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FLORIDA
-MUSEUM
OF NATURAL HISTORYTM
BULLETIN
SKELETOCHRONOLOGY: A METHOD FOR
DETERMINE THE INDIVIDUAL AGE AND GROWTH OF
MODERN AND FOSSIL TORTOISES (REPTILIA: TESTUDINES)
Dana J. Ehret
Vol. 47, No. 2, pp. 49-72
2007
UNIVERSITY OF FLORIDA
GAINESVILLE
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SKELETOCHRONOLOGY: A METHOD FOR DETERMINE THE
INDIVIDUAL AGE AND GROWTH OF MODERN AND FOSSIL TORTOISES
(REPTILIA: TESTUDINES)
Dana J. Ehret'
ABSTRACT
Skeletochronology is a method used to estimate the individual ages of animals by counting lines of arrested growth (LAGs) within skeletal
tissues. This method was applied to evaluate the ages of modem gopher tortoises, Gopherus polvphemus, from north central Florida and fossil
tortoise species, Gopherus laticuneus and Stylemvs nebrascensis, from the White River Group in northwestern Nebraska. Different skeletal
elements were tested for growth lines and the humerus was determined to be the most useful bone for analysis. LAGS in Gopherus polyphemus
were correlated with alternative age estimations based on scute annuli counts, carapace lengths, and plastron lengths. While age estimates were
similar in younger individuals, the alternative aging techniques did not accurately reflect the ages of older individuals. Scute wear, sexual
dimorphism, and decreased growth with age are factors contributing to this discrepancy. Similarly, LAGS were found in the humeri of
Gopherus laticuneus and Stylemys nebrascensis. Fossil specimens range in age from 0 years hatchlingg) to over 40 years old. Fossil samples
provide insight into changing ecological conditions during the Eocene-Oligocene Transition. This critical period appears to show a shift from
a Stylemys nebrascensis to Gopherus laticuneus dominant ecosystem.
Key Words: Gopherus polyphemus, skeletochronology, Stylemys nebrascensis, Gopherus laticuneus, incremen-
tal growth, Eocene, Oligocene.
TABLE OF CONTENTS
Introd auction .............................................................................. ........................ 50
Geological Context of the Nebraska Badlands..................... ..................... 50
W hite River Group Fossil Tortoises................................ ....................... 50
Paleoenvironm ent and Paleoclim ate................................... ....................... 52
M odem Gopherus polyphemus........................................ ....................... 53
M materials ................................................................................... ...................... 53
Gopherus polyphem us Sam ples.......................................... ....................... 53
Fossil Tortoise Sam ples..................................................... ....................... 53
Determining Individual Ages of Tortoises................................. ....................... 54
Modem Gopherus polyphemus For Baseline Data.................................... 54
LAGS in Fossil Tortoises.............. .................................................. 57
LAGS in Gopherus polyphemus....... .... ............................................. 57
R esorption D ata................................................................. ..................... 58
G row th A nalysis..................... ..................................................................... 61
Gopherus polyphemus............. .................................................... 61
Fossil Tortoises .................................................................. .................... 65
D discussion and Sum m ary........................................................... ...................... 65
A cknow ledgem ents........................................................................ ..................... 69
Literature C ited.................................. ..................................................................... 70
'Florida Museum of Natural History, Dickinson Hall, PO Box 117800, University of Florida, Gainesville, Florida 32611-7800
Ehret, D.J. 2007. Skeletochronology: A Method For Determining The Individual Age and Growth of Modern and Fossil Tortoises (Reptilia:
Testudines). Bull. Florida Museum Nat. Hist. 47(2):49-72.
INTRODUCTION
The task of aging long-lived chelonian species has been
a problem for scientists as long as turtles have been
studied. Many techniques are used to accurately esti-
mate the probable age of wild and captive raised indi-
viduals. More popular methods include mark-release-
recapture, scute annuli counts, carapace and/or plastron
measurements, scute wear assessments, and changes
in skeletal morphology (Zug 1991). While some meth-
ods are more successful than others, none consistently
predict accurate age estimates for individuals. How-
ever, the precision of all methods of age estimation is
dependent on having access to known-aged individuals
for reference.
A promising method to estimate age for some rep-
tilian and amphibian species is skeletochronology. This
method counts lines of arrested growth (LAGs) in a
cross-section of a long bone. While it has proven to be
a reliable age indicator in some species, the method has
never been used in Gopherus polyphemus Daudin 1802,
or any fossil chelonian species.
In this study, I use skeletochronology to estimate
the ages of Gopherus polyphemus and the fossil tor-
toise species Gopherus laticuneus (Cope) 1873 and
Stylemys nebrascensis Leidy 1851. Skeletochronological
data collected in this study are also cross-referenced
with three other aging techniques in the extant species
to evaluate similarities and differences in the methods.
While this method has been used in some amphibians,
reptiles, dinosaurs, mammals, and even birds,
skeletochronology has been largely overlooked for ag-
ing tortoises. Published studies using skeletochronology
in various reptiles have shown that one LAG is equal to
one year (Grubb 1971; Castanet & Cheylan 1979;
Germano 1988; Castanet 1994). An accurate method
for aging tortoises is extremely important in demographic
and population studies. Information on age at sexual
maturity, maximum age in the wild, and growth differ-
ences between sexes can all be inferred using
skeletochronology.
The extinct tortoises Gopherus laticuneus and
Stylemys nebrascensis are common in rocks of the
White River Group exposed across the North American
Great Plains. Both species have a geologic range span-
ning the late Eocene and early Oligocene (Chadronian
and Orellan North American land mammal ages, or
NALMAS) (Hutchinson 1992, 1996; Prothero &
Swisher 1992).
Gopherus polyphemus is found exclusively in the
southeastern United States, ranging from southern South
Carolina south to Dade County, Florida, and west to the
eastern portion of Louisiana (Auffenberg & Franz 1982;
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
Franz & Quitmyer 2005). This species is intended to
serve as a modem analog for the fossil species because
of its phylogenetic relationship to the fossil Gopherus
species, the relative similarity between the modern
tortoise's environment and the inferred paleoenvironment
of the fossil species, and the relative abundance and
accessibility of materials.
GEOLOGICAL CONTEXT OF THE NEBRASKA
BADLANDS
The White River Group consists of volcaniclastic
fluvial, eolian, and lacustrine sediments that accumulated
across the mid-continent of North America from the
middle Eocene to the middle Oligocene, 38 to 29 million
years ago (mya). These lithostratigraphic units are most
commonly exposed in Nebraska, South Dakota, Colo-
rado, Montana, and Wyoming (Larson and Evanoff 1998).
Fossil tortoises occur throughout the White River Group
which is divided into the Chamberlain Pass, Chadron,
and Brule formations (LaGarry 1998, Terry 1998, Terry
and LaGarry 1998). The type sections for the Chadron
and Brule formations are located at Toadstool Park in
Sioux county, Nebraska (Figure 1).
The Chadron Formation of northwestern Nebraska
is divided into two distinct members. The lower unit is
the Peanut Peak Member and the upper unit is the Big
Cottonwood Creek Member (Terry 1998, Terry and
LaGarry 1998). The Big Cottonwood Creek Member
also coincides with the Eocene-Oligocene boundary
(Swisher & Prothero 1990). It is in the Chadron Forma-
tion that Stylemys nebrascensis first appears (Figure
2).
