Nest Environment and Hatchling Fitness Correlates in the Sea Turtles Caretta Caretta and Natator Depressus Elizabeth Louise Sim Bsc (Hons)

Home , Chelydridae, Leatherback sea turtle

Nest environment and hatchling fitness correlates in the sea turtles Caretta caretta and Natator depressus Elizabeth Louise Sim BSc (Hons)

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 School of Biological Sciences

ABSTRACT

Marine turtles have a complex lifecycle and face threats in both marine and terrestrial environments. Nesting females lay a large number of eggs, very few of which produce hatchlings that survive to reach breeding age. As hatchlings cross the beach, they are exposed to predation, disorientation, dehydration and debris on the beach. When hatchlings enter the water they are exposed to aquatic predators. Hatchlings do not actively avoid predators, or defend themselves, so simply being able to move quickly through this environment would increase their chance of survival. A number of variables can affect hatchling locomotor performance, and this thesis examines three of these: incubation temperature, scute pattern and rookery location in two species of sea turtle, loggerhead (Caretta caretta) and flatback turtles (Natator depressus).

The first part of this thesis focuses on scute pattern. Scutes cover the carapace of turtles and tortoises, and each species has a modal pattern. Deviations from this modal pattern are more common in hatchlings than in adult turtles, suggesting that hatchlings with non-modal scute patterns have higher mortality rates, but this hypothesis has not been tested previously. Hatchlings with modal scute patterns were larger and heavier than hatchlings with non-modal scute patterns in both species examined, however this size difference did not translate into a difference in terrestrial locomotor performance. However, N. depressus hatchlings with the modal scute pattern produced more thrust than hatchlings with non-modal scute patterns in the first 40 minutes of swimming, which may give them an advantage over hatchlings with non-modal scute patterns.

The second part of this thesis focuses on incubation temperature. Marine turtle eggs successfully incubate within a narrow range of temperatures. Even within the viable developmental temperature range, it has been proposed that hatchlings from eggs incubated close to the thermal tolerance limits may have reduced fitness compared to hatchlings from eggs incubated at intermediate temperatures. In this study, C. caretta hatchlings from hot nests were less likely to emerge from the nest, were smaller, and also performed poorly during crawling and swimming trials compared with hatchlings incubated in moderate temperature nests.

The third part of this thesis focuses on differences in hatchling morphology and locomotor performance between two N. depressus rookeries representing two separate ‘evolutionarily significant units’ (ESUs). Hatchlings at the two sites differed in size, but this size difference did not translate into a difference in either crawling speed or self-righting ability.

This thesis contributes to our understanding of factors that influence marine turtle hatchling fitness, both environmental and morphological. This information can be used in the monitoring of endangered marine turtle populations.

DECLARATION BY AUTHOR

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

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Publications during candidature

Sim, E. L., Booth, D. T and Limpus, C. J. (2014). The effect of incubation temperature on flatback (Natator depressus) turtle hatchlings from Mon Repos, Queensland, Australia. In submission.

Publications included in this thesis

Sim, E. L., Booth, D. T and Limpus, C. J. (2014). Non-modal scute patterns, morphology, and locomotor performance of loggerhead (Caretta caretta) and flatback (Natator depressus) turtle hatchlings. Copeia. 2014(1): 63-69. Incorporated as Chapter 2. Contributor Statement of Contribution Author Elizabeth Sim (Candidate) Designed experiments (70%) Collected data (100%) Wrote the paper (70%) Author David Booth Designed experiments (30%) Wrote and edited the paper (30%) Author Colin Limpus Fieldwork supervision and assistance

Sim, E. L., Booth, D. T, Limpus, C. J. and Guinea, M. L. (2014). A comparison of hatchling locomotor performance and scute pattern variation between two rookeries of the flatback turtle (Natator depressus). Copeia 2014(2): 339-344. Incorporated as Chapter 5. Contributor Statement of Contribution Author Elizabeth Sim (Candidate) Designed experiments (90%) Collected data (100%)

Wrote the paper (70%) Author David Booth Designed experiments (10%) Wrote and edited the paper (30%) Author Colin Limpus Fieldwork supervision and assistance Author Michael Guinea Fieldwork supervision and assistance

Sim, E. L., Booth, D. T and Limpus, C. J. (2014). Incubation temperature, morphology and performance in loggerhead (Caretta caretta) turtle hatchlings from Mon Repos, Queensland, Australia. Biology Open in press. Incorporated as Chapter 3. Contributor Statement of Contribution Author Elizabeth Sim (Candidate) Designed experiments (70%) Collected data (100%) Wrote the paper (70%) Author David Booth Designed experiments (30%) Wrote and edited the paper (30%) Author Colin Limpus Fieldwork supervision and assistance

Sim, E. L., Booth, D. T and Limpus, C. J. (2014). The effect of incubation temperature on flatback (Natator depressus) turtle hatchlings from Mon Repos, Queensland, Australia. In submission. Incorporated as Chapter 4. Contributor Statement of Contribution Author Elizabeth Sim (Candidate) Designed experiments (70%) Collected data (100%) Wrote the paper (70%) Author David Booth Designed experiments (30%) Wrote and edited the paper (30%) Author Colin Limpus Fieldwork supervision and assistance

Contributions by others to the thesis

Advice for experimental design and field set-up was provided by Dr David Booth and Dr Colin Limpus (Chapters 2-5) and Dr Michael Guinea (Chapter 5). Advice on data interpretation and Editorial assistance was provided by Dr David Booth (Chapters 1-6). Fieldwork assistance was provided by Melissa Kelly (Chapters 2-5), volunteers and staff at Mon Repos Conservation Park (Chapters 2-5) and volunteers and staff on Bare Sand Island (Chapter 5).

Statement of parts of the thesis submitted to qualify for the award of another degree

None.

ACKNOWLEDGEMENTS

This thesis would not have been possible without the support of a number of people. Firstly, my academic supervisors David Booth, Col Limpus and Craig Franklin. David, thank you for all of your support, your extremely fast corrections and for putting the shed together every year! Col, thank you for all of the field guidance and for sharing your wealth of turtle knowledge. Craig, thank you for your feedback and help with manuscripts.

The fieldwork component of this project would not have been possible without the help of many people. A big thank you to Melissa Kelly, who helped me out during the busiest season, with many late nights and early mornings. Thank you to Helen and Mike for keeping me sane in the field, and to all other volunteers at Mon Repos between 2010 and 2012, and Bare Sand Island in 2011.

Thanks also to my friends for all of your help, support and welcome distractions. In particular Taryn, Luis and Renee. I couldn’t have done it without you guys. Thanks to all my turtle friends, near and far. It’s great to be a part of such a big community that shares my love of turtles. Thanks to my family for supporting me from afar and for the visits (both here and in Melbourne).

And finally, thank you to the turtles for being so awesome.

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KEYWORDS

Sea turtle, hatchling, incubation temperature, scute patterns, locomotor performance, swimming frenzy

AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS

(ANZSRC)

ANZSRC code: 060604, Comparative Physiology, 50% ANZSRC code: 060809, Vertebrate Biology, 50%

FIELDS OF RESEARCH (FOR) CLASSIFICATION

FoR code: 0606, Physiology, 50% FoR code: 0608, Zoology, 50%

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TABLE OF CONTENTS

Abstract ...... i Acknowledgements ...... vii Keywords ...... viii Australian and New Zealand Standard Research Classifications (ANZSRC) ...... viii

Fields of Research (FOR) Classification ...... viii List of figures ...... xii List of tables ...... xiv List of abbreviations ...... xvi

Chapter 1 ...... 1

General introduction ...... 1 Study species ...... Error! Bookmark not defined. Structure and aims of thesis ...... 12

Chapter 2 ...... 14

The effect of non-modal scute patterns on phenotype and locomotor performance of loggerhead (Caretta caretta) and flatback (Natator depressus) turtle hatchlings ...... 14 Abstract ...... 14 Introduction ...... 15 Methods ...... 16 Study site ...... 17 Egg collection ...... 17 Hatchling collection ...... 17 Hatchling measurements ...... 17 Scute pattern classifications ...... Error! Bookmark not defined. Correlates of fitness ...... 18 Self-righting experiments ...... 18 Crawling ability ...... 19 Swimming ability ...... 19 Nest excavations ...... 19 Statistics ...... 20 Results ...... 20 Hatchling type and scute pattern ...... 20 Scute pattern, size and locomotor performance ...... 21 Discussion ...... 24

Hatchling type and scute pattern ...... 24 Hatchling size ...... 25 Hatchling locomotor performance ...... 26

Chapter 3 ...... 29

Incubation temperature, morphology and performance in loggerhead (Caretta caretta) turtle hatchlings from Mon Repos, Queensland, Australia ...... 29 Abstract ...... 29 Introduction ...... 30 Methods ...... 31 Study site ...... 31 Collection of eggs ...... 31 Hatchling collection ...... 32 Hatchling measurements ...... 32 Self-righting experiments ...... 32 Crawling ability ...... 32 Swimming ability ...... 33 Nest excavations ...... 33 Statistics ...... 33 Results ...... 34 Discussion ...... 39 Nest temperature ...... 39 Hatching and emergence success ...... 39 Hatchling size and mass ...... 40 Terrestrial locomotor performance ...... 41 Swimming performance ...... 42 Conclusions ...... 42

Chapter 4 ...... 44

The effect of incubation temperature on flatback turtle (Natator depressus) hatchlings...... 44 Abstract ...... 44 Introduction ...... 45 Methods ...... 46 Study site ...... 46 Data collection ...... 47 Measurements of hatchling quality ...... 47 Locomotor experiments ...... 47 Nest excavations ...... 48

Data analysis ...... 48 Results ...... 49 Discussion ...... 53 Hatching and emergence success ...... 53 Scute variation ...... 53 Hatchling size and mass ...... 54 Crawling and self-righting performance ...... 55 Swimming performance ...... 55 Conclusions ...... 56

Chapter 5 ...... 57

A comparison of hatchling locomotor performance and scute pattern variation between two rookeries of the flatback turtle (Natator depressus) ...... 57 Abstract ...... 57 Introduction ...... 58 Methods ...... 59 Study sites ...... 59 Hatchling collection ...... 60 Hatchling measurements ...... 60 Locomotor performance tests ...... 60 Statistics ...... 61 Results ...... 61 Discussion ...... 63 Hatchling size ...... 63 Scute pattern variation ...... 63 Locomotor performance ...... 64 Conclusion ...... 64

Chapter 6 ...... 66

General Discussion ...... 66 Non-modal scute patterns ...... 66 Incubation temperature ...... 67 Rookeries ...... 68 Further research ...... 68

Literature Cited ...... 71

LIST OF FIGURES

Figure 1.1 General shape of a thermal performance curve for ectotherms, showing the critical thermal minimum (CTmin), critical thermal maximum (CTmax) and the optimum temperature (Topt). Adapted from Krenek et al. (2012)...... 4

Figure 1.2 The expected relationship between fluctuating asymmetry and quality of an organism (adapted from Leung and Forbes (1997))...... 7

Figure 1.3 Diagram of the carapace of a loggerhead turtle (Caretta caretta) showing the position and arrangement of scutes. N = nuchal scute, V = vertebral scutes, C = costal scutes, M = marginal scutes and PV = post-vertebral scutes. Modified from Coker (1910) ...... 10

Figure 2.1 Examples of C. caretta scute patterns: (A) modal scute pattern, (B) minor non-modal scute pattern (additional nuchal scute) and (C) major non-modal scute pattern (additional vertebral and costal scutes). Modified from Coker (1910) ...... 18

Figure 2.2 Mean thrust produced by (A) C. caretta swimming hatchlings with the modal scute pattern (n = 100) and major non-modal scute patterns (n = 62) and (B) N. depressus hatchlings with the modal scute pattern (n = 28) and major non-modal scute patterns (n = 16)...... 24

Figure 3.1 Daily rainfall (bars) and temperature profiles (lines) experienced by C. caretta nests in (A) 2010-2011 (n = 25), (B) 2011-2012 (n = 12) and (C) 2012-2013 (n = 7). Note that nests in the 2010-2011 and 2011-2012 seasons were obtained over several weeks, whereas nests in the 2012-2013 season were all obtained in the same week...... 35

Figure 3.2 Emergence success (A), Mean carapace width (B), Mean carapace size index (C) and Mean crawling speed (D) of C. caretta hatchlings plotted against the T3dm (mean maximum 3 day temperature)...... 37

Figure 3.3 Mean thrust produced (A), mean stroke rate (B), proportion of time spent power stroking (C) and mean maximum thrust (D) of C. caretta hatchlings from nests with a T3dm (mean maximum 3 day temperature) below 34°C and above 34°C. Data were measured over a four hour period. Error bars indicate standard error...... 38

Figure 4.1 Daily rainfall (bars) and temperature profiles (lines) of N. depressus nests in (A) 2010- 2011 (8 nests) and (B) 2011-2012 (2 Nests). Rainfall data obtained from Bundaberg Aero station, located 20 kilometres from rookery...... 50

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Figure 4.2 Linear regressions showing the effect of mean nest temperature on hatching success, emergence success, mass, carapace length, carapace width, carapace size index, proportion of hatchlings with the modal scute pattern, self-righting propensity, time taken to self-right and crawling speed of 194 N. depressus hatchlings from 10 clutches. The relationship was significant for length (p = 0.02, R2 = 0.58), size index (p = 0.047, R2 = 0.52) and self-righting propensity (p = 0.01, R2 = 0.52)...... 52

Figure 4.3 Mean thrust produced (A), proportion of time spent power stroking (B), mean stroke rate (C), and mean maximum thrust (D) of N. depressus hatchlings from a nest with a mean incubation temperature of 29.6°C (the maximum recorded) and 27.6°C (the minimum recorded) over a forty minute period. Error bars show standard error...... 52

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LIST OF TABLES

Table 1.1 List of studies that have experimentally tested the effect of incubation temperature on size of hatchling reptiles...... 2

Table 1.2 List of studies that have experimentally tested the effect of incubation temperature on locomotor performance of reptile hatchlings...... 5

Table 1.3 List of studies that have experimentally tested the effect of incubation temperature on abnormalities in reptile hatchlings...... 8

Table 1.4 List of studies that have examined the proportion of hatchling and adult turtles with non- modal scute patterns at the same rookery...... 11

Table 2.1. List of studies that have examined the proportion of hatchling and adult turtles with modal scute patterns...... 15

Table 2.2 Mean proportion of C. caretta unhatched embryos, hatchlings in nest and emerged hatchlings with modal, minor non-modal and major non-modal scute patterns (N = 24 clutches). .. 21

Table 2.3 Mean proportion of N. depressus unhatched embryos, hatchlings in nest and emerged hatchlings with modal, minor non-modal and major non-modal scute patterns (N = 9 clutches). .... 21

Table 2.4 Hatchling morphological and locomotor parameters (± SE) for 1407 C. caretta hatchlings with the modal (A), minor (B) and major (C) non-modal scute patterns (N = 34 clutches)...... 22

Table 2.5 Hatchling morphological and locomotory parameters (± SD) for 254 N. depressus hatchlings with the modal scute pattern (A), minor non-modal (B) and major non-modal (C) scute patterns (N = 11 clutches) ...... 23

Table 3.1 Number of C. caretta nests with a mean three day maximum temperature below 34°C and above 34°C in the 2010-2011, 2011-2012 and 2012-2013 nesting seasons...... 36

Table 3.2 Mean ± standard error and range for incubation temperature, emergence and hatching success, hatchling morphological parameters, and hatchling locomotor performance of C. caretta nests with a T3dm (mean 3 day maximum temperature) below 34°C (N = 37) and above 34°C (N = 7)...... 36

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Table 5.1 Mean (±SE) mass, carapace length, width and size index and body temperature of Natator depressus hatchlings from Mon Repos (n = 184 from nine clutches) and Bare Sand Island (n = 129 from seven clutches) rookeries. Significant differences between ESUs are indicated in bold font. . 62

Table 5.2 Mean proportion (±SE) of Natator depressus hatchlings with the modal, minor non-modal and major non-modal scute pattern variation from Mon Repos (n = 9 clutches) and Bare Sand Island (n = 7 clutches) rookeries. Significant differences between ESUs are indicated in bold font...... 62

LIST OF ABBREVIATIONS

ESU Evolutionarily significant unit T3dm Mean three day maximum temperature

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CHAPTER 1

GENERAL INTRODUCTION

The developmental environment can have far-reaching effects on an ectotherm’s survival and fitness. Whereas viviparous organisms can develop in an environment regulated to some degree by their mother’s physiology and behaviour, oviparous organisms are at the mercy of the physical nest environment during embryonic development. Thus, the environment in which these eggs develop can vary significantly in terms of temperature, humidity and other environmental variables. In addition, all of the nutrients required by the developing embryo must be contained within the egg.

Temperature is a major factor in the development of ectothermic embryos. Incubation temperatures that are too low or too high are lethal to the embryo (Hubert, 1985; Miller, 1985), however non-lethal incubation temperatures can also affect a range of hatchling attributes including sex (Valenzuela, 2004; Valenzuela & Lance, 2004), size (Ji et al., 2002; Booth et al., 2004), body shape (Mickelson & Downie, 2010), colouring (Deeming, 2004), metabolic rate (Steyermark & Spotila, 2000), growth rate (Brooks et al., 1991; O'Steen, 1998; Rhen & Lang, 1999; Du & Ji, 2003; Booth et al., 2004), locomotor performance (Janzen, 1993b; Doody, 1999; Du & Ji, 2003; Booth et al., 2004), thermoregulatory behaviour (Blumberg et al., 2002; Goodman & Walguarnery, 2007), incidence of deformities and fluctuating asymmetry (Elphick & Shine, 1998; Brana & Ji, 2000; Ji et al., 2002), survival (Billett et al., 1992) and behaviour (Burger, 1998). For these reasons nest temperature and possible changes to nest temperatures are predicted to have a major influence on life history characteristics and viability of oviparous reptile populations. Global temperatures increased by 0.72 °C between 1951-2012 (Hartmann et al., 2013), and are predicted to rise by 1.5-2.0 °C by 2100 (Collins et al., 2013). Higher surface temperatures will almost certainly result in higher nest temperatures for oviparous ectotherms, causing possible detrimental effects on hatchling phenotypes.

