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Diet-tissue stable isotope (Δ
13
C and Δ
15
N) discrimination factors
for multiple tissues from terrestrial reptiles
Ronnie Steinitz
1
*, Jeffrey M. Lemm
2
, Stesha A. Pasachnik
2
and Carolyn M. Kurle
1
1
Division of Biological Sciences, Ecology, Behavior, and Evolution Section, University of California, San Diego, La Jolla, CA
92093-0116, USA
2
San Diego Zoo Institute for Conservation Research, Behavioral Ecology Division, Escondido, CA 92027-7000, USA
RATIONALE: Stable isotope analysis is a powerful tool for reconstructing trophic interactions to better understand
drivers of community ecology. Taxon-specific stable isotope discrimination factors contribute to the best use of this tool.
We determined the first Δ
13
C and Δ
15
N values for Rock Iguanas (Cyclura spp.) to better understand isotopic fractionation
and estimate wild reptile foraging ecology.
METHODS: The Δ
13
C and Δ
15
N values between diet and skin, blood, and scat were determined from juvenile and adult
iguanas held for 1 year on a known diet. We measured relationships between iguana discrimination factors and size/age
and quantified effects of lipid extraction and acid treatment on stable isotope values from iguana tissues. Isotopic and
elemental compositions were determined by Dumas combustion using an elemental analyzer coupled to an isotope ratio
mass spectrometer using standards of known composition.
RESULTS: The Δ
13
C and Δ
15
N values ranged from –2.5 to +6.5‰and +2.2 to +7.5‰, respectively, with some differences
among tissues and between juveniles and adults. The Δ
13
C values from blood and skin differed among species, but not
the Δ
15
N values. The Δ
13
C values from blood and skin and Δ
15
N values from blood were positively correlated with
size/age. The Δ
13
C values from scat were negatively correlated with size (not age). Treatment with HCl (scat) and lipid
extraction (skin) did not affect the isotope values.
CONCLUSIONS: These results should aid in the understanding of processes driving stable carbon and nitrogen isotope
discrimination factors in reptiles. We provide estimates of Δ
13
C and Δ
15
N values and linear relationships between iguana
size/age and discrimination factors for the best interpretation of wild reptile foraging ecology. Copyright © 2015 John
Wiley & Sons, Ltd.
The best practices for conservation and management of
declining species and their habitats necessitate an
understanding of the natural and anthropogenic processes
driving the disappearance or persistence of a given species.
Therefore, it is important to study species interactions such
as foraging ecology and habitat use to assess their potential
as drivers of community ecology and species decline.
[1,2]
There are many ways to study these, including long-term
behavioral observations and fecal (scat) and stomach content
analyses. However, these methods can be time and labor
intensive,
[3]
resulting in small sample sizes that may not be
representative of larger populations. In addition, stomach
contents and fecal analyses only indicate an animal’s most
recent meal, precluding dietary estimations over longer
temporal scales.
[4]
Stable carbon (
13
C/
12
Corδ
13
C values) and nitrogen
(
15
N/
14
Norδ
15
N values) isotope analyses of predator and
prey tissues can provide a more comprehensive assessment
of animal foraging over variable temporal scales with
minimal disturbance, labor, and cost.
[5,6]
The δ
13
C values
from animal tissues indicate dietary carbon sources allowing
for the distinction between ingestion of marine- or
terrestrial-based primary production, plant or animal
components, and C
3
or C
4
plants, among other things.
[7]
The δ
15
N values from animal tissues largely reflect animal
trophic position as the values from organisms increase
predictably with increasing trophic levels and they can also
indicate the nitrogen processes governing the base of a
food web.
[5,6,8]
Stable isotope analysis can also provide a wide range of
temporal data as isotopic turnover varies depending upon
the protein turnover of a particular tissue.
[9]
Therefore, the
analysis of multiple tissues from a single individual can
provide dietary insights from several time periods. In
addition, stable isotope mixing models can be used to
estimate the proportions that isotopically distinct dietary
items contribute to an animal’s total diet.
[10,11]
However, the
best use of these models for estimating the diets of wild
populations requires accurate model parameters, including
reasonable estimates of the isotope discrimination or trophic
enrichment factors.
[6]
These factors are the differences in
isotope values between a consumer and its dietary items
and are expressed as Δ
13
C=δ
13
C
PredatorTissue
–δ
13
C
Prey
and
*Correspondence to: R. Steinitz, Division of Biological
Sciences, Ecology, Behavior, and Evolution Section,
University of California, San Diego, La Jolla, CA 92093-
0116, USA.