The lower unit of the overlying Brule Formation in
northwestern Nebraska is the Orella member. Two
lithotopes can be distinguished across the exposed out-
crop (LaGarry 1998). The dominant lithotope consists
of volcaniclastic clayey siltstones and silty claystones,
sheet sandstones, and volcanic ashes. The secondary
lithotope consists of single and multistoried channel sand-
stones. Historically, the Big Cottonwood Creek Mem-
ber of the Chadron Formation, along with the Orella
Member of the Brule Formation, was correlated to the
Turtle-Oreodont zone of South Dakota based on the
abundance of fossils of these taxa (Schultz & Stout 1955).
WHITE RIVER GROUP FOSSIL TORTOISES
Tortoises have a relatively extensive fossil record
when compared to other chelonian groups; however, they
are poorly studied (Auffenberg 1974; de Broin 1977;
Hutchinson 1980; Joyce et. al 2004). Basal tortoises
are known from the Eocene of North America and Eu-
rope. Specimens referred to the genus Hadrianus and
EHRET: Determining The Individual Age and Growth of Tortoises
5 0 25 mi
8 0 40 km
Figure 1. Map of northwestern Nebraska field area with permission from Terry and LaGarry (1998 p. 124).
Reprinted with permission from Geological Society of America.
an undescribed species from the Paleocene of Asia are
currently classified as the oldest members of the group
(Joyce et. al 2004). While turtles in general are rela-
tively abundant in the fossil record, complete specimens
(including skulls, shells, and post-cranial elements) that
allow precise identification are extremely rare. There-
fore, the precise taxonomic placement for Hadrianus
is unknown. By the late Eocene, Stylemys and Gopherus
are recognizable as distinct genera (McCord 2002).
The genus Stylemys encompasses a number of
species that span from the late Eocene through the Mi-
ocene (40 to 10 mya; McCord 2002). Stylemys
nebrascensis is one of the most common fossil turtles
in North America. It was the first fossil chelonian de-
scribed from North America (Leidy 1851). Specimens
have been found in North Dakota, South Dakota, Wyo-
ming, Colorado, and Nebraska for over 150 years
(Prothero and Whittlesey 1998). The geologic range of
this species extends from the Chadron Formation through
the Orella Member of the Brule Formation, a period of 4
to 5 million years (Hutchinson 1996).
Stylemys is recognized by a number of diagnostic
characteristics. One of the features used for identifica-
tion is the normal neural formulae (4-6-6-6-6-6-6-6 or 4-
8-4-6-6-6-6-6) for the species. In the neural formula,
each number indicates the number of sides per neural
bone starting with the first neural. In all specimens the
posterior epiplastral excavation is shallow or absent
(Auffenberg 1964). The cervical scale is longer than it
is wide. The anterior lobe of the plastron is wider than it
is long. The shape of the humeral head in Stylemys is
also compressed dorso-ventrally in adults (Auffenberg
1964). A proportionately thicker and more rounded shell
is diagnostic of the species (Hay 1908; Hutchinson 1996).
The carapace of these individuals may reach, or ex-
ceed, lengths of 530 mm. Finally, the square, boxed-off
shape of the gular projection differs from that of
Gopherus.
The first reported appearance of the genus
Gopherus in the fossil record is from the late Eocene
(~- 34 Ma) with Gopherus laticuneus. Although it is
unknown if the genus is descended from Stylemys or
from a common ancestor, the genus shares a number of
morphological characteristics with Stylemys
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
TOADSTOOL PARK, NE
Composite Section
(this volume)
Om
50 oxox NPAZ 30.060 Ma (2)
E XXX)(XX
Ic
cp x
x=ooox UWA 30.58 Ma (2)
LWA b: 31.846 Ma (2)
a: 31.811 Ma (2)
p: 31.673 Ma (2)
"serendipity ash"
0 m
UPW ? --------
LPW?
+87
PPA-1
30m
PETE SMITH HILL
Reference Section 9
(this paper)
Figure 2. Geologic Section of the White River Group at Toadstool Park, NE, from Terry and LaGarry (1998 p.
133). Reprinted with permission from Geological Society of America.
nebrascensis (Hay 1908; Auffenberg 1964). Previously,
these species were closely linked because they share
the synapomorphy of a premaxillary ridge in their upper
jaw (Crumly 1994; McCord 2002). Gopherus
laticuneus is the most primitive described species of
the genus, which includes four extant species (G
polyphemus, G flavomarginatus, G berlandieri, and
G agassizii). Given the basal phylogenetic position of
this taxon within Gopherus, some have placed it in a
separate taxon, Oligopherus (Hutchinson 1996, McCord
2002).
The characteristics that diagnose Gopherus
laticuneus includes the normal neural formulae 6-6-4-
6-6-6-6-6 or 4-8-4-6-6-6-6-6, posterior epiplastron ex-
cavation relatively shallow, nuchal scale rather short and
wide, the shell generally thinner, overly pronounced and
toothed epiplastral extensions, and an extended and
toothed xiphiplastra (Hutchinson 1996).
PALEOENVIRONMENT AND PALEOCLIMATE
The use of skeletochronology relies on seasonal
cycles to preserve lines of arrested growth (LAGs) and
annuli. Therefore, information on the paleoclimate dur-
ing the Eocene-Oligocene transition is important to the
100m-
150m-
200m-
EHRET: Determining The Individual Age and Growth of Tortoises
context of this study. Seasonal climatic variation is very
important for accentuation of LAGs (Castanet & Smirina
1990; Chinsamy-Turan 2005). The Eocene-Oligocene
(34.36 +/- 0.11 Ma) transition shows a major shift in
climatic conditions (Terry and LaGarry 1998). Paleosols
from the Big Cottonwood Creek member (late
Chadronian, North American Land Mammal Age) shows
a gradual change from humid, forested conditions to more
seasonal, semi-arid conditions occurring through the Late
Eocene (Terry 2001). This change was originally inter-
preted as the "Terminal Eocene Event" when it was
first detected. After redefinition of the Eocene-Oli-
gocene boundary, the event has now been termed the
"Early Oligocene Event" or the Eocene-Oligocene Tran-
sition (EOT) (Miller 1992, Prothero & Heaton 1996;
Terry 2001, Kohn et al. 2004).
It was suggested that the temperature during the
White River Group deposition in the early Oligocene was
~ 16 C, which is a decrease from an Eocene green-
house of 25 C (Wolfe 1992, Berggren & Prothero
1992). As mentioned previously, Stylemys nebrascensis
appears during the middle to late Eocene, but does not
become common until the Eocene-Oligocene boundary.
Gopherus, on the other hand, did not appear until the
latest Eocene and can be found in the upper Chadron
(latest Eocene) and lower Brule (early Oligocene) For-
mations. Bramble (1971) and McCord (2002) suggested
that Stylemys is a more mesic-adapted species, while
Gopherus is a more xeric-adapted species. This would
account for a shift in species abundance as the climate
became cooler and drier (Terry 2001).
