Ian_Ives_MSThesis_12.. - Asian Turtle Conservation Network

Conservation of Sulawesi’s Two Endemic Chelonians,
Leucocephalon yuwonoi and Indotestudo forstenii;
An Investigation into In-Situ and Ex-Situ Conservation Concerns:
A Thesis
Presented to the Department of Environmental Studies
Antioch University New England
In Partial Fulfillment
of the Requirements for the Degree of
Masters of Science
By Ian E. Ives
January 2006

© 2006
All Rights Reserved

TABLE OF CONTENTS
Page
LIST OF TABLES………………………………………………………………………..ii
LIST OF FIGURES…………………………………………………………………….…ii
LIST OF APPENDICES………………………………………………………………….iv
ACKNOWLEDGMENTS………………………………………………………………...v
ABSTRACT…………………………………………………………………………….viii
PREFACE: An Overview of the Taxonomic and Conservation Status of Indotestudo
forstenii and Leucocephalon yuwonoi……………………………………1
CHAPTER 1: Field Notes on the Current Distribution and Trade of Two Endemic
Chelonians from Sulawesi Indonesia: Leucocephalon yuwonoi and
Indotestudo forstenii……………………………………………………..10
Introduction………………………………………………………………10
Methods and Study Sites…………………………………………………13
Results for Leucocephalon yuwonoi……………………………………..15
Results for Indotestudo forstenii…………………………………………20
Discussion………………………………………………………………..24
CHAPTER 2: Population Dynamics and Phenotypic Differences in Indotestudo
Forstenii: Are Phenotypic differences between populations indicative
of distinct lineages………………………………………………………28
Introduction………………………………………………………………28
Methods…………………………………………………………………..31
Results……………………………………………………………………33
Discussion………………………………………………………………..34
CHAPTER 3: Is Lack of Reproductive Success by Captive Leucocephalon yuwonoi
Caused by Stress………………………………………………………..40
Introduction………………………………………………………………40
Methods………………………………………………………………… .46
Results……………………………………………………………………49
Discussion………………………………………………………………..52
LITERATURE CITED…………………………………………………………………..61
TABLES…………………………………………………………………………………70
FIGURES………………………………………………………………………………...77
APPENDECES…………………………………………………………………………..88
i

LIST OF TABLES
1. Morphometric comparisons between the Palu and long term captive
Leucocephalon yuwonoi groups……………………………………………………70
2. Morphometric data from captive Indotesdtudo forstenii in holding in
Palu, Sulawesi……………………………………………………………………….71
Page
3. Localities for Indotestudo forstenii with and without a nuchal scute,
suggesting two distinct populations (northern and southern) with
distinguishing phenotypes…………………………………………………………...72
4. Comparison of carapace length and mass measurements between
I. forstenii with and without nuchal scutes…………………………………………..73
5. Uncorrected (“p”) distance matrix for samples of Indotestudo forstenii
from an assurance colony (Turtle Bank, Massachusetts) in the U.S……………… 74
6. Baseline plasma corticosterone concentrations in long term captive
male and female L. yuwonoi…………………………………………………………75
7. Repeated measures MANOVA comparison of CORT levels in longterm
captive female Leucocephalon yuwonoi held in two housing
arrangements, solitary and 3-animal groupings……………………………………...76
ii

LIST OF FIGURES
1. Net export of live I. forstenii from Indonesia between 1985 – 2004………………..77
2. Map of Central Sulawesi, Indonesia showing reported capture
locations of L. yuwonoi mentioned in text. …………………………………………78
3. Map of Central Sulawesi, Indonesia showing localities mentioned in
Text………………………………………………………………………………….79
4. Comparison of nuchal types in Indotestudo forstenii……………………………….80
5. Map of Sulawesi, Indonesia showing areas of endemism as described
by Evens (2003)……………………………………………………………………..81
6. Areas in Sulawesi with different mean annual rainfall……………………………...82
Page
7. Experimental design. Baseline CORT samples were taken on 3
separate occasions (gray blocks) evenly distributed over the course
of a 41-day period……………………………………………………………...…….83
8. Time required to obtain blood samples from long-term captive L. yuwonoi………...84
9. Time required to obtain blood samples from L. yuwonoi as a function
of time of day……………………………………………………………...…………85
10. Least squares mean differences in CORT levels during three sampling
periods between captive female Leucocephalon yuwonoi held in groups
of 3 (G) and alone (A)………………………………………………………………86
11. Differences in CORT levels for females and males between three
Periods during the species active daytime period; A = pre dawn,
B = daytime, and C = after dark…………………………………………………….87
iii

LIST OF APPENDICES
Page
A. Map of Indonesia including Sulawesi……………………………………………….88
B. Palu Valley and surrounding foothills……………………………………………….89
iv

ACKNOWLEDGMENTS
I would like to thank my thesis advisor, Jonathan Atwood, for his thoughtful
support, dedication to my work and guidance throughout this process. Jon’s insistence
that I stick with this relatively esoteric topic rather than one directly related to my
eventual career was insightful, in that it gave me the opportunity to explore unique once
in a lifetime journeys and experiences. The members of my committee, Charlie Innis,
Mike Penko and Phillip Spinks were of great assistance in carrying out and completing
this study. Charlie Innis volunteered his expertise in clinical techniques, specifically
blood collection, and withstood long and tedious sampling bouts at unlikely hours of the
day. Mike Penko generously provided me access to an absolutely astonishing collection
of Asia’s most endangered chelonians and provided me with the necessary infrastructure
and tools to do this work. He also contributed housing for me during long stretches of
data collection. Phiilip Spinks introduced me to the complexities of phylogenetics,
conducted the genetic work for Chapter 2 and contributed directly to the methods and
results sections of that chapter.
Thanks to Brad Shaffer at UC Davis for agreeing to conduct the genetic work for
this study, Penn State University Core Endocrinology lab for carrying out immunoassays
and methods section for chapter 3, the American Museum of Natural History for
supplying me with historical locality information in a very timely and hassle free manner
and Alison Robbins for procuring veterinary supplies for me. Woody Ives generously
provided me with the funds to tackle this endeavor and Peter Erskine both funded my
travels to and accompanied me on my trip to Indonesia. Bill McCord was greatly
supportive in the early stages of this study and provided me with invaluable personal
v

communications. Cris Hagan, Margaret Kinnaird and Robbert Lee provided me with a
variety of invaluable information for my trip to Indonesia. I am thankful to those in
Indonesia who graciously contributed to this work and offered hospitality during my
travels, including Aslan, Danny G, Faudin Taula, Fitra Rinawati and Nurlin Djuni.
I would like to acknowledge the late Dr. Barbara Bonner for her inspirational
lifetime achievements in chelonian therapeutics and for introducing me to Asia’s amazing
chelonians. Her trust in me and endless guidance will never be forgotten.
Lastly, I would like to thank my wife Viola and son Alan, who were immensely
loving, patient and understanding while I was locked behind the living room glass door
during the winter of 2005/2006. Viola contributed to this work in so many ways, both
directly and indirectly, and for this I am very grateful.
vi

Wild-caught adult Leucocephalon yuwonoi awaiting shipment at a commercial
holding compound in Palu Indonesia.
When the well is dry, we know the worth of water.
-- Benjamin Franklin
vii

ABSTRACT
The Sulawesi forest turtle, Leucocephalon yuwonoi and the forstens tortoise,
Indotestudo forstenii represent the only two endemic chelonians from Sulawesi
Indonesia. Both are poorly understood and rapidly declining in numbers throughout their
ranges due to overexploitation for pet, live food, and medicinal purposes. I conducted a
preliminary in-situ investigation of the current distribution and trade of the two species,
investigated population dynamics and phenotypic differences in I. forstenii, and analyzed
baseline levels of the stress hormone corticosterone in a large group of captive L.
yuwonoi. My in-situ investigation provided evidence for previously undocumented
populations for both species in Sulawesi and the surrounding islands, that, if confirmed,
significantly increase the species’ known distribution. Trade of L. yuwonoi over the last
three years appears to have changed little. Anecdotal information obtained from
interviews with hunters suggests that there has not been significant changes in yield per
hunt over the last 3 years. Similarly, large exporters are not finding it necessary to pay
higher prices for individuals caught, as would be expected with a decline in supply.
Comparisons of I. forstenii populations from the north of Sulawesi with those from the
south revealed differences in mass, MCL and the presence of a nuchal scute. To
determine if these phenotypic differences are diagnostic for mtDNA lineages in the
species, an examination of cytb Mitochondrial DNA (mtDNA) sequences in two groups
of wild caught captive I. forstenii, one group with and one without the nuchal scute was
performed. Results showed that the nuchal differences do not correspond to mtDNA
lineages. I therefore suggest that the differences in size are the result of environmental
factors. Corticosterone (CORT) levels in a long-term captive group of L. yuwonoi were
viii

high relative to other reptiles, showed no detectable increase during the first 5 ½ minutes
after capture, were higher in males than in females and were lower in females housed in
groups of 3 than in females housed alone. Future research is required to better understand
the cause and biological significance of the high CORT levels detected in both sexes of
this collection. L. yuwonoi collections experiencing reproductive success, as well as
those experiencing reproductive failure should have CORT assays conducted in order to
determine if stress is a contributing factor to the lack of reproductive success in captivity.
This work provides much needed baseline information for future studies involving range
and distribution of the two species, population studies of I. forstenii, and reproductive
biology of L. yuwonoi.
ix

PREFACE
AN OVERVIEW OF THE TAXONOMIC AND CONSERVATION STATUS OF
INDOTESTUDO FORSTENII AND LEUCOCEPHALON YUWONOI
Sulawesi is the largest island in the biogeographic area known as Wallacea, a
region in the Indonesian Archipelago lying between the islands of Borneo to the west and
New Guinea to the east. Sulawesi’s biota is highly distinctive and has extremely high
levels of endemism. Sulawesi’s reptile endemism in particular is high, with 26% of its
117 reptile species found nowhere else on earth (Whitten et al., 1987). In all of
Indonesia, only Irian Jaya has more endemic reptiles than Sulawesi.
Two mysterious and highly endangered chelonians, the Sulawesi Forest Turtle,
Leucocephalon yuwonoi and the Forsten’s Tortoise, Indotestudo forstenii represent the
only two endemic chelonians from Sulawesi. Although the two species are members of
different families, they have many similarities with regard to their phylogenetic and
conservation status. Both have undergone changes in taxonomic standing which has
increased their conservation concern, and both are poorly understood and rapidly
declining in numbers throughout their ranges. As with most of Asia’s chelonians, L.
yuwonoi and I. forstenii are highly sought after for pet, live food, and medicinal purposes.
L. yuwonoi belongs to the Geoemydidae (Bataguridae), a large and widespread
family of turtles that has undergone many phylogenetic studies and is currently in
taxonomic flux (Spinks et al., 2004). Much of this flux can be attributed to the poor
understanding of the family overall. Fifty six of the 68 species within the family
Geoemydidae inhabit ranges within Asia, a region of the world with little financial
1

esources for conservation of wildlife, many remote and hard to reach habitats, and a
great appetite for turtles and turtle products.
The first formal description of L. yuwonoi was published in 1995 (McCord et al.,
1995), which through cladistic analysis based on morphology placed the species in the
genus Geoemyda. Subsequent taxonomic revisions were made by Fritz and Obst (1996),
and again by McCord et al. (2000). This last revision involved extensive phylogenetic
analysis of gene sequence variation within turtles of the genus Geoemyda. The analysis
revealed Geoemyda as being polyphyletic, and recommended that yuwonoi be considered
to represent a new monotypic genus. Leucocephalon, derived from Greek leukos,
meaning white, and kephale, meaning head was then chosen as the taxonomic designation
for the species, reflecting the sexually dimorphic head coloration of the species (McCord
et al., 2000).
I. forstenii is a member of the Testudinidae, a monophyletic family of tortoises
distributed throughout Africa, Asia and to a lesser degree the Americas and Europe (Ernst
and Barbour, 1998; Spinks et al., 2004). I. forstenii is currently recognized as one of
three species of the genus Indotestudo, the others being I. elongata and I. travancorica.
Yet this has not been the case until recently. I. forstenii was initially described in 1840 as
Testudo forstenii from Halmahera Island in Eastern Indonesia (Schlegel and Müller,
1840). The subgenus Indotestudo was recognized in the late 1920’s (Lindholm, 1929), an
approach that was followed later by Williams (1952) and Loveridge and Williams (1957).
Bour (1980) elevated the subgenus to a full genus, hence Indotestudo forstenii.
Hoogmod and Crumly (1984) conducted morphological examinations of the three species
and surmised that I. forstenii could not be distinguished from I. travencorica, and
2