E-mail: ronnie86@gmail.com
Copyright © 2015 John Wiley & Sons, Ltd.Rapid Commun. Mass Spectrom. 2016,30,9–21
Research Article
Received: 23 June 2015 Revised: 4 September 2015 Accepted: 28 September 2015 Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2016,30,9–21
(wileyonlinelibrary.com) DOI: 10.1002/rcm.7410
9
similarly for nitrogen. Discrimination factors are typically
obtained from studies using captive animals held on known,
consistent diets for an adequate amount of time and they can
aid in the interpretation of stable isotope data from wild
animals (e.g.
[12]
).
It is frequently difficult to collect these data, as access to
animal populations held on known diets is rare.
Consequently, the determination of adequate discrimination
factors is not common,
[6]
especially for terrestrial reptiles
(but see Table 1). Therefore, the Δ
13
C and Δ
15
N values of
~1.0‰and ~3.4‰, respectively, are generally used as
discrimination factors for many studies.
[7,8,13,14]
However,
given the large degree of variation in the Δ
13
C and Δ
15
N
values observed among taxa and even within taxa among
different tissues,
[12,15–19]
the use of these generalized numbers
in stable isotope mixing models can be flawed and lead to
erroneous interpretations.
[20,21]
A primary purpose of our
study was to determine the stable isotope discrimination
factors of captive Rock Iguanas (Cyclura spp.) for use in wild
reptile studies and to better understand isotope fractionation
patterns in reptiles.
Rock Iguanas are found exclusively in the West Indies. They
are at risk due to multiple threats, including habitat
destruction and invasive species, and their IUCN Red List
status ranges from vulnerable to critically endangered.
[22]
They typically inhabit subtropical dry forests, require sandy
or soil conditions in which to burrow and lay eggs, and
depend heavily on the presence of rocky crevices for shelter
as adults.
[23]
They are predominantly herbivorous, consuming
foliage and fruits, but are also known to feed opportunistically
on animal material (e.g.
[25]
). Because of their diet, Rock
Iguanas play a key role in structuring their ecosystems. For
example, Hartley et al.
[26]
demonstrated that seeds passing
through iguana digestive tracts sprout earlier than those not
ingested, and wide dispersal of the seeds by the iguanas
[27]
may lead to advantageous priority effects for these plant
species.
[28]
Where they occur, Rock Iguanas are the largest
native herbivore, making them essential for maintaining
native plant communities in the highly endangered tropical
dry forest ecosystems that they often inhabit.
[23]
Their
threatened status combined with their important role as
ecosystem engineers underscores the priority of the species-
specific management efforts and conservation actions led by
the International Union for Conservation of Nature (IUCN),
Species Survival Commission (SSC), Iguana Specialist Group
(ISG) and the International Iguana Foundation (IIF).
To better understand the foraging-related interactions
between wild terrestrial reptiles in general, and Cyclura spp. in
particular, and their communities, we determined the Δ
13
Cand
Δ
15
N values between diet and scat, blood, and skin components
for females and males and across different age and size classes
from three captive Rock Iguana species. We targeted tissue
samples that were obtained both invasively (blood) and
opportunistically (scat and skin) so as to maximize the
applications of our data to studies of wild reptiles. In addition,
different tissues have different protein turnover, and thus
isotopic turnover times,
[9]
so sampling a variety of tissues from
wild animals allows for reconstruction of foraging ecology at
different time scales. Protein turnover is lower in ectotherms
such as reptiles,
[29]
so the isotopic turnover is slower than in
endotherms, but is probably on the order of days for scat,
[30]
5
to 6 months for blood,
[17]
and greater than 6.5 months for skin.
[17]
Rock Iguanas in the wild are assumed to shed (slough)
their skin annually,
[31]
and this seemed to coincide with
the resumption of rapid growth in the spring. Shedding is
thus affected by growth rate, although accurate data is
difficult to obtain for wild animals.
[32]
Street
[33]
also reported
an annual shedding for Cyclura nubila in the wild, while, in
captivity, C. carinata also shed their skin annually. However,
captive individuals are typically fed more and higher
quality food than wild individuals, and thus may exhibit
higher growth rates and more frequent shedding.
[31,34]
Shedding may require up to several months for
completion, and juveniles appear to shed more rapidly
than adults, although this was reported as an observation
and was not measured.