Based on paleosols, the White River Group repre-
sents a transition from fluvial to eolian sediments that
occur near the top of the Orella Member at Toadstool
Park, Nebraska (LaGarry pers. comm., Terry 2001).
Paleosols show a transition from forested/slightly for-
ested conditions in the late Eocene to more open prairie-
like conditions in the Oligocene (Terry 2001.) River sys-
tems also shifted during this period from meandering to
braided systems with periods of seasonal deposition.
MODERN GOPHERUS POLYPHEMUS
Gopher tortoises (Gopherus polyphemus) are
found most often in sandy upland areas of pine (Pinus
spp.) and oak (Quercus spp.) with an understory of
wiregrass (Aristida spp.), beach scrub, oak hammocks,
or pine flatwoods (Auffenberg & Franz 1982; Ernst et
al. 1994). Annual precipitation levels over the range of
G polyphemus is between 1162-1593 mm (Germano
1994). They are avid burrowers and may keep several
burrows active at any given time. Active and aban-
doned burrows are used by the tortoise and other verte-
brate and invertebrate species. In Florida, tortoises are
active most of the year, retreating to their burrows at
night and only coming out for portions of the day. When
they are above ground, G polyphemus spends most of
its time basking and searching for food or feeding (Smith
1992; Ernst et al. 1994). Although there is little informa-
tion on the longevity of G polyphemus, captive raised
Gopherus berlandieri Agassiz 1857 (the Texas tor-
toise) have been documented to exceed 52 years of age
(Judd & McQueen 1982).
MATERIALS
GOPHERUS POLYPHEMUS SAMPLES
The shells and skeletons of Gopheruspolyphemus
were obtained by Richard Franz and the author through-
out north central Florida under the FLMNH Florida Fish
and Wildlife Conservation Commission collection permit
#WS01058 to salvage mortally wounded specimens on
roads, killed by predators, or burned in wildfires. Gopher
tortoises were sampled from several localities in north
central Florida. Samples were collected from High
Springs, Rattlesnake Island in Fort Matanzas National
Monument near St. Augustine, and Melbourne.
FOSSIL TORTOISE SAMPLES
Fossil tortoises (Gopherus laticuneus and
Stylemys nebrascensis) were collected in the late
Eocene-Oligocene beds of northwestern Nebraska in
the summer of 2001 with the aide of Bruce MacFadden
and volunteers that were assisting the annual Pony Ex-
press trip. Specimens were collected from the Sand
Creek Ranch and on Forest Service land near Toadstool
Park north of Crawford, Nebraska (Figure 1). Speci-
mens designated with FLMNH (UF) numbers are housed
in the Nebraska collection at the museum (Table 1). The
main sites of collection for the summer 2001 collections
include: Horse Hill Low (NE 008), Turkey Foot East
High (NE 001) Figures 3 and 4, Sagebrush Flats (NE
016), Bald Knob High (NE 004) Figure 5, and the
Pettipiece ranch.
As mentioned previously, all fossil materials are
from the Chadronian and Brule formations of the White
River Group (Figure 6). Most samples come from the
"turtle-oreodont" zone in the boundary area between
the Chadron Formation and the Orellan member of the
Brule Formation. All fossils were collected well below
the Whitneyan-Orellan boundary, based on local
lithostratigraphy. Given the geochronology of this inter-
val (see above), these fossils occur within an interval
between 34 and 32 mya.
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
Table 1. Fossil tortoise specimens, identifications, and localities.
Specimen Species Locality Locality Number
UF 226256 G laticuneus Horse Hill Low NE 008
UF 226257 Unknown Horse Hill Low NE 008
UF 226258 G laticuneus Horse Hill Low NE 008
UF 226259 S. nebrascensis Horse Hill Low NE 008
UF 226260 S. nebrascensis Horse Hill Low NE 008
UF 226261 Unknown Horse Hill Low NE 008
UF 226262 S. nebrascensis Bald Knob High NE 004
UF 226263 G laticuneus Bald Knob High NE 004
UF 226264 Unknown Sagebrush Flats NE 016
UF 226265 G laticuneus c.f. Sagebrush Flats NE 016
UF 226266 G laticuneus Sagebrush Flats NE 016
UF 226267 S. nebrascensis Sagebrush Flats NE 016
UF 226268 S. nebrascensis Sagebrush Flats NE 016
UF 226269 S. nebrascensis Turkeyfoot East High NE 001
UF 226270 S. nebrascensis Pasture 33B Low NE 035
UF 226271 S. nebrascensis Pasture 33B Low NE 035
UF 226272 G laticuneus c.f. Pasture 33B Low NE 035
UF 191470 S. nebrascensis Turkeyfoot East High NE 001
UF 201906 G laticuneus Turkeyfoot East High NE 001
UF 209750 G laticuneus Unknown N/A
UF 226273 S. nebrascensis Bald Knob East Butte NE 004
UF 226274 S. nebrascensis Turkeyfoot NE 003
UF 226275 Unknown Turkeyfoot above PWL NE 003
UF 226276 G laticuneus Turkeyfoot NE 003
UF 226277 S. nebrascensis Turkeyfoot East NE 001
UF 226278 G laticuneus Pettipiece West Basin N/A
DETERMINING INDIVIDUAL AGES OF
TORTOISES
MODERN GOPHERUS POLYPHEMUS FOR
BASELINE DATA
Skeletochronology data were collected from
Gopherus polyphemus individuals to determine if the
technique would be a viable method before destructive
analysis of rare fossil specimens. Gopheruspolyphemus
was chosen for the following reasons: 1) it is closely
related to the fossil species, Gopherus laticuneus, used
in this project (Bramble 1971), 2) in a previous study a
closely related species (Gopherus agassizii) provided
positive results for age determination (Germano 1988),
and 3) skeletons of Gopherus polyphemus are rela-
tively abundant in museum collections. The specimens
used in this study are from the Florida Museum of Natu-
ral History (UF) and The Chelonian Research Institute
(PPC).
Different studies have recommended the use of
long bones (e.g. humerus and/or femur) for
skeletochronology, while others use vertebrae or scle-
rotic rings (Zug 1991). Therefore, I examined different
skeletal elements to determine which would be most
advantageous for these tortoises. Bones were taken
from both the left and right sides of these individuals.
Bones from the same side of the body should be used;
however, most specimens in my sample were incom-
plete and alternative bones had to be substituted. Data
suggest that this did not bias the analysis, because simi-
lar bones grow at similar rate, regardless of which side
of the body it comes from (G. Erickson pers. comm).
This assertion was tested on one specimen; UF 143426,
in which the left and right humeri were sectioned and
both consistently showed the same number of LAGs.
Sets of bone elements from individual G.
polyphemus were sectioned. Humeri, femora, scapu-
lae, ilia, and vertebrae were selected for histological
preparation and examined for LAGs. I initially predicted
EHRET: Determining The Individual Age and Growth of Tortoises
? E--
Figure 3. Erosional surfaces of Chadron and Brule for-
mations at Turkey Foot East Badlands, looking west-
ward toward the pine ridge, Toadstool Park and
Roundtop, at Sand Creek Ranch, Sioux County, Ne-
braska. Most of the rough-textured sediments on the
lower slopes represent the turtle-oreodont zone of the
older literature (photography by Shelley E. Franz).
that bones from the pelvic or shoulder girdle might prove
better for this research because those elements are pre-
served more regularly in fossil specimens. Articulated
girdles tend to remain within the shell of dead speci-
mens, thus providing for a better chance for preserva-
tion. Upon examination of the scapula and ilium, I found
that LAGs could be observed, but bone samples had
undergone more reconstruction than other bones. Ver-
tebrae also show a high amount of reconstruction and a
majority of the LAGs are not visible. The humerus and
femur proved the most reliable in maintaining LAGs with
the least amount of reconstruction in G polyphemus.