suggested that all Indonesian populations of I. forstenii were introduced from India. They
ultimately concluded that both species should be synonymized under I. forstenii.
McCord et al. (1995) challenged the assumption that I. forstenii was introduced to
Indonesia, noting zoogeographical correlates with the Geoemydids. Iverson et al. (2001)
conducted a phylogenetic study based on mtDNA sequence variation in all three species
of Indotestudo, and concluded that populations of I. forstenii from Indonesia were not
introduced from India; thus I. elongata, I. forstenii and I. travencorica should be treated
as separate and full species.
The taxonomic revisions that have occurred within L. yuwonoi and I. forstenii
present several conservation implications. Being taxonomically distinct endows a species
with a certain amount of conservation priority, and both species share this feature. In the
case of L. yuwonoi, the recently revised monotypic designation placed the species as the
sole member of the genus, thus setting it apart from the Geoemydids it was once
considered to be related to. For I. forstenii, receiving nominal species status meant a
division of one previously assumed species (I. forstenii) into two separate species (I.
forstenii and I. travencorica). This move is not just a simple adjustment of nomenclature
but also reflects a change in Indotestudo biogeography, population dynamics and rarity.
Through this revision, the whole of I. forstenii’s known populations are now restricted to
the two islands of Sulawesi and Halmahera.
The distinctiveness of the two species is apparent not only at the taxonomic level,
but possibly at a more overriding ecological level. As far as distribution is concerned, I.
forstenii represents the only tortoise east of Wallace’s line, and L. yuwonoi is one of only
two Geoemydids east of the line (Iverson, 1992). Both species may be distinct in terms
3

of their natural history and role in the ecosystem, yet due to the paucity of quantitative
field studies on either, the full extent of their distinctiveness remains unknown.
The status of Sulawesi’s two endemic chelonians in the wild, by all available
measurements, is dire. The 2001 World Conservation Union (IUCN) Red List categorizes
L. yuwonoi as critically endangered, with a known or inferred population decline of 80%
within the last three generations, a similar future decline over the same time period, and a
total, declining wild population numbering less than 250 adults (IUCN 2005). I. forstenii
has been listed by the IUCN as Endangered, with a known or inferred population decline
of 70% within the last three generations and a known or inferred future decline of 50%
over the same time period (IUCN, 2004). These inferred population and population
decline estimates presented by the IUCN are derived primarily from export statistics and
documented occurrences of the two species in the live animal food and pet trade. Because
knowledge of these species’ current distribution, population trends and habitat
availability is incomplete, their true overall population size is uncertain.
The primary reason for both species’ decline is overcollection for the live animal
food and pet trade, both domestically and internationally. With respect to the live animal
food trade, China’s transformation to capitalism and subsequent economic boom, as well
as the growth of mainland Southeast Asian economies, has greatly increased the demand
for live food and animal-derived medicinal products (Compton, 2000). The number of L.
yuwonoi seen in Chinese food markets in the late 1990’s reflects this increasing demand.
L. yuwonoi sightings in a few of China’s largest markets increased from only a few
specimens in the early 1990’s to 2000-3000 animals in 1998, then dropped dramatically
to around 100 animals in 1999 (IUCN TFTSG & ATTWG, 2000). No observation of the
4

species in Chinese markets has been documented since 1999 ( W. McCord, pers. comm.).
Because demand has not changed, it can be assumed that the disappearance of L. yuwonoi
in the Chinese markets is due to minimal supply. Furthermore the sharp decline in L.
yuwonoi numbers from one year to the next suggests a dramatic decreased in the wild
population due to overcollection rather than from habitat loss, which, while certainly a
contributing factor, results in a slower decline of population numbers (CITES, 2000).
Overcollection has contributed to I. forstenii population decline as well, yet in
contrast to L. yuwonoi, documented export of this species has been directed mainly to
Europe, Japan and the United States, where it has been exploited in the pet trade
(Compton, 2000). I. forstenii has been included in The Council on International Trade in
Endangered Species (CITES) Appendix II list since 1975 and therefore has a long export
record. CITES data show numbers of live exports from Indonesia fluctuating greatly
between 1985 and 2004 (Figure 1). Export numbers peaked three times over this 20-year
period (1989, 1993 and 1996) with subsequent drops in 1990 and 1994. In 1997 the
export number was similar to 1996, then dropped to a relatively constant level from 1998
- 2003. In 2004 the export number bottomed out at 6 individuals. Most of the betweenyear
variability in this data is due to fluctuating export quotas. For example, in 1997, the
quota was 900 individuals, thus allowing for the large number of turtles to be exported.
In 1998 the export quota was lowered to 475 and in 2002 lowered again to 400 (UNEP-
WCMC, 2005). Because demand has been consistently high over this span of time,
export numbers followed the mandated quotas consistently whenever the supply allowed.
This data indicates a cyclical trend in the number of I. forstenii legally exported since
1985. Unfortunately, actual trade patterns cannot be quantified due to the unknown
5

volume of illegal export and domestic utilization. Furthermore, an accurate
determination of the extant population based on this information, alone, is impossible,
thus emphasizing the urgent need for data on wild populations.
The life history traits of turtles, which have evolved to increase lifetime
reproductive success under conditions not influenced by humans, place severe pressures
on the ability of populations to respond to human-induced population decline such as
over-harvesting of juveniles and adults (Congdon, 1993). Limited reproductive
observations suggest that females of both species may produce clutches 3 times a year,
with clutch sizes consisting of 3 (I. forstenii) or 2 (L. yuwonoi) eggs (C. Innis, pers.
comm.; Highfield, 1996; pers. obs.). The low annual reproductive outputs of these two
species coupled with assumed (yet undocumented) low nest and juvenile survivorship
place them at a high risk for extirpation when human-caused population decline is
introduced. At present, the continued harvest of all age classes of both L. yuwonoi and I.
forstenii, as seen through export data and documented human use, has likely reduced the
ability of populations of either species to respond and recover. Add the restricted range
of both species, and the vulnerability to other threats increases exponentially.
While not the primary threat to either species, deforestation, human settlement, and
agricultural conversion of natural habitat are also factors (Samedi & Iskandar 2000).
Forest fires, in particular, appear to be a real threat to I. forstenii, in part due to the
species’ tolerance of arid habitats. Populations in the arid Palu Valley of Central
Sulawesi are likely effected by fires, and in Northern Sulawesi, dry season wildfires have
been specifically implicated as a source of mortality in I. forstenii (Platt et al., 2001). In
Central Sulawesi, where the largest documented populations of both species occur,
6

conversion of habitat into agriculture has progressed at an alarming rate; 22% of this
province’s forest cover was lost between 1985 and 1997 (Holmes 2000). Because both
species have been documented in secondary forest habitats, it is unlikely that they are
dependent on pristine habitat for their survival. Continued habitat alteration though, will
certainly have detrimental effects on populations of both species due to increased soil
erosion, and altered water quality and stream flow patterns.
Conservation strategies have been implemented by the international community in
response to the unprecedented loss of chelonians in Indonesia and throughout Asia, and
include actions for conservation of Sulawesi’s two endemic chelonians. The phrase “the
Asian Turtle Crisis” was coined after unanimous international agreement on the scale of
the problem. While identifying the problem, the moniker also serves to galvanize and
motivate the chelonian conservation community. The Turtle Survival Alliance (TSA), a
joint working group of the IUCN/SSC has initiated “A Global Action Plan for
Conservation of Tortoises and Freshwater Turtles”, a long-term initiative designed to
stem the decline of chelonians throughout the world, but particularly in Asia. Because
Asia holds such a large proportion of the world’s chelonian species, as well as some of
the most threatened species, this region has been given the highest conservation priority
by the TSA. While L. yuwonoi and I. Forstenii are considered among the most
threatened chelonians in Asia and are included in the Action Plan, conservation efforts
specifically directed towards either species have, until recently, been limited to
identification of threats, classification of status, animal acquisition and management
strategy planning.
7

One of the conservation strategies undertaken by the TSA involves establishment
of “assurance colonies”, which are assemblages of wild-caught individuals that are held
in captivity for the purpose of maintaining genetic viability through sustainable captive
management programs. These colonies are primarily reserved for the most critically
endangered species, such as L. yuwonoi and I. forstenii. Today there are some 140 L.
yuwonoi and 74 I. forstenii in assurance colonies managed by the Taxon Management
Group (TMG) of the TSA ( B. Zeigler, pers. comm.; C. Innis, pers. comm.). TMG
members include zoos, private breeders, veterinarians, and other qualified individuals.
Their objective is to develop in situ and ex situ management techniques and conduct
research that contributes to the explicit goal of preventing extinction of the world’s
threatened chelonians.
International help notwithstanding, The Indonesian Government’s role in the
conservation of its native flora and fauna will ultimately determine the fate of Sulawesi’s
chelonians. While The Council on International Trade in Endangered Species (CITES)
grants both species protection under Appendix II (CITES, 2000), the Indonesian
government does not legally protect either species. Instead, the government manages the
species as a fisheries resource through the Fisheries Department (Department Kelautan
dan Perikanan - DKP). The Indonesian Government acceded to CITES in 1978, and
therefore uses the listing as grounds for setting annual catch quotas based on the
Indonesian Institute of Science’s (Lembago Ilmu Pengetahuan Indonesia - LIPI)
recommendations.
There are many shortcomings in the established laws that lead to confusion and
mistakes in enforcement. Three major issues include questionable methods for
8

establishing quotas, inability of government officials to identify many species and
thereby effectively administer their quotas and law enforcement (Samedi & Iskandar,
2000; Shepherd and Ibarrondo, 2005). Recent research conducted by TRAFFIC has
drawn attention to an often ignored Indonesia law that requires any transport and
distribution of animal species, whether they be protected or not, to be carried out under a
license and permit (Shepherd and Ibarrondo, 2005). TRAFFIC determined there to be no
such permits held by traders of the endangered Roti Island snake-necked turtle,
Chelodina mccordi, and thus all export of the species since 1980 has not been in
accordance with Indonesian national laws. Given the lack of environmental law
enforcement in Sulawesi, I would suggest that much of the trade and export of L.
yuwonoi and I. forstenii is also being conducted without license and permit.
In order for the Indonesian Government to place a species on its protected list,
proposals must be presented by the Indonesian Institute of Science. Unfortunately, there
has been little population level analysis conducted for either species, and documented
descriptions of life history, distribution, status and population trends in the wild is
severely lacking. Without this information, proposals in favor of granting protection to
the species cannot be made to the Indonesian Government (D.T. Iskandar, pers. comm.).
Therefore, it is the responsibility of the scientific community to gather and present
quantitative data that can shed light on the biology of these two species and promote a
concerted effort by the Indonesian Government to conserve sustainable populations.
9

CHAPTER 1
Field Notes on the Current Distribution and Trade of Two Endemic Chelonians
from Sulawesi Indonesia, Leucocephalon yuwonoi and Indotestudo forsteniiI
Introduction
Biological and conservation assessment of Sulawesi’s two endemic chelonians,
Leucocephalon yuwonoi and Indotestudo forstenii, has historically received scant
attention compared to Sulawesi’s other endemic taxon. This is in part due to the inherent
difficulties of locating a small number of widely dispersed animals within isolated
tropical locations but mainly due to the lack of knowledge of reptiles in general. Much of
the initial work conducted with regard to these species has been taxonomic (relying on
only a few museum specimens). Recent genetic investigations (Iverson, et al., 2001;
McCord, Iverson, Spinks & Shaffer, 2000; Spinks et al., 2004) have helped resolve
phylogenetic relationships within the two species, and in doing so have revealed their true
taxonomic distinctiveness. An ancillary result of genetic findings though has been the
exposure of additional conservation implications not only for these two species but for
other endangered species worldwide. For example, these revisions make I. forstenii the
only tortoise east of Wallace’s line and L. yuwonoi as one of only two Geoemydids east
of the line (Iverson, 1992, McCord, et al., 1995). The genetic investigation described in
Chapter 2 of this thesis into phenotypic differences of I. forstenii has demonstrated that
morphological differences within a species does not necessarily represent genetic
diversity of the magnitude needed to designate a new subspecies or separate species.
10

Therefore, genetic re-examinations may condense or eliminate the number of true
subspecies within a genus, thus redefining the biogeographical state of a species.
To date, only partial knowledge of the distribution of Sulawesi’s two endemic
chelonians exists. The known distribution of L. yuwonoi extends from Gorontalo in North
Sulawesi south to Palu and east to Poso in Central Sulawesi (Platt et al., 2001; Hagan and
Ching, 2005; McCord, 2004; McCord, Iverson & Boeadi, et al. 1995). Two field surveys
focusing on L. yuwonoi documented the occurrence of the species in Cape Santigi as well
as at a local collector’s house outside of Tinombo village in Central Sulawesi (Platt et al.,
2001; Hagan and Ching, 2005). I. forstenii is known to exist on the islands of Halmahera
and Sulawesi, yet specific locality information exists only for Sulawesi (Hoogmoed and
Crumly, 1984; Iverson, 1992, Platt et al., 2001). The documented range of I. forstenii in
Sulawesi is localized and significantly restricted. Known localities exist at Mnt.
Boliahutu and around Buol in North Sulawesi and Santigi in Central Sulawesi
(Hoogmoed and Crumly, 1984; Iverson, 1992, Platt et al., 2001). Additionally, a
population was discovered in the Morowali Reserve in Central Sulawesi (Groombridge,
1982). Because the geological history of the island is very complex and uncertain, the
evolution of the two species’ distribution is unknown. It is unclear whether Sulawesi was
a single landmass that was subsequently fragmented then recently reunited (a vicariance
hypothesis) or whether it was an archipilago that was recently uplifted and united (a
dispersal hypothesis) (Whitten, 1987). Whatever the current distribution of both species
is, it can be attributed to the islands unique geologic history, each species ecological
capacities, routs of dispersal over time, demography, effective population size and time
(Evans, et al., 2003).
11

