ASSESSING THE COMPOSITION OF GREEN TURTLE
(Chelonia mydas) FORAGING GROUNDS IN
AUSTRALASIA USING MIXED STOCK ANALYSES
By
MICHAEL PAUL JENSEN
B.Sc. (University of Aarhus) (2001)
M.Sc. (University of Aarhus) (2005)
Institute for Applied Ecology
Faculty of Applied Science
University of Canberra
Australia
A thesis submitted in fulfilment of the requirements of
the Degree of Doctor of Philosophy at the University of
Canberra.
March 2010
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Assessing the composition of green turtle (Chelonia mydas) foraging grounds in
Australasia using mixed stock analyses.
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This thesis (© by Michael P. Jensen, 2010) may be freely copied or distributed for private and/or commercial use
and study. However, no part of this thesis or the information herein may be included in a publication or referred to
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acknowledged.
ii
ACKNOWLEDGEMENTS
This thesis results from three and a half years of research carried out since I came to Australia in
June 2006. It has been a fantastic ride, with fieldwork at some of the most beautiful places on
earth. I have worked with a great number of people, whose contribution in assorted ways to
both the research and the preparation of this thesis deserve special mention. It is a pleasure to
convey my gratitude to you all in my humble acknowledgment.
In the first place, I would like to express my sincere gratitude to my primary supervisor, Nancy
FitzSimmons, for her advice and guidance from the onset of this research, as well as giving me
extraordinary experiences throughout the whole process. Above all, and most importantly, she
provided me unflinching encouragement, trust and support during my time in Australia. Her
true passion for science and marine turtles in particular, has made her as a constant oasis of
ideas, which exceptionally inspired and enriched my growth as a student, a researcher and as a
person. She became my mentor and my friend, and I am indebted to her more than she can
know.
I gratefully acknowledge also my secondary supervisor, Col Limpus, for his advice, ideas and
support. He contains a wealth of knowledge and wisdom when it comes to marine turtles and he
has a remarkable way of asking questions that has made me rethink my research and ideas,
always to the benefit of the project.
During this work I have collaborated with many colleagues for whom I have great regard. I
wish to extend my warmest thanks to all those who have helped me with my work, from
sending me samples, helping with fieldwork, providing comments and advice, to being a great
inspiration.
Another special thanks goes to my friend and colleague Ian Bell. In June 2007, he somewhat
sceptically, agreed to let a Danish geneticist come along on one of his fieldtrips to the remote
northern Great Barrier Reef. Since then, he has contributed so much and given me unconditional support during several fieldtrips, which made him a vital part of this research and so
of this thesis. I hope he doesn‟t regret his decision. His originality and confronting questions
have triggered and nourished an intellectual maturity that I will benefit from for a long time to
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come. Ian, I am grateful in every possible way and hope to keep up our collaboration in the
future.
It is a pleasure to pay tribute also to all those who helped provide me with samples. To Mick
Guinea and Scott Whiting who have taken time during their own fieldwork to collect genetic
samples and to Nick Pilcher who included me in his interesting research of Malaysian green
turtles. Many thanks also to Mark Hamann, Frank Loban and Steven Ambar and the rest of the
mob up in Torres Strait. I am much indebted to all of them for their valuable contributions, from
the planning of the project to the many days of fieldwork. Thanks to Kelly Pendoley for the
most recent samples from Western Australia and to Bob Prince for the original WA collection.
I would also acknowledge my fellow students and colleagues at the Institute for Applied
Ecology; Anett Richter, Kate Hodges, Stewart Pittard, John Roe, Wendy Dimond, David Wong,
Carla Eisemberg, Alex Quinn, Anna MsDonald, Tariq Ezaz and Marion Hoehn for their moral
support and their friendship. A special thank you goes to Niccy Aitken, first and foremost for
her friendship, but I have also benefited from her advice and guidance. She always kindly
granted me her time, even to answer some of my naive questions about lab work. She also
provided useful comments to the manuscript. I convey special acknowledgement to the office
staff Kerrie, Sam and Tara for all their help dealing with travel funds, administration and
bureaucratic matters during my stay and in particular with my commute between Canberra,
Denmark, the Great Barrier Reef and the rest of the world so I could more easily carry out my
research and travels.
I am very grateful to Alberto Abreu-Grobois, my masters supervisor, who remains my mentor
and provides great inspiration for the way I go about doing research. He always takes time to
provide his advice and his bright thoughts are always very fruitful for shaping my ideas and
research.