In reptiles, eggs incubated at higher temperatures generally produce smaller hatchlings (Table 1.1). These hatchlings may be disadvantaged in terms of crawling or swimming because of their smaller size (Bustard, 1972; Garland, 1985; Arnold & Bennett, 1988; Losos, 1990; Janzen, 1993a; Elsworth et al., 2003). However most reptile embryos complete development without using the entire yolk reserve (Noble, 1991). This residual yolk is internalised and is used as an energy resource for the first days of the hatchling’s life, where it fuels growth (Troyer, 1987; Fischer et al., 1991; Ji et al., 1999; Ji & Sun, 2000), digging out of the nest (Troyer, 1983), over-wintering (Congdon et al., 1983; Nagle et al., 1998; Lance & Morafka, 2001) or dispersal (Kraemer & Bennett, 1981; Werner, 1988). This residual yolk tends to be larger in hatchlings from eggs incubated at warmer temperatures (Allsteadt 1

& Lang, 1995; Booth & Astill, 2001a; Hewavisenthi & Parmenter, 2001; Ji & Du, 2001a, 2001b; Ji et al., 2002; Booth et al., 2004). The mechanism behind this is likely due to longer incubation periods at cooler temperatures, which allow the embryo more time to convert yolk into hatchling tissue (Booth, 2006). Therefore larger hatchlings are able to crawl and/or swim faster, and can potentially escape gape-limited predators, but would have a smaller reserve energy source in their residual yolk, whereas smaller hatchlings with a larger energy reserve in their residual yolk would be at an advantage if they have a long period before they begin to feed.

A few studies reported no effect of temperature on hatchling size in reptiles (Table 1.1). In some cases, a narrow range of temperatures was tested, meaning there could be an effect with a wider range of temperatures. In other cases, other variables such as growth rate were affected, resulting in size differences in juveniles (Nelson et al., 2004). Several studies, particular for snakes, found the largest hatchlings were produced at intermediate temperatures (Table 1.1). A possible explanation could be due to a higher cost of producing hatchlings at extreme high temperatures (Ji & Du, 2001b; Du & Ji, 2003) . Finally, several studies reported the opposite effect, with the largest hatchlings being produced at hotter incubation temperatures (Table 1.1). It is possible in these cases hydric conditions of incubation also differed, because hydric conditions can affect water uptake, and therefore hatchling size in reptiles (Gutzke & Packard, 1987; Packard & Packard, 1988).

Table 1.1 List of studies that have experimentally tested the effect of incubation temperature on size of hatchling reptiles.

Incubation Effect of increased Source temperature incubation temperature range CROCODILIA Aligatoridae Alligator mississippiensis 29-33°C Larger hatchlings Allsteadt and Lang (1995) Caiman latirostris 29-33°C Largest hatchlings at Pina et al. (2007) intermediate temperatures Crocodylidae Crocodylus Oiloticus 28-34°C Smaller hatchlings Hutton (1987) RHYNCHOCEPHALIA Sphenodontidae Sphenodon punctatus 18-22°C No effect Nelson et al. (2004) SQUAMATA Agamidae Calotes versicolor 24-33°C Smaller hatchlings Ji et al. (2002)

Colubridae Coluber constrictor 22-28°C Smaller hatchlings Burger (1990) Elaphe carinata 24-32°C Smaller hatchlings Ji and Du (2001a) Lampropeltis getulus 28-32°C Larger hatchlings Burger (1990) Pituophis melanoleucus 22-32°C Gutzke and Packard (1987) Elapidae 2

Naja naja atra 24-32°C Largest hatchlings at Ji and Du (2001b) intermediate temperatures Iguanidae Iguana iguana 29-31°C Smaller hatchlings Phillips et al. (1990) Lacertidae Podarcis muralis 26-32°C Smaller hatchlings Brana and Ji (2000) 24-35°C Smaller hatchlings Vandamme et al. (1992) Phrynosomatidae Sceloporus undulatus 22-27°C No effect Parker and Andrews (2007) Sceloporus virgatus 15-30°C Larger hatchlings Qualls and Andrews (1999) Scincidae Bassiana duperreyi 25-30°C Smaller hatchlings Booth et al. (2000) 20-27°C Smaller hatchlings Elphick and Shine (1998) 16.8-20.1°C Smaller hatchlings Shine et al. (1997) Lampropholis delicata 20-27°C Smaller hatchlings Downes and Shine (1999) Lampropholis guichenoti 25-30°C No effect Booth et al. (2000) Nannoscincus maccoyi 20-27°C Smaller hatchlings Downes and Shine (1999) Oligosoma suteri 18-26°C Larger hatchlings Hare et al. (2004) Saproscincus mustelina 20-27°C Smaller hatchlings Downes and Shine (1999) TESTUDINES Chelidae Elusor macrurus 26-32°C Smaller hatchlings Micheli-Campbell et al. (2011) Emydura signata 26-31°C Smaller hatchlings Booth (1998) Chelydridae Chelydra serpentina 22.6-25.8°C Smaller hatchlings Packard et al. (1999) 26-31°C Smaller hatchlings Packard et al. (1987), Packard et al. (1988) 24-30°C Smaller hatchlings Booth et al. (2004) Cheloniidae Caretta caretta 29.8-31.7°C Smaller hatchlings Wood et al. (2014) 28.5-32.5°C Smaller hatchlings Chu (2008) 30-32°C No effect Reece et al. (2002) Chelonia mydas 26-30°C Smaller hatchlings Booth and Astill (2001a), Booth et al. (2004), Burgess et al. (2006) 28.5-32.4°C Smaller hatchlings Ischer et al. (2009) Not reported Smaller hatchlings (Glen et al., 2003) 29-32.5°C Smaller hatchlings Weber et al. (2012) Natator depressus 26-32°C Smaller hatchlings Hewavisenthi and Parmenter (2001) Emydidae Chrysemys picta 22-32°C Smaller hatchlings Gutzke et al. (1987) 26-30°C Smaller hatchlings Janzen and Morian (2002) Geoemydidae Chinemys reevesii 24-33°C Smaller hatchlings Du et al. (2006) Testudinidae Gopherus polyphemus 26-32°C Smaller hatchlings Demuth (2001) Trionychidae Apalone mutica 26-30°C Larger hatchlings Janzen (1993b) Apalone spinifera 25-34°C Smaller hatchlings Doody (1999) Pelodiscus sinensis 23-34°C Largest hatchlings at Du and Ji (2003) intermediate temperatures 24-34°C Largest hatchlings at Ji et al. (2003) intermediate temperatures

The larger size of hatchlings produced at low temperatures may be advantageous in terms of moving more quickly (Brana & Ji, 2000), however many studies did not find this effect (Table 1.2). There was an even split between an increase in locomotor performance with higher temperatures, and many studies indicating peak locomotor performance at intermediate temperatures, with no clear pattern for any of the taxonomic groups (Table 1.2). A possible reason for this diversity of findings may be the range of temperatures used in different studies. For example, different studies of green turtles (Chelonia mydas) have reported both an increase and decrease in locomotor performance with increasing incubation temperature (Table 1.2). However, the study that reported a decrease in performance with increased incubation temperature used a thermal range of 28.5-32.4°C, in which the highest temperature used approached the uppermost temperature of viable incubation. Whereas the studies that reported an increase in performance with increased incubation temperature used a thermal range of 26-30°C, in which the lowest temperatures approached the lower temperature limit for viable incubation (Table 1.2).

It is likely that the highest quality hatchlings are produced at intermediate incubation temperatures (Deeming & Ferguson, 1991). Although temperatures at the edges of the viable thermal range can produce live hatchlings, these hatchlings may be sub-optimal due to the costs of producing hatchlings under stressful conditions (Deeming & Ferguson, 1991). The performance of ectotherms is tied to temperature through a parabolic curve (Figure 1.1), and it seems reasonable to assume that incubation temperatures are tied to the quality of the developing embryos in a similar manner. There are certain temperatures above and below which the embryos fail to develop, and there is an intermediate optimal temperature at which the highest quality hatchlings are produced. The mechanisms underlying this decrease in locomotor performance are unknown, but it could be caused by differences in the cardiovascular system and thus the ability to deliver oxygen to muscles (Micheli-Campbell et al., 2011), maturity of neuromuscular signalling pathways (Ischer et al., 2009), thermal stress causing abnormal development (Brana & Ji, 2000), or differences in the physiological properties of muscle fibres (Elphick & Shine, 1998; Freedberg et al., 2004; Chu, 2008).

Table 1.2 List of studies that have experimentally tested the effect of incubation temperature on locomotor performance of reptile hatchlings.

Incubation Measure of Effect of increased Reference temperature locomotor incubation range performance used temperature on locomotion RHYNCHOCEPHALIA Sphenodontidae Sphenodon punctatus 18-22°C Crawling performance No effect Nelson et al. (2006)

SQUAMATA Chamaeleonidae Chamaeleo calyptratus 25-30°C Crawling performance No effect Andrews (2008)

Colubridae Coluber constrictor 22-28°C Crawling speed No effect Burger (1990) Lampropeltis getulus 28-32°C Crawling performance Decrease Burger (1990) Pituophis melanoleucus 21-33°C Crawling performance Decrease Burger (1991)

Lacertidae Podarcis muralis 26-32°C Crawling performance Decrease Brana and Ji (2000) 24-35°C Crawling performance Decrease Vandamme et al. (1992)

Phrynosomatidae Sceloporus undulatus 22-27°C Crawling performance No effect Parker and Andrews (2007) Sceloporus virgatus 15-30°C Crawling performance Decrease Qualls and Andrews (1999)

Scincidae Bassiana duperreyi 20-27°C Crawling performance Increase Elphick and Shine (1998) Lampropholis delicata 20-27°C Crawling performance Increase Downes and Shine (1999) Nannoscincus maccoyi 20-27°C Crawling performance Decrease Downes and Shine (1999) Oligosoma suteri 18-26°C Crawling performance Increase Hare et al. (2008)

Saproscincus mustelina 20-27°C Crawling performance Increase Downes and Shine (1999)

TESTUDINES Chelidae Elusor macrurus 26-32°C Self-righting Decrease Micheli-Campbell et al. performance (2011) 26-32°C Swimming Decrease Micheli-Campbell et al. performance (2011) Emydura signata 24-30°C Swimming Intermediate Booth et al. (2004) performance temperature best

Chelydridae Chelydra serpentina 26.5-30°C Self-righting No effect Steyermark and Spotila performance (2001)

Cheloniidae Caretta caretta 29.8-31.7°C Self-righting Decrease Wood et al. (2014) performance 29.8-31.7°C Crawling performance Decrease Wood et al. (2014) 28.5-32.5°C Crawling performance Decrease Chu (2008) 28.5-32.5°C Swimming Decrease Chu (2008) performance

Chelonia mydas 26-30°C Swimming Increase Booth et al. (2004) performance 28.5-32.4°C Crawling performance Decrease Ischer et al. (2009) 28.5-32.4°C Swimming Decrease Ischer et al. (2009) performance 26-30°C Swimming Increase Burgess et al. (2006) performance

Emydidae Graptemys ouachitensis 24-31°C Self-righting Increase Freedberg et al. (2001) performance 25-30°C Self-righting Increase Freedberg et al. (2004) performance Trachemys scripta elegans 25-30°C Self-righting Increase Freedberg et al. (2004) performance

Testudinidae Gopherus polyphemus 26-32°C Crawling performance No effect Demuth (2001)

Trionychidae Apalone mutica 26-30°C Crawling performance Increase Janzen (1993b) 26-30°C Swimming Increase Janzen (1993b) performance Apalone spinifera 25-34°C Self-righting Intermediate Doody (1999) performance temperature is best 25-34°C Swimming Intermediate Doody (1999) performance temperature is best Pelodiscus sinensis 23-34°C Crawling performance Increase Du and Ji (2003)

As well as affecting hatchling size and locomotor performance, some evidence suggests incubation temperature affects the prevalence of abnormalities or fluctuating asymmetry in reptile hatchlings (Table 1.3). Fluctuating asymmetry is the absolute difference between the left and right sides of a bilateral trait (van Valen, 1962). Fluctuating asymmetry is an indicator of stress during development, 6 including environmental stressors (Ji et al., 2002; Soderman et al., 2007), environmental contaminants (Allenbach et al., 1999), disease (Parris & Cornelius, 2004) and habitat fragmentation (Lauck, 2006). High levels of fluctuating asymmetry generally indicate lower quality organisms (Leung & Forbes, 1997; Moller, 1997; Figure 1.2). In studies on reptiles, high levels of fluctuating asymmetry have been correlated with reduced growth (Moller, 1997), reduced fecundity (Moller, 1997), reduced survival (Moller, 1997) and less exertion during locomotor performance (Vervust et al., 2008). This relationship may be direct, with the abnormalities directly causing reduced fitness (e.g. asymmetry in limbs causing slower locomotion), or indirect, where the abnormality is an indicator of the quality of the organism and may represent internal abnormalities, susceptibility to parasitism or reduced immune function (Moller, 1997).

Fluctuating asymmetry is measured differently in different species. For turtles, the number and arrangement of carapace scutes is often used. For lizards, snakes and crocodiles the arrangement of scales and presence of deformities are used (Table 1.3). Again, there is no clear pattern as to how incubation temperature affects abnormalities – some species show no effect, some species have more abnormalities at higher temperatures, and other species have more abnormalities at lower temperatures (Table 1.3). This lack of a clear pattern may be because of the range of temperatures used in each study. As with locomotor performance, it is likely that abnormalities are more common at temperatures towards the extremes of the viable incubation temperature range, and that

intermediate temperatures are optimal and produce fewer hatchlings with abnormalities. Quality

Fluctuating asymmetry

Figure 1.2 The expected relationship between fluctuating asymmetry and quality of an organism (adapted from Leung and Forbes (1997)).

However it is not known whether incubation temperature is the major cause of hatchling abnormalities. In studies reporting abnormalities during embryonic development, several other causes have been proposed, including other environmental stresses during development (Palmer & Strobeck, 1986; Qualls & Andrews, 1999; Brana & Ji, 2000; Hewavisenthi & Parmenter, 2001), contaminants (Bishop et al., 1991; Bishop et al., 1998; Bell et al., 2006), inbreeding (Olsson et al., 1996), genetic factors (Ayres-Fernandez & Cordero-Rivera, 2004), and relocation or rough handling of eggs (Mast & Carr, 1989).

Table 1.3 List of studies that have experimentally tested the effect of incubation temperature on abnormalities in reptile hatchlings.

Temperature Trait measured Affected by incubation Source range temperature? CROCODILIA Crocodyhs johnstoni 26-34°C Abnormalities of Yes, greater at extreme Webb et al. (1983) the jaws, spine high and low temperatures and tail SQUAMATA Agamidae Calotes versicolor 24-33°C Fluctuating Yes, greater at higher Ji et al. (2002) asymmetry temperatures Colubridae Elaphe carinata 24-32°C Deformities of No effect Ji and Du (2001a) trunk or tail Pituophis melanoleucus 22-32°C Deformities Yes, greater at higher Gutzke and Packard temperatures (1987) Elapidae Naja naja atra 24-32°C Deformities of No effect Ji and Du (2001b) trunk or tail Lacertidae Podarcis muralis 26-32°C Number of Variations in both Brana and Ji (2000) scales, eye directions diameter, hind limb length Phrynosomatidae Sceloporus virgatus 15-30°C Number of Yes, greater at lower Qualls and Andrews scales temperatures (1999) Scincidae Oligosoma suteri 18-26°C Abnormalities of Yes, greater at lower Hare et al. (2002) the tail and feet temperatures TESTUDINES Cheloniidae: Natator depressus 26-32°C Scute pattern Yes, greater at lower Hewavisenthi and variation temperatures Parmenter (2001)

As environmental conditions in the nest can affect a wide range of hatchling traits, different populations of the same species can differ in morphological, locomotor and behavioural traits (How et al., 1996; Tiwari & Bjorndal, 2000; Du et al., 2005). This variation allows a differential allocation

8 of resources to optimise fitness for that population in its environment (Brockelman, 1975; McGinley et al., 1987; Stearns, 1993).

Study species

Marine turtles are ideal organisms to use to test effects of the developmental environment on hatchling phenotypes and by inference fitness. Female turtles lay large clutches of between 50 and 150 eggs (Limpus, 2009), and it is estimated that one hatchling in a thousand survives to breeding age (Heppell et al., 1996), suggesting high initial mortality and strong selection against poor quality hatchlings. Marine turtle eggs require a narrow range of temperatures for incubation, hatchlings display fluctuating asymmetry in the form of non-modal scute patterns, and the species are comprised of several distinct populations, which do not interbreed.

Successful development of marine turtle embryos incubated at a constant temperature throughout incubation occurs over a thermal range of 24°C-33°C (Miller, 1985). Constant temperature incubation outside this temperature range significantly reduces hatching success, causing abnormal development and failure to develop (Bustard & Greenham, 1968; Billett et al., 1992). However, embryos are tolerant to periods of temperature above 33oC, especially during the later part of incubation (Booth & Astill, 2001b; Hewavisenthi & Parmenter, 2002; Booth & Freeman, 2006; Maulany et al., 2012a). Incubation temperature also influences hatchling size, locomotor performance and presence of deformities (Tables 1.1, 1.2 & 1.3).