[31,34]
In reptiles, tissue generation only occurs periodically in the
inner skin layers, and when this happens the layers above
them are replaced in their entirety. About 2 weeks before
shedding, the inner layer begins active growth and a new
layer of skin grows under the old one.
[35–38]
With the growth
of this second, new skin, it follows that the old layer,
composed of keratinized dead skin cells, would consist of
the isotopic signatures incorporated when it was being
generated while the previous layer was being keratinized
and sloughed. Since this is reported to occur annually, the
shed skin should reflect the isotopic signature accumulated
during the previous year.
EXPERIMENTAL
Iguanas sampled
Body tissues and physical measurements (weight (g) and
snout-vent length (mm), SVL) were obtained from 34
individuals of captive populations from three Rock Iguana
species, Cyclura collei,C. lewisi, and C. pinguis (Table 2) held
at the San Diego Zoo Institute for Conservation Research
(ICR) facilities in Escondido (CA, USA). Cyclura collei
historically ranged throughout Jamaica and, although the
population was thought to be extinct in the wild until the
1990s when a trace population was found in the Hellshire Hills
region of southeastern Jamaica. Cyclura lewisi are native to
Grand Cayman Island, and C. pinguis arenativetoAnegada,
Guana, and Norman Islands, in the British Virgin Islands.
The ICR held all individuals on a steady diet of 15 plant
types (Table 3) for over 12 months, an adequate time period
for full stable isotope equilibration of the iguanas’tissue to
their experimental diet.
[39]
Individual iguanas were offered
food once daily and had water available ad libitum. ICR staff
chopped food according to the size of the individual being
fed (adults: ~5 × 5 cm and juveniles: ~2.5 × 2.5 cm) and each
animal was individually provided with all food types for
each meal in the proportions detailed in Table 3. All iguanas
were housed separately for most of the year, further ensuring
that each animal ate the prescribed proportions of all diet
items provided for the time periods represented in this study.
All dietary items from the bulk pile of food that was prepared
for the iguanas were sampled, collecting several replicates
when possible, along with scat and shed skin from individual
iguanas, over two sampling periods, spaced 6 months apart
(February 2013 and August 2013) to account for potential
seasonal variations in the stable isotope values from dietary
R. Steinitz et al.
wileyonlinelibrary.com/journal/rcm Copyright © 2015 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016,30,9–21
10
Table 1. Stable carbon and nitrogen isotope discrimination factors (‰, ±SD when available) for different tissue types from reptiles
Species and diet Tissue Δ
13
CΔ
15
N Reference
Collared Lizards
Crickets raised on C
4
-based dog food Plasma +0.2 ± 0.3 –Warne et al.
[19]
Same as above Red blood cells +1.2 ± 0.6 –Warne et al.
[19]
Desert Box Turtle (younger cohort)
Mealworms raised on a C
3
(wheat)-based diet Plasma +2.9 ± 0.2 –Murray and Wolf
[18]
Same as above Red blood cells +3.2 ± 0.2 –Murray and Wolf
[18]
Same as above Scute keratin +5.1 ± 0.4 –Murray and Wolf
[18]
Mealworms raised on a C
4
(corn)-based diet Plasma +1.0 ± 0.3 –Murray and Wolf
[18]
Same as above Red blood cells –0.5 ± 0.1 –Murray and Wolf
[18]
Same as above Scute keratin –0.2 ± 0.7 –Murray and Wolf
[18]
Desert Box Turtle (older cohort; +1 year)
Mealworms raised on a C
3
(wheat)-based diet Plasma +3.7 ± 0.2 –Murray and Wolf
[18]
Same as above Red blood cells +4.1 ± 0.3 –Murray and Wolf
[18]
Same as above Scute keratin +4.0 ± 0.9 –Murray and Wolf
[18]
Mealworms raised on a C
4
(corn)-based diet Plasma +0.3 ± 0.2 Murray and Wolf
[18]
Same as above Red blood cells –2.5 ± 0.5 –Murray and Wolf
[18]
Same as above Scute keratin –0.9 ± 0.1 –Murray and Wolf
[18]
)
Green Turtles (juveniles)
Pelleted diet Skin (epidermis
a
) +1.9 ± 0.6 +3.8 ± 0.4 Vander Zanden et al.
[69]
Same as above Dermis +2.2 ± 0.6 +4.2 ± 0.5 Vander Zanden et al.