Based on this preliminary assessment, I selected
the humerus as the optimal skeletal element to be exam-
ined. The decision between using the humerus or the
femur is based on the availability of material. In both
modem and fossil samples, there are more humeri than
femora available in the sample. Counts of LAGs in all
bone samples are included in Table 2.
An advantage of using the modem gopher tortoise,
G polyphemus, is the possibility for parallel age esti-
mates obtained from other methods (Halliday & Verrell
1988). The most beneficial specimens would have been
individuals of known-age (Castanet & Smirina 1990).
Unfortunately no such samples of G polyphemus were
located and other aging techniques had to be employed.
Scute annuli counts have been used (quite) exten-
sively in research involving chelonians. They are thought
to be annual in most species and can be viewed and
counted with relative ease (Legler 1960). The draw-
backs, however, include: loss of annuli due to excessive
wear (Figure 7), false annuli being counted as true an-
nual increments, and the difficulty of aging older indi-
viduals because of the closer spacing of the annuli on
the scute (Germano 1988). However, annuli can pro-
vide a method to evaluate and compare the age estima-
tions as determined by LAGs.
Scute annuli were counted for all but one of the
specimens examined. One large female proved to be
too old and its scute annuli were too worn to count. Due
to this problem, evaluated age estimates were compared
with other methods that were used regularly in study of
chelonian species, i.e. measuring straight-line carapace
and/or plastron length to correlate size and age (Landers
et al. 1982; Mushinsky et al. 1994). There are a number
of drawbacks and restrictions however, that should be
addressed.
Given that chelonians are ectotherms, size can vary
based on the environment in which different populations
live. Factors including average temperature, rainfall,
vegetation levels, and nutrition can all influence the
growth rates of individuals (Gibbons 1976). Therefore,
carapace and plastron lengths will vary from population
to population throughout a given range. Plastron lengths
in gopher tortoises are very limiting for another reason:
the epiplastral extension (gular region) of the gopher tor-
toise is a highly variable feature (Figure 8). Males tend
to have a longer gular projection than females, although
this is not a rule. Plastron length can be measured, but
its use in estimating age is likewise equivocal (Mushinsky
et. al. 1994).
Carapace lengths tend to be more precise than plas-
tron length for age reconstruction however; estimates
are still relative and not absolute. Straight-line carapace
length is a popular method among researchers. The
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
Figure 4. Eroded cliffs of Chadron and Brule formations at Turkey Foot East Badlands, looking northwest toward
Sand Creek Road, at Sand Creek Ranch, Sioux County, Nebraska. Upper PWL ash forms a prominent white layer,
adjacent to the grass, in the bottom of the valley (photography by Shelley E. Franz).
Figure 5. Eroded surfaces of the Chadron and Brule formations at Bald Knob Badlands, looking southwest toward
the pine ridge, at Sand Creek Ranch, Sioux County, Nebraska. The upper PWL ash layer forms a prominent white
shelf about mid-slope (photography by Shelley E. Franz).
EHRET: Determining The Individual Age and Growth of Tortoises
Figure 6. A moderate-sized specimen of gopher tortoise, Gopherus laticuneus, excavated from the Chadron Forma-
tion, just above the upper PWL ash layer, at Horse Hill
(photography by Shelley E. Franz).
same environmental factors mentioned above also ap-
ply to carapace length therefore individuals from differ-
ent populations should not be compared to one another
unless they share a common geographic range or envi-
ronment.
LAGS IN FOSSIL TORTOISES
Based on the results for G polyphemus, I chose
the humerus as the appropriate bone element for deter-
mining the age of White River fossil tortoises because
of the high number of humeri present and the observ-
able LAGs. The fossil specimens were prepared in the
Vertebrate Paleontology lab at the Florida Museum of
Natural History, Gainesville (FLMNH). Table 1 shows
all samples and the localities from which they came.
Samples were embedded, cut, and polished following
the methods of Chinsamy and Raath (1992) and exam-
ined for LAGs. The fossil specimens showed growth
marks similar to those documented in G polyphemus.
The LAGs analyzed within the long bones of the fossils
were correlated to plastron and carapace lengths since
keratinous scutes typically do not preserve in the fossil
record. Measurements of shell dimensions were com-
pared; however, there are no known shell length-age
classes for these species.
Badlands, Sand Creek Ranch, Sioux County, Nebraska
In G laticuneus and S. nebrascensis specimens,
the plastron tends to be better preserved in the fossil
record than the carapace. Post-mortem deformation
and distortion during fossilization tends to compress and
misshape the carapace in fossil tortoises. In many cases,
shell length estimates for White River fossils are af-
fected by taphonomic factors. As a result, available
shell fragments were compared with specimens, that
were better preserved. Therefore, a majority of fossil
tortoise shell length estimates are not exact, but provide
sufficient information to allow interpretation.
LAGS IN GOPHERUS POLYPHEMUS
All individuals were cleaned and prepared before
bones were measured and sectioned. Most of the car-
casses had to be skeletonized. Individuals were then
rinsed in a weak solution of industrial strength soap and
bleach and then scrubbed with a soft brush to remove
any remaining tissue. Bones were dried under a heat
lamp. All individual bones were measured for length
and width to the nearest millimeter. I also measured the
diameter of the shaft of the humerus because of its im-
portance when estimating resorption of growth lines in
thin section. However, there is no correlation between
individual bone size or length and age (Castanet &
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
Table 2. Gopherus polyphemus identification, sex, and visible LAG counts.
Specimen Sex* Humerus Femur Scapula Ilium Vertebra
UF 143424 F 18 21 17 21 10
UF 143425 M 10 11 7 8 11
UF 143426A F 9 13 6 6 8
UF143426B F 9 13 6 6 8
UF 143427 M 7 6 7 7 6
UF 143428 J 3 0 0 0 0
UF 144653 J 6 7 6 8 N/A
UF 144657 J 6 8 4 7 N/A
UF 144659 F 8 7 5 7 4
UF 144655 J 7 N/A 6 7 9
UF 150177 F 7 6 6 5 N/A
UF 143430 J 10 7 5 4 N/A
PPC 6669 M 12 14 15 14 N/A
PPC 6674 M 17 21 18 21 16
PPC 3510 H 0 N/A N/A N/A N/A
* M = Male, F = Female, J = Juvenile and H = Hatchling
Cheylan 1979, Chinsamy-Turan 2005).
For preparation of the thin sections, all samples
were sent to Matson's Laboratory, LLC of Milltown,
Montana. Following standard procedures, bones were
cut, decalcified, embedded in paraffin, and stained with
hematoxylin dye (Castanet & Cheylan 1979; Zug et al.