Hatchlings marine turtles exhibit fluctuating asymmetry as variation in the number and arrangement of the carapace scutes. Scutes are large scales which cover the carapace of all turtles and tortoises except Dermochelys coriacea. Typically the scute pattern consists of a median row of unpaired scutes (vertebral scutes), flanked on both sides by paired costal scutes. Anterior to the first vertebral scute is the nuchal scute, and surrounding the costal scutes are the marginal scutes. Posterior to the last vertebral scute are the postvertebral scutes (Pritchard & Mortimer, 1999; Figure 1.3). Typically a greater proportion of hatchlings than adult turtles exhibit non-modal scute patterns in the same population (Table 1.4). However, generally these studies only count nesting females because of logistical difficulties of capturing adult male turtles, whereas they count hatchlings that are (usually) a mix of male and female turtles (Mast & Carr, 1989), so it is theoretically possible that non-modal scute patterns are more common in male turtles. However, nests with warm (female-producing) temperatures do produce hatchlings with non-modal scute patterns (Sim unpublished data 2012), and the few studies that have investigated adult male turtles have found a similar proportion with non- modal scute patterns to adult female turtles (Suganuma et al., 1994). Studies have also shown similar size and locomotor performance of male and female hatchlings produced at the same temperatures, 9 suggesting that factors other than sex affect hatchling mortality (Booth et al., 2004). Because of this discrepancy between hatchlings and adults, it is often assumed that non-modal scute patterns indicate lower quality hatchlings that experience higher initial mortality, thus explaining their lower frequency in the adult population (Türkozan et al., 2001). However, this hypothesis has never been tested.

In many sea turtle rookeries, hatchlings face a high risk of predation as they crawl down the beach to the sea (Hendrickson, 1958; Bustard, 1972), and swim across shallow reefs (Gyuris, 1994, 2000; Pilcher et al., 2000). Sea turtle hatchlings generally do not actively avoid predators, so faster crawling or swimming ability (or the ability to self-right more quickly) would mean hatchlings spend less time in this high-predator environment, and have a greater chance of survival (Salmon & Wyneken, 1987; Gyuris, 1994).

Table 1.4 List of studies that have examined the proportion of hatchling and adult turtles with non- modal scute patterns at the same rookery.

Species Rookery Hatchling n Adult n Greater proportion of Reference turtles with the modal scute pattern

Caretta caretta Turkey 3309 176 Adults Türkozan et al. (2001) South Africa 50 65 Adults Hughes (1974) Chelonia mydas Turkey 917 13 Hatchlings Ergene et al. (2011) South Africa 372 167 Adults Hughes (1974) Eretmochelys Australia 120 12 Adults Limpus et al. (1983) imbricata Lepidochelys South Africa 5 6 Hatchlings Hughes (1972) olivacea Natator Australia 388 14 Adults Limpus (1971) depressus

The loggerhead turtle, Caretta caretta, is an ideal organism in which to test effects of incubation temperature and non-modal scute patterns on hatchling quality. Female C. caretta have a high reproductive output, laying clutches of around 126 eggs (Limpus, 2009). Like other marine turtle species, hatchlings have a low survival rate to breeding age, and the species is listed as Endangered by the IUCN Red List (IUCN, 2011). C. caretta has a global distribution (Hirth, 1971; Dodd, 1988), meaning temperatures are likely to vary in different parts of the world. The hatchlings have a posthatching planktonic phase in which juveniles live in surface waters and feed on macro zooplankton in the open ocean (Limpus, 2009).

The flatback turtle, Natator depressus, differs from other marine turtle species in several key aspects of life history, making it an interesting comparison to the loggerhead turtle. Female N. depressus lay fewer eggs than loggerhead turtles (around 60; Limpus, 2009), however these eggs are significantly larger and produce significantly larger hatchlings in terms of mass and length (Limpus, 2009). N. depressus is endemic to the Australian continental shelf (Limpus et al., 1988) and hatchlings do not have an oceanic dispersal stage, remaining on the Australian continental shelf for their entire lifecycle (Limpus et al., 1994; Bolten, 2003). The flatback turtle is listed as Data Deficient by the IUCN, but is listed as Vulnerable in Australia on the Environment Protection and Biodiversity Conservation Act List of Threatened Fauna (Department of Sustainability Environment Water Population and Communities, 2011).

Structure and aims of thesis

The general aim of my PhD study was to further understanding of the environmental and physiological factors that affect marine turtle hatchling fitness and survival. To achieve this aim, I asked three main questions in my research:

1) Do marine turtle hatchlings with non-modal scute patterns differ from hatchlings with the modal scute pattern in terms of morphology and locomotor performance? 2) How does natural incubation temperature affect the phenotype and locomotor performance of marine turtle hatchlings? 3) Do marine turtle hatchlings from different rookeries differ in phenotype and locomotor performance?

I answered these questions in four data chapters:

Chapter 2 investigates whether hatchlings with non-modal scute patterns differ to hatchlings with the modal scute pattern in terms of morphology and performance in C. caretta and N. depressus. This study is the first to directly test the hypothesis that hatchlings with non-modal scute patterns are smaller in size and/or have reduced locomotor performance compared to hatchlings with the modal scute pattern. This work has been published as: Sim, E. L., D. T. Booth and C. J. Limpus (2014). Non-modal scute patterns, morphology and locomotor performance of loggerhead (Caretta caretta) and flatback (Natator depressus) turtle hatchlings. Copeia 2014(1): 63-69.

Chapter 3 investigates the effect of natural incubation temperatures on C. caretta hatchlings phenotype and locomotor performance. Here I test the hypothesis that high incubation temperature results in reduced hatchling size and locomotor performance of C. caretta in a temperate rookery. This work is in press for publication by Biology Open.

Chapter 4 investigates the effect of natural incubation temperature on N. depressus hatchling phenotype and locomotor performance. Here I test the hypothesis that high incubation temperature results in reduced hatchling size and locomotor performance of N. depressus from the southernmost rookery of this species. This work will be submitted for publication in Marine Biology.

Chapter 5 investigates differences in phenotype and locomotor performance of N. depressus hatchlings from two different rookeries. This study compares hatchling locomotor performance of two different populations of the same species which have distinctly different body sizes, to the test the hypothesis that larger hatchlings have better locomotor performance. This work has been published as Sim, E. L., D. T. Booth and C. J. Limpus (2014). A comparison of hatchling locomotor

12 performance and scute pattern variation between two rookeries of the flatback turtle (Natator depressus). Copeia 2014(2): 339-344.

Chapter 6 summarises the key findings of my PhD thesis work and discusses possible future directions of research stemming from my investigations.

CHAPTER 2

THE EFFECT OF NON-MODAL SCUTE PATTERNS ON PHENOTYPE AND

LOCOMOTOR PERFORMANCE OF LOGGERHEAD (CARETTA CARETTA) AND

FLATBACK (NATATOR DEPRESSUS) TURTLE HATCHLINGS

Abstract

Non-modal scute patterns are observed more frequently in hatchlings than in adult sea turtles, which suggests greater survival of hatchlings with the modal scute pattern. Here I compare morphological parameters and fitness correlates of Caretta caretta and Natator depressus hatchlings with the modal scute pattern against hatchlings with non-modal scute patterns. I found hatchlings with the modal scute pattern were larger and heavier than hatchlings with non-modal scute patterns, however this size difference did not translate into a difference in crawling speed or self-righting ability for either species. There was also no difference in swim thrust produced by C. caretta hatchlings over the first four hours of swimming, however N. depressus hatchlings with the modal pattern produced greater swim thrust during the first 40 minutes of swimming than hatchlings with non-modal scute patterns. This difference may affect the risk of predation and mortality at this early life stage.

Introduction

The carapace of most turtle and tortoise species is covered by large keratinous scales called scutes (Zangerl & Johnson, 1957). The most frequent or modal sea turtle pattern consists of five vertebral scutes along the medial line of the carapace, flanked on both sides by four to seven pairs of costal scutes. Anterior to the first vertebral scute is the nuchal scute, which is followed by 11 or 12 pairs of marginal scutes which flank the costal scutes. Posterior to the final vertebral scutes is one pair of post- vertebral scutes (Pritchard & Mortimer, 1999).

Individual variations on this modal pattern occur relatively often, particularly in hatchlings, and consist of differences in the number, shape or arrangement of scutes, with the most common variation being additional or supernumerary scutes (Zangerl & Johnson, 1957; Mast & Carr, 1989). These variations have been observed in individuals of all species of marine turtles except Dermochelys coriacea (Hill, 1971; Limpus, 1971; Limpus et al., 1983; Mast & Carr, 1989; Türkozan et al., 2001; Ergene et al., 2011; Table 2.1). When non-modal patterns are exhibited, there is no clear trend towards symmetric or asymmetric patterns. In addition, most studies that recorded the scute pattern of adult and hatchling turtles from the same population have found a higher incidence of non-modal scute patterns in hatchling turtles compared to adult turtles (Limpus, 1971; Limpus et al., 1983; Türkozan et al., 2001; Ergene et al., 2011).

Table 2.1. List of studies that have examined the proportion of hatchling and adult turtles with modal scute patterns.

Species Rookery Age class N Proportion with the Reference location modal scute pattern

Caretta caretta Turkey Hatchling 320 0.341 Ergene et al. (2011)

Chelonia mydas Turkey Adult female 8 0.625 Ergene et al. (2011) Turkey Hatchling 917 0.781 Ergene et al. (2011) Cyprus Hatchling 718 0.596 Özdemir and Türkozan (2006) Japan Adult female 1252 0.950 Suganuma et al. (1994) Japan Adult male 661 0.967 Suganuma et al. (1994)

Lepidochelys kempii Mexico Hatchling 5919 0.447 Mast and Carr (1989)

Few sea turtle hatchlings survive to breeding age (Frazer, 1986; Heppell et al., 1996), and it has been hypothesised that the lower frequency of non-modal scute patterns observed in adult turtles is due to higher initial mortality of hatchlings with non-modal scute patterns, resulting in fewer surviving to breeding age (Türkozan & Yilmaz, 2007). It is unlikely that non-modal scute patterns affect survival directly, because variations in the number of scutes generally do not affect the shape or

15 hydrodynamics of the carapace (Pritchard, 1969), however they may be a phenotypic expression of underlying morphological or physiological abnormalities which may adversely affect the survival of the turtle (Mast & Carr, 1989).

Hatchling sea turtles face the highest risk of predation while crossing the beach from the nest to the sea, and while swimming offshore (Bustard, 1972; Gyuris, 1994; Davenport, 1997). The longer a hatchling spends in these environments, the greater the risk of mortality (Glenn, 1998; Harewood & Horrocks, 2008), increasing to almost 50% when the hatchlings must swim over coral reefs (Gyuris, 1994; Pilcher et al., 2000). Since sea turtle hatchlings do not actively defend themselves against predators (Gyuris, 1994), hatchlings which are able to move through these environments more quickly will reduce their risk of predation. I hypothesised that hatchlings with non-modal scute patterns would have disadvantages that would cause them to spend more time in this high-predation environment (e.g. smaller size and/or reduced locomotor performance) compared to hatchlings with the modal scute pattern, thus making them more vulnerable to predation in the early stages of their life.

Most studies concerning scute patterns in hatchling sea turtles have concentrated on reporting the frequency of non-modal scute patterns, or determining the cause of these non-modal scute patterns (Suganuma et al., 1994; Türkozan & Yilmaz, 2007; Margaritoulis & Chiras, 2011). Here I compare morphological parameters and fitness correlates of loggerhead (Caretta caretta) and flatback (Natator depressus) turtle hatchlings with non-modal scute patterns to hatchlings with the modal scute pattern to determine whether differences in the correlates of fitness can explain the apparent differences in survival to breeding age.

Methods

Experimental design

In order to categorise scute patterns I split them up into three groups (Figure 2.1): modal, minor non- modal (variation in the number of nuchal, marginal or post-vertebral scutes) or major non-modal (variation in the number of vertebral or costal scutes). I chose these classifications for two reasons – firstly because the nuchal, marginal and post-vertebral scutes are much smaller in size, and secondly because variation in marginal, nuchal and post-vertebral scutes is more common in both hatchling and adult turtles (Türkozan et al., 2001; Ergene et al., 2011; Margaritoulis & Chiras, 2011) than variation in the costal and vertebral scutes.

Study site

This study was conducted at Mon Repos Conservation Park (24°48’S, 152°27’E) in south east Queensland, Australia. Mon Repos is a major loggerhead rookery, with between 100 and 600 females nesting each year (Limpus, 2009) and a minor rookery for flatback turtles, with between one and 13 females nesting each year (Limpus, 2009). This beach is also the southern-most limit for nesting flatback turtles (Limpus, 2009).

Egg collection

In December 2010 and 2011, I collected 36 clutches of loggerhead and 13 clutches of flatback eggs by locating nesting females on the beach between dusk and dawn. After the female had laid eggs and left the beach, I uncovered the eggs and relocated them to a hatchery area of the beach using a 10L plastic bucket. During relocation I minimised rotation of the eggs and completed the relocation within two hours. Within this time, I weighed (± 0.1g) a random sample of 10 eggs from each clutch using a portable balance (AND model EK-1200A).

Hatchling collection

In February 2011 and 2012 I collected 1496 loggerhead hatchlings from 36 clutches and 265 flatback hatchlings from 13 clutches by placing an enclosure made of plastic mesh around the top of each nest at dusk. These enclosures were checked every half hour between dusk and dawn to ensure that hatchlings were not on the surface long before being tested for locomotory performance. As soon as an emerging clutch was discovered, up to sixty hatchlings were haphazardly selected, and transported to the laboratory in a 10L bucket by foot (a five minute procedure).

Hatchling measurements and scute classification

Once in the laboratory, each hatchling was weighed (± 0.1g) with an electronic balance (AND model EK-1200A) then the straight carapace length and width at the widest point were measured with digital callipers (± 0.1mm). In order to incorporate both of these measurements into one index of hatchling size, I multiplied length by width to get a value for carapace size index, as used by Ischer et al. (2009). The number of each type of scute was then recorded and the hatchlings were photographed as a record.

Correlates of fitness

Three correlates of fitness were chosen: self-righting ability, crawling speed and swimming ability. Self-righting ability was chosen because it has been previously recognised as an indicator of fitness in hatchling sea turtles (Booth et al., 2013) and freshwater turtles (Delmas et al., 2007). As hatchlings crawl down the beach, they often become inverted (Hosier et al., 1981). Until the hatchling has righted itself it remains vulnerable to predation, and if it is unable to right itself, it risks death by dehydration or over-heating. I chose crawling speed for similar reasons, since hatchlings which can crawl more quickly down the beach are exposed to terrestrial predators for a shorter length on time. Finally I chose swimming ability because the near-shore environment can contain a gauntlet of predators (Gyuris, 1994), so hatchlings that can swim more quickly will spend less time in this environment, reducing their risk of predation.

Self-righting experiments

Locomotor experiments were begun within an hour of first collecting emergent hatchlings. I quantified righting performance using the same method as Booth et al. (2013). Each hatchling was placed upside down on its carapace on a flat area of sand and the time taken to self-right was measured with a stopwatch. If a hatchling failed to self-right within 10s, it was returned to its plastron for 10s before the next trial. This experiment was repeated until the hatchling had successfully self-righted three times, or had attempted self-righting six times, whichever came first. I gave each hatchling a

18 righting propensity score from 0 (failed to self-right) to 6 (successfully self-righted three times out of three), then averaged self-righting time across successful self-righting events for each hatchling.

Crawling ability

Immediately following the self-righting experiments, I measured the plastron surface temperature of each hatchling with an infra-red thermometer (Smart Sensor AR300), because performance of reptiles is correlated with body temperature (Adams et al., 1989). I then measured hatchling crawling speed using the same method as Ischer et al. (2009). I placed each hatchling at the landward end of a 2.9m length of black plastic guttering lined with moist, lightly compacted beach sand. This runway was 10cm wide and contained a dim light at the seaward end to attract the hatchling and ensure that it crawled in a straight line. I timed each hatchling crawling along the guttering with a stopwatch, and converted this value to cm/s.

Swimming ability

Immediately following crawling trials I haphazardly selected eight hatchlings from each group of 30 (four with the modal scute pattern and four with major non-modal scute patterns). I measured swimming ability using the same method as Ischer et al. (2009). Hatchlings were fitted with a Lycra harness, which contained a monofilament line which was attached to a force transducer (MLT050 ADInstruments) connected to a bridge amplifier (model ML112 ADInstruments). The output was recorded via a data acquisition system (Power Lab 8/20 ADInstruments) programmed to sample force 40 times per second. Hatchlings were swum in plastic tubs containing sea water maintained at 28°C for four hours. Before and after each trial the transducers were calibrated by hanging a known mass from each. Swimming performance was quantified by calculating mean thrust (mN) for each 10-min period throughout the 4 hour swimming trial.

Nest excavations

Two days after the emergence of the first group of hatchlings, the nest was excavated and all of the eggs and eggshells were removed. Any dead or live hatchlings found inside the nest were scute- counted. Any unhatched eggs were opened and large embryos were also scute-counted. Hatchlings can fail to emerge from the nest for a number of reasons which are unrelated to their quality, for example entanglement in roots, becoming trapped under rocks or hard sand, or predation by crabs and other predators (Limpus, 2009). For this reason, I separated hatchlings found (live or dead) in the nest from embryos that died during development.

Statistics

To test the association of frequencies of hatchlings in each hatchling type (emerged, in nest or embryo) and each scute pattern (modal, minor non-modal and major non-modal) I used a Poisson regression model with clutch included as a random factor. I only included the first clutch for each female and excluded any clutches in which there were no hatchlings or embryos found in the nest.

To test the effect of scute pattern variation (as a categorical variable) on hatchling mass, length, width, size index and plastron surface temperature I used an ANOVA. Hatchlings were used as the data unit, with clutch nested within mother as random factors. I excluded any clutches which did not have at least one hatchling from all three scute pattern groups from the analysis (N = 2 for both species). When there was a significant difference in mass or size between scute pattern categories, I used a Tukey post-hoc test to determine between which groups those differences occurred.

To test the effect of scute pattern on self-righting ability and crawling speed I used an ANCOVA, with hatchlings as the data unit, plastron surface temperature as a covariate and clutch nested within mother as random factors. When there was a difference detected between scute patterns, I added size index to the model as a covariate to determine if the difference could be explained by a difference in size. Where there was a significant difference between scute patterns, I used a Tukey post-hoc test to determine between which groups those differences occurred.

Swim thrust data were analysed using a repeat measures ANCOVA with the thrust produced each ten minute as the data unit and scute pattern as a fixed factor and clutch as a random factor. Again, when there was a difference detected between scute patterns, I added size index to the model as a covariate to determine if the difference could be explained by a difference in size.

