Content uploaded by Steven H Pearson
Author content
All content in this area was uploaded by Steven H Pearson on Jul 22, 2014
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
Content uploaded by Susan S Kilham
Author content
All content in this area was uploaded by Susan S Kilham on Jul 10, 2014
Content may be subject to copyright.
Stable Isotopes of C and N Reveal Habitat Dependent
Dietary Overlap between Native and Introduced Turtles
Pseudemys rubriventris
and
Trachemys scripta
Steven H. Pearson
1
*, Harold W. Avery
2
, Susan S. Kilham
1
, David J. Velinsky
1
, James R. Spotila
1
1 Drexel University, Department of Biodiversity, Earth and Environmental Science, Philadelphia, Pennsylvania, United States of America, 2 Drexel Unive rsity, Department of
Biology, Philadelphia, Pennsylvania, United States of America
Abstract
Habitat degradation and species introductions are two of the leading causes of species declines on a global scale. Invasive
species negatively impact native species through predation and competition for limited resources. The impacts of invasive
species may be increased in habitats where habitat degradation is higher due to reductions of prey abundance and
distribution. Using stable isotope analyses and extensive measurements of resource availability we determined how
resource availability impacts the long term carbon and nitrogen assimilation of the invasive red-eared slider turtle
(Trachemys scripta elegans) and a native, threatened species, the red-bellied turtle (Pseudemys rubriventris) at two different
freshwater wetland complexes in Pennsylvania, USA. At a larger wetland complex with greater vegetative species richness
and diversity, our stable isotope analyses showed dietary niche partitioning between species, whereas analyses from a
smaller wetland complex with lower vegetative species richness and diversity showed significant dietary niche overlap.
Determining the potential for competition between these two turtle species is important to understanding the ecological
impacts of red-eared slider turtles in wetland habitats. In smaller wetlands with increased potential for competition between
native turtles and invasive red-eared slider turtles we expect that when shared resources become limited, red-eared slider
turtles will negatively impact native turtle species leading to long term population declines. Protection of intact wetland
complexes and the reduction of introduced species populations are paramount to preserving populations of native species.
Citation: Pearson SH, Avery HW, Kilham SS, Velinsky DJ, Spotila JR (2013) Stable Isotopes of C and N Reveal Habitat Dependent Dietary Overlap between Native
and Introduced Turtles Pseudemys rubriventris and Trachemys scripta. PLoS ONE 8(5): e62891. doi:10.1371/journal.pone.0062891
Editor: David L. Roberts, University of Kent, United Kingdom
Received December 7, 2012; Accepted March 27, 2013; Published May 13, 2013
Copyright: ß 2013 Pearson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding for this research has come from three sources: PA Fish and Boat Commission SWG Project T-38, DuPont Clear into the Future Student
Fellowship to Steven Pearson, The Betz Chair of Environmental Science at Drexel University. The funders at the PA Fish and Boat Commission and at DuPont Clear
into the Future had no role in study design, data collection and analysis, decision to publis h, or preparation of the manuscript. The Betz Chair of Environmental
Science at Drexel University was involved in study design, data analysis, and preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: shp36@drexel.edu
Introduction
Habitat degradation is the leading cause of extinction and
population declines worldwide [1]. Species richness and species
diversity generally decrease as habitat availability is reduced and
rates of disturbance increase [2,3]. For species in the same guild of
an ecological community, decreases in resource availability can
lead to increases in resource overlap and a narrowing of niche
breadth [4,5] leading to increased risk of resource competition [6].
Competition for shared resources between species often negatively
impacts the growth rates, fecundity rates and/or survivorship of at
least one of the competing species [7]. Disturbed habitats are
susceptible to the establishment of introduced species due to
alteration of community structure with open niches that can be
filled by non-native species [8,9].
Today, naturally evolved and established ecological communi-
ties are being disrupted at unprecedented rates through habitat
degradation and species introductions [1], leading to alterations in
resource availability and changes in community structure [2].
Native species are negatively impacted by introduced species
through predation and competition [10]. Introduced predators
can cause the severe collapse of native faunas that do not adapt
quickly enough to increased predation rates [11,12]. Introduced
competitors cause decline of native species by increasing rates of
exploitative and interference competition [7,13]. When competi-
tion occurs for limited resources the species that more efficiently
utilizes resources will competitively exclude the less efficient
species [14,15]. Co-existence between competing species can
occur if inferior competitors disperse more rapidly or utilize
resources that shift in space and time [16]. Competition between
species may result when dietary resources are not partitioned and
will cause reduced fitness levels of one or all competing species [7].
Ecological studies of diets have historically relied on short term
dietary intake through observations of feeding and/or the
collection of stomach contents through fecal collection, stomach
flushing or dissection [17–19]. Long term diets of organisms have
been studied through the analyses of carbon 13 and nitrogen 15
stable isotopic fractions (d
13
C and d
15
N) [20–22]. Naturally
occurring isotopic fractions of nitrogen (d
15
N) and carbon (d
13
C)
indicate an organism’s trophic level and the source of carbon
assimilated from its diet, respectively [23]. The premise of all
stable isotope studies of animals is that isotopes of the same
element are incorporated at different rates into tissue through
nutrient assimilation by an organism during digestion or other
PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e62891
physiological processes [24,25]. Factors affecting d
13
C and d
15
N
isotope assimilation include tissue metabolism, trophic level,
temperature, C:N ratios in items consumed, taxonomy, body size,
and an organism’s form of eliminating nitrogenous waste [25,26].