Preliminary reports along with my own personal observations suggest that the
distributions of both species are parceled within Sulawesi and surrounding islands (Platt,
Lee & Klemens, 2001; Hagan and Ching, 2005; B. McCord, pers. Comm.; F. Taula, pers.
Comm.; K. Tepedelen). While I. forstenii populations are likely allopatric (assuming the
documented Halmahera Island population still exists), only limited anecdotal evidence
gathered during this study suggests that L. yuwonoi populations are also allopatric (due to
a report of a population on Peleng island). Because the full extents of I. forstenii’s and L.
yuwonoi’s distributions are not clearly known, their status in the wild is uncertain. The
lack of information about these species creates a significant challenge to their
conservation, and confounds effective establishment of management plans (Groombridge,
1982; Hoogmoed and Crumly, 1984; Iverson, 1992, Platt et al., 2001).
While estimates of population decline have been formulated though CITES trade
data, and reports of the species’ occurrence in the marketplace, only two preliminary field
reports document the species’ habitat, life histories, distribution and threats (Platt, Lee &
Klemens, 2001; Hagan and Ching, 2005). No quantitative population studies have been
conducted for either species. Until results from such a study are forthcoming, further
estimates of population size can be determined through ongoing monitoring of collection
and trade of the species within Sulawesi. This report therefore builds upon the
foundation of knowledge established by the afore mentioned field studies and is intended
to serve as a preliminary investigation towards further work by the author.
12

Methods and Study Site
From 19-27 February 2005 I conducted a preliminary field investigation of the
current distribution and trade status of the Sulawesi forest turtle, L. yuwonoi and the
forstens tortoise, I. forstenii. Morphological data was gathered from specimens at a
commercial holding facility in Palu Central Sulawesi where wild caught turtles are kept
in holding for local and international distribution into the pet and food markets. Species
distribution information was gathered through interviews in the Palu Valley of Central
Sulawesi, as well as the Palolo, and Kulawi Valleys adjacent to Lore Lindu National
Park, also in Central Sulawesi. Additionally, a habitat survey was conducted to
determine the presence of L. yuwonoi in Lore Lindu National Park.
The Palu holding facility was currently holding 53 L. yuwonoi, and 42 I. forstenii.
The facility is owned and operated by a turtle trader who has been in the business for
over 20 years. The captive L. yuwonoi were housed communally in an outdoor cement
enclosure measuring 3.5 m x 2 m, The enclosure was partially covered and filled with
several centimeters of water. Turtles were fed sporadically with scraps of vegetables
from the kitchen. The I. forstenii were housed in a 2 m x 2 m enclosure filled with
several potted plants, presumably provided for shade. Food was provided occasionally in
the form of scraps from a nearby kitchen.
I spoke with over 100 people between February 19-27 in villages in the Palu
valley and along the north and west borders of the park. My interviews were an attempt
to gather anecdotal information regarding the existence of L. yuwonoi and I. forstenii
populations in the region. For each person I spoke with, photographs of L. yuwonoi were
presented. Additionally, photographs of the other known extant chelonians of Sulawesi
13

were shown for clarification. Three questions were asked to each individual I met, all
intended to determine how familiar people in this region were with the two species.
Additionally, I interviewed people in order to ascertain the location of local turtle hunters
that could show me captured turtles and take me to known habitats. Lastly, I visited The
Nature Conservancy’s Palu office in order to obtain maps of the region and logistical
information.
Anecdotal information suggests that populations of L. yuwonoi may exist in Lore
Lindu National Park, a 231,000 ha World Heritage Site. Confirmation of L. yuwonoi
populations in the park would be significant, as it would be the first documented
occurrence of the species in a protected area. In an effort to document the presence of L.
yuwonoi in the park, a habitat survey was conducted near the village of Kulawi, Central
Sulawesi on the border of Lore Lindu National Park. I surveyed an unnamed creek on
the western boundary of Lore Lindu National Park in the village of Kulawi at
coordinates: 01º 26’ S, 119º 59 E (Figure 2, flag #1). The village lies along one of many
small rift valleys collectively known as the Kulawi valley. Due to tectonic movement
along this valley, frequent small landslides occur throughout the area, especially in
deforested areas and along roads. The village is at an elevation of approximately 600m
and is within the elevation range of the lowland forest zone (Whitten et al., 1987). Both
young secondary forest as well as primary forest surrounded the village. Land-use
activities in the area include agricultural production and small scale harvesting of timber
and non-timber forest products. Annual rainfall for the region varies between 2000 –
3000 mm, and northern monsunal rains occurred daily during the study period. The
survey area encompassed a 5 km stretch of the creek between 1900 and 2430 hours on a
14

day in which no significant rainfall had occurred (all previous attempts to survey the
creek after significant rainfall proved impossible – due to heavy silting of the water).
Results
▪Leucocephalon yuwonoi
Morphological data was taken from all 53 L. yuwonoi at the Palu holding facility.
Midline carapace length, maximum width (±0.1mm) and weight (kg) were recorded. Age
classes recognized in this study were juvenile and adult. Juveniles were differentiated
from adults based on the absence of secondary sexual characteristics. Several immature
turtles were classified as adults when these characteristics were clearly present. One
immature turtle though, with a midline carapace length of 133.4 was classified as a
juvenile due to the absence of a chin stripe, plastral concavity or discernable tail
characteristics. There were no hatchlings in the collection. Juvenile-to-adult age class
ratio was 1.0:7.8, while the female-to-male sex ratio was 1.0:2.1. Several individuals of
both sexes were very old, as their plastra were worn almost entirely smooth. One old
female was missing claws on every foot, and appeared to be in poor health. One very old
male had a tether hole drilled into his carapace and was quite emaciated.
The overall condition of the group appeared to be good. Morphometric data for
this group and a long term captive group are compared in Table 1. Comparisons of
health index’s (turtles weight divided by length) for both males and females from this
group and a long-term captive group in the U.S. revealed significant differences in males
but not in females. The health index from the U.S. males was significantly higher (equals
15

healthier) than that of Palu males (t-test: t = 3.090, P = 0.003), while females from both
groups showed no significant difference (t-test: t = 1.376, P = 0.184).
Each female at the holding facility was palpated for the presence of shelled eggs.
The presence of an egg could be felt in 20% of the females or 3 of the 15 females
examined. Remnants of at least one egg were clearly observed in the enclosure. It was
unclear whether there was a successful hatching from the egg(s). No sexual aggression
was observed between males, yet one male was seen mounting a female and attempting
to copulate. He then noticed the observer and dismounted.
The owner of the facility, the primary L. yuwonoi dealer in Sulawesi, was
interviewed in order to gain information regarding trends in utilization and trade of this
species. Although anecdotal, this information contributes to the sparse information
currently available for species population ranges and demographics. The 53 individuals
observed had reportedly been captured within the last month. Although coordinates
representing the precise capture locations of each turtle could not be obtained, several
locations were identified as collection sites. These sites included; Santigi (298 km north
of Palu, Ongka (approximately 285 km north of Palu), the village of Bankit
(approximately 295 km north of Palu), Tate (approximately 85 km north of Palu), and
Tompe (approximately 80 km north of Palu), all on the Minahasa Peninsula, in Central
Sulawesi (Figure 2). L. yuwonoi were also reportedly captured around the village of
Balantak on the westernmost tip of Central Sulawesi and on Peleng Island in the Banggai
Island chain South East of Luwuk, Central Sulawesi (Figure 2). If L. yuwonoi accounts
can be confirmed around Balantak or on the Banggai Islands, this would significantly
increase the species’ known distribution, and represent a new population. Moreover, the
16

presence of L. yuwonoi on an isolated island could have important management
implications considering the possibility of genetic isolation.
The dealer identified several villages from which certain age groups were
collected. Many of the adult males from the current collection were captured in Ongka
and Santigi, while the juveniles were taken from Tompe near km marker 89. Adult L.
yuwonoi of both sexes were reportedly captured in Peleng Island. Additionally, the
dealer stated that many of turtles captured on the Minahasa Peninsula were found within
a few kilometers of the coast. The presence of L. yuwonoi in close proximity to the coast
is consistent with locality accounts from previous field reports (see Platt et. al., 2001;
Hagan and Ching, 2005).
According to the dealer, approximately 50 L. yuwonoi arrive at his facility per
month. In the past the dealer himself would drive long distances to retrieve turtles from
the towns in which they were captured, yet in the last few years, the dealer reports having
the turtles delivered to Palu by the hunters themselves. According to this dealer, local
hunters are paid 35,000 Rupiah (~$3.50 USD in February of 2005) for each adult L.
yuwonoi of either sex. He claims that this amount has not changed in recent years.
Subsequently, the dealer then receives 150,000 Rupiah (~$15.00 USD) per adult from an
international reptile exporter in Jakarta. Hagen and Ching (2002) reported the same Palu
dealer paying hunters 40,000 Rupiah per adult and in return receiving 150,000 Rupiah
from the same exporter in February of 2002. Based on these figures, it can be assumed
that collectors have not realized significant changes in yield per hunt over the last 3 years.
Similarly, large exporters are not finding it necessary to pay higher prices for individuals
caught, as would be expected with a decline in supply.
17

Turtles brought to the facility above and beyond the annual quota are sold locally,
according to the dealer. With a reported 50 L. yuwonoi arriving at the facility per month,
many more turtles are being taken from the wild than IUCN estimates. More
investigation is necessary to determine the accuracy of this account, as well as the extent
of domestic use of L. yuwonoi.
My interviews of some 100 local villagers from the Palu, Kulawi and Palolo
Valley’s of Central Sulawesi revealed a surprising result. While there was certainly a fair
amount of information lost in translation, the overall conclusion can be drawn that people
in these valleys are unfamiliar with L. yuwonoi. For each person I spoke with,
photographs of L. yuwonoi were presented. Additionally, photographs of the other
known extant chelonians of Sulawesi were shown for clarification. Three questions were
asked to each individual I met; have you ever seen a kura kura doun (vernacular name for
L. yuwonoi), do you know of someone who has turtles, and have you ever eaten turtles
Surprisingly, “no” was usually the answer to all of these questions. The vast majority of
persons interviewed had never seen a L. yuwonoi, and had never eaten turtles. However,
many persons said they knew of people who either had turtles or ate turtles regularly.
I also met and spoke with several staff members from the Nature Conservancy’s
Palu office in order to gather logistical support for my trip into Lore Lindu National Park
and to confirm reports of L. yuwonoi in the park. Interestingly, staff members I spoke
with had no personal accounts of the species in general, and were unaware of
observations of the species in the park.
I was taken to a creek on the western boundary of Lore Lindu National Park by a
local rattan collector, who reported seeing “small turtles” while on harvesting trips into
18

Lore Lindu. The creek was similar physiographically to tributary creeks where L.
yuwonoi had been found during studies in 1998 and 2002 (Platt et al., 2001, Hagan and
Ching, 2005). The creek met microhabitat criteria one might expect in a creek supporting
L. yuwonoi, namely, cool, clear and fast flowing water, pools, steep terrain and dense
foliage at the understory level. While much of the creek was surrounded by secondary
forest and various plantations, there were no clearings in the canopy and the landscape
was completely undeveloped. This habitat is similar to previously documented L.
yuwonoi localities in the Minahasa Peninsula of Sulawesi (Platt et al., 2001, Hagan and
Ching, 2005). This location was ultimately chosen as the study site in part because it met
the criteria of known L. yuwonoi habitat, but primarily because reports indicated that
there was a likelihood of finding the species there.
The survey was conducted on a 5 km stretch of the creek between 1900 and 2430
hours on a day in which no significant rainfall had occurred (all previous attempts to
survey the creek after significant rainfall proved impossible – due to heavy silting of the
water). The creek’s channel varied in width from approximately 4 m to less that 1 meter
depending on the slope of the adjacent hillside. Pools encountered varied in depth from
over 1 meter to less than 15 cm. The average water temperature (taken from 6 points
along the stream) was 24.1° C, while air temperature varied from a high of 27.5° C at
1900 hours to a low of 26.6° C at 2330 hours. Numerous fig trees, Ficus sp., were
identified along the stretch of creek surveyed. Mites and leaches were found along the
creek's edge and on surrounding debris. Several Bufo celebensis and an undetermined
species of skink were also seen in the creek, yet no L. yuwonoi could be found.
Furthermore, I did not obtain any concrete evidence suggesting the presence of L.
19

yuwonoi populations in the Palolo, Palu or Kulawi valleys, nor Lore Lindu National Park
itself.
I can report the presence of another chelonian whose existence is assumed but
undocumented in Sulawesi, the soft-shell turtle Amyda cartilaginea. I was brought to the
home of a Chinese family in the village of Palolo, Donggala County, Central Sulawesi
(Figure 3, flag #1). Several skeletal remains of soft-shell turtles were scattered around
their yard. The wife, a mother of four children, prepared dishes out of turtle meat once a
week. Their oldest son would trap soft-shell turtles in a nearby pond and local turtle
hunters would sell I. Forstenii to them, but they had never seen or eaten a L. yuwonoi.
The family provided a picture of a soft-shell taken at the nearby pond that has since been
confirmed as Amyda cartilaginea. I visited several other homes with turtles during my
time in Lore Lindu, and in all instances, the turtles I encountered were either I. forstenii,
or the ubiquitous Malayan box turtle, Cuora amboinensis.
▪Indotestudo forstenii
The afore mentioned commercial holding facility in Palu was also the temporary
home for 42 I. forstenii. Morphological data was taken from all 42 I. Forstenii at the
facility (Table 2). Age classes recognized in this study were juvenile and adult.
Juveniles were differentiated from adults based on the absence of secondary sexual
characteristics including plastral concavity, carapace width and discernable tail
characteristics. A total of 10 immature turtles were classified as adults, as such
characteristics were clearly present. There were no hatchlings in the collection.
Juvenile-to-adult age class ratio was 1.0:6.0, while the female-to-male sex class ratio was
20