Data analysis was performed using R (R Development Core Team, version 2.15.0, 2013). Data are reported as means and standard errors of means or as least squares covariate means and statistical significance was assumed if p < 0.05.

Results

Hatchling type and scute pattern

For C. caretta, there was an association between hatchling type (dead embryo, trapped in nest, 2 emerged) and frequency of scute pattern type (modal, minor non-modal, major non-modal) (X 4 = 23.88, P < 0.001). Closer examination of the data revealed a greater proportion of in-nest and emerged hatchlings with the modal scute pattern, whereas two thirds of unhatched embryos had either major

20 or minor non-modal scute patterns (Table 2.2). There was no difference between the N. depressus 2 hatchling groups in terms of proportion of hatchlings with each scute pattern (X 4 = 3.94, P = 0.41; Table 2.3).

Table 2.2 Mean proportion of C. caretta unhatched embryos, hatchlings in nest and emerged hatchlings with modal, minor non-modal and major non-modal scute patterns (N = 24 clutches).

Modal Minor non-modal Major non-modal

Unhatched embryos 0.330 0.336 0.334

Hatchlings in nest 0.578 0.226 0.195

Emerged hatchlings 0.574 0.161 0.266

Table 2.3 Mean proportion of N. depressus unhatched embryos, hatchlings in nest and emerged hatchlings with modal, minor non-modal and major non-modal scute patterns (N = 9 clutches).

Modal Minor non-modal Major non-modal

Unhatched embryos 0.528 0.111 0.361

Hatchlings in nest 0.507 0.081 0.412

Emerged hatchlings 0.451 0.177 0.371

Scute pattern, size and locomotor performance

C. caretta hatchlings with the modal scute pattern were heavier and had wider carapaces (and as a consequence had a greater size index) than hatchlings with major non-modal scute patterns (Table 2.4). There were no differences in size between the normal and minor non-modal, or minor and major non-modal groups (Table 2.4).

N. depressus hatchlings with the modal scute pattern were heavier, longer, wider and larger than hatchlings with major non-modal scute patterns (Table 2.5). Hatchlings with minor non-modal scute patterns were also wider (and as a consequence had a larger size index) than hatchlings with major non-modal scute patterns (Table 2.5).

Table 2.4 Hatchling morphological and locomotor parameters (± SE) for 1407 C. caretta hatchlings with the modal (A), minor (B) and major (C) non-modal scute patterns (N = 34 clutches).

Attribute Modal Minor non- Major non-modal F statistic P value Comparison of modal treatments (N = 821) (N = 366) (N = 220)

Mass (g) 19.9 ± 0.1 19.8 ± 0.1 19.7 ± 0.3 5.60 P < 0.001 A = B, B = C, A > C

Length (mm) 43.4 ± 0.1 43.3 ± 0.1 43.3 ± 0.2 2.62 P = 0.07 A = B = C

Width (mm) 35.2 ± 0.1 35.1 ± 0.1 35.0 ± 0.2 3.36 P = 0.03 A = B, B = C, A > C

Size index (mm2) 1529 ± 4 1523 ± 6 1517 ± 13 3.87 P = 0.02 A = B, B = C, A > C

Righting time (s) 2.9 ± 0.04 2.9 ± 0.1 2.9 ± 0.1 0.0002 P = 0.99 A = B = C

Righting 5.7 ± 0.01 5.6 ± 0.01 5.6 ± 0.01 0.58 P = 0.56 A = B = C propensity score

Crawling speed 5.3 ± 0.1 5.3 ± 0.1 5.4 ± 0.1 Significant interaction between scute pattern and body (cm/s) temperature.

Plastron surface 26.3 ± 0.1 26.3 ± 0.1 26.4 ± 0.3 1.49 P = 0.22 A = B = C temperature (ºC)

Despite these size differences there was no difference in righting propensity, average time taken to self-right or crawling speed between the hatchlings with the modal scute pattern and hatchlings with minor or major non-modal scute patterns in either C. caretta or N. depressus (Tables 2.4 & 2.5). There was also no difference in plastron surface temperature between the scute groups (Tables 2.4 & 2.5).

Attribute Modal Minor non- Major non- F P value Comparison of modal modal statistic treatments (N = 119) (n = 54) (N = 81)

Mass (g) 43.1 ± 0.3 42.9 ± 0.4 42.0 ± 0.8 4.63 P = 0.01 A = B, B = C, A > C

Length (mm) 60.8 ± 0.3 60.7 ± 0.3 60.2 ± 0.6 3.14 P = 0.045 A = B, B = C, A > C

Width (mm) 53.6 ± 0.3 53.5 ± 0.4 52.4 ± 0.5 6.84 P = 0.001 A = B, B > C, A > C

Size index (mm2) 3262 ± 28 3256 ± 35 3162 ± 59 6.56 P = 0.002 A = B, B > C, A > C

Righting time (s) 3.9 ± 0.2 3.8 ± 0.2 3.9 ± 0.2 0.46 P = 0.63 A = B = C

Righting propensity 4.6 ± 0.2 4.7 ± 0.2 4.8 ± 0.2 0.03 P = 0.97 A = B = C

Crawling speed (cm/s) 6.7 ± 0.2 7.1 ± 0.3 6.9 ± 0.3 1.27 P = 0.28 A = B = C

Plastron surface 26.0 ± 0.2 26.0 ± 0.2 26.1 ± 0.5 0.65 P = 0.52 A = B = C temperature (ºC)

Mean thrust produced by swimming hatchlings decreased over time for both C. caretta and N. depressus hatchlings (Figure 2.2). There was no difference in the thrust produced between C. caretta hatchlings with the modal scute pattern and hatchlings with major non-modal scute patterns (F1,140 = 0.25, p = 0.62; Figure 2.2). N. depressus hatchlings with the modal scute pattern produced greater thrust than hatchlings with major non-modal scute patterns, but only for the first forty minutes of swimming (p = 0.005; Figure 2.2). This difference persisted even after size was controlled for by using carapace size index as a covariate (p = 0.01).

Discussion

Hatchling type and scute pattern

Sixty per cent of Mon Repos C. caretta emerged hatchlings exhibited the modal scute pattern, which is higher than that reported for C. caretta hatchlings in Turkey (34%; Table 2.1), but well within the range recorded for other species of sea turtle all over the world (44.7% - 78.1%; Table 2.1).

Unfortunately, no substantial data were available in the literature with which to compare the data collected from the emerged N. depressus hatchlings; however, the proportion (49%) of hatchlings

24 with the modal scute pattern was toward the lower end of the range exhibited by other species (Table 2.1). Some evidence suggests low incubation temperatures cause a greater proportion of hatchlings with non-modal scute patterns in N. depressus (Hewavisenthi & Parmenter, 2001), and since Mon Repos is the southernmost limit for nesting of this species, this rookery may have a greater proportion of hatchlings with non-modal scute patterns than other rookeries.

Several studies suggest that non-modal scute patterns can be caused by nest relocation or rough handling of the eggs (Mast & Carr, 1989; Türkozan & Yilmaz, 2007), however none of the authors have suggested a mechanism by which this occurs, or compared relocated and in situ eggs from the same clutch. All nests in this study were relocated with minimal rotation and all within two hours of being laid, as per Limpus et al. (1979).

C. caretta that died during embryonic development had a much higher incidence of non-modal scute patterns than both the in-nest and emerged hatchlings, which suggests that hatchlings with non-modal scute patterns are more likely to die during development. It is unlikely that the non-modal scute patterns themselves are the cause of death, however they may be indicative of low-quality hatchlings with other internal abnormalities (Mast & Carr, 1989). I didn’t find a difference in frequency of non- modal scute patterns between hatchlings and embryos that died during development in N. depressus, however this is likely due to the much smaller sample size in terms of number of clutches and also number of eggs in each clutch.

A previous study which investigated non-modal scute patterns in Kemp’s ridley turtles (Lepidochelys kempi) did not find a difference in proportion of hatchlings with non-modal scute patterns between emerged hatchlings and dead in-nest and unhatched embryos (Mast & Carr, 1989). However, the authors did not separate dead emerged hatchlings and unhatched embryos into two separate groups like I did, which may have masked a difference between hatched and unhatched hatchlings.

Hatchling size

In both species hatchlings with the modal scute pattern were heavier than hatchlings with major non- modal scute patterns. I suggest two possible explanations for this observation; either smaller eggs are more likely to produce hatchlings with non-modal scute patterns, or hatchlings with non-modal scute patterns leave more material behind in the eggs at hatching. The first possibility could be tested by looking at whether there is a correlation between egg size and frequency of non-modal scute patterns. The second possibility could be tested by weighing the remaining shell and its remnants after the turtle has hatched and correlating this mass with the frequency of non-modal scute patterns. A heavier

25 mass may mean a larger yolk to provide energy for the first few days of the hatchling’s life, therefore increasing probability of survival (Gyuris, 1994; Booth et al., 2004).

Whereas there was no difference in carapace length between C. caretta hatchlings with modal and non-modal scute patterns, N. depressus hatchlings with the modal pattern were on average 0.7mm longer than hatchlings with major non-modal scute patterns. Larger hatchlings have the advantage of evading gape-limited predators (Bustard, 1972; Janzen, 1993b) and previous work on green turtle hatchling predation has shown that hatchlings with a carapace length of greater than 51.0mm were less likely to be depredated than hatchlings with a carapace length of less than 47.0mm (Gyuris, 2000). Since N. depressus hatchlings spend their entire life in the near-shore environment (Bolten, 2003), being larger would be more important to them than to C. caretta.

C. caretta hatchlings with the modal scute pattern had a wider carapace than hatchlings with major non-modal scute patterns, by an average of 0.2mm, whereas N. depressus hatchlings with the modal scute pattern were on average 1.2mm wider than hatchlings with major non-modal scute patterns. Although the C. caretta difference is statistically significant, it is so small that it is unlikely to be biologically relevant. Natural variations in hatchling width within the same clutch of greater than 0.6mm have been reported for C. caretta hatchlings (Chu, 2008). However, the difference for N. depressus hatchlings is greater than the maximum range of variation in carapace width reported in Hewavisenthi and Parmenter’s (2001) study (0.8mm).

Hatchling locomotor performance

Despite the size differences I observed, particularly for N. depressus, I did not detect any difference in self-righting ability or crawling speed between modal and major and minor non-modal hatchlings in either species. In the case of the C. caretta hatchlings, this was expected because the size differences between the groups were reasonably small, however I expected to see a difference in N. depressus hatchlings due to the greater size difference.

The first 30-60 minutes of a sea turtle hatchling’s life are when it is most vulnerable to predation, as it crawls from the nest to the sea and then swims through the shallow coastal waters (Gyuris, 1994; Pilcher et al., 2000). Therefore the ability to quickly self-right after becoming inverted, to crawl quickly and to swim quickly should all affect hatchling survival because they will minimise the time that hatchlings are exposed to these high-risk environments.

Sea turtle hatchling terrestrial locomotion has been described as “inefficient and stereotypic” (Davenport, 1997), and several studies have observed hatchlings becoming inverted by beach flotsam and depressions in the sand (Hosier et al., 1981; Davenport, 1997; Steyermark & Spotila, 2001;

Triessnig et al., 2012). C. caretta hatchlings in Turkey overturned on average 2.1 times during the crawl from the nest to the sea, which increased the time spent crawling by an average of 40.5 seconds (Triessnig et al., 2012). Previous studies have shown larger C. caretta hatchlings are faster and more likely to self-right within 10 s (Wood et al., 2014), however that study investigated a larger range of sizes than I observed in this study.

Although other studies have found that hatchling size is positively correlated with crawling speed (Janzen, 1993b; Wren et al., 1998; Janzen et al., 2000a; Janzen et al., 2000b), I did not find a difference in this study. This could be because of the small range of sizes observed in this study, or due to other factors that affect crawling speed. Since I didn’t observe any difference between the modal and non-modal groups, selection against non-modal scute patterns is likely either occurring after the hatchlings leave the beach, or is due to an attribute I did not measure.

Hatchling size has been positively correlated with swimming force in C. caretta (Chu, 2008) and C. mydas hatchlings (Burgess et al., 2006; Ischer et al., 2009). However, similarly to self-righting ability and crawling speed, there was no difference in swim thrust produced in the first four hours between C. caretta hatchlings with the modal scute pattern and hatchlings with major non-modal patterns. However N. depressus hatchlings with the modal pattern produced significantly more swimming thrust than hatchlings with major non-modal scute patterns, but only for the first forty minutes of swimming. This might be explained by a difference in life history: C. caretta hatchlings have an oceanic migration period, whereas N. depressus remain on the continental shelf (Bolten, 2003). N. depressus hatchlings also have a less intense frenzy period, and their swimming effort decreases more quickly than C. caretta and C. mydas hatchlings (Pereira et al., 2011).

The first forty minutes is likely to be the most important in terms of predation avoidance, since predation risk decreases as the hatchling moves further away from the shore. (Salmon et al., 2009; Pereira et al., 2011). By swimming more quickly during the first forty minutes, hatchlings with the modal pattern will not only be more likely to be able to out-swim predators, but also move out of the predator-rich near shore environment more quickly. The lack of a difference in swimming performance of C. caretta hatchlings suggests that if there is selection against non-modal scute patterns during the swimming frenzy, it happens after the first four hours of swimming.

Conclusions

Hatchlings with non-modal scute patterns were larger and heavier than hatchlings with non-modal scute patterns, however this only translated into a difference in locomotor performance in swimming of N. depressus hatchlings. This may be ecologically relevant, as the risk of predation and mortality

27 is high as the hatchlings swim out to sea. Because of the higher proportion of hatchlings than adult turtles with non-modal scute patterns, it is likely that selection against non-modal scute patterns occurs at a later stage in the lifecycle. A number of other factors influence hatchling survival over this period, and future studies could investigate whether hatchlings with the modal scute pattern are better or more efficient foragers, grow faster or are better at navigating ocean currents.

CHAPTER 3

INCUBATION TEMPERATURE, MORPHOLOGY AND PERFORMANCE IN

LOGGERHEAD (CARETTA CARETTA) TURTLE HATCHLINGS FROM MON REPOS,

QUEENSLAND, AUSTRALIA

Abstract

Marine turtles are vulnerable to climate change because their life history and reproduction are tied to environmental temperatures. The egg incubation stage is arguably the most vulnerable stage, because marine turtle eggs require a narrow range of temperatures for successful incubation. Additionally incubation temperature affects sex, emergence success, morphology and locomotor performance of hatchlings. Hatchlings often experience high rates of predation in the first few hours of their life, and increased size or locomotor ability may improve their chances of survival. Between 2010 and 2013 I monitored the temperature of loggerhead (Caretta caretta; Linnaeus, 1758) turtle nests at Mon Repos Rookery, and used these data to calculate a mean three day maximum temperature (T3dm) for each nest. I calculated hatching and emergence success for each nest, then measured the mass, size and locomotor performance of hatchlings that emerged from those nests. Nests with a T3dm greater than 34°C experienced a lower emergence success and produced smaller hatchlings than nests with a T3dm lower than 34°C. Hatchlings from nests with a T3dm below 34°C performed better in crawling and swimming trials than hatchlings from nests with a T3dm below 34°C. Thus even non-lethal increases in global temperatures have the potential to detrimentally affect fitness and survival of marine turtle hatchlings.

Introduction

Many marine turtle populations have experienced drastic population declines over the past thirty years due to anthropogenic disturbances, habitat loss, marine debris and predation (Lutcavage et al., 1997; M. Hamann et al., 2007; Wallace et al., 2011). With impending rises in global temperatures (IPCC, 2007), marine turtles are likely to face threats to many stages of their life cycle. The oceanic currents that drive hatchling dispersal may become disrupted (Mark Hamann et al., 2011; Van Houtan & Halley, 2011), food availability may be affected (Hawkes et al., 2009) and shifts in latitude may occur as a response to oceanic warming (Witt et al., 2010). Nesting grounds face threats such as sea level rise (Fish et al., 2005; M. Hamann et al., 2007), increased cyclonic activity (Poloczanska et al., 2009; Fuentes & Abbs, 2010) and increased nest temperatures (Booth et al., 2004; Glen & Mrosovsky, 2004; Hawkes et al., 2007; Fuentes et al., 2010; Booth & Evans, 2011; Booth et al., 2013).

Nest temperature is determined by a combination of sand temperature and metabolic heating produced by developing embryos (Broderick et al., 2001; Booth & Freeman, 2006). Metabolic heat can raise the nest temperature to 2-6°C above sand temperature in the final weeks of incubation (Broderick et al., 2001; van de Merwe et al., 2006; Zbinden et al., 2006). Constant incubation temperature experiments performed in the laboratory have shown that marine turtle eggs fail to hatch when incubated below 24°C or above 34°C (Bustard & Greenham, 1968; Bustard, 1971; Blanck & Sawyer, 1981; Miller, 1985; Ackerman, 1997; Matsuzawa et al., 2002; Carthy et al., 2003). However, field- based studies have shown that unhatched embryos can survive temperatures above 34°C for short periods of time, particularly during later stages of development (Ewert, 1979; Limpus et al., 1985; Miller, 1985; Maulany et al., 2012a, 2012b; Booth et al., 2013).

Even sub-lethal incubation temperatures can affect emergence, morphology and locomotor performance of marine turtle hatchlings (Reece et al., 2002; Glen & Mrosovsky, 2004; Burgess et al., 2006; Ischer et al., 2009; Booth & Evans, 2011). Nests with higher incubation temperatures produce smaller hatchlings with a larger residual yolk (Reece et al., 2002; Booth, 2006; Burgess et al., 2006), which may be more susceptible to predation by gape-limited predators (Bustard, 1972; Gyuris, 2000; Burgess et al., 2006). The risk of predation is highest as hatchlings crawl down the beach and swim across the shallow reef (Gyuris, 1994) (Davenport, 1997). Hatchlings generally do not actively avoid or defend themselves against predators (Gyuris, 1994), which means hatchlings that can move more quickly through this “gauntlet” of predators may have a greater chance of surviving the initial few hours.