Stable isotopes have been used in determining the C and N
sources in organisms’ diets [27], trophic position in food webs [28]
and in comparative studies of species feeding ecology between
study sites [29–31].
We used stable isotope analyses to quantify the diets and extent
of resource overlap between the native red-bellied turtle (Pseudemys
rubriventris) and the introduced red-eared slider turtle (Trachemys
scripta elegans) in two southeastern Pennsylvania wetland complexes
that differed in ecological characteristics. Red-eared slider turtles
have been introduced globally and negatively impact basking
behavior and growth rates of European pond turtles (Emys
orbicularis galloitalica) and the Spanish terrapin (Mauremys leprosa)
under experimental and natural conditions [32,33]. We relate the
results of stable isotope analyses to wetland characteristics and the
potential for competition between red-bellied turtles and red-eared
slider turtles.
Study Sites
We carried out our research at two wetland complexes that
differed in size, extent of connectivity, and the species richness and
diversity of vegetative communities. One wetland complex was
located at the Silver Lake Nature Center (SLNC), Bristol, PA and
consisted of two lakes each greater than nine hectares which were
connected by a creek and surrounded by protected lowland forest
and parkland. The second wetland complex was at Fort Mifflin
(FM), Philadelphia, PA and consisted of three small wetlands, each
less than 0.8 hectares separated by steep banks and paved roads,
and surrounded by mowed lawns and narrow patches of forest
(Table 1).
Materials and Methods
Ethics Statement
We collected all animals and tissue samples under the Drexel
University Institutional Animal Care and Use Committee
approved protocol # 18487 and Pennsylvania Fish and Boat
Commission Scientific Collecting Permits # 121 issued to HWA
and #345 issued to SHP. Permission to collect at SLNC and FM
was granted by the land managers.
Calculation of Wetland Size
We calculated wetland size using ArcGIS 9.3 by digitizing
aquatic habitat boundaries using aerial photographs. Digitized
boundaries were converted into polygons and area was calculated
using Hawths Tools [34,35]. We summed the total area of
individual wetlands to calculate the total amount of aquatic habitat
available for turtles to use within study sites.
Availability of Vegetation Resources
We determined vegetative community composition through
monthly vegetation surveys performed between June and Septem-
ber 2010. We used a hybridized quadrat-belt transect sampling
technique [36]. At each wetland within a wetland complex we
chose 10 littoral zone transect sites by randomly selecting
10 points along the wetland’s perimeter using ArcGIS software.
We located survey points using handheld Garmin GPS units and
then determined final location randomly [37,38]. Each transect
was 3 m long and ran perpendicular to the wetland edge. Along
each transect, three 0.5 m
2
quadrats were sampled with 1.5 m
center spacing. This sampling technique enabled determination of
species composition in each quadrat and an estimate of percent
cover for terrestrial plants and submerged, emergent and floating
macrophytes across a 3 m gradient of water depth. We used these
data to determine species richness and species diversity of riparian
vegetation at each wetland studied. Species diversity was
determined using the Shannon Wiener Diversity Index in which
H’ is the diversity
H
0
~
X
S
i~1
(Pi)(log2Pi)
index, s = the number of species and Pi = the proportion of total
samples belonging to the i
th
species [39].
Sampling for Stable Isotopes
Sample Collection –Turtles. We captured turtles by hand,
basking traps and baited hoop net traps and took tissue samples
from individual sexually mature adult turtles during the active
season (June through September) over a three year period
(2008,2009,2010). Red-eared slider turtles are sexually dimorphic
so we sampled sexually mature male red-eared slider turtles
greater than 100 mm straight plastron length (SPL) and females
greater than 175 mm SPL [40,41]. Red-bellied turtles exhibit less
pronounced sexual dimorphism so all turtles sampled were greater
than 175 mm SPL [40,42]. Tissues included blood drawn from the
forelimb [43], tail tissue from the posterior most 3 mm of the
turtle’s tail and shell filings collected during ID code notching.
Stable isotope sample sizes are presented in Table 2. We stored
blood on ice or in a freezer for up to 12 hours until we separated
blood plasma and red-blood cells by centrifugation. We took
samples of tail tissue with sterile scalpel blades. Using clean half
round files, we produced shell filings and collected them in sealable
plastic bags. Carbon and nitrogen stable isotopes of blood tissue
for red-eared slider turtles have a turnover rate of 3–6 months [44]
and are representative of short term nutrient assimilation. Shell
and tail tissue turnover rates are unknown for adult turtles but we
assume that isotopic composition of these tissues represent diet
assimilation over many years.
Sample Collection – Plants. We collected each plant species
encountered during the monthly vegetative resource availability
Table 1. Wetland characteristics at the Silver Lake Nature Center (SLNC) and Fort Mifflin (FM), Pennsylvania, USA.