1.0:3.0. This relatively large male-biased sex ratio may be attributed to females of the
species being more sedentary and less likely to be caught roaming out in the open. Mean
midline carapace length for males was larger than that of females. Several large males
were recorded including five with midline carapace lengths (MCL) exceeding 240mm.
Additionally, 6 juveniles estimated at between two and six years of age were recorded,
the smallest measuring 71mm MCL (Table 2).
The condition of the 42 individuals held in captivity at the Palu holding facility
was good. Comparisons of health index’s for both males and females from the Palu
group and a the long-term captive group revealed no significant differences (t-test: t =
1.859, P = 0.092 males; t = 0.171, P = 0.865 females). A rudimentary examination was
performed on 10 tortoises to note the appearance of ulcerative lesions of the mouth. This
condition, observed in many exported I. forstenii, is believed to be caused by a herpes
virus, although this has not been proven (C. Innis, pers. comm.). Additionally, tortoises
were examined for obvious fluid discharge around the mouth. Lesions similar to ones
found in the mouths of a group of 38 long term captive adults in the U.S. were apparent
on 3 of the 10 individuals examined, while none displayed any apparent fluid discharge.
The appearance of lesions on individuals that had been in captivity for only a few weeks
suggests that the pathological condition exists in certain wild populations of I. forstenii.
If this is true, then the high incidence of this condition in captive I. forstenii could be
attributed in part to transmission of the disease from individuals of affected populations
to those from unaffected populations during export.
Each female was palpated for the presence of shelled eggs. The presence of eggs
could be felt in 1 of the 9 females at the facility. The gravid female, reportedly from the
21

Minahasa Peninsula, measured 226.5 mm in midline carapace length, 150.2 mm in
maximum carapace width, and weighed 1.5 kilograms. No sexual aggression or
reproductive behavior was observed from the group.
From February 19-27, 2005 I investigated reports of a localized I. forstenii
population in the hills to the east of the Palu Valley. I spoke with over 50 people in the
Palu and Kulawi Valleys in an effort to find local turtle hunters that could show me I.
forstenii catches and take me to known habitats. Generally, locals in this region were
unfamiliar with the species, yet there were a few exceptions. On a lead from a local
fisherman, I stopped at a turtle hunter’s house in Bora Village (Figure 3, flag #2) at
kilometer marker 25 on the main road from Palu to Gimpu. The man had in his
possession two I. forstenii and reported catching the species on a regular basis, 5
kilometers from his house in the hills east of the Palu Valley. Turtles were captured
through the use of a hunting dog. On the hunt, the dog reportedly relies on its sense of
sight as well as its sense of smell. Once the dog has discovered a turtle, the hunter will
scour the area for additional individuals, often discovering nests in the immediate
vicinity. This hunter claimed that he did not train his dog for this endeavor, but rather
relied on this dog’s mother to teach it the art of “turtling”.
Another turtle hunter who’s house was located two Kilometers south of Bora
Village in Watunonju Village (Figure 3, flag #5), also used a dog. He reportedly catches
I. forstenii for a local Chinese cook who served turtles in his restaurant. On Saturday’s,
the cook would come to the hunters house and pay 15,000 Rupiah (~$1.50 USD) for each
turtle caught. The hunter claimed to consistently produce at least one turtle per week for
22

the cook. On the day I visited, the hunter had one small I. forstenii that he had caught 10
kilometers from his house in the Hills east of the Palu Valley.
I was taken to I. forstenii habitat in the foothills on the eastern edge of the Palu
Valley. These hills represent a climatic transition zone between the hot and dry Palu
valley and the cooler humid mountains of Lore Lindu National Park to the south east.
This particular region is considered slightly seasonal for rainfall and receives between
1,500 – 2,000 mm of rain annually depending on location (Whitten, 1987). This
uncharacteristically dry region (for Indonesia) is also a highly disturbed area, with
heavily eroded soils. Many drought tolerant plants exist including Acacia farnesiana and
the gum tree Eucalyptus deglupta, known to locals as leda (Whitten, 1987; The Nature
Conservancy, 2001). There are many introduced plant species including the prickly pear
cactus Opuntia nigricans, the shrub Kalanchoe pinnata, and the tamarind Tamarindus
indica (Whitten, 1987).
In villages along the western border of Lore Lindu National Park, I encountered
two captive I. forstenii. I was shown a male I. forstenii found near a stream on the border
of Lore Lindu National Park in Kulawi (km marker 71 south of Palu). The capture
location was 5m upstream from the main road in a plot of land cleared for a mixed cocoa,
banana and palm plantation (Figure 3, flag #3). The coordinates of the capture location
are: 01º26’S, 119º59’E. This turtle was reportedly eaten two days after being shown to
me. In the village of Lempelero, (km marker 98 south of Palu) I was shown a male I.
forstenii serving as a family pet. The family had kept the turtle tethered to a tree for the
last 5 months. The turtle was reportedly found on the bank of a stream in the village at
coordinates: 01°39’S, 120°02’E (Figure 3, flag #4).
23

These reports represent the first documented accounts of I. forstenii populations in
this region of Central Sulawesi. Although there is no confirmation from this study of I.
forstenii populations inside the boundaries of Lore Lindu National Park, the reports of
individuals found within 1 km of the boundary suggests a strong likelihood of their
occurrence. If indeed confirmation can be made, a thorough demographic study should
be conducted to determine the extent of this population.
Discussion
While drawing conclusions about the overall population size of both species based on the
information gathered from the encountered specimens is difficult, some preliminary
observations can be made. First, numbers of both species reportedly captured and
brought to the Palu holding facility in one month in 2002 and again in one month in 2005,
suggest that actual island wide population numbers greatly surpass official IUCN and
CITES estimates (Hagan and chin, 2005; pers. obs.). Second, there appears to be a
relatively robust demand for these species locally, because all turtles caught above the
annual export quota are sold (Taula, pers. com.). Third, the reported capture localities for
both species suggests very dispersed yet fragmented populations.
Field studies are urgently needed to determine the extent of local consumption,
confirm capture localities, investigate population demographics and to determine the true
status of the species’ in the wild. While information obtained from my interviews was
important and a good starting point, it is limited in its scientific value. Locals were very
eager to give an affirmative answer to my questions as a way of showing interest and
24

knowledge of their natural history. Additionally, spending time with a foreigner was a
pleasant diversion from every day life, regardless of the necessity.
As far as I. forstenii is concerned, information from this study suggests there are
still enough individuals in the hills surrounding the Palu Valley to make hunting for them
profitable to those engaged in the trade. Some hunters I spoke with are supplementing
their primary income by hunting. Therefore their catch-per- unit effort must be
reasonably high to justify continued hunting, especially with such little compensation.
My information suggests that if collection could be eliminated, domestic consumption
reduced, and enforcement of international trade strengthened, I. forstenii populations
might be able to thrive in the Kulawi Valley and the hills to the east of the Palu Valley
under present habitat conditions.
Establishing whether or not populations of L. yuwonoi or I. forstenii exist in Lore
Lindu National Park or other protected areas in Sulawesi is critical. If populations of
either species are shown to exist there they would most certainly benefit from a greater
level of protection than those outside the park, due to Sulawesi’s very high rate of
deforestation. From 1985 to 1997, Central Sulawesi (the province where Lore Lindu
National Park is situated) has lost approximately 959,100 hectares (72%) of its forests
(World Bank, 2001). Due to transmigration schemes, more and more people are living in
close proximity to the park. In many cases, agriculture crops and plantations have sprung
up illegally inside park boundaries. Poachers, rattan collectors, hunters and others
relying on forest products for their livelihood, move largely unimpeded throughout the
park due to lack of enforcement (F. Rinawati, pers. com.). The demand for additional
land by transmigrants has jeopardized protected areas. In early 2000, the government
25

chose to turn over roughly 2,000 hectares of land inside the park to locals for plantations
(Indonesian Observer, 2000). These human activities have threatened the integrity of
Lore Lindu National Park and jeopardized the government’s credibility for establishing
truly protected areas in Sulawesi.
Documenting the existence of endangered species in Lore Lindu is one of many
ways to increase enforcement and promote the park as a refuge for the islands natural
treasures. Recently, the Indonesian government has taken the initiative to actively
manage another National Park, Bunaken National Marine Park. Through participation
from all those who have a stake in the park, the government has promoted wise resource
use while preventing overexploitation. With enough attention focussed on Lore Lindu’s
threatened biodiversity and overall ecological value, the government may be convinced to
do the same for it as well.
There is no question that the ongoing systematic collection of Sulawesi’s two
endemic chelonians is reducing the viability of wild populations IUCN TFTSG &
ATTWG, 2000; Platt et al., 2001). The degree to which deforestation and habitat
fragmentation compromises I. forstenii and L. yuwonoi populations is unclear though.
Lowland forests in Sulawesi, areas in which both species are known to inhabit, have been
disappearing for decades. As of 1997, only a small fraction of Lowland Plains forest
(0.2%) remains forested (Holmes, 2002). Despite this dramatic loss of forests and
amongst continuous pressures from overcollection, populations of both species persist,
albeit at an unknown level (Platt et al., 2001; Hagan and Chin, 2005; Hagan pers.
comm.). With this in mind, it seams possible that the elimination of hunting pressures
alone could reverse population declines significantly.
26

Successful conservation of Sulawesi’s two endemic chelonians requires a
community-based approach to their management by involving local people in protected
area management decisions, promoting income sources that do not jeopardize endangered
species and emphasizing an end to collection for the live animal food and pet trade.
Curtailing overcollection pressures would greatly diminish the rate of population decline
by removing the most severe cause of the species’ decline.
27

CHAPTER 2
Morphological and Genetic Variation in Indotestudo forstenii: Evidence of Distinct
Lineages
Introduction
Because conservation of Asia’s endangered chelonians often requires captive
breeding and reintroductions, it is critical to determine the genetic variation both within
and between populations and then define how this variation is geographically structured.
In doing so, managers of captive specimens can maintain the genetic constitution of
populations, and effectively pursue in situ and ex situ conservation actions that will
maintain the genetic viability of the species of concern.
Since the mid 1990’s, molecular systematic analyses have resulted in various
taxonomic revisions of many groups of Asian chelonians (Honda et al., 2002a, 2002b;
Spinks et al., 2004; Stuart and Parham, 2004; Guicking et al., 2002). The primary goal in
determining phylogenetic relationships is to construct monophyletic groups which
include all the descendants of the most recent common ancestor and deconstruct those
groups that are paraphyletic (groups that contain some but not all of the descendants of
the most recent ancestor) or polypheletic (groups that include species that have been
incorrectly assigned to a group due to evolutionary convergence). Based on our current
understanding, the Testudinidae, which includes I. forstenii, is thought to be
monophyletic (Shaffer et al. 1997; Spinks et al., 2004).
Despite the apparent resolution of the Testudinids at the interspecific level, there
has been some ambiguity at the intraspecific level. This ambiguity concerns the
28

morphologic and genetic variation within I. forstenii. Phenotypic differences in this
species exist in the nuchal (cervical) scute, which may be present or absent, and
individual differences in size between nuchal types (Hoogmoed and Crumly, 1984;
Iverson, et al., 2001; Platt et al., 2001; C. Hagan, pers. comm.; Figure 4). In a study of
phylogenetic relationships within the genus Indotestudo, Iverson, et al. (2001) described
variation among the mitochondrial cytochrome b gene (cytb) of two captive I. forstenii
specimens, one with and one without a nuchal scute. The very low variation that was
detected was considered to be mainly an artifact of sampling, and the possibility of
separate lineages within the species was not ruled out.
Our understanding of phenotypic or morphological variation is beginning to come
into focus for some Asian chelonian species i.e. Cuora (Stuart and Parham 2004; Iverson
and McCord 1992), and Cyclemys and Mauremys (Fritz et al., 1997 and Iverson and
McCord 1997, 1994, respectively). Such variation is often a result of vicariance, in
which a species’ distribution is broken by a geographic barrier, thereby allowing
subsequent differentiation of the resultant allopatric groups. Whether the level of
differentiation is of the magnitude necessary to designate subspecies or separate species
is often the central question being asked by taxonomists.
Increasingly, the type of data used in molecular studies is dependent on the
phylogenetic groups being examined and the specific questions being asked (Lamb and
Lydeard, 1994; Sanderson and Shaffer, 2002). The use of cytb markers for determining
genetic divergence among Asian chelonians is widespread, and is largely a result of its
reliability and utility (Shaffer et al., 1997; Engstrom et al. 2002; Spinks et al. 2004). The
accumulation of cytb data, also allows researchers to make standardized comparisons
29