Most studies of the effects of incubation temperature on hatchling performance have used either controlled constant temperature incubation in the laboratory or the mean incubation temperature for 30 naturally-incubated nests on beaches. Recently some studies have shown that the mean three day maximum temperature (T3dm) may be a more accurate predictor of emergence success and hatchling locomotor performance in olive ridley turtles (Lepidochelys olivacea) (Maulany et al., 2012a, 2012b) than mean temperature. Despite having a mean temperature in the normal range, nests with a T3dm above 34°C had a lower emergence success, and these nests produced hatchlings that performed poorly in locomotor performance trials (Maulany et al., 2012a, 2012b). That study was conducted in Indonesia, which is a tropical environment, and accordingly has high sand temperatures and high rainfall (Maulany et al., 2012b). Studies in sub-tropical environments have not been conducted, nor have studies on other marine turtle species. With impending climate change, it is important to understand how increasing temperatures will affect sea turtle nests worldwide.

In this paper I investigate the effect of T3dm on emergence success hatchling size and hatchling locomotor performance using the loggerhead turtle (Caretta caretta) at a sub-tropical rookery in Queensland, Australia. I predicted that nests with a T3dm above 34°C would have a lower emergence success, and produce hatchlings that are smaller and exhibit decreased locomotor performance compared to hatchlings from nests with a T3dm below 34°C.

Methods

Study site

I conducted this study at Mon Repos Conservation Park (24°48’S, 152°27’E) in south east Queensland, Australia. Mon Repos is a major sub-tropical C. caretta rookery, with between 100 and 600 females nesting each year (Limpus, 2009).

Collection of eggs

In December 2010, 2011 and 2012 I located nesting female C. caretta by patrolling the beach between dusk and dawn. I opportunistically and randomly selected 44 clutches of eggs laid by 37 females (25 in 2010, 12 in 2011 and 7 in 2012). I counted the eggs in each clutch and randomly selected a sample of 10 eggs, which I then weighed (± 0.1 g) using an electronic balance (model EK-1200A, A&D, Tokyo, Japan). I relocated the clutches to a hatchery area located in the dunes of Mon Repos beach within two hours of oviposition. To generate a range of incubation temperatures I placed these relocated clutches either in full sun, half shade or full shade, to simulate the range of shade conditions of dune vegetation on natural nests (Wood et al., 2014). I calibrated temperature data loggers (ibutton model DS1922L, Maxim Integrated, San Jose, CA, USA) in a water bath with a certified thermometer

31 and determined they were accurate to ± 0.2°C. I then set them to record the temperature every two (at a resolution of 0.0625°C) hours and placed one in the centre of each nest.

Hatchling collection

After each nest had been incubating for 50 days, I placed a plastic enclosure around the top of the nest at dusk. I checked these enclosures every half hour between dusk and dawn to ensure that hatchlings were not on the surface long before being tested for locomotor performance. As soon as I discovered an emerging nest, I randomly selected between eight and sixty hatchlings, depending on how many there were in the first emergence. I transported the hatchlings to the laboratory in a 10L bucket. I collected a total of 1653 hatchlings.

Hatchling measurements

Once in the laboratory, I weighed each hatchling (± 0.1 g) with an electronic balance (model EK- 1200A, A&D, Tokyo, Japan). I then measured the straight carapace length and width at the widest point (± 0.1 mm) with digital callipers (model 06915, Sontax, Peth, WA, Australia). I calculated carapace size index by multiplying length by width to give a value in mm2.

Self-righting experiments

I began locomotor experiments within an hour of first collecting emergent hatchlings. To quantify righting performance I used the same method as Booth et al (2013). I placed each hatchling upside down on its carapace on a flat area of sand and timed how long it took to self-right. If a hatchling failed to self-right within 10s, I returned it to its plastron for 10s before the next trial. I repeated this procedure until the hatchling had successfully self-righted three times, or had attempted self-righting six times, whichever came first. I then assigned the hatchling a righting propensity score from 0 (failed to self-right) to 6 (successfully self-righted three trials out of three attempts) as used by Booth et al. (2013).

Crawling experiments

In order to control for the effect of body temperature on performance in ectotherms, I measured the plastron surface temperature of each hatchling with an infra-red thermometer (model AR300, Smart Sensor, Houston, TX, USA). I then measured hatchling crawling speed using the same method as Ischer et al (2009). I placed each hatchling at the landward end of a 2.9m length of black plastic guttering lined with moist, lightly compacted beach sand. The runway was 10cm wide and contained a dim light at the seaward end to attract the hatchling and ensure that it crawled in a straight line. I timed each hatchling crawling along the guttering with a stopwatch and converted this value to cm/s. 32

Swimming experiments

Immediately following crawling trials I selected eight hatchlings from each sample. I measured swimming ability using the same method as Ischer et al (2009), by fitting hatchlings with a Lycra harness that contained a monofilament line and was attached to a force transducer (model MLT050, ADInstruments, Sydney, NSW, Australia). This was connected to a bridge amplifier (model ML112 ADInstruments, Sydney, NSW, Australia) and the output was recorded via a data acquisition system (PowerLab model 8/20, ADInstruments, Sydney, NSW, Australia) programmed to sample force 40 times per second. Hatchlings swam in plastic tubs containing sea water maintained at 28°C for four hours. I calibrated the transducers before and after each trial by hanging a known mass from each one. I quantified swimming performance for each individual hatchling turtle by using LabChart v7.0 to calculate mean thrust (mN), stroke rate, proportion of time spent power-stroking and mean thrust per power stroke for each 10-min period throughout the 4 hour swimming trial, as per Pereira et al (2011).

Nest excavations

Between two and five days after the emergence of the first group of hatchlings, I excavated each nest and retrieved the data logger. I counted the number of hatched and unhatched eggs found in the nest and determined the hatching success and emergence success. I downloaded the data logger and calculated the mean temperature for the three warmest days of incubation (T3dm). I then split the nests into two groups: those with a T3dm below 34°C, and those with a T3dm above 34°C, as used by Maulany et al. (2012a).

Statistics

I averaged all variables across hatchlings sampled to get a mean value for each nest. I used an ANOVA to test for a difference between the T3dm above and below 34°C groups. Specifically, to test the effect of T3dm above or below 34°C on hatching success and emergence success I used a nested ANOVA with clutch nested within maternal identity as a random factor. To test the effect of T3dm on hatchling mass, carapace length, carapace width and carapace size index, I used a nested ANCOVA with egg mass as a covariate and clutch nested within maternal identity as a random factor. Finally, to test for differences in righting ability or crawling speed, I used an ANCOVA with nests as the data unit, plastron surface temperature as a covariate and clutch nested within mother as a random factor. I removed non-significant interactions. If there was a significant difference I added carapace size index to the model as a covariate to determine whether these differences were solely due to a

33 difference in hatchling size. To test for the effect of T3dm on all swimming attributes, I used a repeated measures ANOVA.

I performed data analysis using R (R Development Core Team, version 2.15.0, 2013) and Statistica (Version 12). Data are reported as means and standard errors of means or as least squares covariate means and I assumed statistical significance if p < 0.05.

Results

Mean nest temperatures were similar in the 2010-11 and 2011-2012 seasons (mean (± SE) of 29.0°C ± 0.1 (range of 27.9-30.9°C) in 2010-11 and 29.1°C ± 0.2 (range of 27.9-30.9°C) in 2011-12; Figure

3.1), but the 2012-13 season was warmer (mean of 31.6°C ± 0.2 (range of 31.0-32.6°C); F1,60 = 34.05, p < 0.001; Figure 3.1). Nests in the shaded hatchery were approximately 1°C cooler than nests on the open beach (t1,49 = 8.44, p < 0.001). Nest temperatures decreased after heavy rainfall events in all three seasons (Figure 3.1). There were nests with a T3dm above and below 34°C in all years (Table 3.1).

300 36

250 34

200 32

30 150

28 100 (mm) rainfall Daily

Mean temperature (ºC) temperature Mean

26 50

24 3000 36

250 34

200 32

30 150

28 100 (mm) rainfall Daily

Mean temperature (ºC) temperature Mean

26 50

24 0300 36

250 34

200 32

30 150

28 100 (mm) rainfall Daily

Mean temperature (ºC) temperature Mean

26 50

24 0 03/12 17/12 31/12 14/01 28/01 11/02 25/02

Date

2010-2011 and 2011-2012 seasons were obtained over several weeks, whereas nests in the 2012-2013 season were all obtained in the same week.

Table 3.1 Number of C. caretta nests with a mean three day maximum temperature below 34°C and above 34°C in the 2010-2011, 2011-2012 and 2012-2013 nesting seasons.

Nesting season Number of nests with a T3dm below 34°C Number of nests with a T3dm above 34°C 2010-2011 24 1 2011-2012 11 1 2012-2013 2 5

The mean incubation temperature was higher in the T3dm > 34ºC nests than the T3dm < 34°C nests (Table 3.2). Nests with a T3dm > 34°C had a lower emergence success than nests with a T3dm < 34°C, but there was no difference in hatch success (Table 3.2). There was no difference in body mass or carapace length between hatchlings from the T3dm < 34°C and T3dm > 34°C nests (Table 3.2). 2 There was no correlation between the clutch size and T3dm (F1,60 = 0.29, P = 0.60, R = 0.005).

Parameter measured < 34°C > 34°C F-statistic P value Mean incubation temperature 29.1 ± 0.1 (27.9 – 31.3) 31.3 ± 0.5 (30.3 – 32.6) 60.31 < 0.001 (°C) Hatching success (%) 78.5 ± 6.2 (31.4 – 98.0) 74.4 ± 5.8 (56.7 – 90.5) 0.03 0.86 Emergence success (%) 70.8 ± 7.0 (35.3 – 95.6) 46.7 ± 6.6 (27.4 – 70.3) 3.59 0.004 Body mass (g) 19.9 ± 0.2 (17.3 – 22.0) 19.5 ± 1.1 (17.5 – 22.6) 0.45 0.53 Length (mm) 43.4 ± 0.3 (41.7 – 45.6) 42.3 ± 1.5 (40.8 – 44.8) 2.08 0.20 Width (mm) 35.5 ± 0.4 (32.8 – 37.3) 33.2 ± 1.6 (31.7 – 34.4) 15.56 0.008 Size index (mm2) 1531.6 ± 9.7 (1373.3 – 1406.8 ± 54.1 (1296.8 – 10.65 0.01 1671.0) 1536.5) Self-righting propensity score 5.6 ± 0.1 (4.0 – 6.0) 5.1 ± 0.3 (4.6 – 5.8) 5.36 0.05 Self-righting time (s) 2.9 ± 0.1 (1.8 – 4.7) 3.4 ± 0.3 (2.7 – 4.2) 3.51 0.10 Crawling speed (cm/s) 5.1 ± 0.2 (2.3 – 8.4) 3.6 ± 0.3 (2.7 – 4.4) 6.18 0.02 Plastron surface temperature 26.4 ± 0.3 (21.7 – 31.2) 27.1 ± 0.3 (26.2 – 28.3) 0.67 0.42 (°C)

Hatchlings from the T3dm < 34°C nests had a wider carapace than hatchlings from the T3dm > 34°C nests and consequently a larger carapace size index (Table 3.2). This difference in size did not translate into a difference in either self-righting propensity or time taken to self-right (Table 3.2). However hatchlings from the T3dm < 34°C nests were faster crawlers than hatchlings from the T3dm > 34°C nests (Table 3.2). When I added size index as a covariate, the difference between the groups remained (F1,19 = 6.56, p = 0.02).

Figure 3.2 (A) Emergence success (p = 0.11, R2 = 0.03), (B) Mean carapace width (p < 0.001, R2 = 0.24), (C) Mean carapace size index (p < 0.001, R2 = 0.18) and (D) Mean crawling speed (p = 0.01, R2 = 0.12) of C. caretta hatchlings plotted against the T3dm (mean maximum 3 day temperature).

For individual hatchlings, plastron surface temperature was negatively correlated with mean time 2 taken to self-right (equation: SRT = 4.69 – 0.06*PST, F1,1609 = 16.51, p < 0.001, R = 0.009) and

37 positively correlated with crawling speed (equation: CS = 0.08*PST + 3.04; F1,1589 = 13.26; p < 0.001, R2 = 0.007). There was no correlation between plastron surface temperature and self-righting propensity (F1,1621 = 2.85, p = 0.09). When I plotted data for emergence success, carapace width, carapace size index and crawling speed continuously against the T3dm there was a clear decline in carapace width and carapace size index when the T3dm was above 34°C (Figure 3.2).

Swimming parameters decreased as time spent swimming increased in both temperature categories (Figure 3.3). Hatchlings from nests with a T3dm < 34°C produced more thrust than hatchlings from nests with a T3dm > 34°C for the first twenty minutes of swimming (p = 0.03-0.04; Figure 3.3). There was no difference in stroke rate, proportion of time spent power stroking or mean maximum thrust between the two groups (Figure 3.3).

Discussion

Nest temperature

Mean nest temperatures recorded in this study fell within the range observed in this rookery in the recent past (30.0-33.1°C in the 2005-2006 season and 28.9-32.7°C in the 2006-07 season (Chu et al., 2008), 28.9-32.7°C in the 2009-10 season (Wood et al., 2014) and 28.1-30.6°C in the 2010-11 season (Read et al., 2012)). Nest temperatures followed a similar pattern across years, becoming warmer towards the end of incubation due to metabolic heating (Zbinden et al., 2006). Thus, the T3dm was usually towards the end of the incubation period when the hatchlings were well developed. Clutch size did not correlate with T3dm, suggesting metabolic heating affected all nests in a similar way, and did not contribute to thermal differences between nests.

Several environmental factors appeared to affect nest temperature in this study. Nests in the shaded hatchery were 1°C cooler on average than nests on the open beach, and most nests experienced a drop in temperature following heavy rainfall. These environmental effects have been shown in other studies (Houghton et al., 2007; Wood et al., 2014), suggesting that the effects of climate change are not limited to increased temperatures. Although all the nests in this study were relocated, it is unlikely that relocation affected nest temperatures, since nests were relocated to the same beach (Pintus et al., 2009; Tuttle & Rostal, 2010; DeGregorio & Williard, 2011).

Hatching and emergence success

As I did not find an effect of T3dm on hatchling success, it is likely that temperatures in this study were not consistently high enough to negatively affect hatching success. However, laboratory constant incubation temperature experiments have shown high temperatures decrease hatching success (Bustard & Greenham, 1968; Bustard, 1971). Other studies with a similar temperature range have also shown no correlation between temperature and hatching success (Hewavisenthi & Parmenter, 2002; Chu et al., 2008). However, similar mean nest temperatures have been correlated with a reduction in emergence success in other C. caretta nests (Chu et al., 2008; Read et al., 2012). More specifically, nests which experienced high temperatures in the last few days of incubation experienced a lower emergence success than those which did not (Matsuzawa et al., 2002; Maulany et al., 2012b).

The effect of T3dm on emergence success but not hatching success in this study suggests that increased nest temperatures are affecting hatchlings directly, either by the heat causing death (Matsuzawa et al., 2002) or decreasing hatchling activity and preventing the hatchlings from

39 emerging (Mrosovsky, 1968; Drake & Spotila, 2002). Critical thermal maxima for other sea turtle species have been measured at between 37.1 and 41.4°C (Drake & Spotila, 2002), which is higher than the sand temperatures measured in this study, however hatchlings began to display uncoordinated movements at temperatures of 33.4°C (Drake & Spotila, 2002). Similarly critical threshold surface temperatures (above which hatchlings will not emerge) of between 32.4°C and 37.5°C have been calculated for sea turtle hatchlings (Moran et al., 1999; Drake & Spotila, 2002). It is likely above this temperature coordinated muscle movement is inhibited (Matsuzawa et al., 2002).

Although all nests in this study were relocated, it is unlikely that relocation affected the emergence of hatching success independently of temperature. Other studies have found no difference between relocated and in situ nests (Wyneken et al., 1988; Kornaraki et al., 2006; Tuttle & Rostal, 2010). One study has shown that hatching success is lower in relocated nests than in in situ nests (Pintus et al., 2009), however these eggs were relocated up to six hours after oviposition, so may have been subject to movement-induced mortality (Limpus et al., 1979). All clutches in this study were relocated within two hours of oviposition.

Hatchling size and mass

A negative correlation between incubation temperature and sea turtle hatchling size has been documented in all species of sea turtle studied (Hewavisenthi & Parmenter, 2001; Booth et al., 2004; Ischer et al., 2009; Mickelson & Downie, 2010; Booth & Evans, 2011; Maulany et al., 2012a; Read et al., 2012; Wood et al., 2014). Similarly, I found that hatchlings from nests with a T3dm above 34°C had smaller carapaces than hatchlings from nests with a T3dm below 34°C. This size difference is likely due to warmer nests experiencing shorter incubation duration, meaning the hatchlings produced have less time to convert their yolk into tissue before hatching. Thus they are smaller in dimensions, but have a larger residual yolk compared to hatchling from cooler nests (Booth & Astill, 2001a; Booth, 2006; Ischer et al., 2009). This likely explains why I found a difference in hatchling size, but not mass, as the total mass of the hatchling and the internal yolk sac would be similar.

Previous studies following a similar relocation protocol have found no difference in size or between relocated and in situ nests (Pintus et al., 2009; Tuttle & Rostal, 2010). Another study found differences in the mass and width of hatchlings from relocated vs. in situ nests, but these differences were so small, they are unlikely to biologically relevant (Türkozan & Yilmaz, 2007).

Larger hatchlings likely have a greater chance of survival, due to their ability to travel more quickly through the predator-rich beach and near-shore environments (Chu, 2008). Larger hatchlings also may be ignored by gape-limited predators (Bustard, 1972), however smaller hatchlings with larger 40 yolk stores will have greater energy stores and will be able to travel further without feeding (Ischer et al., 2009). Thus there is a trade-off and the effect on fitness and survival will vary depending on predation pressure at rookeries.