Wetland Wetland Area Species Richness Shannon- Wiener Diversity Index
SLNC 0.21 km
2
51 1.348
FM 0.04 km
2
30 0.93
Species richness and diversity of vegetation was greater at Silver Lake Nature Center than at Fort Mifflin.
doi:10.1371/journal.pone.0062891.t001
Habitat and Stable Isotopes of Two Turtle Species
PLOS ONE | www.plosone.org 2 May 2013 | Volume 8 | Issue 5 | e62891
surveys described above. We also collected vegetation opportu-
nistically throughout the season to ensure that we sampled all of
the potential dietary items. Plants analyzed were processed as
whole plants, flowers or fruits.
Sample Preparation and Processing. We processed turtle
tissues (Table 2) following techniques described by Seminoff et al.
[44]. All tissues were dried at 60uC for 24 to 48 hours. Vegetation
samples (Table 3) were rinsed with water to ensure that animal
material was removed and dried at 60uC for 24 hours. We did not
extract lipids or mathematically normalize d
13
C values because of
the relatively low lipid content in the tissues we analyzed. Turtle
blood has a low lipid content compared to birds for which lipid
extraction of blood has been determined to be unnecessary
[27,45,46]. Furthermore, we determined the percent lipid of tail
tissue by lipid extraction with dichloromethane to be below the 5%
threshold that Post et al. (2007) suggest lipid extraction or
mathematical normalization of d
13
C be performed on [47]. All
samples were sealed and stored frozen until prepared for mass
Table 2. Mean d
13
C and d
15
N values and sample sizes for all tissues collected from red-bellied turtle (Pr) and red-eared slider
turtles (Ts) between 2008 and 2010 at the Silver Lake Nature Center (SLNC) and at Fort Mifflin (FM).
Wetland Year Tissue Type
nMeand
13
C(%) Mean d
15
N(%)
C
p-value
N
p-value
Pr Ts Pr Ts Pr Ts
Plasma 12 5 218.19 225.92 6.91 9.49 0.002 0.03
SLNC 2008 RBC 10 6 219.18 226.63 5.56 8.33 0.0002 0.004
Tail 14 7 218.26 224.88 6.57 9.80 0.00007 0.00007
Plasma 76227.28 226.20 11.50 12.03 0.11 0.65
FM 2008 RBC 95226.66 225.80 10.31 9.53 0.18 0.23
Tail 96226.44 224.88 10.14 10.81 0.018 0.43
Filings 76226.67 225.35 11.27 10.37 0.004 0.26
SLNC 2009 Plasma 15 4 219.52 224.10 7.34 11.60 0.029 0.001
RBC 12 6 220.33 224.30 5.97 9.98 0.001 0.0009
Plasma 16 15 226.45 226.90 9.74 10.55 0.42 0.22
FM 2009 RBC 10 14 227.14 226.42 9.74 9.26 0.21 0.35
Tail 10 12 225.65 226.91 9.63 11.12 0.026 0.001
SLNC 2010 Plasma 10 10 218.46 223.80 8.25 11.14 0.0002 0.0003
FM 2010 Plasma 10 9 221.43 224.87 10.85 11.62 0.19 0.5
P-values below the 0.05 significance level are highlighted in bold.
doi:10.1371/journal.pone.0062891.t002
Table 3. Carbon and Nitrogen stable isotope values for vegetation at Fort Mifflin (FM) and Silver Lake Nature Center (SLNC) during
2010.
Wetland Complex Species Common Name Mean d
13
C(%)Meand
15
N(%)
Peltandra virginica Arrow Arum 228.50 5.15
Lemna minor
Duckweed 227.85 10.09
FM
Myriophyllum spp.
Water milfoil 216.50 2.24
Nuphar advena
Spatterdock 226.86 1.03
Wolffia spp Watermeal 223.64 7.27
Amorpha fruticosa False Indigo 227.13 0.20
Hibiscus moscheutos Swamp Rosemallow 227.09 8.26
Lemna minor
Duckweed 226.49 10.08
Myriophyllum spp.
Water milfoil 224.73 9.20
Nuphar advena
Spatterdock 226.25 6.10
SLNC Parthenocissus quinquefolia Virginia Creeper 228.24 5.67
Solanum dulcamara Bittersweet Nightshade 228.24 10.53
Viburnum dentatum. Arrowwood 226.76 5.39
Vitis vulpina Frostgrape 226.47 7.45
Lyngbia spp. Filamentous Algae 219.92 11.02
The values presented are the mean value for all tissue sampled from these plant species. Plants species that were sampled at both wetlands are in bold.
doi:10.1371/journal.pone.0062891.t003
Habitat and Stable Isotopes of Two Turtle Species
PLOS ONE | www.plosone.org 3 May 2013 | Volume 8 | Issue 5 | e62891
spectrometry. We pulverized dried samples into a homogenous
powder with an agate mortar and pestle, with a glass stirring rod
or with a liquid nitrogen SPEC Certiprep freezer mill. Pulverized
samples weighing 0.6 mg to 1 mg for turtle tissues and 1 mg to
1.5 mg for vegetation samples were placed in 3.565 mm and
569 mm pressed tin capsules respectively, sealed and analyzed at
the Patrick Center for Environmental Research, the Academy of
Natural Sciences, Philadelphia, PA, using a Finnigan Delta Plus
coupled to a NA2500 Elemental Analyzer (EA-IRMS). Cross
contamination was avoided by cleaning all processing equipment
before and after each sample. Samples were run in duplicate or
triplicate and analytical variability was generally less than 3%
RSD. Multiple in-house standards were analyzed for each run to
assess comparability over time. Samples were reported in the
standard d (%) notation:
dX~ Rsample
=
RstandardðÞ{1Þ|1000
where X is either
13
Cor
15
N and R is either
13
C/
12
Cor
15
N/
14
N.