1986; Chinsamy & Raath 1992, Chinsamy-Turin 2005).
The dye is an important aide in making the annual growth
marks more distinguishable. The sections were then
embedded in plastic and mounted on microscope slides
(also see www.matsonslab.com). For each specimen,
the actual age was withheld until the author also had a
chance to make an independent age estimate. This was
done to evaluate the precision of the estimates.
I counted the LAGs using a compound microscope;
counts were repeated two times. The average of the
two counts provided the age estimates for all individu-
als. Professional technicians at Matson's Laboratory
also provided counts. The two sets of LAG counts var-
ied no more than 1-2 LAGs, suggesting consistency in
the interpretation. In previous studies, the occurrence
of two LAGs per year has been observed (Castanet &
Smirina 1990). These non-periodic lines, which can re-
sult from a double annual growth cycle, were not found
in specimens analyzed during this study. In addition to
counting the number of LAGs, the width of all incre-
ments was also measured. These measurements were
used to estimate resorption of early LAGs.
RESORPTION ESTIMATION
In order to measure the LAGs and account for
resorption, the average layer-thickness calculation was
used. All counts and measurements involved the LAGs
found on the ventral side of the bone. Due to biome-
chanical function and resorption, LAGs are not equally
spaced around the circumference of the humerus
(Parham & Zug 1997; Chinsamy-Turan 2005). When
looking at a bone in thin section, the LAGs persist longer
on the dorsal and ventral sides of the bone (also known
as the short axis). Therefore, the radius of the humerus
was recorded as half the diameter of the resorption core
plus the sum of the LAGs on the ventral half of the
bone.
LAGs and periosteal layers lost into the cancel-
lous core of the bone are a major problem in age estima-
tion (Francillon-Vieillot et al. 1990, Chinsamy-Turan
2005). In bone growth, early periosteal layers tend to
be tightly packed followed by a number of layers that
are more widely spaced. These widely spaced layers
continue until the animal reaches sexual maturity, at which
point they tend to become closely spaced again. The
variation in growth line width may lead to an exaggera-
tion in the number of total LAGs (both present and re-
absorbed) when only the mean width of all LAGs is
calculated.
The average layer-thickness back-calculation uses
the mean width of the three existing innermost layers,
EHRET: Determining The Individual Age and Growth of Tortoises
A B
Figure 7. (A) Scute of young individual showing well-defined annuli (B) Scute of an older individual showing wear and
loss of annuli.
Figure 8. Differences in gular projections of Gopherus polyphemus (A) Juvenile (B) Adult male.
which is then divided into the radius of the bone's short
axis, to provide an estimate of the number of resorbed
marks (Castanet & Cheylan 1979; Zug et al. 1986; Casta-
net & Smirina 1990; Parham & Zug 1997; Erickson &
Tumanova 2000). While most research indicates this to
be an appropriate protocol, Parham and Zug (1997) cau-
tion that this equation may also yield an overestimate of
LAGs. (The latter authors' work on skeletochronology
in sea turtles uses an alternate method to recount for
resorption however it is beyond the intended scope of
this paper to make comparisons in methods.)
As an independent age estimate, scute annuli mea-
surements were also collected and correlated with those
derived from skeletochronology. On the shells of many
chelonians, concentric growth increments form on each
individual scute. It has been found that these rings are
annual in some species and can be positively correlated
with age up until a given point, usually 20 years (Cagle
1946; Sexton 1959; Castanet & Cheylan 1979; Judd &
Rose 1983; Galbraith & Brooks 1987; Germano 1988).
The second costal scute of the carapace was chosen to
count annuli following Germano (1988). This scute was
chosen for two reasons: the carapace receives much
less wear than the plastron, allowing for preservation of
scute annuli. Also, the second costal is much more even-
sided than others, making the annuli easier to distinguish.
True annual rings annulii) were distinguished from
false or double annuli based on descriptions by Legler
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
(1960) and Landers et al. (1982.) These authors de-
fined true annuli as those rings that formed a deep groove
around the entire scute (Figure 9). Two counts were
made on (two) separate occasions, and the average (of
the two counts) was used as the age estimate. These
results were then matched with the LAG counts taken
from long bones.
The other set of measurements taken from G
polyphemus specimens include shell dimensions. Mea-
surements taken included: straight-line carapace length
(SCL), straight-line plastron length (PL) along suture,
and the length of hyoplastron at the suture. Some stud-
ies have shown that carapace and/or plastron length are
suitable for age/size relationships (Landers et al. 1982;
Mushinsky et al. 1994). The latter measurement
(hyoplastron) was taken in order to check the accuracy
of calculating carapace length based on plastron ele-
ments published in Franz and Quitmyer (2005).
Straight-line carapace and plastron lengths were
recorded by placing large calipers at either end of the
tortoise shell at the midline. This length was taken in-
stead of an over-the-shell measurement using a mea-
suring tape, because of the increased chance of error in
broken or misshapen shells. The length of the
hyoplastron was also taken down the midline suture, as
per Franz and Quitmyer (2005).
I prepared all specimens to recover humeri, took
shell measurements, and identified individuals to species
when possible. Preparations were done using a dremel
tool, dental picks, an air scribe, and various adhesives in
the prep lab at the FLMNH with the help of Russ
McCarty.
Before sectioning, fossil bones were measured in
the same way as modem samples, specifically, the lengths
and widths of bones or the remaining portions of bones.
No discrimination was made as to whether the right or
left humerus was used due to the extreme rarity of fos-
sil tortoise limb bones. Rough cuts were made on a
rock saw prior to embedding to produce a clean surface
Shield
at birth
SCALE
05
Figure 9. Measurement of scute annuli on the plastron
of Gopherus polyphemus from Landers et al. (1982
pg. 84). Reprinted with permission from FLMNH.
Figure 10. A mid-diaphyseal cut made on the humerus.
EHRET: Determining The Individual Age and Growth of Tortoises
in the mid dyaphseal shaft (Figure 10). Sections are cut
from the mid-shaft to avoid remodeling that may occur
near the proximal or distal end of the long bone (Parham
& Zug 1997, Chinsamy-Turan 2005). Humeri were then
put into molds and embedded in Por-a-Kast clear plastic
produced by the Synair Corporation. Samples were then
allowed to cure for a few days to ensure hardening.
A slow speed Buehler Isomet saw was used to cut
1-3 mm sections from the humeri. The Por-A-Kast
plastic used in the embedding process did not allow for
finer cuts, as section warping was visible in thinner
samples. These sections were then mounted on petro-
graphic slides using a two-part epoxy manufactured by
Logitech. Slides were then allowed to cure for 2-3 days
before grinding.
Thin-section and slide grinding was performed at
the laboratory of Dr. Gregory Erickson at Florida State
University Tallahassee, Fl. Slides were sanded on table-
top grinders using different grits in a fining up sequence.
A coarser paper (600 grit) was used initially to remove
excess material, and finer papers (800-1200 grit) were
used in preceding succession to remove any coarse
grooves or imperfections left behind. For fossil slides,
most were sanded down to a thickness of about 100
micrometers or less. Slides were then viewed under a
compound microscope (using polarized light) to count
and measure LAGs. Measurements were taken in an
identical manner to those described for G polyphemus.