Terrestrial locomotor performance

Crawling speed has been negatively correlated with mean incubation temperature before in several field-based studies on hatchling sea turtle species (Chu, 2008; Ischer et al., 2009; Maulany et al., 2012a; Read et al., 2012; Booth et al., 2013; Wood et al., 2014). In C. caretta hatchlings this difference remained, even after the authors added hatchling size as a covariate (Chu, 2008; Read et al., 2012), suggesting that increased crawling speed is not solely due to the difference in size. My study showed that hatchlings from nests with a T3dm below 34°C crawled more quickly than hatchlings from nests with a T3dm above 34°C, supporting the above trend. As in the other study, the difference remained even after controlling for the larger body size of hatchlings from cooler nests. This difference suggests that even though the time spent above 34°C in the late stages of incubation was not enough to be fatal, it may have had a detrimental effect on the physiology of the developing embryo. Sea turtle embryos are almost fully developed at this stage of development (Miller, 1985), so high temperatures at this stage are unlikely to have affected the formation of structural components of tissues such as muscle fibres, but might have caused some damage once they have formed, resulting in decreased muscle performance in hatchlings. Crawling speed affects the amount of time the hatchling spends on the beach, with longer on beach periods increasing the chance of terrestrial predation, or becoming dehydrated or overheated (Dial, 1987; Burgess et al., 2006; Janzen et al., 2007).

Despite differences in size, there was no difference in either self-righting propensity or time taken to self-right between the two groups. Two previous studies using the mean incubation temperature of relocated nests found that hatchlings from warmer nests were less likely to self-right within 10 seconds, and take longer to self-right than hatchlings from cooler nests (Read et al., 2012; Wood et al., 2014). The range of mean incubation temperatures were similar in all studies (27.9-32.6°C in this study and 29.6 – 32.2°C (Wood et al., 2014), 28.1-32.5°C (Read et al., 2012)), and so was the range of body sizes, which suggests that self-righting ability is not strongly related to nest temperatures. Also, since turtle hatchlings self-right by flexing their head against the substrate (Booth et al., 2013), neck length may be important to self-righting ability, but has not been measured in self-righting studies.

Swimming performance

Swimming effort of sea turtle hatchlings generally decreases with time spent swimming, with the largest decreases occurring within the first two hours of entering the water (Pilcher & Enderby, 2001; Booth et al., 2004; Booth, 2009; Ischer et al., 2009; Booth & Evans, 2011; Pereira et al., 2011). Similarly, hatchlings in this study decreased their swimming effort in terms of thrust produced, stroke rate, proportion of time spent power-stroking and mean maximum thrust over the four hour testing period. Hatchlings are most vulnerable to predation in the shallow near-shore environment (Salmon & Wyneken, 1987; Gyuris, 1994) and by maximising their swimming effort at the beginning of their swim, they will move out of this zone rapidly. The swimming effort then decreases as the hatchlings fatigue.

Hatchlings from Mon Repos rookery must swim approximately 5-10km offshore to reach the south- east Australian current, which then carries them to their juvenile feeding grounds (Walker, 1994). Hatchlings from nests with a T3dm below 34°C produced more thrust than hatchlings from nests with a T3dm above 34°C in the first twenty minutes of swimming. This period is likely the most important in terms of survival, since the near shore environment is the most predator-dense (Gyuris, 1994; Pilcher et al., 2000; Salmon et al., 2009). Although I did not measure swimming speed directly, hatchlings were of a uniform shape and would have a similar hydrodynamic resistance. Therefore thrust can be considered a proxy for swim speed (Booth & Evans, 2011). Previous work on sea and freshwater turtle hatchlings has suggested that hatchlings from a mid-range incubation temperature perform better in swimming trials than those from either high or low mean incubation temperatures (Booth et al., 2004; Burgess et al., 2006; Chu, 2008). Again, this might be due to adverse effects of extreme high or low temperatures on the physiology of the developing embryo. With impending global climate change, global air temperatures (and therefore nest temperatures) will increase over the next few years (Hays et al., 2003; Hays, 2008; Fuentes et al., 2011) resulting in hatchling sea turtles emerging from nests with reduced locomotor performance ability.

Conclusions

This study demonstrates that sub-lethal incubation temperatures late in incubation can have negative effects on both morphology and locomotor performance of marine turtle hatchlings. Sub-lethal temperatures can affect hatchling emergence, size, and crawling and swimming ability. As global temperatures rise, the proportion of nests experiencing extreme high temperatures is likely to increase, which may affect hatchling survival rates. One of the limitations of this study was the smaller number of nests with a T3dm above 34°C compared to nests with a T3dm below 34°C, and the fact that these were mostly concentrated in one 42 season. This arose from my decision to incubate nests on a beach with variable thermal conditions, rather than control conditions in a laboratory. I suggest a long term study over several years, in order to sample nests from seasons with a variety of thermal and climactic conditions.

CHAPTER 4

THE EFFECT OF INCUBATION TEMPERATURE ON FLATBACK TURTLE (NATATOR

DEPRESSUS) HATCHLINGS.

Abstract

Sea turtles rely on the physical environment to incubate their eggs, and require temperatures between 24°C and 33°C for successful development of embryos. Incubation temperature has also been shown to affect hatchling attributes such as emergence success, size and locomotor performance in green, olive ridley and loggerhead turtles. I investigated the effect of incubation temperature of in situ nests on hatching and emergence success, scute pattern, mass, size and locomotor performance in the flatback turtle (Natator depressus). Flatback turtles differ from other sea turtle species in a number of life history traits – they produce larger eggs and hatchlings for their size, and they lack the oceanic development stage present in all other sea turtle species. Between 2010 and 2012 I monitored the temperature of ten in situ N. depressus nests. I calculated hatching and emergence success for each nest, and measured the mass, width, length, scute pattern and locomotor performance of hatchlings that emerged. I found a positive correlation between hatchling length and incubation temperature, a positive correlation between incubation temperature and self-righting propensity, and positive correlations between mean swim thrust produced and proportion of time spent power-stroking and mean incubation temperature. Thus, low incubation temperatures may affect hatchling survival through their effect on hatchling locomotor performance. The nest temperatures I observed were low compared to other sea turtle studies, so it would be interesting to collect data from N. depressus nests that have experienced higher temperatures.

Introduction

Oviparous reptiles generally rely on the physical environment to incubate their eggs (Packard & Packard, 1988). If the incubation temperature is too low, embryos cease developing and die, and if it is too high, the embryos die as well (Bustard & Greenham, 1968; Hubert, 1985). Incubation temperature can also affect hatchlings in a variety of ways including sex, body size and shape, amount of yolk converted into tissue, scale or scute pattern, locomotor performance, behaviour and post-hatch growth rate (Hewavisenthi & Parmenter, 2001; Deeming, 2004; Valenzuela, 2004; Valenzuela & Lance, 2004; Booth, 2006). In sea turtles, a negative correlation between incubation temperature and hatchling size has been reported (Reece et al., 2002; Glen et al., 2003; Booth et al., 2004; Stokes et al., 2006; Ischer et al., 2009; Maulany et al., 2012a), and incubation temperature can affect hatchling locomotor performance, through its effect on hatchling size, and through a direct effect (Booth et al., 2004; Burgess et al., 2006; Ischer et al., 2009; Booth & Evans, 2011; Maulany et al., 2012a; Wood et al., 2014).

The temperature of sea turtle nests depends on air temperature, shading, rainfall, depth, orientation and sand colour (Fuentes et al., 2010; Maulany et al., 2012a; Wood et al., 2014). Sea turtle eggs successfully incubate between constant temperatures of 24 - 33°C (Miller, 1985), but can withstand variable temperatures above 33°C late in incubation (Booth & Astill, 2001b; Hewavisenthi & Parmenter, 2002; Maulany et al., 2012b). Although these high temperatures do not directly kill hatchlings, a growing body of evidence suggests hatchlings from nests that experience high temperatures towards the end of incubation are likely to experience lower survival rates due to reduced hatchling size and poorer locomotor performance.

Sea turtles experience high mortality early in their lifecycle (Crouse et al., 1987), particularly as developing embryos or as newly-emerged hatchlings. After the hatchlings emerge from the nest, they must crawl to the water, a journey which leaves them vulnerable to predation, desiccation and disorientation (Bustard, 1972; Janzen et al., 2007). Hatchlings often become inverted on this crawl, and must right themselves before continuing (Hosier et al., 1981; Triessnig et al., 2012). Once the hatchlings reach the water, they engage in a “swim frenzy” (Lohmann et al., 1997) lasting around 24 hours. The purpose of this swim is to transport hatchlings to offshore currents, after which frenzy swimming is no longer necessary (Salmon & Wyneken, 1987; Putman et al., 2010). While swimming, hatchlings alternate between bouts of “power-stroking”, in which the front flippers move together in a lift-based movement to produce thrust, and bouts of “dog-paddling” in which all four flippers move in a paddle-like motion, while a breath of air is also taken (Salmon & Wyneken, 1987; Wyneken & Salmon, 1992).

Although the effects of incubation temperature on hatching success and hatchling locomotor performance have been studied in many sea turtle species (Burgess et al., 2006; Chu, 2008; Ischer et al., 2009; Segura & Cajade, 2010; Maulany et al., 2012a; Wood et al., 2014), the flatback turtle (Natator depressus) has not been studied. Flatback turtles differ from all other sea turtle species in a number of key life history traits: (1) they are endemic to the Australian continental shelf and remain in the neritic zone for their entire life cycle (Walker & Parmenter, 1990; Bolten, 2003). Although this life history strategy may offer more food resources to hatchlings, there is also a greater risk of predation (Walker, 1994). (2) Flatback turtles lay larger eggs for their body size than all other sea turtle species (Bustard & Limpus, 1969), which produce larger hatchlings, that may be able to avoid predation by gape-limited predators (Hirth, 1980; Walker & Parmenter, 1990; Walker, 1994). (3) Compared to green, loggerhead and leatherback turtle hatchlings, flatback hatchlings have a less intense, but longer “swim frenzy”, which is consistent with them remaining in the neritic zone (Salmon et al., 2009; Pereira et al., 2012).

A previous study investigated temperature of in situ flatback nests (Hewavisenthi & Parmenter, 2002). The authors suggest that flatback nests experience a lower rate of metabolic heating than other sea turtle species, due to a smaller clutch size (Hewavisenthi & Parmenter, 2002). Another laboratory- based study investigated the effect of incubation temperature on flatback hatchling size and scute pattern abnormalities (Hewavisenthi & Parmenter, 2001), and found that flatback hatchlings from eggs incubated at a mid-range temperature were larger than hatchlings produced at low or high temperatures. They also found that eggs incubated at low temperatures produced more hatchlings with non-modal scute patterns, but only in one of the two years investigated (Hewavisenthi & Parmenter, 2001).

In this study I examine the effects of in situ incubation temperature on emergence success, hatching success, prevalence of non-modal scute patterns, hatchling mass, hatchling size and locomotor performance in the flatback turtle. I investigated whether the general effects of incubation temperature on hatchling morphology and locomotor performance found in other sea turtle species are also true for flatback turtles.

Methods

Study site

I conducted this study at Mon Repos Conservation Park (24°48’S, 152°27’E) in south east Queensland, Australia, which is the southernmost rookery for this species on the east coast of Australia (Limpus, 2009). 46

Data collection

In December 2010 and 2011 I randomly selected 10 clutches of eggs. After laying, I excavated the eggs and counted them. I then randomly selected a sample of 10 eggs to weigh (± 0.1 g) using an electronic balance (model EK-1200A, A&D, Tokyo, Japan). I relocated the clutches to a hatchery area of the beach within two hours of oviposition. I placed temperature data loggers (ibutton model DS1922L, Maxim Integrated, San Jose, CA, USA) set to record the temperature every two hours in the centre of each clutch.

Fifty days after laying, I placed a plastic enclosure around the top of each nest at dusk to catch emerging hatchlings. I checked these enclosures every half hour between dusk and dawn. As soon as I discovered an emerging clutch, I randomly selected up to thirty hatchlings from the first emergence cohort, and transported them to the laboratory in a bucket by foot. I sampled a total of 194 hatchlings from ten clutches.

Measurements of hatchling quality

Once in the laboratory, I weighed each hatchling (± 0.1 g) with an electronic balance (model EK- 1200A, A&D, Tokyo, Japan), then measured the straight carapace length and width at the widest point (± 0.1 mm) with digital callipers (model 06915, Sontax, Peth, WA, Australia). I calculated carapace size index by multiplying length by width to give a value in mm2.

I then counted the number of each type of scute on the carapace and photographed each hatchling. I classified hatchlings into three different scute pattern groups: modal, minor non-modal (variation in the number of nuchal, marginal or post-vertebral scutes) or major non-modal (variation in the number of vertebral or costal scutes) as per Sim et al. (2014).

Locomotor experiments

I began locomotor experiments within an hour of first collecting emergent hatchlings. To quantify self-righting ability I used the same method as Booth et al (2013). I placed each hatchling upside down on a flat area of sand and timed how long it took to self-right. If a hatchling failed to self-right within 10s, I returned it to its plastron for 10s before the next trial. I repeated this procedure until the hatchling had successfully self-righted three times, or had attempted self-righting six times, whichever came first. I then assigned the hatchling a righting propensity score from 0 (failed to self- right) to 6 (successfully self-righted three trials out of three attempts) as used by Booth et al. (2013).

Immediately following the self-righting experiments, I measured the plastron surface temperature of each hatchling with an infra-red thermometer (model AR300, Smart Sensor, Houston, TX, USA). I 47 then quantified hatchling crawling speed using the same method as Ischer et al (2009). I placed each hatchling at the landward end of a 2.9m length of black plastic guttering lined with moist, lightly compacted beach sand. The runway contained a dim light at the seaward end to attract the hatchling and ensure that it crawled in a straight line. I timed each hatchling crawling along the guttering with a stopwatch, and converted this value to cm/s.

Immediately following crawling trials I selected eight hatchlings from each clutch, four with the modal scute pattern and four with non-modal scute patterns. I measured swimming ability using the same method as Ischer et al (2009), by fitting hatchlings with a Lycra harness that contained a monofilament line and was attached to a force transducer (model MLT050, ADInstruments, Sydney, NSW, Australia). The force transducer was connected to a bridge amplifier (model ML112 ADInstruments, Sydney, NSW, Australia) and the output was recorded via a data acquisition system (PowerLab model 8/20, ADInstruments, Sydney, NSW, Australia) programmed to sample force 40 times per second. Hatchlings swam in plastic tubs containing sea water maintained at 28°C for four hours. I calibrated the transducers before and after each trial by hanging a known mass from each one. I quantified swimming performance by calculating mean thrust (mN), stroke rate, proportion of time spent power-stroking and mean thrust per power stroke for each 10-min period throughout the 4 hour swimming trial, as reported in Pereira et al (2011).

Nest excavations

Data analysis

I averaged all variables across hatchlings sampled from a nest to obtain a mean value for that nest. I used linear regressions to test for correlations between mean incubation temperature and the various response variables. To test for a correlation between mean incubation and hatching success, emergence success and proportion of hatchlings with the modal scute pattern, I used a linear regression. To test for a correlation between mean incubation temperature and hatchling mass, carapace length, carapace width and carapace size index, I used a linear regression with egg mass as a covariate. To test for a correlation between mean incubation temperature and self-righting ability or crawling speed, I used a linear regression with plastron surface temperature as a covariate. I removed non-significant interactions. If there was a significant correlation I added carapace size index

48 to the model as a covariate to determine whether these differences were solely due to a difference in hatchling size. To test for a correlation between mean incubation temperature and all swimming attributes, I used a repeated measures ANOVA.

I performed data analysis using R (R Development Core Team, version 2.15.0, 2013). Data are reported as means and standard errors of means or as least squares covariate means and I assumed statistical significance if p < 0.05.

Results

Mean nest temperatures ranged from 27.6°C to 29.6°C (Figure 4.1). Nests experienced dial variation in temperature, and nest temperatures decreased following heavy rainfall (Figure 4.1). Mean incubation temperature was not correlated with either hatching or emergence success (Figure 4.2). Neither hatchling mass nor width were correlated with mean incubation temperature, however there was a positive correlation between mean incubation temperature and hatchling length, and consequently size index (Figure 4.2). The proportion of hatchlings with the modal scute pattern was not correlated with incubation temperature (Figure 4.2). Hatchling self-righting propensity was positively correlated with mean incubation temperature, with hatchlings from cooler nests less likely to self-right within ten seconds (Figure 4.2). Neither hatchling self-righting time nor crawling speed were correlated with mean incubation temperature (Figure 4.2).

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In the swimming experiments there was a positive correlation between mean incubation temperature and mean thrust produced (F1,21 = 5.66, p = 0.049) and mean incubation temperature and proportion of time spent power-stroking (F1,21 = 9.66, p = 0.02). There was no correlation between mean 52 incubation temperature and either powerstroking rate or mean maximum thrust produced. When I plotted the data for the warmest nest (mean temperature of 29.6°C) and the coolest nest (mean temperature of 27.6°C), values were always higher at the higher temperature (Figure 4.3).

Discussion

Nests in this study experienced dial variation in temperature, as has been observed for flatback nests previously (Hewavisenthi & Parmenter, 2002) as well as other sea turtle species with relatively shallow nests (approximately 40-60cm) (e.g.Spotila et al., 1987; Maloney et al., 1990). The nests in this study also experienced decreases in temperature following heavy rainfall events, as has been observed in other species (e.g. Booth & Astill, 2001b; Matsuzawa et al., 2002). The range of mean nest temperatures (2.3°C) was similar to the range of mean temperatures recorded in other studies (Booth & Astill, 2001b; Ischer et al., 2009; Read et al., 2012).

Hatching and emergence success

Hatching success was unaffected by mean incubation temperature, a finding consistent with that found in constant temperature experiments on the same species (Hewavisenthi & Parmenter, 2001), and in situ experiments in which hatchlings experienced temperatures of above 35°C late in the incubation period (Hewavisenthi & Parmenter, 2002). I did not observe a correlation between mean incubation temperature and emergence success, as has been found in a previous study on N. depressus using similar temperatures (Blamires & Guinea, 2000). However, studies that investigated a greater range of temperatures in other sea turtle species have found a negative correlation between incubation temperature and emergence success (e.g.Chu et al., 2008; Maulany et al., 2012b; Read et al., 2012).