The d
15
N standard was air (d
15
N = 0), and the d
13
C standard was
the Vienna PeeDee Belemnite (VPDB) limestone that was assigned
a value of 0.0%. Analytical accuracy was based on standardization
of scientific grade N
2
and CO
2
used for continuous flow-IRMS
with International Atomic Energy Agency’s (IAEA) N-1, N-3, and
USGS 26 for nitrogen and IAEA’s sucrose, National Institute of
Standards and Technology’s (NIST) NBS 19, and NIST’s NBS 22
for carbon, respectively.
Data Analysis
We analyzed results of stable isotope analysis by first averaging
d
13
C and d
15
N values for individual samples with replicated
tissues. Averaged values were then used for all subsequent
analyses. We analyzed isotopic values within year and by tissue
in R using standard t-tests (unequal variance assumed) with species
as the grouping factor. We accepted statistical significance at the
p = 0.05 level. In this study, a significant difference between species
within a year was representative of isotopic niche partitioning. We
analyzed isotopic values between years and by tissue using fixed
effect ANOVAs with year as the treatment and isotopic means as
the response variable with program R [48]. All comparisons
between years were significantly different and we did not combine
data between years. Significant differences between years may not
be representative of dietary shifts due to the temporal variations in
d
13
C and d
15
N signatures of aquatic vegetation [49].
Results
Wetland Size, Vegetative Species Richness and
Vegetative Species Diversity
Aquatic habitat at SLNC was 5.75 times larger than that at FM.
Plant species richness at SLNC was 1.26 times greater than at FM
and plant species diversity using the Shannon-Wiener Diversity
Index was 1.45 times greater at SLNC (Table 1). After four
monthly surveys the cumulative number of species surveyed at FM
had leveled off while at SLNC the number of species was still
increasing (Figure 1). Species documented at each wetland are
presented in Table S1.
Stable Isotope Values
At SLNC there were significant differences between species for
d
13
C and d
15
N values for all turtle tissues representing short term
and long term diets (Table 2). At FM no significant differences in
d
13
C and d
15
N values existed for turtle tissue that represented
short term diets (Plasma/RBC) (Table 2). In 2008 and 2009 there
were significant differences in d
13
C values in turtle tissues that
represented long term diets (tail/shell filings), with red-eared slider
turtles having significantly higher d
13
C values in 2008 and
significantly lower values in 2009. In 2009 there was a significant
difference in d
15
N values from turtle tissues that represented long
term nitrogen assimilation with red-eared slider turtles having
significantly higher d
15
N values (Table 2). At FM d
13
C values of
plant tissue ranged between 228.5% and 216.5% and d
15
N
Figure 1. Species accumulation curves of aquatic vegetation for the two study sites in 2010. After 4 months of vegetative surveys the
number of new species being found at Fort Mifflin (FM) had leveled off while at Silver Lake Nature Center (SLNC) the number of new species had not
leveled off. Additional sampling at FM would likely not have found many new species while at SLNC additional sampling would likely result in higher
species richness.
doi:10.1371/journal.pone.0062891.g001
Habitat and Stable Isotopes of Two Turtle Species
PLOS ONE | www.plosone.org 4 May 2013 | Volume 8 | Issue 5 | e62891
values ranged between 1.03% and 10.09 % (Table 3). At SLNC
d
13
C values of plant tissue ranged between 228.24% and
219.92% and d
15
N values ranged between 0.20% and 11.02%
(Table 3).
Lipid Values
Percent lipids of tail tissue were found to be low, with a mean of
1.24%, lipid for all samples, n = 6. Red-bellied turtles had a mean
of 1.32% and standard deviation of 1.06, n = 3, while red-eared
slider turtles had a mean percent lipid of 1.15% and a standard
deviation of 0.62, n = 3. A two-tailed t-test showed no significant
difference between species (p = 0.8).
Discussion
Potential for competition in different wetlands
To our knowledge this is the first study comparing the isotopic
niches of native and introduced species at different sites with
measured differences in ecological characteristics. At our study
sites anthropogenic impacts resulted in different habitat patch
sizes. Historically, both of our study sites were either tidal creeks/
floodplains (SLNC) or associated tidal wetlands of the Delaware
River (FM). However, anthropogenic activities created impound-
ments and protected habitat at SLNC while they degraded the
wetlands at FM to remnant impounded patches. These anthro-
pogenic impacts may be the driving force behind our findings that
at SLNC the d
13
C and d
15
N niches of red-bellied turtles and red-
eared slider turtles did not overlap while the d
13
C and d
15
N niches
did overlap at FM. In anthropogenic altered habitats, shifts in the
d
15
N niche of sailfin mollies (Poecilia latipinna ) led to reduced
growth rates in the altered habitat [29].