I humbly thank my thesis reviewers that in the midst of all their activity, they accepted to be
members of the review committee.
A big thank you to the “turtle gang”, Suzanne Livingstone, Jason van de Merwe, Mark Trodoir,
Colette Wabnitz, and Dave Wayers for their friendship and for their advice and support. To Sam
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Emerick, thank you for your support and for all your fruitful comments on the manuscript. I
would like to thank the sea turtle community for making me part of a very inspirational group of
people.
Collective and individual acknowledgments are also owed to the many volunteers who took
time to help with fieldwork. Thanks to Brian McNeill, Lachlan Duff, Sam Dibella, Tony
Mitchell (Barney), Sarah Vargas, Anelise Hahn, Stewart Pittard, Klaus Karlsen, Maren
Lyngsgaard and many others too numerous to name. They made fieldwork fun and many new
friendships were formed during the many weeks of turtle work.
Where would I be without my family? My parents deserve special mention for their steady
support. My Father, Henning, is the person who gave me the passion for nature from when I
was a child. My Mother, Ulla, is the one who sincerely raised me with her loving support in
everything I do. Thanks to my sister Susanne, her husband Cristòbal, and my niece and nephew
Isabel and Cris, for being supportive and caring. Without their encouragement and
understanding it would have been impossible for me to finish this work.
Finally, I would like to thank everybody who contributed to the successful realisation of this
thesis, as well as expressing my apology that I could not mention personally each of you one by
one.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...................................................................................................... i
TABLE OF CONTENTS ........................................................................................................ iv
LIST OF FIGURES ................................................................................................................. vi
LIST OF TABLES .................................................................................................................. vii
ABSTRACT .............................................................................................................................. 1
1. Introduction to the Thesis ....................................................................................................... 4
1.1 Sea turtles of the world..................................................................................................... 4
1.1.1. Lifecycle of Sea Turtles ....................................................................................... 5
1.1.2. Green turtles (Chelonia mydas)............................................................................ 8
1.1.3. Green turtle population genetics .......................................................................... 8
2. Review: Mixed Stock Analysis and marine turtles .............................................................. 12
2.1 Introduction .................................................................................................................... 12
2.2 Mixed Stock Analysis .................................................................................................... 14
2.3 MSA and Marine turtles ................................................................................................. 15
2.3.1. Loggerhead turtles (Caretta caretta).................................................................. 17
2.3.2. Hawksbill turtles (Eretmochelys imbricata) ...................................................... 18
2.3.3. Green turtles (Chelonia mydas).......................................................................... 19
2.3.4. Complex life history ........................................................................................... 20
2.3.5. Rookery size and distance .................................................................................. 20
2.3.6. Ocean Currents ................................................................................................... 25
2.3.7. Temporal differences.......................................................................................... 26
2.3.8. Gender differences ............................................................................................. 27
2.3.9. Size class differences ......................................................................................... 28
2.4 Limitations with MSA .................................................................................................... 28
2.4.1. Sample size ......................................................................................................... 29
2.4.2. Source populations ............................................................................................. 29
2.4.3. Resolution of genetic markers ............................................................................ 30
2.5 Conservation and Management ...................................................................................... 32
3. Influences of larger sample sizes and longer sequences for defining Management Units and
estimating stock composition of green turtle foraging grounds ............................................... 34
3.1 Introduction .................................................................................................................... 34
3.2 Materials and Methods ................................................................................................... 37
3.2.1. Sample collection ............................................................................................... 37
3.2.2. Characterization of mtDNA haplotypes ............................................................. 38
3.2.3. Molecular analysis.............................................................................................. 39
3.2.4. Mixed Stock Analysis ........................................................................................ 42
3.3 Results ............................................................................................................................ 42
3.3.1. Rookery diversity ............................................................................................... 42
3.3.2. Population differentiation ................................................................................... 44
3.3.3. Foraging aggregations ........................................................................................ 51
3.3.4. MSA ................................................................................................................... 52
3.4 Discussion ...................................................................................................................... 54
3.4.1. Rookery diversity and differentiation ................................................................ 54
3.4.2. Foraging aggregations and MSA........................................................................ 56