among different taxa, and enables various phylogenetic or biogeographic hypotheses to
be tested using a common marker.
Empirical field data, as well as anecdotal evidence, suggests that I. forstenii
populations are patchily distributed throughout the western part of both Northern and
Central Sulawesi (Hoogmoed and Crumly, 1984; Iverson, 1992; Platt et al., 2001; Samedi
and Iskandar. 2000). Although no fossil records are available, and only a few localities
of recent occurrence are known, information gathered in this study suggests that I.
forstenii may have undergone vicariance, a common mode of evolution in Sulawesi’s
flora and fauna (Evens et al., 2003; Whitten, 1987). The extent of vicariance and
resultant species diversification in Sulawesi is comparable to that of the Caribbean,
Hawaiian and Philippine Islands (Evens, et al., 2003). However, it is unclear whether
Sulawesi was once a single landmass that was subsequently fragmented and then recently
reunited (a vicariance model), or whether it was historically an archipelago that has been
recently united through uplift (a colonization model) (Evens et al., 2003; Whitten, 1987).
In either case, this fragmentation could have disrupted a once continuous distribution and
may have caused I. forstenii to become allopatrically distributed throughout the island.
Determining whether variability in the nuchal scute of I. forstenii actually
distinguishes populations requires a thorough biogeographic survey with numerous
samples from known localities throughout the range of the species. While outside the
scope of this thesis, examining genetic differences among phenotypically distinct
individuals is not. Accumulated evidence from molecular genetics studies has
demonstrated the need for genetic analysis along with morphological analysis in order to
accurately classify chelonian taxa. For example, genetic studies of the alligator snapping
30

turtle, Chelydra serpentina (Roman et al., 1999) and ridley sea turtles, Lepidochelys
olivacea, (Bowen et al., 1998) have demonstrated that an apparent lack of phenotypic
variation within a species does not necessarily reflect a lack of genetic diversity.
In this study, we collected morphometric measurements and generated nucleotide
sequence data from the cytb gene from I. forstenii with and without nuchal scutes and
asked whether readily observed phenotypic differences are indicative of distinct
mitochondrial lineages.
Methods
▪Determinations from the Field
Evidence suggesting phenotypic variability in wild populations of I. forstenii was
derived from information gathered from my fieldwork in the Palu Valley of Central
Sulawesi in February 2005. Reports I obtained from local turtle hunters, and a turtle
dealer in the Palu Valley, indicated that individuals captured in the north on the Minahasa
Peninsula do not have nuchal scutes, while individuals captured in the south in the Palu
Valley and other neighboring valleys have a nuchal scute (Figure 4). Although I lacked
resources to collect wild specimens from populations in the north of the island, I worked
to substantiate these claims by examining specimens captured in the south and reviewing
specimen material collected on the Minahasa Peninsula in 1872, 1896, and 1999.
I also assessed whether turtles with the two nuchal types also differ in size, by
comparing means of midline carapace length (MCL) and mass for I. forstenii of both
nuchal types. Analyses were performed by sex (males, n = 39; females, n = 32), using
JMP-IN 5.1 statistical software (Sall et al., 2005), with α = 0.05. Depending on
31

distributions of the measurement data, I used either a t-test or a Wilcoxon signed-ranks
test. Of the 39 males, 25 were from a commercial holding facility in Palu and 14 were
from a large captive assurance colony located at the Turtle Bank, a non-profit
conservation facility located in Massachusetts. Of the 32 females, nine were from the
holding facility and 23 from the assurance colony. All measurements were straight-line
measurements to the nearest 0.1 mm and all weights were in grams.
▪Sampling Techniques
Turtles used for genetic analysis were members of the captive assurance colony at
the Turtle Bank. All individuals were wild caught and were brought to the United States,
either through confiscation by U.S. customs officials or through legal purchases from a
licensed reptile exporter between 1997 and 2001. Twelve adult I. forstenii including six
with nuchal scutes and six without were sampled for genetic analyses. One ml of whole
blood was drawn from the subcarapacial vein of each turtle using a 23 gauge, 1”
heparinized needles. The blood collection technique was modified from that of
Hernandez-Divers et al. (2002). The subcarapacial vein was chosen because of the large
volume obtainable from this site and for its accessibility, regardless of the position of the
turtle’s head. Blood was preserved in a lysis buffer composed of 100mM Tris (pH 8),
100mM EDTA, 10mM NaCl and 1% SDS. Samples were shipped to the University of
California at Davis and stored at -20 0 C until analysis.
▪Genetic Analysis
Mitochondrial DNA (mtDNA) sequence variation was examined in two groups of
wild caught captive I. forstenii (n=12), one group with and one without the nuchal scute.
32

Our nucleotide sequence data set consisted of up ≈ 1100 base pairs (bp) of the
mitochondrial cytochrome b (cytb) gene for all 12 individuals. Genomic DNA was
obtained from the blood via proteinase K digestion followed by a salt extraction protocol
(Sambrook and Russell 2001). For the sequences generated here, we used the cytb
primers and PCR protocol from Spinks et al. (2004). Gene products were sequenced on
ABI 3100 automated sequencers in the UC Davis Division of Biological Sciences DNA
sequencing facility. Cytochrome b sequences were aligned within individual turtles using
SeQed (Applied Biosystems) and converted into amino acid sequences using MacClade
4.06 (Maddison and Maddison 2003). Alignments across taxa were made by eye in
PAUP* V4.0b10 (Swofford, 2001). No insertions or deletions were detected and all
nucleotide sequences translated into amino acid sequences. Pairwise uncorrected "P"
genetic distances were calculated using PAUP*4.0b10 (Swofford, 2001), and are shown
in Table 3. Sequences generated from this study will be deposited in GenBank.
Results
▪Field Data
Five of the six I. forstenii museum specimens with locality data were examined to
determine the presence or absence of a nuchal scute (Table 3). One specimen was on
loan and unavailable for examination. Each specimen was reportedly captured on the
Minahasa Peninsula, and all lacked a nuchal scute. While one field survey reported a
population of I. forstenii outside the Minahasa Peninsula (Groombridge, 1982), no
locality data were available. I observed 23 I. forstenii with nuchal scutes and 19
individuals without the scute while visiting a commercial turtle holding facility in Palu.
33

The owner of the facility, and long time collector of I. forstenii in the region, stated that
only individuals south of Palu have a nuchal scute, although this could not be confirmed.
Locality data could be confirmed for two of the five individuals I encountered in the Palu
and Kulawi Valleys (Table 3).
Mean comparisons of nuchal types revealed significant differences for both mass
and MCL in both sex’s. Turtles lacking nuchal scutes were longer in MCL than those
with nuchal scutes (P = 0.003, males; P = 0.018, females). Additionally, turtles lacking
nuchal scutes were heavier than those with nuchal scutes (P = 0.003, males; P = 0.024,
females; Table 4).
▪Genetic Analysis
We generated up to 1113 base pairs (bp) of cytb for 12 individuals. We recovered
very low levels of sequence variation within this group (Maximum uncorrected "P"
sequence divergence = 0.2%, Table 5), and found no correlation between cytb divergence
and presence or absence of the nuchal scute. For example, tortoises I1 and I8, which lack
nuchal scutes, were not divergent from I9 and I16 both of which have nuchal scutes, at
least with this marker (Table 5).
Discussion
The results of our cytb Mitochondrial DNA (mtDNA) sequence data revealed that
individual I. forstenii with and without the nuchal scute had nearly identical cytb
haplotypes, suggesting that nuchal differences do not reflect mtDNA lineages. The
maximum uncorrected "P" sequence divergence of 0.2% in this sample strongly suggests
34

that presence or absence of the nuchal scute is not correlated with mtDNA divergence.
Yet because cytb is merely one informative locus among a limitless number of possible
loci, it should not be exclusively relied upon to assess the possible existence of multiple
lineages. There is a chance of finding genetic differences between the two nuchal types
using a different gene, specifically nuclear DNA. Nuclear DNA’s utility for
phylogeographic and population genetics studies has been documented (Zhang and
Hewitt, 2003; Ballard and Whitlock, 2004) and was recently applied to the molecular
analysis of Emys marmorata in order to examine the nuclear gene aspects of this species’
evolutionary history (Spinks and Shaffer, 2005). Yet recent studies have shown that it is
difficult to find informative nuclear gene sequences within chelonians, so the use of this
marker may be limited. We plan on using nuclear DNA (nDNA) as a complimentary
source of data for this analysis. The nuclear markers would help us to determine if the
pattern from the mtDNA analysis were the result of mtDNA introgression. It is possible
that two divergent types of I. forstenii exist, each corresponding to the presence or
absence of the nuchal scute, yet subsequent contact between these types resulted in a few
nearly identical mtDNA haplotype becoming fixed within both types. If this were the
case, then the nDNA could unveil the hidden differences.
While nuclear genes can be used in determining intraspecific variation, mtDNA
are utilized more often. It appears that in turtles, the rate of nucleotide sequence evolution
in nDNA is far slower than that of mtDNA, thus reducing its usefulness in evaluating
intraspecific divergences (Zhang and Hewitt, 2003). Additionally, mtDNA is still the
preferred loci for phylogenetic analysis due to its reliability, utility and high incidence of
success in these types of analyses (Shaffer et al., 1997). Nonetheless, it is still important
35

to realize that further technical developments in this field such as multi-locus single
nucleotide polymorphism (SNP) analyses might change our view of population
substructure within this species.
The intraspecific phylogeny of I. forstenii is a mystery, particularly at the
phylogeographic level. Similarly, hypotheses explaining the geological and
palegeographical history of Sulawesi have only recently gained wide acceptance (Wilson
and Moss, 1998). Current evidence suggests that geologic events on Sulawesi caused
range fragmentation of the islands’ flora and fauna, whereby the biodiversity of unrelated
taxa with different life histories were compartmentalized (Evens, et al, 2003). Though
there are certainly many intertwined factors that have affected the diversity of organisms
on Sulawesi, Evens (2003) identified seven concordant “areas of endemism” in which
different taxa share the same distributions (Figure 5). Taxa found in these areas of
endemism likely diverged as a result of vicariance events (Whitten, 1987). The most
famous example of allopatry on Sulawesi is that of the island’s macaques (Macaca sp.),
whereby the putative division of a once continuous distribution isolated multiple
populations which then diverged into multiple subspecies (Bynum et al., 1997; Evens et
al., 1999; Evens et al., 2001).
Despite the finding that nuchal differences do not apparently correspond to
mtDNA lineages, my morphometric results support the existence of two morphologically
distinct populations of I. forstenii. This variability has three possible explanations based
on established geologic hypotheses: a vicariance scenario, a dispersal scenario and habitat
variation scenario. A vicariance event could have created two present populations
concordant with the Northwest and West central areas of endemism described by Evens,
36

one encompassing the Minahasa Peninsula and one from Palu south (Figure 5). Given
the narrowness of the southern extent of the Minahasa Peninsula, this section of land
could have acted as a barrier for dispersal, dividing the once contiguous distribution
roughly in half.
Alternatively, the regional variation within the species could also be a result of
dispersal. In this scenario, two distinct populations occurring on once isolated islands
within an archipelago could have dispersed and established sympatry following the
unification of the archipelago as a result of uplifting. Genetic drift, and founder effects,
both of which are more significant in small populations, could have affected the
population genetics within this species at present. Founder effects, in particular, could
explain the limited genetic variation observed in the mtDNA of I. forstenii. Despite an
incomplete understanding of Sulawesi’s complex paleogeographic evolution, each of
these hypothesis need to be tested. To make further progress on this issue, wild
specimens from each of these two proposed populations will need to be genetically
tested.
Another explanation for the apparent differences in morphology between
populations involves climate variability between the Minahasa Peninsula and the Palu
Valley. While temperatures throughout most of I. forstenii’s known range are relatively
constant, there is marked variation in rainfall. The hills adjacent to the Palu Valley,
where I documented the species presence, receive < 2,000 mm of rain annually (Whitten,
1987), while many of the localities on the Minahasa Peninsula where the species has been
recorded receive > 2,500 mm ( Figure 6). Limited resources as a result of a dryer climate
could result in reduced growth rates and smaller body size of tortoises in and around the
37