[69]
Same as above Plasma +1.2 ± 0.6 +4.1 ± 0.4 Vander Zanden et al.
[69]
Same as above Red blood cells +0.5 ± 0.6 +2.4 ± 0.4 Vander Zanden et al.
[69]
Green Turtles (juveniles)
Gelatin amalgamate with fixed portions of a pelleted diet Skin (epidermis
a
) +0.2 ± 0.1 +2.8 ± 0.3 Seminoff et al.
[70]
Same as above Dermis –– Seminoff et al.
[70]
Same as above Plasma –0.1 ± 1.0 +2.9 ± 1.0 Seminoff et al.
[70]
Same as above Red blood cells –1.1 ± 0.2 +0.2 ± 1.0 Seminoff et al.
[70]
Green Turtles (adults)
Pelleted diet Skin (epidermis
a
) +1.6 ± 0.6 +4.0 ± 0.4 Vander Zanden et al.
[69]
Same as above Dermis +2.6 ± 1.2 +4.9 ± 0.6 Vander Zanden et al.
[69]
Same as above Plasma +0.2 ± 0.6 +4.2 ± 0.4 Vander Zanden et al.
[69]
Same as above Red blood cells +0.3 ± 0.6 +2.5 ± 0.4 Vander Zanden et al.
[69]
Leatherback Turtles (juveniles)
Gelatin amalgamate of Pacific Ocean Squid Skin (epidermis
a
) +2.3 ± 0.6 +1.9 ± 0.5 Seminoff et al.
[71]
Same as above Dermis –– Seminoff et al.
[71]
Same as above Plasma –0.6 ± 0.5 +2.9 ± 0.8 Seminoff et al.
[71]
Same as above Red blood cells +0.5 ± 0.4 +1.5 ± 0.8 Seminoff et al.
[71]
Loggerhead Sea Turtle (hatchlings)
Pelleted, soy-protein-based diet Skin (epidermis
a
) +2.6 ± 0.3 +1.7 ± 0.1 Reich et al.
[39]
Same as above Dermis –– Reich et al.
[39]
Same as above Plasma +3.0 ± 0.2 +0.3 ± 0.1 Reich et al.
[39]
Same as above Red blood cells –0.6 ± 0.7 –0.3 ± 0.3 Reich et al.
[39]
(Continues)
Stable C and N isotope discrimination factors for reptiles
Rapid Commun. Mass Spectrom. 2016,30,9–21 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm
11
Table 1. (Continued)
Species and diet Tissue Δ
13
CΔ
15
N Reference
Loggerhead Sea Turtle (juveniles)
Pelleted, soy-protein-based diet `Skin (epidermis
a
) +1.1 ± 0.2 +1.6 ± 0.1 Reich et al.
[39]
Same as above Dermis ––Reich et al.
[39]
Same as above Plasma –0.4 ± 0.2 +1.5 ± 0.2 Reich et al.
[39]
Same as above Red blood cells +1.5 ± 0.2 +0.2 ± 0.1 Reich et al.
[39]
Pond Sliders
Soy-based commercial pellet food Plasma +3.8 ± 0.1 +2.5 ± 0.8 Seminoff et al.
[17]
Same as above Red blood cells +1.9 ± 0.3 –Seminoff et al.
[17]
Same as above Whole blood +2.2 ± 0.2 –0.8 ± 0.8 Seminoff et al.
[17]
Same as above Liver +3.0 ± 0.3 +0.4 ± 0.5 Seminoff et al.
[17]
Same as above Brain +2.9 ± 0.3 –Seminoff et al.
[17]
Prairie Lizards
Crickets raised on C
4
-based dog food Plasma –0.5 ± 0.3 –Warne et al.
[19]
Same as above Red blood cells –1.1 ± 0.8 –Warne et al.
[19]
Same as above Liver –1.0 ± 0.2 –Warne et al.
[19]
Same as above Skin –0.8 ± 0.5 –Warne et al.
[19]
Same as above Muscle –1.9 ± 0.2 –Warne et al.