At SLNC the potential for competition for dietary resources was
low as the extent of dietary resource overlap was low. The
partitioned d
13
C and d
15
N niches were likely a factor of larger
wetland size, greater vegetative species richness and greater
vegetative species diversity which enabled a wider niche base for
species to partition. Another potential factor impacting the isotopic
niches may have been invertebrate species richness and diversity.
We recognize that animal matter is important to turtle diets but
red-bellied turtles and red-eared slider turtles are primarily
herbivorous and are known to feed on animal matter opportunis-
tically [40,50]. Higher d
15
N levels (Figure 2) of red-eared slider
turtles at SLNC may have indicated that animal matter was an
important driver of the dietary niche partitioning found at SLNC.
Fecal sample examination from both species indicated a tenfold
increase in the percent volume of animal matter in the diets of red-
eared slider turtles compared to red-bellied turtles at SLNC
(Pearson, unpublished data). The higher volume of animal matter
in red-eared slider turtle diets is reflected by the significantly
greater d
15
N values compared to red-bellied turtles at SLNC
(Table 2/Figure 2, SLNC).
At FM the potential for competition in the short term was
greatly increased as the d
13
C and d
15
N niche axes did not
significantly differ between species. Whether or not red-bellied
turtles are weaker competitors than red-eared slider turtles for
limited resources is yet to be determined. However, when a shared
dietary resource between red-bellied turtles and red-eared slider
turtles becomes limiting, competition will occur and the species
better suited to obtain that resource will negatively impact the
growth, fecundity or survivorship of the weaker competitor [7].
Our study showed that under certain conditions (i.e. in smaller
wetlands) the potential for competition between red-bellied turtles
and red-eared slider turtles did exist. If overlap for resources
occurs over extended periods of time it is likely that these species
will compete for resources and that this competition will have
negative impacts on long term population growth of one of the
species [6].
Figure 2. d
13
C (x-axis) and d
15
N (y-axis) results for blood plasma sampled from red-bellied turtles (closed squares) and red-eared
slider turtles (open squares) at the Silver Lake Nature Center (SLNC, bottom row) and Fort Mifflin (FM, top row) for the years 2008,
2009 and 2010. Error bars represent the standard error of the mean. At SLNC there were significant differences for d
13
C and d
15
N across all three
years. At FM no significant differences were found in d
15
N values and in 2008 and 2009 no significant differences in d
13
C values were found.
doi:10.1371/journal.pone.0062891.g002
Habitat and Stable Isotopes of Two Turtle Species
PLOS ONE | www.plosone.org 5 May 2013 | Volume 8 | Issue 5 | e62891
Differences in Wetland Characteristics
At SLNC stable isotope signatures indicated that red-eared
slider turtles and red-bellied turtles did not utilize the same dietary
resources on either a short term or long term basis. This was
consistent between years for all tissues sampled. The high
vegetative species richness enabled these species to partition diets
by consuming different plants at SLNC. At FM stable isotope
signatures revealed no significant differences between diets of the
two turtle species on a short term basis but indicated differences on
a long term basis. These differences between wetland complexes
can be due to several factors. One explanation could be that the
range of available carbon and nitrogen stable isotopes at FM was
narrower. However, this was not the case as the breadth of stable
isotope values at FM was not collapsed in comparison to SLNC
(Table 3). For the same set of plant species the widest breadth of
carbon and nitrogen stable isotope values was found for vegetation
sampled from FM. A second explanation could be differences in
wetland size. Aquatic habitat available at SLNC was 5.75 times
the size of aquatic habitat at FM (Table 1). Smaller habitat size
reduces space available to forage which can increase the likelihood
that two species will consume the same resources. At FM the
depressed niche differentiation between species may have been
due in part to a reduction in available habitat. A third possibility
for differences in long term dietary niche overlap between wetland
complexes could have been differences in dietary resources
available. Adult red-bellied turtles and red-eared slider turtles
are primarily herbivorous but will eat available animal material
[40]. At FM the overlap for diets was due in part to FM having
fewer plant species to partition (Table 1) while at SLNC the red-
eared slider turtles added invertebrates to their diet causing a
greater separation in dietary niches between the species.
Our research occurred at two wetland complexes that
represented different disturbance histories. We recognize that we
did not replicate these studies in other wetlands with similar sizes
and ecological characteristics. However, our results are valid as an
example of how wetland characteristics can impact the assimila-
tion of an introduced species into native communities with
different disturbance histories. This ‘‘natural experiment’’ [3,51]
was designed to determine how wetland characteristics relate to
dietary niche overlap between red-eared slider turtles and red-
bellied turtles. An increase in vegetative species richness, like that
seen at SLNC, may enable red-bellied turtles and red-eared slider
turtles to partition dietary resources while a narrower resource
base, like that seen at FM, may lead to an increase in dietary
resource overlap. Our findings are similar to those of Luiselli et al.
who report that differences in diets of the west African mud turtle
(Pelusios castaneus) and the west African black turtle (Pelusios niger)at
a pristine site and an oil-polluted site in the Nigeria Delta are due
to a change in dietary resource availability at the disturbed site
[52]. Similarly, Kamler et al. report that diets of swift foxes (Vulpes
velox) are altered based on resource availability in continuous and
anthropogenically altered prairie habitats [53].