Scute variation

I did not find a correlation between mean incubation temperature and proportion of hatchlings with the modal scute pattern. However, in constant temperature experiments, N. depressus hatchlings from eggs incubated at 26°C had more carapace abnormalities than hatchlings from eggs incubated at 29°C. Since nests in this study had a mean temperature of at least 27.6°C, incubation temperatures were probably not cold enough to affect scute patterns. Causes of non-modal scute patterns have not been established, with factors including genetics, inbreeding depression, pollution, relocation and rough handling of eggs being suggested as causes (Mast & Carr, 1989; Ayres-Fernandez & Cordero-Rivera, 2004; Türkozan & Yilmaz, 2007).

In the current study, an average of only 40% of hatchlings in a nest had the modal scute pattern, a proportion much lower than observed at another N. depressus rookery (Sim et al., 2014), and also 53 lower than is typical for other sea turtle species (0.59 - 0.87; Mast & Carr, 1989; Suganuma et al., 1994; Özdemir & Türkozan, 2006; Ergene et al., 2011). The low proportion of N. depressus hatchlings with the modal scute pattern at Mon Repos rookery may be caused by low sand temperatures experienced during this study or relocation of nests.

Hatchling size and mass

Most sea turtle species show a correlation between incubation temperature and hatchling size, but not incubation temperature and hatchling mass (Reece et al., 2002; Glen et al., 2003; Booth et al., 2004; Stokes et al., 2006; Ischer et al., 2009; Booth & Evans, 2011; Maulany et al., 2012a) . Similarly, I found no correlation between mean incubation temperature and hatchling mass, but a correlation between hatchling size and next temperature.

Generally, sea turtle hatchlings produced by warmer nests are smaller than hatchlings produced by cooler nests, a finding that may be explained by warmer temperatures reducing the incubation period, resulting in less of the yolk being turned into hatchling tissue, producing smaller hatchlings with a larger residual yolk (Booth & Astill, 2001a; Booth, 2006; Ischer et al., 2009; Booth & Evans, 2011). However, my results with N. depressus were contrary to this general trend, since I observed a positive correlation between incubation temperature and hatchling length. Similarly, N. depressus eggs incubated at 29°C produced hatchlings with longer and wider carapaces than hatchlings from eggs incubated at 26°C (Hewavisenthi & Parmenter, 2001). Hatchlings incubated at 29°C were also longer than hatchlings from eggs incubated at 32°C (Hewavisenthi & Parmenter, 2001), and hatchlings from eggs incubated at 26°C and 29°C were wider than hatchlings incubated at 32°C (Hewavisenthi & Parmenter, 2001).

Flatback hatchlings are larger than the hatchlings of other sea turtle species, and also have a less streamlined and more circular carapace, so their carapace size may be restricted by the dimensions of the egg. In reality, the size differences experienced by hatchlings produced at different temperatures may not be large enough to be biologically relevant. The current theory is that larger hatchlings may be able to travel through predator rich beach and nearshore environments more quickly (Gyuris, 2000; Chu, 2008), reducing risk of predation, as well as avoiding gape-limited predators (Gyuris, 2000). However, if there is a trade-off between carapace size and the amount of residual yolk in hatchling, then smaller hatchlings will have larger yolk reserves which could sustain them for longer without having to feed (Gutzke et al., 1987; Ischer et al., 2009). The range of nest temperatures observed in the current study was small and hatchlings from a wider range will need to be examined to establish whether the positive relationship between nest temperature and carapace size found in the current study persists. 54

Crawling and self-righting performance

Hatchling self-righting propensity was positively correlated with mean incubation temperature, meaning hatchlings from warmer nests were able to self-right more readily. This correlation is contradictory to what has been recorded in other species, in which hatchlings from warmer nests have lower self-righting propensity scores (Read et al., 2012; Wood et al., 2014). However, it is likely that sub-lethal extreme temperatures have a detrimental effect on hatchling performance at both low and high temperatures. The temperatures I observed in this study were low compared to other in situ sea turtle studies (Maulany et al., 2012a; Read et al., 2012; Wood et al., 2014), due to low air temperatures and high rainfall, so the positive correlation between righting propensity and nest temperature compared to the negative relationship found in previous studies could be due to a difference in the range of nest temperatures experienced in different studies. I did not notice a correlation with either self-righting time or crawling speed, likely due to the narrow range of temperatures in this study.

Sea turtle hatchlings generally do not avoid or fight predators (Gyuris, 1994), therefore moving through the predator gauntlet more quickly may reduce their risk of predation. Hatchlings that can self-right quickly will spend less time on the beach and vulnerable to predation, thus increasing their chance of survival. If a hatchling becomes inverted and cannot successfully self-right, it is likely to die from dehydration or desiccation. Similarly, hatchlings that can crawl more quickly will pass through this predator “gauntlet” more quickly and reduce their risk of predation. Although I did not find any correlation between mean incubation temperature and crawling speed, other studies on other species of sea turtle have found that hatchlings from eggs that experienced high mean temperatures were slower crawlers (Ischer et al., 2009; Maulany et al., 2012a; Wood et al., 2014). It would be interesting to investigate warmer flatback nests to determine whether high nest temperatures (i.e. greater than 32°C) have the same effect.

Swimming performance

In other sea turtle species, mean thrust, mean maximum thrust, time spent powerstroking and powerstroke frequency during a powerstroking bout all decreased as time spent swimming increases (Booth & Evans, 2011; Pereira et al., 2011). I observed a similar pattern in N. depressus. I also found correlations between mean incubation temperature, mean thrust produced and proportion of time spent power-stroking. Therefore it is likely that the difference in mean thrust produced by hatchlings from different nests is caused by the difference in proportion of time spent power-stroking, with hatchlings from cooler nests spending less time power-stroking than hatchlings from warmer nests.

In other sea turtle species, hatchlings from cooler nests are generally better swimmers (Chu, 2008; Booth & Evans, 2011), but again, these studies included higher nest temperatures than I investigated in this study. One constant temperature study found that green turtle hatchlings from eggs incubated at 26°C were consistently poorer swimmers than hatchlings from eggs incubated at 28°C or 30°C (Booth et al., 2004), suggesting that low temperatures, as well as high temperatures, produce inferior hatchlings.

Whereas all other sea turtle species cross the nearshore high-predation “gauntlet” into the safety of the open ocean relatively quickly (Gyuris, 2000; Pilcher et al., 2000), flatback hatchlings remain in the nearshore environment for their entire life (Bolten, 2003). Whereas other species have a vigorous frenzy phase which ensures they move offshore quickly and away from the relatively high density of aquatic predators, flatback hatchlings have a lower stroke rate, proportion of time spent power stroking and mean maximum thrust per powerstroke than green and loggerhead turtles (Pereira et al., 2011). Unlike green and loggerhead turtles, flatback turtles may use their energy more constantly over the first day, since predation pressure may remain relatively constant throughout their swimming frenzy (Pereira et al., 2011). When motivated, flatback turtle hatchlings can swim more quickly than green turtle hatchlings, suggesting they do actively avoid predators (Salmon et al., 2009; Salmon et al., 2010). Flatback hatchlings experienced a sharp decline in the mean maximum thrust within the first two hours of swimming, much more of a decline than loggerhead and green turtles (Pereira et al., 2011). Flatback turtles also spend a smaller proportion of time power-stroking than loggerhead or green turtles, decreasing to only 20% by the end of an 18 hour period – as opposed to loggerhead and green turtles, which continued to spend around 70% of the time power-stroking (Pereira et al., 2011). However, the frenzy phase in flatback turtles lasts 3-4 days (Salmon et al., 2009), compared with 24- 28 hours in other sea turtle species (Wyneken & Salmon, 1992).

Conclusions

This study explores the effect low incubation temperatures on in situ nests, and the results show that low nest temperatures may be just as detrimental to sea turtle locomotor performance as high nest temperatures. It would be interesting to include some high temperature flatback nests to determine whether the effects are the same for this species as for other species.

CHAPTER 5

A COMPARISON OF HATCHLING LOCOMOTOR PERFORMANCE AND SCUTE

PATTERN VARIATION BETWEEN TWO ROOKERIES OF THE FLATBACK TURTLE

(NATATOR DEPRESSUS)

Abstract

Marine turtle species consist of several genetically discrete ‘evolutionarily significant units’ (ESUs) which do not interbreed. I studied flatback turtle (Natator depressus) hatchlings from two rookeries (Mon Repos Conservation Park and Bare Sand Island, Australia) representing two separate ESUs. Turtles from these ESUs differ in several key life history traits, including body size, and I predicted hatchlings would also differ in locomotor performance. I also investigated the proportion of hatchlings with non-modal scute patterns to determine whether this varies between ESUs. I collected newly-emerged hatchlings, and measured mass, carapace length and width, and recorded the scute pattern. I then measured self-righting ability and crawling speed. My results confirmed a difference in hatchling size between the two ESUs, with Mon Repos rookery hatchlings being larger. However the size difference did not translate into a difference in self-righting ability or crawling speed. Mon Repos rookery also produced a larger proportion of hatchlings with major non-modal scute pattern compared to Bare Sand Island rookery. The differences suggest hatchling survival rates may differ between ESUs, and that ESUs should be studied separately when implementing conservation measures.

Introduction

Many species exhibit variation in life history traits among populations (How et al., 1996; Tiwari & Bjorndal, 2000; Du et al., 2005). This variation is often attributed to a differential allocation of resources to optimise fitness for that population in its environment (Brockelman, 1975; McGinley et al., 1987; Stearns, 1993). Most marine turtle species have a wide distribution, comprising many nesting populations which do not interbreed (Norman et al., 1994; Bowen, 2003; Wallace & Saba, 2009). These discrete populations, referred to as ‘evolutionary significant units’ (ESUs (Moritz, 1994)), can be distinguished from each other on the basis of differences in genetic factors, body size, timing of nesting and clutch size (Tiwari & Bjorndal, 2000; Limpus, 2009; Wallace & Saba, 2009).

Female marine turtles produce a large number of eggs, few of which survive to breeding age (Gyuris, 1994; Heppell et al., 2003; Chaloupka & Limpus, 2005). Differences in life history traits between ESUs may influence hatchling survival, resulting in different survival rates. Direct measurements of fitness and survival are difficult to obtain in marine turtles, due to their long lifespan, widely dispersed habitat use, and the difficulty of tracking them through their successive age classes (Booth et al., 2004). Therefore correlates of fitness such as body size and hatchling locomotor performance (self- righting ability, crawling speed and swimming attributes) have been used in hatchling turtles (Booth et al., 2004; Freedberg et al., 2004; Ischer et al., 2009). Measures of terrestrial locomotor performance are used as fitness correlates because they can influence the amount of time a hatchling spends on the beach (Paitz et al., 2010), and increased time on the beach can lead to increased risk of desiccation and predation (Bustard, 1972; Steyermark & Spotila, 2001; Delmas et al., 2007). Consequently, hatchlings that spend more time on the beach may have low survival rates (Dial, 1987; Janzen et al., 2007). Although several studies have investigated fitness correlates of hatchling sea turtles (Booth & Astill, 2001a; Burgess et al., 2006; Pereira et al., 2011), these studies have focussed on single ESUs.

Another proposed indicator of fitness is variation in the number of scutes on the carapace. The modal scute pattern for flatback turtles (Natator depressus) is 1 nuchal, 5 vertebral, 4 pairs costal, 11 pairs of marginal and 1 pair of post-vertebral scutes (Limpus, 1971). Non-modal scute patterns have been reported for all marine turtle species (Hill, 1971; Limpus, 1971; Mast & Carr, 1989), and usually include supernumerary scutes (Zangerl & Johnson, 1957). Non-modal scute patterns are generally more common in hatchling turtles than in adult turtles (Limpus, 1971; Mast & Carr, 1989; Türkozan et al., 2001). The decrease in the proportion of individuals with non-modal scute patterns in breeding adults suggests that fewer turtles with non-modal scute patterns survive to breeding age (Türkozan et al., 2001). It is likely that non-modal scute patterns do not influence survival directly, but are indications of greater internal abnormalities (Mast & Carr, 1989). Non-modal scute patterns may be

58 caused by genetics, environmental conditions during egg incubation or handling of eggs (Hewavisenthi & Parmenter, 2001; Türkozan & Yilmaz, 2007; Velo-Anton et al., 2011), and therefore have the potential to vary among different rookeries.

N. depressus is endemic to the Australian continental shelf (Limpus, 1971) and has been classified into four different ESUs: the Eastern Australian ESU, the Gulf of Carpentaria and Torres Strait ESU, the Western Northern Territory ESU and the North-West Shelf ESU (Limpus, 2009). Several key differences separate these ESUs in terms of size of eggs, hatchlings and females, and timing of nesting. For example, the Western Northern Territory ESU consists of smaller adult females, that lay smaller eggs, that produce smaller hatchlings than the Eastern Australian ESU (Limpus, 1971; Whiting & Guinea, 2003). In addition, turtles in the Western Northern Territory ESU nest in the Austral winter (June - August; Whiting & Guinea, 2003), whereas turtles in the Eastern Australian ESU nest in the Austral spring/summer (November - January; Limpus, 1971).

This paper compares hatchling fitness correlates and proportion of non-modal scute patterns of N. depressus from the Eastern Australian and Western Northern Territory ESUs. I predicted that Eastern Australian ESU hatchlings would be faster at crawling than Western Northern Territory ESU hatchlings, because larger hatchlings have been found to crawl faster than smaller ones from the same clutch (Wren et al., 1998; Chu, 2008; Ischer et al., 2009). I predicted that Western Northern Territory ESU hatchlings would be able to self-right more quickly and more often due to their smaller size making them more manoeuvrable (Booth et al., 2013). Finally, I predicted that the proportion of hatchlings with non-modal scute patterns would differ between the two rookeries because of genetic and environmental differences. Both of these factors have previously been proposed as causes of non- modal scute patterns (Hewavisenthi & Parmenter, 2001; Velo-Anton et al., 2011)

Methods

Study sites

I sampled the Eastern Australian ESU at Mon Repos Conservation Park in south-east Queensland (24°48’S, 152°27’E). This beach is a minor N. depressus rookery, with between one and 13 nesting females per year (Limpus, 2008) and is also the southern limit of nesting for N. depressus in eastern Australia (Limpus, 1971). I sampled the Western Northern Territory ESU on Bare Sand Island in the Northern Territory (12°32’S, 130°25’E). This island is a major N. depressus rookery, with up to 20 nesting females per night during peak nesting months of June and July (Whiting & Guinea, 2003).

Hatchling collection

At Mon Repos I relocated nine clutches laid by seven females into a hatchery area in December 2010. After the nests had been incubating for 50 days, I placed a plastic enclosure around the top of each nest each night at dusk. I checked the enclosures every half hour between dusk and dawn. As soon as I discovered an emerging clutch, I randomly selected up to 30 hatchlings from the first emergence, which I transported to the laboratory in a bucket by foot (a five minute journey). When two clutches emerged simultaneously, I kept each clutch in a separate bucket. I collected a total of 184 hatchlings from these clutches during February 2011.

At Bare Sand Island I sampled 129 hatchlings from seven emerging clutches that I located by patrolling the beach between dusk and dawn during July 2011. Because the clutches emerged over an 10-day period, and the mean renesting interval for this ESU is 14.8 days (Hope & Smit, 1998), I assumed that they were laid by different females. I transported hatchlings in separate buckets for each clutch by foot to a central location on the beach, a journey of less than 10min.

Hatchling measurements

At both rookeries, I weighed the hatchlings (± 0.1g) with a portable balance (AND model EK-1200A), measured the straight carapace length, and width (± 0.1mm) at the widest point with digital callipers (Sontax 150mm digital calliper, China), and calculated the carapace size index (length x width; Ischer et al., 2009). I counted the number of carapace scutes, and photographed the carapace scute pattern. I classified hatchlings into three groups as per Sim et al. (2014): modal scute pattern, minor non- modal patterns (variation in the number of nuchal, marginal and/or post-vertebral scutes) or major non-modal scute patterns (variation in the number of vertebral and/or costal scutes).

Locomotor performance tests

Within the first hour of collection, I began locomotor performance tests. I placed each hatchling upside down on its carapace on a flat area of sand and, using a stopwatch, measured the time taken to self-right. Following previous experimental protocol, hatchlings that failed to self-right within 10s were returned to their plastron for 10s, a period long enough for the hatchling to become re-oriented and begin vigorous crawling again, before the next trial (Booth et al., 2013). Trials continued until the hatchling had successfully self-righted three times, or had attempted self-righting six times, whichever came first. I gave each hatchling a righting propensity score from 0 to 6 using the same method as Booth et al. 2013, in which 0 = no self-rightings in six attempts, 1 = one self-righting in six attempts, 2 = two self-rightings in six attempts, 3 = three self-rightings in six attempts, 4 = three self-rightings in five attempts, 5 = three self-rightings in four attempts and 6 = three self-rightings in 60 three attempts. I averaged self-righting time across successful self-righting events for each hatchling. Twelve hatchlings from nine clutches failed to self-right at all, and they were excluded from the self- righting time data.

Immediately following the self-righting experiment, I measured the plastron surface temperature of the hatchling with an infra-red thermometer (Smart Sensor AR300, ± 1.5°C) to control for the effect of body temperature on locomotor performance (Hutchison et al., 1966; Adams et al., 1989; Elnitsky & Claussen, 2006). I then measured crawling speed using a 2.9m length of black plastic guttering as a raceway (Ischer et al., 2009). The raceway was lined with moist, lightly compacted beach sand and contained a dim light at the seaward end to attract the hatchling and ensure that it crawled in a straight line. If a hatchling did not begin to move within three minutes, I aborted the trial and excluded that hatchling from the analysis. A total of two hatchlings from one clutch were excluded. I timed each hatchling crawling along the guttering. I subjected each hatchling to self-righting and crawling tests (2-3 minutes) before moving on to the next hatchling to ensure plastron surface temperature measurements remained relevant throughout the trials.