Palu Valley. This effect has been hypothesized for other turtle species, including Emys
orbicularis (Fritz and Schulze, 2003) and Testudo weissingeri (Bringsoe et al., 2001;
Fritz et al., 2005 in press). T. weissingeri, a dwarf population of T. marginata restricted
to the Manai Peninsula of Greece was recently designated a nominal species primarily do
to size and color differences to that of T. marginata. Subsequent use of mitochondrial
and nuclear genomic markers by Fritz et al. (2005) determined that T. weissingeri was not
a distinct evolutionary lineage. The reduced size of T. weissingeri was suggested to be a
result of suboptimal environmental conditions. A thorough survey of wild I. forstenii
populations could reveal additional variation in the species population demographics,
habitat utilization and natural history.
In summary, the phenotypic variation of I. forstenii, while not demonstrative of
mtDNA clades, does indicate a level of diversity within the species range and may
represent varied selection in different habitats as a result of environmental factors or
genetic bottlenecking. Our genetic results support recent findings by Fritz and
colleagues’ (2005) that phenotypic variability in Testudines, whatever its origin, is not
necessarily diagnostic of mitochondrial genetic variability. We stress the use of
“mitochondrial” here, because we realize that this is but one loci. Additional power
though the use of nDNA and gene frequencies will allow us to make stronger inferences
about the geneology of I. forstenii, and may yet reveal further differences. None the less,
it is becoming increasingly clear that one must take into account traditional morphology –
based systematics, offering well-accepted methodology as well as molecular systematics,
offering new rapidly evolving methods in the classification of Asian chelonians. The
traditional approach, depends on the phenotype and behavior of the organism, and is
38

more likely to use morphological differences as grounds for classification or revisions of
taxa, while the molecular systematic approach will rely on the genotype or gene
frequencies. As efforts to determine the phylogeography of Asia’s endangered
chelonians continues, it is critical to use an inclusive approach, whereby cladistics backed
by molecular sytematics are used in conjunction with morphological methods of
classification.
Due to the continued harvest of this species from the wild and the urgent need for
protection, immediate conservation focus should be placed on determining the species
distribution. A concerted effort by Indonesian and international organizations to protect
and effectively manage the remaining hotspots for biological diversity within Sulawesi’s
West Central and Northwest areas of endemism, would be an important starting point.
Subsequently, efforts to protect the genetic variation of I. forstenii throughout the entire
range of the species can commence.
39

CHAPTER 3
Is Lack of Reproductive Success by Captive Leucocephalon yuwonoi Caused by
Stress
Introduction
One of the primary goals of those working to combat the Asian Turtle Crisis has
been to successfully manage and re-introduce into the wild large numbers of healthy,
reproductively active turtles. This is achieved through establishment of “assurance
colonies”, assemblages of wild caught individuals held in captivity for purposes of
maintaining genetic viability though sustainable captive management programs.
Effectiveness of these efforts obviously depends, among other things, on the ability to
establish successful breeding programs.
The TSA has created a TMG for L. yuwonoi, and a Captive Management Plan
(TMP) has been drafted. At this early stage in development of the TMP, accumulation of
baseline data concerning all aspects of L. yuwonoi’s reproductive biology and life history
is of great importance. This information will help inform decisions concerning
husbandry, breeding trials, incubation techniques, neonate rearing, genetic management
of collections, and health issues, all factors that are critical to successful ex-situ
management and reintroduction.
Little is known about the reproductive biology of L. yuwonoi, and there is no
published information on the species’ reproduction in the wild, growth rates,
developmental morphology or age of sexual maturity. For unknown reasons the species
does not reproduce well in captivity; to date, there has been only one successful hatching
40

from a TMG captive breeding trial, and it occurred without any manipulation or even
knowledge by the keeper (C. Innis, pers. comm.). Several theories might explain this low
hatching success, yet determining the cause or combination of causes has proven difficult
due to the numerous variables associated with captive management and the successful
development of chelonian embryos. Innis (2003) hypothesized that embryos may be
killed while in the oviduct during a turtle’s transportation from Asia to captivity. Gravid
females that have been administered oxitocin to induce laying of eggs carried in transport
have produced highly calcified eggs that have ultimately proved unviable (pers. obs.).
Embryo mortality in these cases could be due to prolonged retention in the oviduct during
the long holding and shipping process, or to direct impacts on the embryo related to the
stress of shipment (B. Bonner, pers. comm.).
Various poorly-understood aspects of the captive breeding environments could be
responsible for the low reproductive success by long-term captives of L. yuwonoi. Such
“stressors”, defined by Guillette et al. (1995) as “any external forces applied to animals
that threaten their homeostasis” could include improper housing conditions, poor diet,
inappropriate temperatures, or disturbance by humans. Again following Guillette et al.’s
(1995) definitions, stressors elicit “stress responses”, which refer to “the combination of
responses mounted by an animal toward such a stressor that involve increased activity of
the adrenal gland”. In particular, adrenal secretions that are produced in response to
stress include the glucocorticoids cortisol (in fish and mammals) and corticosterone (in
birds, amphibians and reptiles) (Greenberg and Wingfield, 1987). These hormones play
central roles in maintaining the animal’s homeostasis (Axelrod and Reisine, 1984).
41

Assessment of corticosterone levels has recently become a standard technique for
evaluating stress in birds, amphibians and reptiles. However, this method requires some
knowledge of baseline levels of corticosterone for a species; such “unstressed titers”
provide background values to which data collected under the influence of various
stressors may be compared.
Unfortunately, knowledge of how corticosterone levels vary, in the absence of
any stressors, is limited for virtually all taxa. Baseline concentrations of corticosterone
may fluctuate in response to numerous environmental factors, including temperature,
humidity, food intake and habitat quality (Kitayski et al., 1999; Romero, 2002).
Corticosterone levels also vary according to circadian rhythms (Breuner et al., 1999;
Joseph and Meier, 1973; Lauber et al., 1987; Weinert et al., 1994). In studies of captive
American alligators, Alligator mississippiensis (Lance and Lauren, 1984), three diurnal
lizards (Chan and Callard, 1972; Dauphin-Villemant and Xavier, 1987; Summers and
Norman, 1988), and green sea turtles, Chelonia mydas (Jessop et al., 2002), maximum
corticosterone levels all coincided with the animals’ daily active periods. I am unaware
of any documented information on rhythms in captive freshwater turtles.
Research on various vertebrate taxa indicate there is an approximately 2 minute
time lag between presentation of a stressful stimulus and appearance of measurable levels
of corticosterone in an animal’s blood (Romero, 2002; Romero and Reed, 2005). By
attempting to draw blood samples as soon as possible after capture, most investigators
assume that such corticosterone levels represent baseline values (“unstressed titers”).
Baseline corticosterone concentrations among reptile species is highly variable, ranging
42

from 0.36 – 1.08 ng/ml in American alligators, 0.55- 2.0 ng/ml in sea turtles, and 80- 150
ng/ml in several squamates (Tyrrell and Cree 1998).
Stress, as indicated by elevated corticosterone levels, has been shown in reptiles
to adversely influence food intake, metabolism of essential nutrients, and growth
(Guillette, et al., 1995). Selye (1936, 1937) early described three anatomical and
physiological responses to stressors including alarm, resistance and exhaustion. In the
alarm stage, there is an acute, short-term response; if the stress becomes chronic, the
animal enters the resistance stage, which may be characterized by an inhibition of
gonadal development and function (Greenberg and Wingfield 1987; Rivier, et al., 1986;
Sapolski, 1986).
In captive female American alligators increased levels of corticosterone correlate
with lower nesting success (Elsey et al., 1990) and in the lizard Anolis carolinensis,
stressful laboratory conditions inhibit reproduction (Jones et al., 1983). Guillette et al.
(1995) showed that stressors can delay or preclude oviposition or parturition, and
suggested that elevated levels of progesterone and corticosterone are critical components
in egg retention in reptiles.
Few studies have investigated whether reptiles held in long-term captivity exhibit
chronic stress as evidenced by elevated corticosterone levels. Elsey et al. (1991) showed
that corticosterone were elevated in long-term captive alligators, thus suggesting that the
animals were suffering from chronic stress. In contrast, Tyrrell and Cree (1994)
concluded that tuataras Sphenodon punctatus held in long-term captivity were not
suffering from chronic stress; while corticosterone levels in captive juvenile females were
higher than those of their wild counterparts during the non-breeding season, these long-
43

term captive females showed lower levels of corticosterone than recently-captured
individuals that had been held in cloth bags for 3 hours.
Studies examining effects of stress on chelonians have been limited to
descriptions of corticosterone concentrations in wild individuals that have been acutely
stressed by capture and handling. Two recent investigations found that changes in
corticosterone levels associated with capture and handling followed a typical reptilian
pattern, with low concentrations at “time-0”, followed by a dramatic increase with
subsequent blood sampling, and, finally, a decline at a critical point in time (Cash, et al.,
1997; Gregory, et al., 1996).
Two studies of free-living chelonians have used different methods for estimating
actual unstressed titers. In their study of red-eared sliders, Trachemys scripta elegans,
Cash et al. (1997) found no relationship between initial corticosterone levels and
handling time, and concluded that unstressed titers could be obtained from samples
drawn within 10 minutes of capture. Greggory et al. (1996) reasoned, somewhat
circularly, that since the levels of corticosterone they observed in loggerhead sea turtles,
Caretta caretta, were among the lowest reported by several comparable studies, these
values must therefore reflect an unstressed condition in the animals.
Because of the complex suite of variables that may potentially influence
corticosterone levels, conclusively determining what effect stress may have on L.
yuwonoi’s ability to reproduce in captivity is beyond the scope of this study.
Nonetheless, this work provides important information concerning basal corticosterone
levels observed in the species under captive conditions; I also examine daily fluctuations
in corticosterone levels during the species’ diurnal active period. Because herpetological
44

medicine is still in its infancy, and because turtles in assurance colonies often require
blood collection for diagnostic purposes, any information that can increase the safety and
effectiveness of this invasive procedure is of great importance. Consequently, I also
report information regarding the effects of time of day, sex, and size of turtle on the time
required to successfully obtain blood samples, and describe an effective location for
venipuncture.
Behavioral observations of captive yuwonoi in this study suggest that individuals
may be sensitive to various stressors. Individuals of both sexes are acutely aware of their
surroundings, and appear visually attuned to movements both within and outside their
cages. Long-term captives, especially males, assume defensive postures and hiss when
closely approached by humans, and resist handling by flailing their legs, biting, and
defecating profusely. Here I provide a preliminary evaluation of yuwonoi’s sensitivity to
stress by comparing whether animals housed in captivity exhibit different levels of
corticosterone under differing social conditions.
All of this information will lay a foundation for future research and management
efforts specifically directed toward this endangered species. More broadly, these data
also add to the growing body of basic information needed to facilitate comparative
studies of stress in other turtles, and reptiles in general.
45

Methods
▪Study Turtles
The L. yuwonoi used for this study were captive turtles held as an assurance
colony at the Turtle Bank in Upton, Massachusetts. All turtles have been present at this
location for a minimum of 3 years, and represent a significant proportion of the world’s
healthy, acclimated, captive L. yuwonoi. All individuals were adults, as determined by
morphological characteristics. Sex was determined through observation of primary and
secondary sex characteristics, which are easily discernable in this species (McCord et al.,
1995; McCord et al., 2000).
All animals were kept in their respective enclosures and room locations for at
least 11 months prior to the study. Females were housed either individually or in groups
of three; all males were housed individually due to aggressive interactions when kept
together. Enclosures for males and 3-animal groups of females measured 153 cm x 92
cm; enclosures for solitary females measured 40 cm x 60 cm. Newspaper lined each
enclosure, and water was provided ad libitum. Turtles were fed a complete diet of
tortoise chow (Mazuri/ PMI), supplemented with mixed fruits and vegetables, once a
week; during the course of this study, feeding occurred three days prior to sampling.
All turtles were exposed to consistent photoperiod (12L:12:D light cycle), diet,
and temperature (approximately 27° C) for at least 3 months prior to, and throughout, the
study period. These environmental conditions approximate the climate of L. yuwonoi’s
known range.
46