3.4.3. Management implications .................................................................................. 57
4. Origin of immature green turtles (Chelonia mydas) at two foraging grounds in Sabah,
Malaysia. .................................................................................................................................. 59
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4.1 Introduction .................................................................................................................... 59
4.2 Materials and Methods ................................................................................................... 63
4.2.1. Study site and sample collection ........................................................................ 63
4.2.2. Characterisation of mtDNA haplotypes ............................................................. 63
4.2.3. Mixed Stock Analysis ........................................................................................ 64
4.3 Results ............................................................................................................................ 65
4.3.1. Mixed Stock Analysis ........................................................................................ 67
4.4 Discussion ...................................................................................................................... 69
5. Stock composition of green turtle (Chelonia mydas) foraging grounds of the Great Barrier
Reef: implications of variation across latitude and size classes ............................................... 73
5.1 Introduction .................................................................................................................... 73
5.2 Materials and Methods ................................................................................................... 75
5.2.1. Study site and sample collection ........................................................................ 75
5.2.2. Sample collection ............................................................................................... 77
5.2.3. Characterisation of mtDNA haplotypes ............................................................. 78
5.2.4. Mixed Stock Analysis ........................................................................................ 79
5.2.5. Migration data from mark-recapture studies ...................................................... 80
5.3 Results ............................................................................................................................ 81
5.3.1. Haplotype diversity along the GBR foraging grounds ....................................... 81
5.3.2. Mixed stock analysis and mark-recapture .......................................................... 86
5.4 Discussion ...................................................................................................................... 92
5.4.1. MSA and mark-recapture tagging data .............................................................. 92
5.4.2. Latitudinal comparisons ..................................................................................... 93
5.4.3. Age class variation ............................................................................................. 94
5.4.4. Conservation implications .................................................................................. 96
6. Synopsis. .............................................................................................................................. 98
Conservation management and implications.................................................................. 100
Future research ............................................................................................................... 101
Modelling ....................................................................................................................... 102
REFERENCES ..................................................................................................................... 103
APPENDIX 1......................................................................................................................... 118
APPENDIX 2......................................................................................................................... 127
APPENDIX 3......................................................................................................................... 129
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LIST OF FIGURES
Figure 1.1. Generalised lifecycle for most species of marine turtles. Species and populations
vary mainly in the duration of the different phases. The figure is divided into
hatchling/juvenile (light grey line), female (dark grey line) and, male (black line) migration.
6
Figure 2.1. Model of random mixing of pelagic turtles and subsequent random recruitment
into benthic foraging grounds that are genetically similar.
22
Figure 2.2. Model of random mixing of pelagic turtles and subsequent selective recruitment
into benthic foraging grounds near natal rookeries, leading to genetic structure among
foraging grounds.
23
Figure 2.3. Model of non-random mixing of pelagic turtles due to oceanic currents and
subsequent selective recruitment into benthic foraging grounds near natal rookeries, leading to
genetic structure among foraging grounds and among pelagic turtles in different regions.
24
Figure 2.4. Simulation of a hypothesised foraging aggregation where a) all rookeries are
completely differentiated and b) rookeries share common haplotypes.
31
Figure 3.1. Schematic of the mtDNA control region in marine turtles and the location of the
short sequence used in Dethmers et al. (2006) (primers TCR5 and TCR6) and the long
sequence used in this study (primers LTEi9 and H950).
39
Figure 3.2. Sampling locations from nesting sites in eastern Indian Ocean and Southeast Asia
with the UPGMA tree of the genetic relationship between rookeries.
41
Figure 3.3. Haplotype network based on maximum parsimony for the short a) and the long b)
sequence.
45
Figure 3.4. Rarefraction curves for each Mus. X-axis shows the sample size and Y axis the
number of haplotypes. The graph shows the mean and the SD.
50
Figure 4.1. Map showing the 17 genetic stocks used as reference for tracing back the origin of
turtles (adapted from Dethmers et al. 2006) and the location of the two study areas, Mantanani
Island and Layang Layang Islands.
62
Figure 5.1. Map showing the location of 17 genetically differentiated breeding stocks initially
included in the MSA analysis and the six foraging grounds analysed for stock composition
76
Figure 5.2. Map showing the location of the six foraging grounds and the mean relative
contribution of nGBR, sGBR/Coral Sea and “other” stocks for each group sampled within the
foraging grounds.
90
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LIST OF TABLES
Table 2.1. Studies using Mixed Stock Analysis of marine turtle foraging aggregations;
categorised by species and region.
16
Table 3.1. Distribution of C. mydas haplotypes sampled at 15 rookeries in the eastern Indian
Ocean and Southeast Asia.