LAGs were more difficult to discern in fossil speci-
mens due to the ineffectiveness of staining in fossils and
also the required increased thickness of the prepared
thin-sections. Mineral replacement and diagenesis also
destroyed some bone microstructure, which made counts
difficult.
Both extant and fossil tortoise LAG counts were
correlated and analyzed. Modem G polyphemus LAGs
were matched with scute annuli counts in order to test
the validity of both methods. Plastron and carapace
lengths were also matched with LAG estimates in both
fossil and extant species to compare size-age relation-
ships. Published size-age correlations were used to es-
timate ages of the tortoise specimens (Landers et al.
1982; Mushinsky et al. 1994).
GROWTH ANALYSIS
GOPHERUS POLYPHEMUS
LAGs were visible in some of the skeletal elements
analyzed for Gopherus polyphemus (n=15). Table 2
shows the sex and the LAG counts for each skeletal
element. The number of visible LAGs was not uniform
in all elements for each individual. Those elements that
did not show LAGs, or showed fewer LAGs, were ei-
ther too remodeled or the individual was under 1 year of
age. While both the femur and humerus retain the
most visible LAGs, the humerus was chosen based on
availability of specimens.
Resorption and age estimates were calculated us-
ing the average layer-thickness back-calculation (Table
3). The average thickness of the innermost three LAGs
(in millimeters) was used to calculate those LAGs that
Table 3. Gopherus polyphemus age estimates including resorption calculation.
Specimen Avg. Width of Inner 3 Annuli (mm) ResorbedAnnuli Age (yrs)
UF 143424 0.10 18 36
UF 143425 0.35 1 11
UF 143426A 0.30 4 13
UF 143426B 0.22 4 13
UF 143427 0.10 1 8
UF 143428 N/R 0 3
UF 144653 N/R 0 6
UF 144657 N/R 0 6
UF 144659 0.34 6 14
UF 144655 N/R 0 7
UF 150177 0.35 4 11
UF 143430 0.23 5 15
PPC 6669 0.26 5 17
PPC 6674 0.14 8 25
PPC 3510 N/R 0 0
were resorbed. Specimens marked N/R showed no re-
sorption of LAGs in my calculations. The numbers of
resorbed LAGs, where present, are listed for all speci-
mens. Older individuals have more resorbed LAGs as a
result of enlarged medullar cavities. The combination
of resorbed and visible LAGs was then added together
to determine an age for each tortoise. Ages range from
0 years, for a hatchling, to 36 years, for a large, gravid
female. The ages listed are 1 year, because the sea-
son of death is not known for most individuals.
Scute Annuli. The scute annuli were counted for
separate age estimations in all specimens (Table 4). Two
specimens did not have annuli, in UF 143424 the scutes
were worn smooth, and PPC 3510 only had a natal plate
visible. The skeletochronology age estimates were com-
pared to the scute annuli age estimates using the Wilcoxon
signed rank test. This is a non-parametric test used to
test the median difference in paired data. This was done
to determine if there is a significant difference between
the two age estimations. A two-sided P-value was gen-
erated using age estimations and was calculated at
P<0.002. Obtaining a P-value smaller than P<0.05 is
considered statistically insignificant therefore the medi-
ans of the two age estimates are significantly different.
Based on the problematic conditions concerning scute
annuli, this test provides evidence that scute annuli counts
are not accurate for Gopherus polyphemus.
Shell Allometry. Other methods that are used to
estimate ages in chelonians include determinations of
carapace and plastron lengths. Landers et al. (1982)
published a study linking plastron length to age in G
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
polyphemus (Figure 11). Although their tortoise popu-
lation was from southern Georgia, the climate is similar
enough to north central Florida to allow for comparison
with the specimens studied here. For individuals where
an accurate plastron measurement is available (n=13),
Table 5 compares plastron length ages estimates based
on the growth curve published in Landers et al. (1982).
The actual plastron lengths can be seen in Table 6. Plas-
tron age estimates were then compared with
skeletochronology age estimates. A Wilcoxon signed
rank test comparing this data yielded a two-sided P-
value of P<0.002. As with the scute annuli age esti-
mates, this result shows that the medians of the two
different age estimates vary significantly. I believe this
low P-value indicates that plastron length is not a good
indicator of age in G polyphemus. Sexual dimorphism
in the form of epiplastral extensions, tend to exaggerate
plastron lengths in many specimens making length vs.
age estimations suspect (Mushinsky et al. 1994).
Carapace measurements were also taken from all
specimens where it was possible (n=13). Unfortunately,
many skeletons, when collected, were disarticulated and
exact straight-line carapace lengths had to be approxi-
mated. Shells were pieced together for measuring and
the calculation methods of Franz and Quitmyer (2005)
were implemented to validate measurements. Franz and
Quitmyer found that the hyoplastron bone length along
the midline suture scales allometrically to body size. This
allometric relationship can be described using a straight-
line regression that they derived: Log y= a + b (log X).
Table 4. Gopherus polyphemus scute annuli counts and skeletochronology age estimates.
Specimen Scute Annuli Count Skeletochronology Age (yrs)
UF 143424 Worn Smooth 36
UF 143425 11 11
UF 143426 12 13
UF 143427 9 8
UF 143428 5 3
UF 144653 7 6
UF 144657 10 6
UF 144659 8 14
UF 144655 10 7
UF 150177 13 11
UF 143430 12 15
PPC 6669 11 17
PPC 6674 16 25
PPC 3510 0 0
EHRET: Determining The Individual Age and Growth of Tortoises
250
200
150
100
50o
* NC Florida
o SW Georgia
* *
0 00
. o
* ** 0 ,
*
** *
000 0 o
.
* 00
o 0
.** o
0b
o
00 *
00 *"
*o
I I I I I I I
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
Estimated Age I Years I
Figure 11. Gopherus polyphemus age estimations based on plastron lengths taken from Landers et al. (1982 pg.
101). Reprinted with permission from FLMNH.
Table 5. Gopherus polyphemus plastron and skeletochronology age estimates.
Specimen Age Estimate Using Plastron Length (yrs)a Skeletochronology Age (yrs)
UF 143424 15 36
UF 143425 9 11
UF 143426 13 13
UF 143427 11 8
UF 143428 6 3
UF 144653 5 6
UF 144657 8 6
UF 144659 10 14
UF 144655 7.5 7
UF 150177 12 11
UF 143430 N/A 15
PPC 6669 10.5 17
PPC 6674 10.5 25
PPC 3510 0 0
a after Landers et al. (1982)
8 0 o o
o
0 o
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
Table 6. Gopherus polyphemus shell allometrya.