Long-term Carbon and Nitrogen Isotopic Niche
Partitioning at FM
Over the three year period of our study, red-bellied turtles and
red-eared slider turtles at FM consistently overlapped in short term
diets but their long term diets differed in the d
13
C and d
15
N values.
These data suggest that the turtle populations may be highly
transient. This is consistent with findings of inter-wetland
movement by marked animals from FM [54]. Since short term
d
13
C and d
15
N values overlapped but long term did not, these
species were feeding on similar resources while at FM but had
different diets while in other wetlands. Due to the small size of
these wetlands it is likely that turtles did not reside in these
wetlands for their full lifetime. Therefore, the d
13
C and d
15
N
represent long term net diet assimilated from other habitats. Our
study site at FM was adjacent to the Delaware River which may
have provided access to a broader watershed for immigrating
turtles to find the site or for emigrating turtles to disperse. In
addition to the Delaware River acting as a source or sink of turtles
for our study site there was a mosaic of remnant wetlands dotting
the landscape between our study site and the John Heinz National
Wildlife Refuge [54]. These wetlands may also have acted as a
source or sink for turtles to/from our study site.
Alternate explanations are that red-eared slider turtle long term
diet assimilation may be representative of a history of living in
captivity or different responses to high protein ephemeral
resources. If the red-eared slider turtles that we sampled were
released pets we would expect their long term stable isotope
signatures to reflect the higher protein signature of domestic turtle
food or human food rather than that of wild turtle populations. If
red-eared slider turtles respond more rapidly to ephemeral protein
sources such as carrion or fluxes of insect larvae their long term
isotopic signatures would also reflect a higher protein diet. As seen
in Table 2 the nitrogen signature for tail tissue of red-eared slider
turtles was higher than those for red-bellied turtles indicating
greater rates of protein consumption by these turtles.
Conservation Implications
The potential for competition between species can increase as
anthropogenic impacts become more severe [4–6]. When compe-
tition occurs between species the negative impacts are not
immediate [55] and in long lived species, such as turtles, would
likely result in reduced growth rates and decreased body condition
[33]. Shifts in growth rates and body condition of turtles can lead
to delayed maturity and decreased lifetime fecundity [56–58], in
turn negatively affecting population size and growth [59,60]. If
red-eared slider turtles negatively impact red-bellied turtles in
Pennsylvania or native species elsewhere, then their introduction
may have long term consequences on the structure of turtle
communities worldwide. The continued introduction of red-eared
slider turtles may lead to decreased population size or extirpation
of native turtle species. As a cautionary measure the sale and
release of red-eared slider turtles should be prohibited outside their
native range while pre-existing owners should be required to
register existing pets to further reduce the number of released
animals. If continued introductions of red-eared slider turtles are
prevented, then targeted control programs may be successful at
stemming this species continued invasion.
Supporting Information
Table S1 Plant species documented during the 2010 resource
availability surveys. Plant species found only at FM and SLNC are
on the left and right, respectively, while species found at both
wetlands are in the center. We documented 31 species at FM and
51 species at SLNC.
(DOCX)
Acknowledgments
We thank Robert Mercer, Lorraine Skala of SLNC and Wayne Irby of FM
for facilitating research on the properties that they manage. We thank M.
Schafer and L. Zaoudeh for help with determining the percent lipids and
A. Byrne, S. Brooks, M. Cunningham and numerous other staff and
volunteers for assistance in completing this research. In addition, we thank
the reviewers whose comments greatly improved our original manuscript.
Habitat and Stable Isotopes of Two Turtle Species
PLOS ONE | www.plosone.org 6 May 2013 | Volume 8 | Issue 5 | e62891
Author Contributions
Drafted part of manuscript: DJV. Provided edits and assistance: SSK DJV
JRS HWA. Conceived and designed the experiments: SHP SSK DJV JRS
HWA. Performed the experiments: SHP. Analyzed the data: SHP SSK
DJV JRS HWA. Contributed reagents/materials/analysis tools: SHP DJV
JRS HWA. Wrote the paper: SHP.
References
1. Wilcove DS, Rothstein D, Dubow J, Phillips A, Losos E (1998) Quantifying
threats to imperiled species in the United States. Bioscience 48: 607–615.
2. Fahrig L (2003) Effects of habitat fragmentation on biodiversity. Annual Review
of Ecology Evolution and Systematics 34: 487–515.
3. Shenko AN, Bien WF, Spotila JR, Avery HW (2012) Effects of disturbance on
small mammal community structure in the New Jersey Pinelands, USA.
Integrative Zoology 7: 16–29.
4. Luiselli L (2006) Food niche overlap between sympatric potential competitors
increases with habitat alteration at different trophic levels in rain-forest reptiles
(omnivorous tortoises and carnivorous vipers). Journal of Tropical Ecology 22:
695–704.