▪Sampling Procedure
Every effort was made to ensure that blood samples reflected baseline levels of
corticosterone for L. yuwonoi’s typical existence at this facility. No unusual activities
were conducted in, or around, the holding areas on sampling days. Otherwise, daily
keeper routines, including cleaning, enclosure manipulations and periodic handling for
health checks were followed as usual.
One sample for each turtle (n=39) was collected during each of 3 time periods
(06:00-09:00, 12:30-15:30, and 19:00-22:00 h) within the species’ active daylight period,
for a total of 117 samples. This schedule was designed to incorporate blood samples
from lights-out periods both in the morning and evening (Figure 7). Three sample groups
(13 individually-housed females, 13 group-housed females, and 13 individually-housed
males) were used to compare corticosterone levels between the sexes and, for females,
between two housing arrangements (alone vs. grouped). Ample time was provided to
allow the animals to recover from stress of the previous sampling, as sampling of the
active period was evenly distributed over the course of 41 days. It is generally accepted
that 36 hours is required for CORT levels to return to basal levels following a stressful
event (Romero and Remage-Healey, 2000). While collecting samples during lights-off
periods (20:00 – 08:00 hrs), care was taken to avoid exposing the turtles to lights prior to
blood collection.
Blood samples were collected from the subcarapacial vein using either 25 gauge,
5/8" or 23 gauge, 1” heparinized needles, depending on the turtle’s size. The blood
collection technique was modified from that of Hernandez-Divers et al. (2002). The
subcarapacial vein was chosen because of the speed with which blood can be drawn, the
47

large volume obtainable, and accessibility of this site regardless of the position of the
turtle’s head. Approximately 1 ml of blood was collected in Microtainer tubes with
lithium heparin and plasma separator (Becton Dickinson Co.). Samples were centrifuged
for 5 min within 15 min of collection, and the resulting plasma frozen at –15° C until
shipment on dry ice to the Core Endocrine Lab at Pennsylvania State University for
processing.
All turtles were measured and weighed after samples were collected.
Measurements were taken of midline carapace length, maximum width and maximum
height with electronic calipers (±0.1 mm); weights were obtained from a digital balance
(±0.1 kg).
▪Sample Processing and Analysis
Corticosterone levels were measured in plasma using a competitive solid-phase
radioimmunoassay method (Soldberg et al., 2001), in which corticosterone-specific
antibodies are immobilized to the wall of polypropylene tubes. 125I radiolabeled
corticosterone (ICN Pharmaceuticals, Costa Mesa, California) competes with
corticosterone in the sample, standard or control for binding to the corticosterone
antibody. Separation of free corticosterone from the antibody bound fraction is
accomplished by decanting. The tubes are then counted in a gamma counter and the
amount of corticosterone present in the sample is determined from a calibration curve
ranging from 10 - 2000 ng/ml using logit transformation of B/Bo. Samples which fell
below the lower range of this curve were represented in this analyses as concentrations of
5 ng/ml (the mean of 0 and 5). Intra-assay precision of this RIA at a concentration of 45
48

ng/ml averaged 4.4%, while inter-assay precision at a concentration of 48 ng/ml was
5.7%. No appreciable cross reactivity of the corticosterone antibody was seen with
cortisol, progesterone or deoxycortisol.
▪Data Analysis
All statistical tests were implemented with JMP IN (version 5.1) software (Sall et
al., 2001), using a significance level of α = 0.05. Results are reported as mean ± SE.
Times required to obtain blood draws were log 10 -transformed to improve normality of
distribution, as were corticosterone levels. Univariate linear regression was used to
evaluate time required to draw blood as a function of time of day, both within and among
each of the three sampling periods. The relationship between the likelihood of obtaining
lymph during the blood draw and turtle size was examined with logistic regression.
Repeated measures MANOVA was used to assess whether corticosterone levels differed
between (a) males and solitary females and, (b) females housed as 3-animal groups or
solitarily. In each MANOVA, time required to obtain the blood draw, and the associated
interaction terms with sex or housing arrangement, were included as covariates.
Results
▪Sampling Procedure
There were no significant differences between the sexes in the time required to
draw blood during each of the three collection periods (Wilcoxon rank sum: morning: Z =
0.939, P = 0.348; mid-day: Z = -1.221, P =0.222; evening: Z = 0.581, P = 0.561).
49

Consequently, I pooled data from the sexes in all subsequent analyses of sampling
procedure.
The mean time required to draw blood across all sampling periods was 114.64 ±
5.48 seconds (n=117), or approximately 1 minute 55 seconds (Figure 8). There was no
relationship between time required to draw blood and time of day during the morning
(linear regression: F = 3.02, P = 0.091) or evening periods (F = 0.10, P = 0.754); during
the mid-day sampling period, time required to obtain blood declined slightly from 12:00
– 15:00 h (F = 4.54, P = 0.040). Blood draws were obtained most quickly (92.5 ± 4.43
sec) during the evening sampling period, and most slowly (138.41 ± 12.23 sec) during
the morning (Figure 9). Lymph was more common in blood samples collected from
smaller turtles than those taken from larger turtles (logistic regression; x² = 0.048, P =
0.035).
▪Basal Corticosterone
A single individual required an exceptionally long time period (7 min 15 sec)
before a blood draw could successfully be obtained; because the corticosterone value
from this sample was the highest recorded in this study (17.4 ng/ml), I excluded this
record from the following analyses. There was no significant effect of time after capture
on baseline corticosterone levels in either sex when values from the three time periods
and, for females, housing status, were pooled (males; F 1, 37 = 0.39, P = 0.539; females;
F 1,76 =3.09, P = 0.083).
Baseline corticosterone concentrations were compared between males and
females in each of the three time slots (Table 6). There was a significant difference in
50

corticosterone levels between males and grouped females in each of the three time slots
(Wilcoxon rank sum: Z = 2.937, P = 0.003 morning; Z = 3.024, P = 0.002 afternoon; Z =
3.102, P = 0.001 evening. Conversely, corticosterone levels did not differ significantly
between males and females kept in individual enclosures (MANOVA, F 1,23 = 0.097, P =
0.150). Mean concentration of basal corticosterone in males (n=38, all housed
individually) was 11.24 (± 0.52) ng/ml, with values ranging from 5.0 (assigned here to
represent values less that the lowest detectable value available to this assay) - 16.3
ng/ml. Mean concentration of basal corticosterone in females kept in individual
enclosures (n=39) was 9.56 (± 0.56) ng/ml, with values ranging from 5.0 – 14.9 ng/ml.
Corticosterone levels in females housed in groups of 3 individuals were
significantly lower than in individually-housed females (P = 0.002; Table 7). There was
no relationship between corticosterone levels and the time required to draw the blood
samples (P = 0.433), or with the interaction term between housing type and time required
for blood draw (P = 0.767). Differences between the two housing arrangements were least
pronounced during the mid-day sampling period (Figure 10). Mean corticosterone
concentration in 3-individual groups (n=13 across all time periods) was 6.64 (± 0.43)
ng/ml, with values ranging from 5.0 – 12.0 ng/ml; females kept alone (n=13) showed
mean corticosterone values of 9.56 (± 0.56) ng/ml, with values ranging from 5.0 – 14.9
ng/ml.
There was a difference in CORT levels for females between the pre-dawn (6:30-
8:00), daytime (8:00-20:00), and after dark periods (20:00-22:00) during the species
active daytime period (Kruskal –Wallace: r² = 8.674, P = 0.013) and a Tukey Kramer
HSD revealed that CORT levels from the pre-dawn period were significantly different
51

from both the daytime and after dark periods. Conversely, there was no difference in
CORT levels for males between the three periods (Kruskal –Wallace: r² = 3.646, P =
0.164: Figure 11).
52

Discussion
▪Sampling Procedure
Because turtles in assurance colonies often require blood collection for
therapeutics and rehabilitation, information that can increase the safety and effectiveness
of blood collection is of great value. Poorly executed blood collections in which forceful
restraint of the animal results in undo stress can have detrimental effects on the animal.
Likewise, collection sites ill suited for the animal can result in excessive collection times,
contamination with lymph, and hematomas. Therefore clinical techniques that increase
the ease with which blood is drawn can increase the safety and efficiency of the
procedure. In this study, safety and efficiency are of particular importance. The value of
the turtles, from conservation, ethical and monetary standpoints requires great care in
assuring the safety of the animals. Efficiency was critical in this study in order to
minimize the effect the procedure had on corticosterone, but also necessary in terms of
adhering to the experimental design schedule.
Lack of a significant difference between the sexes in the time required to draw
blood indicates that there are no anatomical or physiological differences between the
sexes that would affect the amount of time required to complete this procedure. These
results suggest that, despite the substantial difference in size between the sexes, the
length and position of the needle at the collection point can be the same for both sexes.
The mean draw time of 1 minute 55 seconds for all samples taken from both sexes
virtually assures that the average corticosterone levels I obtained were indicative of
baseline levels for this captive group, and not a reflection of stress resulting from the
blood draw procedure. Blood drawn within 2-3 min has been assumed to reflect pre-stress
53

aseline CORT concentrations for most vertebrate species studied thus far (Romero,
2002; Romero and Reed, 2005). In reptiles, however, a window of as much as 10 min
may exist before increases in corticosterone are detectable (see review by Tyrrell and
Cree, 1998). Thus there is a high likelihood that even samples which required draw times
of up to 5.5 min in this study are representative of baseline corticosterone levels. This is
an important determination, given the relatively high corticosterone levels obtained from
these turtles, and is critical first step in assessing whether or not L. yuwonoi suffers from
chronic stress in captivity.
There was a significant relationship between the time required to draw blood and
time of day, with draw times highest in the morning and decreasing during each
subsequent sample period. In this analysis, blood draw times over 2 minutes 56 seconds
were excluded in order to avoid the potential effect of outliers, which might suggest a
pattern where none exists. While a relationship was found, there was substantial
variability in the time required to draw blood, reflected by low (0.105) r² value. Human
performance can vary with procedures such as this that require a steady hand and
prolonged concentration for long periods of time, and it is possible that the investigator
executed blood draws more quickly in the evening than earlier in the day. Also, basking,
coupled with greater activity levels, would increase the temperature of the turtles
throughout the day, thus increasing blood perfusion and blood pressure allowing faster
collection as the day progressed. This could very well explain the disproportionately
high morning draw times of males whose larger surface areas require a longer time to
reach a given temperature.
54

Because the large sample size necessitated an experimental design in which 3
hours were needed to draw blood within each time slot, there was a possibility of
obtaining a relationship between time required to draw blood and time of day within each
of the three periods. However, the test results show that this was not the case, suggesting
that the rate of change in blood perfusion and/or blood pressure is slow within the turtle’s
active daylight period.
Lymph was more common in blood samples collected from smaller turtles than
those taken from larger turtles. Although this fact may not have any biological
significance, it was relevant to the study, as lymph was a possible contaminant of the
blood samples.
▪Basal Corticosterone
The assurance colony of L. yuwonoi examined in this study has higher baseline
corticosterone levels than many other reptiles (captive and wild) that have been studied to
date. Mean corticosterone levels across all time periods were 11.38 ng/ml (males) and
8.09 ng/ml (females). In comparison, multiple studies of captive alligators obtained
mean values no higher than 1.08 ng/ml, levels of captive green sea turtles peaked at
approximately 0.7 ng/ml, captive male desert tortoises, Gopherus agassizii, showed
levels no higher than 6.48 ng/ml, and captive tuataras had mean levels of 4.65 ng/ml
(Elsey, et al., 1989, Tyrrell and Cree, 1994; Jessop et al., 2002; Lance et al., 2001).
Wild-captured red-eared sliders showed mean levels of 3.5 ng/ml, and loggerhead sea
turtles had mean levels of 0.55 ng/ml (Gregory et al., 1996; Cash et al., 1997). While
comparisons between studies must be made with caution due to the living status of the
55

animals tested (wild vs captive) and varied radioimmunoassay techniques utilized, it
appears that the baseline corticosterone levels from this study are an order of magnitude
greater than those of many other reptiles, including other chelonians, and therefore should
be considered high. The values obtained in this study will now have to be compared to
values of both baseline and stressed levels of wild L. yuwonoi before any conclusions can
be made regarding the effect these elevated levels may have on this collection or on the
reproductive biology of the species.
L. yuwonoi showed no significant increase in corticosterone levels within the first
5 min 30 sec of capture. Similarly, red-eared sliders showed no significant increase in
corticosterone levels within the first 10 min of capture (Cash et al., 1997) and the lizard
Sceloporus cyanogenys showed no change within the first 3 min of capture (Manzo et al.,
1994). In contrast, corticosterone levels in the tree lizard, Urosaurus ornatus,
significantly increased within the first 10 min of capture and likely showed some increase
in half that time (Moore et al., 1991). These results demonstrate the variability in
secretion rates of corticosterone levels within reptilian species, and suggest that L.
yuwonoi has a moderate rate of corticosterone levels secretion compared to other reptiles.
Furthermore, the lack of a significant increase in corticosterone within 5 min 30 sec of
capture is consistent with my contention that these are truly baseline values for the
species, rather than values resulting from the stress of the blood draw procedure.
The significantly higher corticosterone levels in males than in females in this
study lends support to the observation that male L. yuwonoi are more prone to stress in
response to the presence of humans and other stimuli in captivity than females. In
captive desert tortoises and American alligators, males also had significantly higher
56

corticosterone levels than females, although no explanation was provided as to why
(Elsey, et al., 1990; Lance et al., 2001). The elevated corticosterone levels in male L.
yuwonoi could also reflect higher metabolism and energy demands than that of females,
under a captive environment. Field tests will need to be undertaken in order to determine
if corticosterone levels observed in captivity are analogous to those of wild individuals.
Interestingly, females that were individually housed had significantly higher
corticosterone levels than females maintained in groups of 3 animals, but not
significantly lower than the individually housed males. These results suggest that
housing females of this species in groups decreases overall stress. One explanation could
be that individuals housed together habituate one another to movements, sounds and
other stimuli that may cause alarm to individually-housed turtles, thus decreasing their
level of stress. Similarly, there may be an element of sociality that somehow reduces
stress in a grouped environment. This finding is significant in that it provides evidence
that housing females together is preferable to housing them individually. This finding
may be of particular relevance to assurance colonies, in which facility space is often at a
premium. Interestingly, tests of the effects of group housing on stress in the highly social
American alligator, showed that, at least with juveniles, high density housing resulted in
chronically elevated corticosterone levels (c. 13 ng/ml) that was three times that observed
under low density conditions (Lance and Lauren, 1984). Consequently, grouping female
L. yuwonoi may only be advantageous up to a certain number of individuals; more study
is needed.
Gregory, et al. (1996) found that large variability in corticosterone levels can exist
within treatment groups, and such was the case in this study. For example, one female
57

whose blood was collected in 1 min 22 sec had a corticosterone value of 13.0 ng/ml,
while another female whose blood collection took 3 min 21 sec had a value of 10.6
ng/ml. Males showed even greater variability; one male produced a corticosterone
reading of 14.1 ng/ml within 1 min of capture, while another produced a reading of 10.3
ng/ml from a collection that took 5 min. This variability could result from any number
of factors not considered in this study, including genetic differences, previous
experiences with stressful stimuli, or overall health.
Identifying the exact point in the daily cycle that corticosterone peaks was not
possible in this study because no sampling was conducted between 22:00 - 6:00 h. Yet
by comparing blood that was collected during the pre-dawn hours (before lights came on
in the morning) to blood collected during daylight and after dark, it was possible to see if
there were significant differences in corticosterone levels between dark and light periods.
For females, corticosterone levels in the pre-dawn hours were significantly different to
both the daytime and after dark periods. In males, although there was a decrease in
corticosterone from the pre-dawn to the after dark periods, there was no statistically
significant difference between the periods. This could well have been due to a small
sample size for this comparison. The result for females though parrellells the findings of
Breuner which show that levels of corticosterone in mammals and birds have a “preactive
peak” just before daylight, followed by a drop in levels from that point forth
(Breuner et al., 1999). So while a pre-active peak in corticosterone can not be proven
here, the results are highly suggestive of a similar 24 hour corticosterone profile in L.
yuwonoi.
58

