are certainly present in my heart, even if you have not been listed on these pages (in alphabetical order). I am truly humbled by—and grateful for—your time and help, and I will always be indebted to each of you. Even if I have thanked each of the you in the past, I thank you here again, and I will continue to thank you in the future. ADVISORS – I owe much of what has gone into this study (the good parts, at least) to my advisors at Griffith University (GU): Jean-Marc Hero – I allegedly met Marc and his wife, Narinder, in Sri Lanka in 2001—an event of which I have no recollection. Fast forward ten years, and I know that I will never forget Marc, especially for everything he has done with and for me during my degree. The supervision, support, and patience he has shown has been invaluable, and has positively influenced my approach to science. Jane Hughes – Part of my interest in this project was that it had a molecular component—a topic that held absolutely no appeal for me as an undergraduate student. Jane can explain complicated subjects in a way which makes them sound both fascinating and surprisingly simple. If I had Jane as my genetics and microbiology professor as an undergraduate, I would have actually attended the lectures! Steve Phillips – Through sheer coincidence (serendipity?), I first made contact with Steve soon after he decided to start thinking about Pseudonaja again. It was at his suggestion that I began this project, and only through his guidance and confidence in my abilities that I was able to complete it. His role as a personal mentor is perhaps his greatest contribution to the thesis... COLLEAGUES – The people listed below have all gone out of their way to help me, whether it be talking science, teaching me how to catch elapids or amplify their DNA, or attempting to answer my questions (about anything).

Acknowledgments

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Peter Baverstock, Brian Bush, Helen and John Cann, Tim Caro, Ray Carthy, Hal and Heather Cogger, Naomi Doak, Angus Emmott, Ed Ferrero, Mark Fitzgerald, Mike Gillam, ‗Geeks‘ from the Nathan Campus (Andrew Bentley, Giovannella Carini, Kat Dawkins, Ana Dobson, Rod Eastwood, James Fawcett, Jane Hughes, Tim Page, Dan Schmidt, Jemma Somerville), Harry Hines, Robert Kimsey, Jodie Kuncoro, Greg Mengden, Peter Mirtschin, Leonard Pearlstine, Ben Phillips, Brad Shaffer, Barbara Triggs, Richard Wells EDUCATION – Several organisations provided space to work, partial funding, travel bursaries, access to equipment, and the opportunity to teach. Their staffs were always professional, extremely helpful, accommodating during the tribulations encountered during the course of my thesis, and downright nice people. GU, DNA Sequencing Facility – Fraxa Caraiani, Nicole Hogg GU, Environmental Futures Centre (née Centre for Innovative Conservation Solutions) – Jean-Marc Hero, Darryl Jones, Dianna Woods, Zhihong Xu GU, Gold Coast Association of Postgraduates – Joel McInnes GU, Graduate Research School – Minerva Capati, Loree Joyce, Vanessa Langton, Razia Osman, David Rounsevell, Rita Wockner GU, Library Services – Kathryn Marcantelli, Noelene Mendoza, Shirley Spiller GU, School of Environment – Michael Arthur, Margie Carsburg, Sonya Clegg, Tony Carroll, Rod Connolly, Petney Dickson, Naomi Doak, James Furse, Jean-Marc Hero, Mariola Hoffmann, Jane Hughes, Darryl Jones, Jutta Masterton, Hamish McCallum, John Robertson, Luke Shoo, Meredith Stewart, Peter Teasdale, Jan Warnken, Carmel and Clyde Wild GU, Student Guild – Jessica Brown, Michelle Brown, Joel McInnes GU, Various – Tony Farrell, Graham Fitzpatrick, Debbie Haynes, Michael Holder, Mona Kazoun, Sarah Simpson International Student Volunteers – Narelle Best, Randy Sykes Peter Rankin Trust Fund for Herpetology – Ross Sadlier FAMILY AND FRIENDS – The people listed below have all gone out of their way to help me, whether it be giving me a place to stay, providing a meal, donating money to