5. Swihart RK, Gehring TM, Kolozsvary MB, Nupp TE (2003) Responses of
‘resistant’ vertebrates to habitat loss and fragmentation: The importance of niche
breadth and range boundaries. Diversity and Distributions 9: 1–18.
6. Bellgraph BJ, Guy CS, Gardner WM, Leathe SA (2008) Competition potential
between saugers and walleyes in nonnative sympatry. Transactions of the
American Fisheries Society 137: 790–800.
7. Polis GA, McCormick SJ (1987) Intraguild predation and competition among
desert scorpions. Ecology 68: 332–343.
8. D’Antonio CM, Vitousek PM (1992) Biological invasions by exotic grasses, the
grass/fire cycle, and global change. Annual Review of Ecology and Systematics
23: 63–87.
9. Mack RN, Simberloff D, Lonsdale WM, Evans H, Clout M, et al. (2000) Biotic
invasions: Causes, epidemiology, global consequences, and control. Ecological
Applications 10: 689–710.
10. Alison C, Daugherty CH, Hay JM (1995) Reproduction of a rare New Zealand
reptile, the Tuatara Sphenodon punctatus, on rat-free and rat-inhabited islands.
Conservation Biology 9: 373–383.
11. Sibly RM, Atkinson D (1994) How rearing temperature affects optimal adult size
in ectotherms. Functional Ecology 8: 486–493.
12. Rodda GH, Fritts TH, Chiszar D (1997) The disappearance of Guam’s wildlife.
Bioscience 47: 565–574.
13. Amarasekare P (2002) Interference competition and species coexistence.
Proceedings of the Royal Society of London, Series B: Biological Sciences
269: 2541–2550.
14. Tilman D (1981) Tests of resource competition theory using four species of Lake
Michigan algae. Ecology 62: 802–815.
15. Cadotte MW (2007) Competition-colonization trade-offs and disturbance effects
at multiple scales. Ecology 88: 823–829.
16. Amarasekare P, Hoopes MF, Mouquet N, Holyoak M (2004) Mechanisms of
coexistence in competitive metacommunities. American Naturalist 164: 310–
326.
17. Rowe JW (1992) Dietary habits of the Blanding’s turtle (Emydoidea blandingi)in
northeastern Illinois. Journal of Herpetology 26: 111–114.
18. Hansen RM (1976) Foods of free-roam ing horses in southern New Mexico.
Journal of Range Management 29: 347.
19. Paoletti G, Puig S (2007) Diet of the lesser rhea (Pterocnemia pennata)and
availability of food in the Andean Precordillera (Mendoza, Argentina). Emu 107:
52–58.
20. Peterson BJ, Fry B (1987) Stable isotopes in ecosystem studies. Annual Review of
Ecology and Systematics 18: 293–320.
21. Wallace BP, Avens L, Braun-McNeill J, McClellan CM (2009) The diet
composition of immature loggerheads: Insights on trophic niche, growth rates,
and fisheries interactions. Journal of Experimental Marine Biology and Ecology
373: 50–57.
22. Reich KJ, Bjorndal KA, Bolten AB (2007) The ‘lost years’ of green turtles: Using
stable isotopes to study cryptic lifestages. Biology Letters 3: 712–714.
23. Post DM (2002) Using stable isotopes to estimate trophic position: Models,
methods, and assumptions. Ecology 83: 703–718.
24. Fry B (2006) Stable isotope ecology. New York, NY: Springer Science. 308 p.
25. Kilham SS, Hunte-Brown M, Verburg P, Pringle CM, Whiles MR, et al. (2009)
Challenges for interpreting stable isotope fractionation of carbon and nitrogen in
tropical aquatic ecosystems. Verh Internat Verein Limnol 30: 749–753.
26. Vanderklift MA, Ponsard S (2003) S ources of variation in consumer-diet d 15N
enrichment: A meta-analysis. Oecologia 136: 169–182.
27. Bulte G, Blouin-Demers G (2008) Northern map turtles (Graptemys geographica)
derive energy from the pelagic pathway through predation on zebra mussels
(Dreissena polymorpha). Freshwater Biology 53: 497–508.
28. Bluthgen N, Gebauer G, Fiedler K (2003) Disentangling a rainforest food web
using stable isotopes: Dietary diversity in a species-rich ant community.
Oecologia 137: 426–435.
29. Kemp SJ (2008) Autecological effects of habitat alteration: trophic changes in
mangrove marsh fish as a consequence of marsh impoundment. Marine
Ecology-Progress Series 371: 233–242.
30. Alves-Stanley CD, Worthy GAJ, Bonde RK (2010) Feeding preferences of West
Indian manatees in Florida, Belize, and Puerto Rico as indicated by stable
isotope analysis. Marine Ecology-Progress Series 402: 255–267.
31. Miranda NAF, Perissinotto R (2012) Stable isotope evidence for dietary overlap
between alien and native gastropods in coastal lakes of northern KwaZulu-Natal,
South Africa. Plos One 7: e31897.
32. Polo-Cavia N, Lopez P, Martin J (2009) Competitive interactions during basking
between native and invasive freshwater turtle species. Biological Invasions 12:
2141–2152.