Statistics

I used an ANOVA to test for a difference in hatchling size and mass between the two ESUs, with clutch nested within mother as random factors. To test for a difference in locomotor performance between the two ESUs I used an ANCOVA with ESU as a fixed factor, clutch nested within mother as random factors and plastron surface temperature as a covariate. I removed non-significant interactions from the model. To determine whether there was a learning response at the population level I used a paired t-test to determine whether there was a difference between the timing of the first and third self-righting trials.

To test for differences in the proportion of hatchlings with non-modal scute patterns between the two ESUs, I calculated the proportion of hatchlings with the modal, minor non-modal and major non- modal scute patterns for each clutch. I transformed the data using an arcsine transformation and used an ANOVA with ESU as the fixed factor and mother as a random factor. I performed data analysis using R (R Development Core Team, version 2.15.0, 2013). I report data as means and standard errors of means or as least squares covariate means and assume statistical significance if p < 0.05.

Results

N. depressus hatchlings from Mon Repos were greater in mass (F1,297 = 39.59, p < 0.001), and longer

(F1,297 = 14.15, p = 0.002) and wider (F1,297 = 10.77, p = 0.006) in carapace than hatchlings from Bare Sand Island (Table 5.1). Plastron surface temperature, self-righting propensity, mean self-righting 61 time and crawling speed did not differ between the two ESUs (Table 5.1). There was no difference in self-righting time between the first and the third trial (t(256) = 1.42, p = 0.16).

Mon Repos Bare Sand Island F statistic p value

Mass (g) 42.2 ± 1.4 33.1 ± 1.0 39.59 < 0.001

Carapace length (mm) 60.5 ± 0.8 57.4 ± 0.6 14.15 0.002

Carapace width (mm) 53.2 ± 1.1 49.6 ± 0.8 10.77 0.006

Carapace size index (mm2) 3227 ± 103 2855 ± 74 12.94 0.004

Body temperature (°C) 26.2 ± 1.0 27.3 ± 0.7 1.28 0.280

Self-righting propensity 4.7 ± 0.1 5.0 ± 0.1 1.35 0.267

Self-righting time (s) 3.5 ± 0.1 3.3 ± 0.1 0.76 0.401

Crawling speed (m/s) 7.5 ± 0.2 6.9 ± 0.3 0.58 0.461

A higher proportion of hatchlings from Mon Repos nests exhibited major non-modal scute patterns than at Bare Sand Island (F1,12 = 13.42, p < 0.01; Table 5.2), whereas there was no difference in the proportion of hatchlings with minor non-modal scute patterns and proportion of hatchlings with the modal scute pattern between the ESUs (Table 5.2).

Mon Repos Bare Sand Island F statistic P value

Modal scute pattern 0.42 ± 0.08 0.65 ± 0.08 3.64 0.08

Minor non-modal scute patterns 0.17 ± 0.05 0.32 ± 0.09 2.41 0.15

Major non-modal scute patterns 0.41 ± 0.08 0.04 ± 0.03 13.45 0.003

Discussion

Hatchling size

N. depressus hatchlings from Mon Repos were significantly heavier and had longer, wider carapaces than hatchlings from Bare Sand Island, something that has been reported previously (Limpus, 1971; Whiting & Guinea, 2003). Similar differences in hatchling size have been found between different ESUs of loggerhead (Caretta caretta), green (Chelonia mydas) and leatherback (Dermochelys coriacea) turtles (Tiwari & Bjorndal, 2000; Limpus, 2009; Eckert et al., 2012). The difference in body size between the ESUs may be due increased predation pressure at Mon Repos Conservation Park causing selection for larger-sized turtles, or a greater amount or more nutritious food available to the Mon Repos nesting turtles, allowing them to grow larger and produce larger eggs and hatchlings.

Hatchling size can affect survivorship due to its effect on locomotor performance or avoidance by gape-limited predators. For example, when C. caretta and N. depressus turtle hatchlings occur on the same beach, the smaller C. caretta hatchlings are depredated by silver gulls (Chroicocephalus novaehollandiae) whereas the larger N. depressus hatchlings are ignored (Limpus, 1973). Experiments on C. mydas have suggested that larger hatchlings are less likely to be depredated by fish in the near-shore environment (Gyuris, 2000). N. depressus hatchlings are unique in that they remain in the near-shore environment instead of migrating into the open ocean like other sea turtle species (Limpus, 1971; Walker & Parmenter, 1990). If predation pressure is similar at both rookeries, then the predation rate may be higher for Bare Sand Island hatchlings, resulting in lower survivorship. Larger hatchlings may also be able to maintain their position in coastal waters better, and avoid being swept away by currents. Again, I would need to investigate the environmental conditions at each site to determine which group would be at an advantage.

Scute pattern variation

Nests from Bare Sand Island had a lower proportion of hatchlings with major non-modal scute patterns than nests from Mon Repos. Several hypotheses have been suggested to explain non-modal scute patterns, including genetic factors, inbreeding, disturbance to eggs, pollution and incubation temperature (Mast & Carr, 1989; Hewavisenthi & Parmenter, 2001; Velo-Anton et al., 2011). In this study the Mon Repos clutches were relocated and the Bare Sand Island ones were not. There is some evidence that non-modal scute patterns are more common in relocated nests (Mast & Carr, 1989) which could explain the discrepancy between rookeries. Incubation temperature may also be a factor, as constant temperature experiments on N. depressus have suggested that non-modal scute patterns

63 are produced at lower temperatures (Hewavisenthi & Parmenter, 2001). Mon Repos Conservation park is the southern-most limit for N. depressus on the east coast of Australia (Limpus, 2009), making it likely that incubation temperatures were lower at Mon Repos than at Bare Sand Island during my study, which could explain the discrepancy.

There is evidence to suggest that non-modal scute patterns indicate lower quality hatchlings, meaning fewer survive to reach maturity (Türkozan et al., 2001). A previous study showed that N. depressus hatchlings with the modal scute pattern out-performed hatchlings with major non-modal scute patterns during the first twenty minutes of swimming (Sim et al., 2014). This suggests a greater number of hatchlings from Mon Repos are succumbing to predation or exhaustion during their swim through the near-shore environment. However, additional research needs to be done on how non- modal scute patterns affect long-term fitness and survival of hatchlings.

Locomotor performance

Despite differences in hatchling size between the two rookeries, I found no difference in either self- righting ability or crawling speed between the two ESUs. Sea turtle hatchlings self-right by flexing their heads against the substrate, which allows the carapace to be raised off the ground, and the hatchling to flip upright (Booth et al., 2013). Although a weak negative correlation between carapace size and time taken to self-right has been observed in C. mydas (Booth et al., 2013), several other studies have suggested incubation temperature or maternal effects are the main drivers of self-righting ability (Steyermark & Spotila, 2001; Delmas et al., 2007; Booth et al., 2013). It is likely that self- righting ability is governed by physiological, morphological and behavioural components and cannot be solely attributed to one variable like body size.

Larger turtle hatchlings typically crawl more quickly, presumably due to their greater stride length (Wren et al., 1998; Chu, 2008; Ischer et al., 2009). However all of these studies focussed on a single ESU only, rather than comparing ESUs that differ in body size like I did in this study. The lack of a difference in crawling speed in this study suggests that the smaller hatchlings from Bare Sand Island compensate for their smaller stride length, most likely by having a greater stride rate.

Conclusion

Despite differences in hatchling size between the two ESUs, I didn’t observe any differences in terrestrial locomotor performance. This suggests that hatchlings from each ESU deal with terrestrial locomotion differently. The difference in the proportion of hatchlings with major non-modal scute patterns is also an interesting finding, although more research is needed into the causes. Because of

64 these differences it is important to study several ESUs of a species before implementing large-scale conservation efforts.

CHAPTER 6

GENERAL DISCUSSION

Fitness and survival of oviparous reptile hatchlings is influenced at the embryonic stage by environmental incubation conditions. The natural environment in which eggs develop can vary significantly in terms of temperature, humidity and other environmental variables. Incubation temperature is one of the most pervasive environmental factors at the incubation stage, and has been demonstrated to affect hatchling fitness through size (Ji et al., 2002; Booth et al., 2004), body shape (Mickelson & Downie, 2010), growth rate (Brooks et al., 1991; O'Steen, 1998; Rhen & Lang, 1999; Du & Ji, 2003; Booth et al., 2004) and locomotor performance (Janzen, 1993b; Doody, 1999; Du & Ji, 2003; Booth et al., 2004). Environmental conditions vary at different rookeries, giving the potential for hatchlings from different rookeries to have differing fitness (Hays et al., 2001; Read et al., 2012). One proposed indicator of hatchling quality is the level of fluctuating asymmetry, or deviation from bilateral symmetry (Leung & Forbes, 1997). In this thesis, I investigated three factors that influence hatchling quality in marine turtles: non-modal scute patterns as an example of fluctuating asymmetry, incubation temperature and rookery origin. The key results of these studies are summarised below.

Non-modal scute patterns

Fluctuating asymmetry is an indicator of stress during development (Allenbach et al., 1999; Ji et al., 2002; Soderman et al., 2007). High levels of fluctuating asymmetry generally indicate lower quality organisms (Moller, 1997; Figure 1.1). In chelonians non-modal scute patterns are a common example of fluctuating asymmetry. In marine turtles the proportion of turtles with non-modal scute patterns is greater in hatchlings than in adult turtles in the same population in almost all cases studied (Limpus, 1971; Hughes, 1972, 1974; Limpus et al., 1983; Türkozan et al., 2001; Ergene et al., 2011). Because of this discrepancy, it has been suggested that non-modal scute patterns indicate low quality hatchlings, fewer of which survive to adulthood (Türkozan & Yilmaz, 2007). In Chapter 2, I tested this hypothesis in Caretta. caretta and Natator depressus hatchlings. The proportions of hatchlings with non-modal scute patterns that I observed in this study were similar to those observed in other species all over the world (34.1 - 78.1%; Mast & Carr, 1989; Özdemir & Türkozan, 2006; Ergene et al., 2011). While the proportion of turtles with non-modal scute patterns is generally greater in hatchling turtles than in adult turtles, unfortunately there is no substantial data in the literature on post-hatchling, juvenile and sub-adult turtles, so it is difficult to determine when the discrepancy occurs.

I show that hatchlings with non-modal scute patterns are more likely to die during development, and have a reduced body size and mass, compared to hatchlings with the modal scute pattern. Thus, non- modal scute patterns may be indicators of underlying abnormalities or otherwise low quality hatchlings. Smaller sized hatchlings may be slower locomotors, and are more vulnerable to gape- limited predators (Gyuris, 2000). Hatchling locomotor performance trials indicate, however, that there are no differences in self-righting ability or crawling speed between hatchlings with non-modal scute patterns and hatchlings with the modal scute pattern for both C. caretta and N. depressus, although non-modal scute pattern hatchling swimming performance was lower than that of modal scute pattern hatchlings for N. depressus. As a consequence, there may be differential survival of N. depressus hatchlings with hatchlings with non-modal scute patterns being exposed to near-shore predators longer than hatchlings with the modal scute pattern. Hence, a combination of smaller size and slower swimming may result in hatchlings non-modal scute patterns having a proportionally greater mortality and thus result in a relative decease in their abundance in the adult population. This adds to the body of knowledge on both hatchling quality and fluctuating asymmetry.

Incubation temperature

In Chapters 3 and 4, I investigated the effect of in situ incubation temperature on hatchling quality in both C. caretta and N. depressus. For C. caretta, I tested the hypothesis that hatchlings from nests with mean three day maximum temperatures (T3dm) above 34°C would be lower quality compared to hatchlings from nests with T3dm below 34°C. In Chapter 3, I provide support for this prediction; hatchlings from nests with a T3dm above 34°C possessed smaller carapaces than hatchlings from nests with a T3dm below 34°C. In locomotor performance trials, hatchlings from nests with a T3dm below 34°C crawled faster than hatchlings from nests with a T3dm above 34°C, even after body size was controlled for. In swimming performance trials hatchlings from nests with a T3dm below 34°C produced a greater thrust than hatchlings from nests with a T3dm above 34°C during the first twenty minutes of swimming, which is the most dangerous time in terms of predation (Gyuris, 1994; Pilcher et al., 2000; Salmon et al., 2009). These findings suggest that even though time spent above 34°C in the late stages of incubation may not be fatal, it can have a detrimental effect on the physiology of the developing embryo, causing decreased locomotor performance and ultimately could lead to decreased survival (Dial, 1987).

Chapter 4 of this thesis focused on the effects of incubation temperature on N. depressus hatchling fitness. In this study there were no nests with a T3dm above 34°C, so mean temperature over the entire incubation period was instead used as a proxy for relative incubation temperature. I tested the hypothesis that hatchlings from warmer nests would be lower quality than hatchlings from cooler

67 nests. A positive correlation, however, was identified between hatchling size and mean incubation temperature; these results are contrary to my hypothesis and previous work on other sea turtle species (Ischer et al., 2009; Wood et al., 2014). The range of mean nest temperatures observed in Chapter 4 was, however, small and hatchlings from nests that experience a wider range of temperatures will need to be examined to establish whether the positive relationship between mean incubation temperature and carapace size persists. When locomotor performance was tested, hatchling self- righting propensity, mean swimming thrust produced and proportion of time spent power-stroking were positively correlated with mean incubation temperature. Thus, the results of this study show that low nest temperatures may be just as detrimental to sea turtle locomotor performance as high nest temperatures.

Both chapters 3 and 4 demonstrate that incubation temperatures towards either boundary of the viable thermal range can have detrimental effects on hatchling quality. With projected anthropogenic climate change, air temperatures will increase (Collins et al., 2013), causing nest temperatures to increase (Hawkes et al., 2009; Fuentes et al., 2011). However, climate change will also cause more frequent extreme storm events and increased rainfall (Bonebrake & Mastrandrea, 2010; Schwalm et al., 2011), which cause nest temperatures to decrease (Houghton et al., 2007). Thus, in the future it is likely that more sea turtle nests will experience temperatures close to the edge of the viable thermal range, producing sub-optimal hatchlings.

Rookeries

Variation in life history traits among populations occurs in many species (How et al., 1996; Du et al., 2005), and may be due to a differential allocation of resources to optimise fitness for that population in its environment (Brockelman, 1975; Stearns, 1993). In Chapter 5 I tested the hypothesis that hatchlings from two N. depressus rookeries, representing two different ‘evolutionary significant units’ (ESUs) would differ in terms of size, locomotor performance and proportion with the modal scute pattern. I found differences in proportion with non-modal scute patterns and hatchling size, but no differences in terrestrial locomotor performance. Thus, hatchlings locomotor performance may not be solely size-dependent and smaller hatchlings can potentially compensate for smaller stride length by increasing stride rate.

Further research

For future studies, it would be useful to compare other attributes that may affect hatchling survival, such as growth rate and prolonged swimming performance between hatchlings with the modal scute pattern and hatchlings with non-modal scute patterns. These factors may explain differences in the

68 proportion of turtles with non-modal scute patterns as adults and hatchlings, because selection may be occurring after the first few hours. It would also be useful to investigate the proportion of turtles with the modal scute pattern at the different life stages between hatchling and breeding adult. Another potential avenue of research would be to dissect the hatchlings to determine whether non-modal scute patterns are indicative of abnormalities in the internal anatomy of hatchlings with non-modal scute patterns (Mast & Carr, 1989).

Chapters 3 and 4 investigated low to moderate nest temperatures in N. depressus and moderate to high nest temperatures in C. caretta. It would appear that there is a classic reaction norm of exercise performance in sea turtle hatchlings, with low and high temperatures resulting in sub-optimal performance compared to moderate temperatures. Hence, experiments need to be continued, investigating low to moderate nest temperatures in C. caretta and moderate to high nest temperatures in N. depressus. I would expect low temperatures to be detrimental to C. caretta hatchlings, because this is the case in Chelonia mydas (Booth et al., 2004) and those two species share many life history traits (Limpus, 2009). Since the positive correlation between incubation temperature and hatchling size that I found in Chapter 4 was contrary to all other sea turtle studies (Booth et al., 2004; Ischer et al., 2009; Wood et al., 2014), an investigation as to the effects of extreme high incubation temperatures on N. depressus would determine whether this correlation continues even at extreme high temperatures.

Finally, I found differences between N. depressus hatchlings from two rookeries representing two different ESUs. This highlights the need for studies on other sea turtle species at multiple rookeries in order to understand species inter-rookery differences and drivers of selection in different environments.

In all of the experiments in this thesis I have used attributes at hatching or within hours of hatching as a proxy for hatchling fitness. Cheloniid sea turtles spend between 21 and 34 years at sea before returning to breed (Limpus, 2009), so it is likely that selection is also acting on turtles after the initial post nest emergence period. It is logistically difficult to investigate parameters of fitness in juvenile turtles, because they live in the open ocean. Long-term captive experiments, for example extended swimming performance trials, or growth rate studies are a possibility and could provide some information (Stokes et al., 2006). Incubation temperature has been shown to affect hatchling growth rate in freshwater turtles (Brooks et al., 1991; Booth et al., 2004), so it would be useful to study growth rate in sea turtles, because this may affect survival later in life.

The mechanisms underlying incubation temperature induced differences in locomotor performance of sea turtle hatchlings are currently unknown, but several possibilities have been suggested, 69 including physiological impairments of muscle physiology, tissue aerobic capacity or metabolic efficiency due to effects on the hypothalamus (Micheli-Campbell et al., 2011), differences in the maturity of neuromuscular signalling pathway due to decreased incubation period (Ischer et al., 2009), or differences in the physiological properties of muscles (Freedberg et al., 2004; Chu, 2008). If we could determine the mechanism(s), we may be able to extrapolate and determine the effects on juvenile and adult turtle survival. This information could be obtained by controlled incubation temperature experiments in which eggs from several different clutches (to control for possible clutch effects) are incubated across a range of temperatures and the cardiovascular and muscle physiology and morphology of resultant hatchlings investigated. Sex hormones and sex-hormone inhibitors should also be applied to some of the eggs during incubation so that any potential physiological/morphological effects can clearly be assigned to incubation temperature effects and not sex difference effects since sea turtles exhibit temperature dependent sex determination which would confound each other if only incubation temperature was manipulated.

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