Specimen Carapace Hyoplastron Carapace Estimate Plastron
UF 143424 278.00 63.70 225.50 266.00
UF 143425 224.00 60.10 215.85 210.00
UF 143426 265.00 65.40 229.98 244.00
UF 143427 258.00 64.80 228.39 227.00
UF 143428 150.00 30.80 130.74 128.00
UF 144653 130.00 33.70 139.87 116.00
UF 144657 174.00 42.00 164.98 167.00
UF 144659 218.00 60.10 215.85 199.00
UF 144655 187.00 47.80 181.79 169.00
UF 150177 220.00 49.30 186.05 210.00
UF 143430 242.00 N/A N/A N/A
PPC 6669 244.00 65.10 229.18 223.00
PPC 6674 234.00 59.30 213.69 224.00
PPC 3510 45.50 N/A N/A 46.50
"after Franz and Quitmyer (2005)
Where b = the slope of the line
a = the y intercept
x = the independent variable (hyoplastron length
along suture)
y = the dependent variable (estimated body size/
carapace length)
Based on this formula, Franz and Quitmyer found
that a = 1 and a slope of the line (b) = 0.75 in modem
gopher tortoises can be used. Using this equation,
straight-line carapace estimations for disarticulated shells
were obtained. Table 6 shows my estimated straight-
line carapace lengths, hyoplastron lengths, and carapace
lengths based on Franz and Quitmyer's work. The ac-
tual carapace measurements compared favorably with
those that were estimated using Franz and Quitmyer's
regression.
Based on my carapace length calculations, ages
were determined using the growth curve published by
Mushinsky et al. (1994). These curves were based on
studies of tortoise populations in central Florida (Figure
12) which are similar geographically to individuals in this
study and are considered appropriate correlations. These
age estimates were compared to skeletochronology age
estimates which can be seen in Table 7. A Wilcoxon
Signed Rank Test performed on this data yielded a two-
sided P-value ofP<0.0332. As stated previously, there
are a number of factors that make carapace length age
'u FEMALES
S----- CL=336(i -0.84e-.ot)
0 CL=301 /(1 +4.73e-O.27t)
10-
0 2 4 6 8 10 12 14 16 18 20 22 24
10 MALES
0
10 -
10 ----- CL304(1-0.83e-0.12t)
60 CL=278/(1 +4.30e-0.3Ot)
10
-A
0 2 4 6
8 1 1,2 14 16 18 20 22 24
AGE(yr)
Figure 12. Gopherus polyphemus age estimates based
on carapace lengths, from Mushinsky et al. (1994 pg.
122). Reprinted with permission of H. Mushinksy.
EHRET: Determining The Individual Age and Growth of Tortoises
Table 7. Gopherus polyphemus carapace and skeletochronology age estimates.
Specimen Age Estimate Using Carapace Length (yrs)a Skeletochronology Age (yrs)
UF 143424 16 36
UF 143425 9 11
UF 143426 13 13
UF 143427 14 8
UF 143428 5 3
UF 144653 5 6
UF 144657 7 6
UF 144659 10 14
UF 144655 8 7
UF 150177 11 11
UF 143430 12 15
PPC 6669 12 17
PPC 6674 11 25
PPC 3510 0 0
1 after Mushinsky et al. (1994)
estimates suspect. Therefore, the low P-value obtained
shows that skeletochronology age estimates are more
precise than carapace lengths. This fact becomes ac-
centuated after the animal has reached sexual maturity
and growth slows.
FOSSIL TORTOISES
Based on the results from the Gopherus
polyphemus specimens, the humeri of the two fossil
tortoise species (n=21) were sectioned and examined
for LAGs (Table 8). A hatchling sized specimen (UF
226265) yielded no rings because of it was under 1 year
old. Another specimen, UF 226272 yielded no rings due
to an advanced age. This specimen had a very high
amount of remodeling in its bones, also known as
Haversion reconstruction, that is most likely attributed
to fatigue repair (Figure 13). The reason for this recon-
struction is not well known; however, it makes aging of
the individual impossible (Chinsamy-Turan 2005). It has
been determined, based on the aging of other individuals
that this specimen was well over 50 years.
Age estimates for fossil specimens were also com-
pared to carapace and plastron lengths. Most of the
shell measurements are based on estimates from the
portions of the shells that were collected. Taphonomic
factors caused a majority of the shells to be distorted
and to break apart. Distortion is much more apparent in
the carapace because it is much more curved than the
plastron. For this reason, plastron length estimates are
more accurate than carapace estimates (Dodd 1995).
The morphometric analyses used on the modern G
polyvphemus could not be applied to fossil specimens
due to differences in size and growth. Therefore, shell
lengths were estimated by comparing partial shells with
complete specimens housed in the FLMNH. Table 9
shows carapace and plastron lengths compared to age
estimates for all specimens. As predicted, smaller tor-
toises yielded younger ages, while older specimens were
larger in size. Growth curves were not speculated upon
in this study because I believe more specimens of dif-
ferent age classes are needed, particularly hatchling sized
or very young individuals.
DISCUSSION AND SUMMARY
A suite of different skeletal elements were taken from
G polyphemus specimens for sectioning (Figure 14).
In all samples, the scapula and ilium were inconsistent in
contrast to the number of LAGs found in the humerus
and femur. With regard to vertebrae, there is a very
high degree of resorption and remodeling in tortoise ver-
tebrae that is unparalleled in other elements. Chinsamy-
Turan (2005) noted that organization of a bone tissue
composition and geometry is dependent on the weight
of the animal and the biomechanical properties of that
bone. Long bones, such as the humerus and femur, with
their more cylindrical shafts tend to have slower and
steadier rates of reconstruction and resorption (Klinger
& Musick 1992). The humerus was primarily used be-
cause of the abundance of elements in the fossil collec-
tion. The relative abundance of humeri as opposed to
femora seems to result from the protection offered by
the tortoises' shells. When a tortoise tucks into its shell,
the front limbs can be tucked more completely and tightly
into the shell.
Skeletochronology estimates were compared to
three other aging techniques that have been used in pre-
vious studies however Wilcoxon Signed Rank tests show
that estimates were not comparable. Scute annuli counts
are not consistent after the animal reaches its early twen-
ties, at which point scute wear and condensation of ring
makes for unreliable age estimates (Germano 1988, 1992;
Mushinsky et al. 1994; Aresco & Guyer 1998). False
annuli can also over inflate age estimations. Using plastra
lengths to age tortoises can yield inaccurate ages as well.
The epiplastral extension in the gular region of the plas-
tron is misleading when aging individuals, therefore this
measurement should not be used for aging G.
polyphemus (Mushinsky et al. 1994). Skeletochronology
also appears to be more accurate than carapace lengths
as changes in resources and environment can greatly
affect carapace growth in individual tortoises.
Caution should also be used when comparing shell
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
dimensions from different populations. Separate popu-
lations across a given species range may have different
body dimensions due to changes in habitat, food avail-
ability, and climatic conditions (Gibbons 1976; Mushinsky
et al. 1994). To determine ages of individuals from cara-
pace and plastron lengths in this project, previously pub-
lished growth trajectories were used from a geographi-
cally similar population.
Interestingly, UF 143424 yielded an age around 15-
16 years based on shell dimensions and, an age of 36
years based on skeletochronology. This discrepancy may
be attributed to the fact that it was a gravid female with
two eggs present when the individual was necropsied.
Additionally, the scutes on the shell were worn smooth,
and no annuli could be counted. In a number of reptile
species, gravid females utilize mineral deposits from bone
to produce eggshell (Wink & Elsey 1986; Wink et al.
1987). After the eggs are produced, the mineral depos-
its are restored within the bone, which leads to a high
amount of reconstruction. Thin-sections of this speci-
men revealed large, open vacuities that may have been
a result of calcium and phosphate utilization (Figure 15).