33. Cadi A, Joly P (2004) Impact of the introduction of the red-eared slider
(Trachemys scripta elegans) on survival rates of the European pond turtle (Emys
orbicularis). Biodiversity and Conservation 13: 2511–2518.
34. Beyer HL (2004) Hawth’s Analysis Tools for ArcGIS. Available at http://www.
spatialecology.com/htools.
35. Steiniger S, Hay GJ (2009) Free and open source geographic information tools
for landscape ecology. Ecological Informatics 4: 183–195.
36. Titus JE (1993) Submersed macrophyte vegetation and distribution within lakes:
Line transect sampling. Lake and Reservoir Management 7: 155–164.
37. Ervin G (2007) An experimental study on the facilitative effects of tussock
structure among wetland plants. Wetlands 27: 620–630.
38. Seastedt TR, Briggs JM, Gibson DJ (1991) Controls of nitrogen limitation in
tallgrass prairie. Oecologia 87: 72–79.
39. Krebs CJ (1999) Ecological methodology. Menlo Park, CA: B enjamin/
Cummings. 620 p.
40. Ernst CH, Lovich JE, Barbour RW (1994) Turtles of the United States and
Canada. Washington: Smithsonian Institute Press. 578 p.
41. Gibbons JW, Greene JL (1990) Reproduction in the slider and other species of
turtles. In: Gibbons JW, editor. Life History and Ecology of the Slider Turtle.
Washington, DC: Smithsonian Institution Press. pp. 124–134.
42. Graham TE (1971) Growth rate of the red-bellied turtle, Chrysemys rubriventris,at
Plymouth, Massachusetts. Copeia: 353–356.
43. Avery HW, Vitt LS (1984) How to get blood from a turtle. Copeia: 209.
44. Seminoff JA, Bjorndal KA, Bolten AB (2007) Stable carbon and nitrogen isotope
discrimination and turnover in pond sliders (Trachemys scripta): Insights for trophic
study of freshwater turtles. Copeia: 534–542.
45. Chaikoff IL, Entenman C (1946) The lipids of blood, liver and egg yolk of the
turtle. Journal of Biological Chemistry 166: 683–689.
46. Cherel Y, Hobson KA, Hassani S (2005) Isotopic discrimination between food
and blood and feathers of captive penguins: Implications for dietary studies in
the wild. Physiological and Biochemical Zoology 78: 106–115.
47. Post DM, Layman CA, Arrington DA, Takimoto G, Quattrochi J, et al. (2007)
Getting to the fat of the matter: models, methods and assumptions for dealing
with lipids in stable isotope analyses. Oecologia 152: 179–189.
48. R Development Core Team (2011) R: A language and environment forstatistical
computing. Vienna, Austria: R Foundation for Statistical Computing. ISBN 3-
900051-07-0, URL http://www.R-project.org/.
49. McCutchan JH Jr, Lewis WM Jr (2001) Seasonal variation in stable isotope
ratios of stream algae. Verh Internat Verein Limnol 27: 3304–3307.
50. Parmenter RR, Avery HW (1990) The feeding ecology of the slider turtle. In:
Gibbons JW, editor. Life History and Ecology of the Slider Turtle. Washington
DC: Smithsonian Institution Press. pp. 257–266.
51. Baum JK, Worm B (2009) Cascading top-down effects of changing oceanic
predator abundances. Journal of Animal Ecology 78: 699–714.
52. Luiselli L, Akani GC, Politano E, Odegbune E, Bello O (2004) Dietary shifts of
sympatric freshwater turtles in pristine and oil-polluted habitats of the Niger
Delta, southern Nigeria. Herpetological Journal 14: 57–64.
53. Kamler JF, Ballard WB, Wallace MC, Gipson PS (2007) Diets of swift foxes
(Vulpes velox) in continuous and fragmented prairie in northwestern Texas.
Southwestern Naturalist 52: 504–510.
54. Avery HW, Spotila JR, Bien WF (2006) Final report: Red-bellied turtle
population study, Philadelphia International Airport.
55. Schoener TW (1983) Field experiments on int erspecific competition. American
Naturalist 122: 240–285.
56. Litzgus JD, Bolton F, Schulte-Hostedde AI (2008) Reproductive output depends
on body condition in spotted turtles (Clemmys Guttata). Copeia 2008: 86–92.
57. Congdon JD, Gibbons JW (1985) Egg components and reproductive
characteristics of turtles: Relationships to body size. Herpetologica 41: 194–205.
58. Avery HW, Spotila JR, Congdon JD, Fischer RU Jr, Standora EA, et al. (1993)
Roles of diet protein and temperature in the growth and nutritional energetics of
juvenile slider turtles, Trachemys scripta. Physiological Zoology 66: 902–925.
59. Congdon JD, Dunham AE, Van Loben Sels RC (1993) Delayed sexual maturity
and demographics of Blanding’s turtles (Emydoidea blandingii): Implications for
conservation and management of long-lived organisms. Conservation Biology 7:
826–833.
60. Heppell SS (1998) Application of life-history theory and population model
analysis to turtle conservation. Copeia 1998: 367–375.
Habitat and Stable Isotopes of Two Turtle Species
PLOS ONE | www.plosone.org 7 May 2013 | Volume 8 | Issue 5 | e62891