46
Table 3.2. Diversity estimates for 17 green turtle MUs and rookeries, including sample size
(n), number of haplotypes (H) haplotype diversity (h) and nucleotide diversity (π).
47
Table 3.3. Analysis of molecular variance (AMOVA) results for 11 green turtle east Indian
Ocean Management Units and the Coburg Island rookery
47
Table 3.4. Genetic differentiation (FST) among MUs and rookeries based on haplotype
frequencies using the long sequence (below diagonal) and estimates of the number of
migrants per generation (Nm; above diagonal).
48
Table 3.5. Estimated P values from exact test of population differentiation among east Indian
Ocean MUs and the Coburg Peninsula rookeries based on haplotype frequencies using the
short sequence (above diagonal) and the long sequence (below diagonal).
49
Table 3.6. Distribution of C. mydas haplotypes sampled at two foraging grounds, Shark Bay
and Cocos (Keeling) Islands.
51
Table 3.7. Contribution of regional stocks to two foraging aggregations at Shark Bay and
Cocos (Keeling) using the long (L) and short (S) sequence.
53
Table 4.1. Haplotype frequencies of nine nesting populations that either had a mean
contribution of (≥ 1%) in mixed stock analysis to the two foraging grounds (FG) or shared
several haplotypes with the FGs.
66
Table 4.2. Estimates of the rookery origin of immature green turtles foraging at Mantanani
Island and Layang Layang Island, based on short sequence.
68
Table 4.3. Estimates of the rookery origin of immature green turtles foraging at Mantanani
Island and Layang Layang Island based on long sequence.
69
Table 5.1. Haplotype composition of the eight stocks used for the MSA and the six foraging
grounds analysed; TS = Torres Strait, CR = Clack Reef, HG = Howicks Group, EB =
Edgecombe Bay, SB = Shoalwater Bay, and MB = Moreton Bay, separated by sampling year
and size class (A = adults, SA = sub-adults and J = juveniles).
82
Table 5.2. Estimates of haplotype (h) and nucleotide diversity (π) within eight green turtle
genetic stocks used for the MSA and six foraging grounds for groupings of adult and
immature turtles.
85
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Table 5.3. Analysis of molecular variance (AMOVA) results for the southern and northern
groups of green turtle foraging and nesting sites.
86
Table 5.4. Results from the Bayesian MSA for six green turtle foraging grounds (FG); TS,
Torres Strait; CR, Clack Reef; HG, Howicks Group; EB, Edgecombe Bay; SB, Shoalwater
Bay and MB, Moreton Bay.
88
viii
ABSTRACT
Understanding the population dynamics in both breeding and foraging habitats is a vital part
of assessing the long-term viability of any species, especially those that are highly migratory.
This is particularly true for green turtles, Chelonia mydas, which are long-lived marine turtles
that undergo migrations for several years as post hatchlings until they select foraging grounds,
and as adults, migrate between their foraging grounds and nesting beaches. Monitoring of
populations at the foraging grounds may help detect early signs of population trends that
would otherwise take decades to be observed at the nesting beach. In order to gain such
insights the connectivity between nesting and foraging habitats must be established. Genetic
analysis of rookeries to define discrete populations (stocks), in combination with Mixed Stock
Analysis (MSA) based on data from molecular markers, provides an effective approach for
estimating the origin of turtles sampled away from their nesting beach.
In this thesis, new investigations into the genetic structure of green turtle populations in
Australasia were conducted using longer (~780 bp) mitochondrial (mt) DNA sequences,
larger sample sizes and new locations. This information provided the baseline data used in
Mixed Stock Analyses of the composition of foraging grounds in three regions of Australasia
including Western Australia, the Great Barrier Reef (GBR) and Malaysia.
In chapter 2, I review what has been learned since the first MSA studies in marine turtles
more than a decade ago. Since the early 1990‟s, numerous studies used this method to
elucidate the rookery origins of young pelagic stage turtles and of older turtles in benthic
foraging grounds, in fisheries by-catch and in strandings. These studies have all shown how
Mixed Stock Analysis has provided valuable new insights into the distribution of marine
turtles, although in most cases the estimates are affected by large uncertainty. Several issues
in the effective use of MSA need to be addressed concerning study design, sample sizes and
the resolution provided by the genetic marker. Nonetheless, Mixed Stock Analysis holds great
potential for monitoring population trends at oceanic and coastal foraging grounds for all size
classes. Comparisons of adults and juveniles provide an opportunity to pick up early signs of
shifts in the contributions of populations that may indicate population decline (or increase)
(e.g., Chapter 5).