Acknowledgments

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the project, assisting with data collection, helping to translate Australian English, making me smile, putting this thesis into its proper perspective, or dropping off (another?!?) snake to measure. Jordyn de Boer, Vincent Byrne, Naomi Doak, Angus Emmott, the Frenzels (Hilde, Katrin, Lothar), Jan Gilroy, the GreenDoggs, Linda Gregory (without whom, literally, this would not have been possible), Marama Hopkins, Apple and David Losada, the Lucky Shots, Glen Mara, Claire Morrison, everyone from the Nepal trip, Felicia Pereoglou, Sue Phillips, everyone in the Postgraduate student offices of G24, Leah Robbie, Clay Simpkins, the old Southport Stadium Thursday through Saturday basketball crews, Kirsty Sullivan (who will be a far better PhD student than I ever was), the Gold Coast Virdees (Diva, Narinder, Priya), the Birmingham Virdees (Balbir, Charan, Harjinder, Rani), and everyone else who should be here but for space, time, and memory constraints MUSEUMS – I have received a tremendous amount of assistance from the staff of a number of museums worldwide. The people listed here provided me space to work, no time limits in which to complete the work, seemed to be genuinely interested when answering my many questions, and, as above, are genuinely good people. I am grateful for the opportunity to examine collections from their respective institutions. AM – Rebecca Johnson, Andrew King, Robert Mason, Ross Sadlier ANWC – Leo Joseph, Rob Palmer, John Wombey BMNH – Colin McCarthy CAS – Ricka Stoelting, Jens Vidnum FMNH – Kathleen M. Kelly, Alan Resetar MNHP – Ivan Ineich NMV – Dianne Brae, Martin Gomon, Jane Melville, Rhyll Plant NTM – Gavin Dally, Paul Horner, Dane Trembath QM – Andrew Amey, Patrick Couper, Jessica Worthington Wilmer SAM – Steve, Donnellan, Mark Hutchinson, Carolyn Kovach, Adam Skinner WAM – Paul Doughty, Brad Maryan, Clare Stevenson ZMB – Mark-Oliver Rödel ZMH – Jakob Hallermann

TABLE OF CONTENTS

I. Title page .........................................................................................................................i II. Copyright ..................................................................................................................... ii III. General Abstract .........................................................................................................iv IV. Ethics and Originality.............................................................................................. viii V. Acknowledgements .....................................................................................................ix VI. Table of Contents ..................................................................................................... xii VII. List of Figures .........................................................................................................xiv VIII. List of Tables ...................................................................................................... xviii IX. Chapter 1: Taxonomic Review of Oxyuranus and Pseudonaja ...................................1 A. Introduction ....................................................................................................2 B. Pseudonaja History ......................................................................................... 8 C. Oxyuranus History ........................................................................................ 18 D. Thesis Aims ..................................................................................................26 X. Chapter 2: A Systematic Method for Systematic Taxonomy .....................................29 A. Introduction ..................................................................................................30 B. Methods ........................................................................................................ 33 C. Results........................................................................................................... 42 D. Discussion.....................................................................................................69 XII. Chapter 3: Optimising DNA Recovery from Chemically-Treated Tissue ............... 81 A. Introduction ..................................................................................................82 B. Methods ........................................................................................................ 86 C. Results........................................................................................................... 94 D. Discussion...................................................................................................109 XIII. Chapter 4: Genetic Analyses ................................................................................ 116 A. Introduction ................................................................................................ 117 B. Methods ...................................................................................................... 122 C. Results......................................................................................................... 136 D. Discussion...................................................................................................160 XIV. Chapter 5: Morphological Analyses and A Review of Taxonomic Hypotheses ..167 A. Introduction ................................................................................................ 168 B. Methods ...................................................................................................... 171

Table of Contents

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C. Results and Taxonomic Summaries............................................................ 175 D. Discussion...................................................................................................234 XV. Chapter 6: General Discussion and Conclusions ................................................... 243 XV. Literature Cited ...................................................................................................... 250 XVI. Appendix 1: Timeline of Pseudonaja taxonomy ................................................. 289 XVII. Appendix 2: Timeline of Oxyuranus taxonomy ................................................. 298 XVIII. Appendix 3: Abbreviations and their definitions ............................................... 301 XIX. Appendix 4: List of external morphological measurements.................................302 XX. Appendix 5: Head scale names, locations, and measurements .............................. 314 XXI. Appendix 6: Summary of DNA extraction protocols ........................................... 321 XXII. Appendix 7: Modeltest NEXUS block................................................................ 349 XXIII. Appendix 8: MrModeltest NEXUS block ......................................................... 355 XXIV. Appendix 9: Sample MrBayes instruction block............................................... 359 XXV. Appendix 10: Full-length haplotypic sequences ................................................. 361 XXVI. Appendix 11: Fragmented haplotypic sequences .............................................. 366 XXVII. Appendix 12: List of external characters examined by previous authors ........ 371 XXVIII. Appendix 13: Final, predictive discriminant function analysis results for type specimens ...................................................................................................................... 375 XXIX. Appendix 14: Summary of continuous measurements for all species-level taxa recognised ...................................................................................................................... 382 XXX. Appendix 15: Summary of non-continuous measurements for all species-level taxa recognised .............................................................................................................. 411