No other gravid specimens examined were gravid, and
no other specimens (male or female) exhibited similar
Table 8. Fossil tortoise age estimates including resorption calculation.
Specimen Avg. Width of Inner 3 Annuli (mm) Resorbed Annuli Age (yrs)
UF 226256 0.46 6 31
UF 226257 N/R 0 8
UF 226258 0.30 7 29
UF 226259 0.28 9 19
UF 226262 N/R 0 8
UF 226263 0.33 12 41
UF 226265 N/R 0 0
UF 226266 0.32 2 17
UF 226267 N/R 0 8
UF 226268 0.22 10 40
UF 226271 N/R 0 9
UF 226272 N/A N/A UNKNOWN
UF 191470 N/R 0 5
UF 201906 N/R 0 8
UF 209750 0.19 11 28
UF 226273 N/R 0 8
UF 226274 0.21 2 17
UF 226275 0.41 6 17
UF 226276 0.22 9 25
UF 226277 N/R 0 9
UF 226278 0.53 1 30
EHRET: Determining The Individual Age and Growth of Tortoises
Figure 13. UF 226272 humerus cross-section showing high level of reconstruction (scale bar = Imm).
Table 9. Carapace and plastron lengths of fossil tortoises compared to skeletochronology age estimates.
Specimen Carapace Length (mm) Plastron Length (mm) Age (yrs)
UF 226256 -560 -480 31
UF 226257 -95 -80 8
UF 226258 -530 -450 29
UF 226259 -260 -240 19
UF 226262 -230 -210 8
UF 226263 562 481.5 41
UF 226265 -85 -90 0
UF 226266 -307 -282 17
UF 226267 240 -210 8
UF 226268 566 500 40
UF 226271 -165 -145 9
UF 226272 -600 -530 UNKNOWN
UF 191470 97.9 82 5
UF 201906 124 111.5 8
UF 209750 366 360 28
UF 226273 158 -142 8
UF 226274 -350 -340 17
UF 226275 -425 -405 17
UF 226276 -425 -405 25
UF 226277 156 132 8
UF 226278 --430 -410 30
BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(2)
A 14. Slides depicting ilium (A) and scapula (B) cross-sections. Arrows show visible LAGs (scale bar =
Figure 14. Slides depicting ilium (A) and scapula (B) cross-sections. Arrows show visible LAGs (scale bar = lmm).
Figure 15. UF 143424 humerus cross-section showing open vacuities (scale bar = Imm).
EHRET: Determining The Individual Age and Growth of Tortoises
vacuities. I believe that this individual was likely much
older than shell dimension age estimates showed, based
on its size and advanced scute wear. However, poten-
tially gravid females should be regarded with caution
when using skeletochronology to determine the age these
animals. More information on mineral utilization of rep-
tiles as suggested in Wink and Elsey (1986) and Wink et
al. (1987) should also be gathered.
LAGs in S. nebrascensis and G laticuneus were
visible in most specimens (Figure 16). With a larger
sample size, growth rates are attainable for the two fos-
sil tortoise species reviewed in this study. In order to
predict growth rates, a growth series including speci-
mens ranging from hatchlings and juveniles up to large
adults is needed. Complete growth series for the two
fossil species identified were not available. Instead, a
composite growth series involving individuals from two
separate species is represented. This does not provide
enough data points for growth rate predictions for the
two species. However, based on specimen sizes and
age calculations, it can be inferred that growth rates
were very similar for both species. Based on shell mea-
surements, it appears that both species attained adult
sizes over 0.5 m in length in and could have taken as
long as 40 years to attain this size.
These findings are of particular importance for the
gopher tortoise in the southeastern United States. Popula-
Figure 16. UF 226278 humerus cross-section with ar-
rows showing well defined LAGs (scale bar = Imm).
tion declines throughout its range have prompted state
and federal protection throughout a majority of its range.
In areas where tortoises have been exterminated due to
human development, disease, or natural disaster,
skeletochronology performed on carcasses can be an
invaluable tool for determining the group structure, the
carrying capacity of the habitat, information regarding
sexual maturity, and individual longevity within the popu-
lation. Where relocation of tortoises is possible, this in-
formation can be used for developing more precise re-
patriation goals. In areas where relocation is not fea-
sible, this data will preserve historical records for the
future when repatriation may be a viable option.
Use of fossil tortoise material for skeletochronology
is also an acceptable method for studying the paleoecol-
ogy of extinct species. The results reported are due, in
part, to the excellent preservation of fossil materials.
Historically, the badlands of South Dakota and Nebraska
have been known for the excellent preserved fossils found
in that region, and the fossil tortoise specimens studied
here were no exception.
Most of the fossil specimens appear to be in or
about the same age class. This artifact is a result of two
factors: 1) smaller individuals do not preserve as well in
the fossil record, and 2) larger individuals are more dif-
ficult to collect and transport. Therefore, it is difficult to
ascertain demographic information for S. nebrascensis
and G laticuneus based on these initial findings.
However, population information gathered is im-
portant for examining the shift in genera during the EOT.
As discussed previously, the climatic conditions and habi-
tat of the region change over this period in time. It ap-
pears that the genus Stylemys was in decline as
Gopherus began to become more abundant in the fossil
record (R. Franz, pers. comm.). Information on the age
and structure of the fossil tortoise species can provide
insight into why this change occurred. The genus
Gopherus appears to be more suitably adapted for liv-
ing in the badlands habitat but we still do not know why.
Faster growing Gopherus individuals, shorter time to
reach sexual maturity, or longer lifespans are all viable
scenarios that can now be tested using skeletochronology.
ACKNOWLEDGEMENTS
This paper represents the work completed for the M.S.
degree in Geological Sciences at the University of
Florida. I thank my committee, especially Bruce J.
MacFadden and Dick Franz, for all of their support and
guidance. I also thank Greg Erickson at Florida State
University for the use of his lab and discussions about
skeletochronology and methods. Several individuals
donated specimens for my research: Ray Ashton (Ashton
Biodiversity Research and Preservation Institute, Inc.),
Peter Pritchard (Chelonian Research Institute), Karen
Frutchey (University of Central Florida), Boyd Blihovde
(Wekiwa Springs State Park), Joan Berish (Florida Fish
and Wildlife Conservation Commission), Craig Guyer
(Auburn University), Dave Parker (Ft. Matanzas Na-
tional Monument #FOMA-2002-SCI-0001). I would also
like to thank Barbara, Reed and Jim Toomey for their
hospitality; Marcia Wright, Helen Cozzini, and members
of the 2001 Pony Express collecting trip for their field
assistance; and G. Erickson, Walter Joyce, and H. E.
LaGarry for reviewing the manuscript. Special thanks
also go to Russ McCarty for his help with fossil prepa-
ration and moral support. I thank Marisol Amador and
Shelley Franz for assistance with images. For their fi-
nancial support I thank the Gopher Tortoise Council, the
Southwest Florida Fossil Club, and the Lucy Dickinson
Fellowship from the Florida Museum of Natural His-
tory. Publication costs were supported by theToomey
Foundation for the Natural Sciences and the Vertebrate
Paleontology Fund at the Florida Museum of Natural
History. This is University of Florida Contribution to
Paleobiology 597.
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