1
Recent increases in industrial development of coastal island and offshore habitats in Western
Australia (WA) have highlighted the need to better understand the dynamics of marine turtle
populations in these areas. An analysis of previously sampled populations (Management
Units; MUs) and four new rookeries identified two possible new Management Units in this
region at Cobourg Peninsula and Cocos (Keeling) Island and grouped Browse Island with the
existing MU at Scott Reef and Barrow Island to the large North West Shelf MU. These
analyses used a 780 bp sequence of the mtDNA control region that encompassed the 386 bp
sequence used in a previous study. The longer sequence, larger sample sizes and new
locations revealed more than doubled the number of haplotypes (n = 39) than previously
observed. However, this made little difference to the population genetic structure as common
haplotypes were still shared among population. MSA showed that the majority (>90%) of
turtles foraging at Shark Bay were from neighbouring North West Shelf rookeries, while the
Cocos (Keeling) foraging ground was composed of turtles mainly from Cocos (~70%), but
with some contributions from North West Shelf and possibly Scott Reef MUs.
In an investigation of foraging populations in Malaysia, mtDNA sequence data were analysed
from 81 immature green turtles at two foraging grounds at Mantanani Island and Layang
Layang Island located northwest of Sabah, Malaysia. Previously published data from 17
Australasian green turtle populations were used as the baseline data for tracing back the origin
of turtles at the two foraging grounds. The majority of these turtles originated from major
rookeries in the Malaysia and Philippine Turtle Islands (~30%), and Sarawak (~60%) in
north-western Borneo. These same rookeries have a long tradition of using unshaded beach
hatcheries that has resulted in the production of mostly female hatchlings. This may have
contributed to the 1:4 female biases seen at the two foraging grounds. The implications of
hatchery practises at nesting beaches are discussed and the importance of continued
monitoring and research at these foraging areas is highly recommended to improve the
management of marine turtles in the region.
Detailed MSA of green turtle aggregations at six major foraging grounds along the east coast of
Australian were combined with data from more than 30 years of mark–recapture efforts along
the Great Barrier Reef. Overall, the MSA in combination with the mark-recapture data supports
a model in which the foraging aggregations are composed of individuals from the two Great
Barrier Reef stocks (nGBR, sGBR) with small contributions from other stocks. The north/south
2
transect of foraging grounds analysed spanned ~2300 km. Along this transect the main
contributor shifted from being predominantly the nGBR stock at foraging grounds in Torres
Strait, Clack Reef and the Howicks Group to predominantly the sGBR stock at Edgecombe
Bay, Shoalwater Bay and Moreton Bay. At the most northern foraging ground in the Torres
Strait, significant shifts in haplotype frequencies between juveniles and adults resulted in major
shifts in the estimated stock contributions for these groups. Fewer juveniles originated from the
nGBR stock and higher proportion originated from the sGBR and „other‟ stocks in comparison
to adults. This trend was apparent in the four most northern foraging grounds, even in
Edgecombe Bay, which had a predominance of turtles from the sGBR stock. Point estimates of
contributions from the nGBR stock dropped from 0.89 in adults to 0.53 in juveniles in Torres,
Strait, from 0.69 to 0.49 at Clack Reef, from 0.66 to 0.49 in the Howicks Group and from 0.10
in adults to 0.01 in juveniles at Edgecombe Bay. In contrast, at the Shoalwater Bay foraging
ground the opposite was observed, with a drop in contribution from the sGBR stock from 0.98
in adults to 0.84 and 0.85 in juveniles and sub-adults, respectively, and an increase in
contributions from „other‟ stocks in juveniles and sub-adults. The observed patterns at the
various foraging grounds likely resulted from several causes and four possible explanations are
explored, the mostly likely of which were that (i) juveniles have shifted foraging grounds as
they mature, or that (ii) reduced hatching success from the main nGBR rookery at Raine Island
for more than a decade has resulted in reduced recruitment into the nGBR foraging ground. The
later possibility suggests a need to take action to conserve the nGBR population The combined
strength of data derived from mark-recapture studies, demographic studies to determine sex,
maturity and breeding status of the turtles, genetic studies to determine stock composition and
satellite telemetry, are needed to provide informed assessments of foraging populations
necessary for guiding sustainable management of marine turtles.
3