Males and female L. yuwonoi pooled together showed a fluctuation in baseline
corticosterone during the active daytime period (between 8:00 and 20:00 hrs) whereby
corticosterone levels peaked in the morning and dropped as the day progressed. When
sex’s were examined separately, females showed this profile while males did not.
Furthermore, when females were separated by housing status, only the grouped females
showed this profile. Not all reptiles show daily fluctuations in baseline corticosterone.
Within chelonians, the green sea turtle displayed fluctuations yet both the loggerhead sea
turtle and the red-eared slider did not (Gregory et al., 1996; Cash et al., 1997; Jessop et
al., 2002). In other reptilians, marine iguanas (Amblyrhynchus cristatus), American
alligators, desert iguanas, green anoles and viviparous lizards show corticosterone
fluctuations while the tuatara does not (Chan and Callard, 1972, Lance and Lauren, 1984;
Dauphin-Villemant and Xavier 1987; Summers and Norman, 1988; Tyrrell and Cree,
1998, Woodley et al., 2003). For those species showing daily fluctuations in
corticosterone, the highest levels are during the animals active period when energy and
metabolic demands are at their greatest. L. yuwonoi’s high corticosterone values
exhibited during their active daylight period seams to correspond with this type of profile.
While these findings beg for comparison, certain factors reduce their comparability
including but not limited to living status (wild vs captive), feeding regimes, sampling
time and temperature, all variables that influence corticosterone levels. Therefore
comparisons should be made with caution.
In summary, corticosterone levels in this captive group of L. yuwonoi were
relatively high compared with many other reptiles, showed no obvious increase within
the first 5.5 min after capture, were higher in males than in females, were lower in
59

grouped females than in females housed alone, peaked in the morning and dropped as the
day progressed, and were significantly different in the pre-dawn hours than in the
daylight and after dark hours. The paucity of data on the relationship between
corticosterone and stress in captive chelonians has made interpreting these results a
challenge. Future research is required to better understand both the cause and biological
significance of the high corticosterone levels detected in this assurance colony. There
may well be a threshold value of corticosterone which must be exceeded before
detrimental effects on reproduction occur. L. yuwonoi collections experiencing
reproductive success, as well as those experiencing reproductive failure should have
corticosterone assays conducted. In doing so, this threshold may be determined.
60

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69

TABLES
Measurement Mean ± 1 SD (range) n
Palu Group - females Body Mass (kg) 1.2 ± 0.423 (0.600–1.8) 15
Midline Carapace Length (mm) 185.6 ± 23.4 (142.2–210.0) 15
Palu Group – males Body Mass (kg) 1.8 ± 0.342 (1.0–2.45) 15
Midline Carapace Length (mm) 231.3 ± 16.5 (180.0–250.0) 15
U.S Group – females Body Mass (kg) 1.2 ± 0.344 (0.483-1.6) 15
Midline Carapace Length (mm) 193.4 ± 19.2 (152.8-218.0) 15
U.S Group – males Body Mass (kg) 2.0 ± 0.678 (0.435-2.7) 15
Midline Carapace Length (mm) 240.9 ± 33.8 (143.0-271.9) 15
Table 1. Morphometric comparisons between the Palu and long term captive Leucocephalon yuwonoi groups.
70

Measurement Mean ± 1 SD (range) n % of total
Males Body mass (kg) 1.1± 0.510 (0.400-2.250) 27 64.2
Midline carapace length (mm) 197.8 ± 35.2 (134.2-260.8) 27
Maximum carapace width (mm) 136.6 ± 22.1 (100.4-177.0) 27
Females Body mass (kg) 0.916 ± 0.464 (0.300-1.6) 9 21.4
Midline carapace length (mm) 177.0 ± 39.9 (115.4-227.0) 9
Maximum carapace width (mm) 126.4 ± 20.1 (94.3-151.7) 9
Juveniles Body mass (kg) 0.191 ± 0.091 (0.100-0.350) 6 14.2
Midline carapace length (mm) 90.4 ± 18.6 (71.0-116.4) 6
Maximum carapace width (mm) 80.1± 9.4 (69.3-90.5) 6
Table 2. Morphometric data from captive Indotesdtudo forstenii in holding in Palu, Sulawesi.
71

Museum Specimens
Museum number Date Location Nuchal Present
R-145099¹ 1999 Cape Santigi, Sulawesi No
R-145100¹ 1999 Cape Santigi, Sulawesi No
R-145101¹ 1999 Cape Santigi, Sulawesi No
1872.4.6.116² 1872 Boliahoeta, Celebes No
1896.12.9.1² 1896 Buol, North Celebes No
USNM 52973³* 1915 Boliahoeta, Celebes did not examine
In situ Specimens†
Location Coordinates Nuchal scute (Y or N)
Kulawi, Sulawesi 01º26’S, 119º59’E Yes
Lempelero, Sulawesi 01°39’S, 120°02’E Yes
Table 3. Localities for Indotestudo forstenii with and without a nuchal scute, suggesting two distinct populations (northern and
southern) with distinguishing phenotypes. Museum specimens were collected on the Minahasa Peninsula, and in situ specimens were
observed in the Kulawi Valley by the author. ¹ - American Museum of Natural History, NY, ² - The Natural History Museum,
London, ³ - National Museum of Natural History, Washington D.C.
* - Specimen on loan and unavailable for examination.
† - Coordinates for in situ specimens represent second hand reports.
72

Measurement Sex Nuchal scute absent¹ Nuchal scute present¹ t² P
Carapace length M 214.6, 27.0 (164.6-260.8), n= 19 190.1, 21.6 (145.2-235.2), n=20 3.14 0.003
Carapace length F 208.3, 12.8 (189.7-227.3), n=17 186.5, 33.4 (115.4-223.2) n=15 2.489 0.018
Mass M 1362.6, 449.3 (682-2250) n= 19 942.2, 229.9 (600-1374) n= 20 2.593 (Z test) 0.003
Mass F 1253.05, 197.2 (980-1650) n= 17 1003.9, 378.3 (300-1615) n= 15 2.376 0.024
Table 4. Comparison of carapace length and mass measurements between I. forstenii with and without nuchal scutes. Total sample of
71 turtles was comprised of individuals from a commercial holding facility in Palu, Sulawesi and an assurance colony in the U.S.
¹ Mean, standard deviation (minimum – maximum), sample size.
² t-test or Wilcoxon rank-sum test.
73

Nuchal
Pairwise uncorrected "P" genetic distance
Scute Taxon I1 I2 I4 I5 I8 I9 I11 I15 I16 I19 I20
absent I1 -
absent I2 0.002 -
present I4 0 0.001 -
present I5 0 0.002 0 -
absent I8 0 0.002 0 0 -
present I9 0 0.001 0 0 0 -
absent I11 0.002 0 0.001 0.002 0.002 0.001 -
absent I15 0.001 0.001 0 0.001 0.001 0 0.001 -
present I16 0 0.001 0 0 0 0 0.001 0 -
absent I19 0.001 0 0.001 0.001 0.001 0.001 0 0.001 0.001 -
present I20 0.001 0 0.001 0.001 0.001 0.001 0 0.001 0.001 0 -
present I21 0 0.001 0 0 0 0 0.001 0 0 0.001 0.001
Table 5. Uncorrected (“p”) distance matrix for samples of Indotestudo forstenii from an assurance colony (Turtle Bank,
Massachusetts) in the U.S.
74

Sample period Males Females grouped Females alone
Morning (6:00 – 9:30) 12.15 ± 0.85 7.61 ± 0.82 10.66 ± 0.97
n 12 13 13
Afternoon (12:30-15:00) 11.76 ± 0.95 6.86 ± 0.81 8.78 ± 1.06
n 13 13 13
Evening (19:00 – 22:30) 9.81 ± 0.97 5.42 ± 0.43 9.23 ± 0.83
n 13 13 13
Mean Total: 11.24 ± 0.52 6.63 ± 0.42 9.55 ± 0.55
Table 6. Baseline plasma corticosterone concentrations (mean ± SE; ng/ml) in long
term captive male and female L. yuwonoi.
75

Test Value Exact F NumDF DenDF Prob>F
Intercept 9.17 201.72 1 22

FIGURES
1400
1200
1000
800
600
400
200
0
1985
1987
1989
1991
# turtles exported
1993
1995
1997
1999
2001
2003
Year
Figure 1. Net export of live I. forstenii from Indonesia between 1985 - 2004 (from
CITES/United Nations Environment Program World Conservation Monitoring Center
data).
77

Bankit
#
Ongka
# #
Santigi Gorontalo
Tate
Tompe
#
#
Palu
Poso
#
Balantak
Gimpu
1
#
%
Peleng
Lore Lindu National Park
Figure 2. Map of Central Sulawesi, Indonesia showing reported capture locations of L. yuwonoi mentioned in text. Number
corresponds to location of habitat survey conducted on western boundary of Lore Lindu National Park.
78

$
r
%
Gorontalo
#
#
2
5
3
#
Palu
1
#
#
#
Poso
Morowali Reserve
Gimpu
4
# Lore Lindu National Park
Figure 3. Map of Central Sulawesi, Indonesia showing localities mentioned in text. Numbers correspond to A.
cartilaginea and I. forstenii localities. Symbols correspond to documented localities of wild I. forstenii.
79

Figure 4. Comparison of nuchal types in Indotestudo forstenii. Evidence from this study suggests that populations of
this species from the Minahasa Peninsula lack nuchals scutes and are larger (left), while those form the Palu Valley to
the south have nuchals and are smaller (right). Further in-situ populations surveys are required to confirm this
hypothesis.
80

Northwest
Northeast
North Central
East Central
West Central
Southeast
Southwest
Figure 5. Map of Sulawesi, Indonesia showing areas of endemism as
described by Evens (2003).
81

Figure 6. Areas in Sulawesi with different mean annual rainfall. Figure
taken from Whitten, et al., 1987.
82

Lights on
1:00
2:00
3:00
4:00
5:00
6:00
7:00
8:00
9:00
10:00
11:00
12:00
13:00
14:00
15:00
16:00
17:00
18:00
19:00
20:00
21:00
22:00
23:00
0:00
1:30
2:30
3:30
4:30
5:30
6:30
7:30
8:30
9:30
10:30
11:30
12:30
13:30
14:30
15:30
16:30
17:30
18:30
19:30
20:30
21:30
22:30
23:30
Fig. 7. Experimental design. Baseline CORT samples were taken on 3 separate occasions
(gray blocks) evenly distributed over the course of a 41-day period. 3.5 hr intervals
separated the sample periods.
83

80
# of Individuals
60
40
20
0
30 90 150 210 270 330 450
Midpoint Time Req'd (sec)
Figure 8. Time required to obtain blood samples from long-term captive L. yuwonoi
(n=117).
84

log10(Time Req'd, sec)
2.6
2.4
2.2
2.0
1.8
800 1200 1600 2000
Time at Completion
Figure 9. Time required to obtain blood samples from L. yuwonoi as a function of time
of day. F= 15.34, P = 0.0002; r 2 = 0.12.
85

12
Housing Status
( LS Means)
10
8
6
4
A
G
600 1230 1900
Time Period
Figure 10. Least squares mean differences in CORT levels during three sampling periods
between captive female Leucocephalon yuwonoi held in groups of 3 (G) and alone (A).
86

15
18
CORT Level (ng/ml) females
14
13
12
11
10
9
8
7
6
5
CORT Level (ng/ml) males
16
15
13
11
9
7
6
4
A B C
4
A B C
Daylight period
Daylight period
A)
B)
Figure 11. Differences in CORT levels for females and males between three periods
during the species active daytime period; A = pre dawn, B = daytime, and C = after dark.
(A) Females showed a significant difference in CORT levels between the three periods, P
= 0.013. Additionally, period A was significantly different from periods B and C at α
0.05. (B) Males did not show a significant difference though, P = 0.165. Width of mean
diamonds is in relation to sample size. Dashes represent Std Dev. Grey line represents
grand mean.
87

Appendice A. Map of Indonesia including Sulawesi (below arrow).
88

Appendice B. Palu Valley and surrounding foothills.
89