LIST OF FIGURES

1.1:

Distribution map showing the known locations of Pseudonaja specimens held by various museums ............................................................................................ 6

1.2:

A distributional map showing the known locations of Oxyuranus specimens held by various museums .................................................................................... 7

1.3:

Images of holotype material for all described Pseudonaja (sub)species........... 19

1.4:

Images of holotype material for all described Oxyuranus (sub)species ............ 27

2.1:

Image series showing process of museum snake selection ............................... 36

2.2:

Bubble plot combining infralabial counts with counts of mid-body dorsal scale rows for six Pseudonaja species ........................................................................ 44

2.3:

Mirrored jitter plot of ventral scale counts for seven commonly-recognised species of Pseudonaja ....................................................................................... 45

2.4:

Random-scatter jitter plot of ratios of the posterior frontal scale width compared to the width to the side of the head for four species of Pseudonaja .... ...........................................................................................................................46

2.5:

Bubble plot comparing the position of the lowest part of the postocular scales in relation to the position of the lowest eye orbits between P. affinis and P. nuchalis.............................................................................................................. 47

2.6:

Explanation of counts of infralabial scales ........................................................ 49

2.7:

Explanation of counts of ventral scales (as per Dowling 1951 ......................... 49

2.8:

Explanation of counts of dorsal scales at mid-body .......................................... 50

2.9:

Explanation of couplet four: the ratio of the distance between the frontal and rostral scales by the length of the parietal suture............................................... 51

2.10:

Explanation of couplet five: the ratio of the posterior width of the frontal scale by the width (at roughly the same plane) from the frontal scale to the edge of the head .............................................................................................................. 52

2.11:

Explanation of couplet six: the comparison of the lowest edge of the lower postocular scale with the lowest edge of the eye orbit ...................................... 53

2.12:

Comparison of potential effort and actual effort expended by authors of modern-day taxonomic reviews of Pseudonaja ................................................ 73

List of Figures

xv

3.1:

Relative location and directionality of each primer used in this chapter.......... 91

3.2:

Images of ethidium bromide stains of DNA extracts taken immediately after extraction ........................................................................................................ 100

3.3:

Images of ethidium bromide stains of DNA extracts taken one month after extraction ........................................................................................................ 101

3.4:

Images of ethidium bromide stains of DNA extracts taken two months after extraction ........................................................................................................ 102

3.5:

Images of ethidium bromide stains of DNA extracts (from three final and two reference protocols for DNA extraction) taken immediately after extraction 106

3.6:

Sample image of successful PCR products ..................................................... 107

3.7:

Sample comparison of selected PCR additives .............................................. 107

4.1:

Distributions of samples from previous phylogenetic studies ......................... 124

4.2:

Relative location and directionality of each primer used in this chapter......... 129

4.3:

Images of ethidium bromide stains of DNA extracts and PCR products ........ 130

4.4:

Screen capture of aligned sequences ............................................................... 134

4.5:

Comparisons of trees created from analyses of variable-length Pseudonaja modesta sequences ........................................................................................... 135

4.6:

The 50% majority-rule consensus tree of a Maximum Parsimony analysis ...137

4.7:

The best tree found from a Maximum Likelihood analysis............................. 139

4.8:

Plots of generation time vs. log-likelihood values from Bayesian analyses .... 140

4.9:

Plots of posterior probabilities of clades compared between all Bayesian runs .. .........................................................................................................................141

4.10:

Plots of cumulative posterior probabilities of each split vs. generation time and posterior probabilities of clades compared between all runs ........................... 142

4.11:

The 50% majority-rule consensus tree from Bayesian analyses ..................... 145

4.12:

Comparative results of Doughty et al. (2007) and this study .......................... 147

4.13:

Comparative results of Skinner et al. (2005) and this study ........................... 148

4.14:

Recovered fragmented sequence lengths of known ages separated by various factors .............................................................................................................. 156

4.15:

Storage ages of formalin-fixed specimens....................................................... 157

4.16:

Distributions of all informative DNA sequences ............................................ 158

4.17:

Illustration of typical results from phylogenetic analyses of morphological data .........................................................................................................................159

List of Figures

xvi

4.18:

Summary graphs of snake size organised by clade position ........................... 161

5.1:

Examples of removing the effects of size and shape from all snakes ............ 174

5.2:

Summary of historical character and specimen use in systematic reviews of Pseudonaja ...................................................................................................... 176

5.3:

Summary of historical character and specimen use in systematic reviews of Oxyuranus ........................................................................................................ 177

5.4:

Generalised results combining all size classes and genders for commonly recognised Oxyuranus and Pseudonaja species .............................................. 182

5.5:

Multivariate analyses of Pseudonaja affinis (geography) ............................... 185

5.6:

Multivariate analyses of Pseudonaja affinis (morphology) ............................ 186

5.7:

Geographic distribution of Pseudonaja affinis MBDSR .................................188

5.8:

Multivariate analyses of Pseudonaja guttata (geography and morphology) ..190

5.9:

Geographic distribution of Pseudonaja guttata .............................................. 191

5.10:

Multivariate analyses of Pseudonaja inframacula (geography)...................... 194

5.11:

Multivariate analyses of Pseudonaja ingrami (geography) ............................ 196

5.12:

Geographic distribution of Pseudonaja ingrami ............................................. 197

5.13:

Multivariate analyses of Pseudonaja modesta (morphology) ......................... 201

5.14:

Geographic distribution of Pseudonaja modesta wide body bands ................ 202

5.15:

A comparison of multivariate results varying the sample sizes, the characters examined, and the types of analysis undertaken.............................................. 206

5.16:

Multivariate analyses of Pseudonaja nuchalis (sub-clades)............................ 207

5.17:

Multivariate analyses of Pseudonaja nuchalis (geography)............................ 207

5.18:

Geographic distribution of Pseudonaja textilis ............................................... 211

5.19:

Multivariate analyses of Pseudonaja textilis (geography and sub-clades) ...... 212

5.20:

Geographic distribution of Oxyuranus microlepidotus MBDSR .................... 216

5.21:

Multivariate analyses of Oxyuranus microlepidotus (geography and morphology) .................................................................................................... 218

5.22:

Multivariate analyses of Oxyuranus scutellatus (geography and morphology) .........................................................................................................................221

5.23:

Geographic distribution of Oxyuranus scutellatus MBDSR ........................... 223

5.24:

Final distribution map of measured, non-damaged Pseudonaja affinis .......... 226

5.25:

Final distribution map of measured, non-damaged Pseudonaja guttata ......... 227

5.26:

Final distribution map of measured, non-damaged Pseudonaja inframacula. 228

List of Figures

xvii

5.27:

Final distribution map of measured, non-damaged Pseudonaja ingrami ........ 229

5.28:

Final distribution map of measured, non-damaged Pseudonaja modesta ....... 230

5.29:

Final distribution map of measured, non-damaged Pseudonaja ‘nuchalis’ .... 231

5.30:

Final distribution map of measured, non-damaged Pseudonaja textilis .......... 232

5.31:

Final distribution map of measured, non-damaged Oxyuranus microlepidotus and Oxyuranus temporalis ............................................................................... 233

5.32:

Final distribution map of measured, non-damaged Oxyuranus microlepidotus and Oxyuranus temporalis ............................................................................... 234

LIST OF TABLES

2.1:

Sample identification key used to demonstrate accuracy indices ..................... 40

2.2:

Raw data for analysis as presented in Table 2.1 ................................................ 41

2.3:

New identification key to the seven currently recognised species of Pseudonaja ...........................................................................................................................48

2.4:

Identification key to the seven currently recognised species of Pseudonaja, as presented by Cogger (2000). ............................................................................. 55

2.5:

Identification key to the seven currently recognised species of Pseudonaja, as presented by Kinghorn (1964) ........................................................................... 57

2.6:

Identification key to the five currently recognised species of Pseudonaja found in the Northern Territory, as presented by Gillam (1979) .................................58

2.7:

Identification key to the three currently recognised species of Pseudonaja found in Western Australia, as presented by Storr et al. (1986) ....................... 59

2.8:

Identification key to the two currently recognised species of Pseudonaja found in Victoria, as presented by Coventry and Robertson (1991) ........................... 60

2.9:

Identification key to the five currently recognised species of Pseudonaja in Queensland, as presented by Wilson (2005) ..................................................... 61

2.10:

Comparison of accuracies for four regional keys with accuracy of the new key (using regional data) .......................................................................................... 62

2.11:

Comparison of results from three distribution-wide keys using data from all available type specimens ................................................................................... 63

2.12:

List of type specimen designations after being identified using my new key ...64

3.1:

A list of mitochondrial primer pairs used in this chapter ..................................90

3.2:

Comparisons of relative efficacy of DNA extraction protocols at the time of extraction, one month after extraction, and two months after extraction .......... 95

3.3:

Summary of protocols showing visible DNA under UV fluorescence ............. 99

3.4:

Summary of protocols showing visible DNA (extracted from 3 mm3 tissue) under UV fluorescence .................................................................................... 102

3.5:

Comparison of DNA extraction protocols as determined by UV spectrometry (DNA yield and purity one year after extraction) and UV fluorescence (PCR outcomes immediately after extraction and two months after extraction) ...... 103

List of Tables 3.6:

xix

Comparison of three final and two reference DNA extraction protocols as determined by UV spectrometry (DNA yield and purity immediately after extraction) ........................................................................................................ 106

4.1:

A summary of phylogenetic studies (with sample sizes) involving Oxyuranus and Pseudonaja ............................................................................................... 123

4.2:

A list of mitochondrial primers used in this chapter ....................................... 128

4.3:

Estimated marginal likelihoods for five Markov Chain Monte Carlo Bayesian analyses ............................................................................................................ 143

4.4:

Model parameter summaries over all five Markov Chain Monte Carlo Bayesian analyses ............................................................................................................ 143

4.5:

Uncorrected (full-length) sequence divergences within and between genera .151

4.6:

Uncorrected (full-length) sequence divergences between commonly-defined species of Oxyuranus and Pseudonaja ............................................................ 152

4.7:

Uncorrected (full-length) sequence divergences within and between genera (modified to reflect phylogram results) ........................................................... 153

4.8:

Uncorrected (full-length) sequence divergences between operational taxonomic units of Oxyuranus and Pseudonaja ................................................................ 154

4.9:

Comparison of published bootstrap support for the relationships between Oxyuranus and Pseudonaja ............................................................................. 164

5.1:

Summary of historical character and specimen use in systematic reviews of Pseudonaja ...................................................................................................... 178

5.2:

Summary of historical character and specimen use in systematic reviews of Oxyuranus ........................................................................................................ 180

5.3:

Support for predicted species and clade classification based on initial discriminant function analyses ........................................................................ 181

5.4:

Summary results of diagnostic characters of insular Pseudonaja affinis ........ 184

5.5:

Characters which separate eastern and western populations of Pseudonaja guttata by more than 50% of total variation .................................................... 191

5.6:

Characters which separate geographic populations of Pseudonaja ingrami by more than 50% of total variation ..................................................................... 196

5.7:

Characters which separate geographic populations of Pseudonaja textilis by more than 50% of total variation ..................................................................... 212

List of Tables

xx

5.8:

Nomenclatural priorities for geographic populations of Pseudonaja textilis ..214

5.9:

Morphological characters which separate mitochondrial clades of Oxyuranus microlepidotus by more than 50% of total variation ....................................... 217

5.10:

Summary results of three diagnostic characters put forth to describe northwestern Oxyuranus scutellatus by Hoser (2009) ............................................. 220

5.11:

Summary of final discriminant function analysis predictions for all examined material ............................................................................................................ 225

5.12:

Final identification key for Oxyuranus and Pseudonaja species recognised ..236

5.13:

Final identification key Pseudonaja affinis subspecies ...................................238

5.14:

Final identification key Pseudonaja guttata subspecies..................................238

5.15:

Final identification key Pseudonaja inframacula subspecies ......................... 239

5.16:

Final identification key Pseudonaja modesta subspecies................................ 240

5.17:

Final identification key Oxyuranus scutellatus subspecies ............................. 240

6.1:

Summary of final taxa recognised as valid ...................................................... 247

6.2:

Summary of previous evolutionary studies of Australo-Pacific Elapidae ....... 248