[19]
Rock Iguanas (juveniles)
Various leafy greens, root vegetables and fruit. See Table 3 Scat –0.6 ± 0.7 +2.7 ± 1.0 This study
Same as above Whole blood +1.7 ± 0.2 +2.9 ± 0.6 This study
Same as above Skin +2.3 ± 0.3 +4.2 ± 0.8 This study
Rock Iguanas (adults, overall)
Various leafy greens, root vegetables and fruit. See Table 3 Scat –1.3 ± 1.0 +3.4 ± 0.7 This study
Same as above Whole blood +2.5 ± 0.6 +4.1 ± 0.4 This study
Same as above Skin +4.5 ± 1.4 +6.0 ± 0.6 This study
a
Tissue referred in cited literature as ’epidermis’, and in this study as ’skin’.
[72]
R. Steinitz et al.
wileyonlinelibrary.com/journal/rcm Copyright © 2015 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016,30,9–21
12
items.
[40]
Blood was sampled only once in August 2013 due
to animal husbandry constraints. We sampled tissues from
adult males and females from all three species and from both
juvenile and adult C. lewisi (Table 2).
Different species of Rock Iguanas mature at different ages,
ranging from 2 to 7 years.
[41]
However, within species there
are contradicting estimates of the age at maturity.
[27,34,41]
There are few long-term population studies of iguanas in
the wild (but see
[42]
), so many observations come from
captive individuals, and therefore the estimated age of sexual
maturity may vary from the actual age in wild counterparts.
It is generally accepted that C. collei and C. pinguis mature
near the upper end of the 2–7 year age range, whereas
C. lewisi can lay eggs as early as their second year.
[34,43]
The
C. collei and C. pinguis individuals that were sampled were
all adults (all ages ≥7 years), whereas the C. lewisi consisted
of 15 juveniles (born Aug. 2011; age <2 years), and five adults
(ages ≥2 years).
Stable isotope analysis
Scat samples were collected either directly from the iguanas if
they defecated when handled, or from their captive enclosure
if they defecated earlier in the day, and any external debris
was removed either upon collection or later in the lab. We
collected skin samples directly from individuals as it was
naturally shed, washed the samples with several drops of
dish soap diluted in ~500 mL deionized (DI) water to break
down any surface oils and remove debris,
[44]
and rinsed them
thoroughly with DI water. All scat and skin samples were
frozen at –20 °C until they were processed for isotope analysis
(see below). Approximately 200 μL of whole blood was
collected from each individual from the ventral coccygeal
vein using a butterfly needle during routine husbandry blood
collection to minimize stress.
[41]
The blood was transferred
onto pre-combusted Whatman GF/F glass microfiber filter
papers (GE Healthcare, Little Chalfont, UK) that were dried
Table 2. Species, mean weight (kg), mean snout-vent length (SVL; mm), mean age (years), and sex ratio (F/M) for all Cyclura
species included in the study. Means are reported ± SD. All animals were adults except where otherwise noted
Species N Weight SVL Age Sex
C. lewisi 5 1.4 ± 1.3
a
293.2 ± 111.1
a
5.8 ± 4.4
e
1/4
C. lewisi (juvenile) 15 0.3 ± 0.4
b
187.3 ± 51.8
b
1.0 ± 0.0 5/10
C. collei 7 3.9 ± 1.0
c
433.3 ± 367.7
c
12.1 ± 6.4
e
4/3
C. pinguis 7 5.5 ± 0.9
d
484.0 ± 32.9
d
23.0 ± 6.7
e
2/5
Adult Total 19 3.5 ± 2.1 398.9 ± 112.6 14.5 ± 9.2 7/12
Total w/ juveniles 34 1.9 ± 2.2 293.1 ± 137.9 8.5 ± 9.6 12/22
Due to sampling constraints, weights and SVLs were obtained only for the following number of individuals of each species
and these were used to calculate overall mean weights and SVLs:
a
5 adults (age 2–11 years).
b
13 juveniles (age 1 year).
c
3 adults (age 7–19 years).
d
5 adults (age 15–28 years).
e
The age and sizes were statistically different among the adults of each species; ANOVA, F
2,16
,p≤0.001.
Table 3. The mean (±SD) stable isotope ratio values (‰) and C:N ratios from dietary components offered weekly to captive
Rock Iguanas (Cyclura spp.) in this study. The % source refers to the percentage that each dietary source contributed to the
overall weekly diet budget. See text for the equation used to determine the δ
13
C and δ
15
N values for the overall diet
Diet item
Portion
weight (g)
Times fed
per week
Total weekly food
budget (g) % source N δ
13
Cδ
15
N C:N
Dandelion greens 125 5 625 23.19 3 –28.8 ± 1.1 1.6 ± 1.4 8.6 ± 1.2
Collard greens 125 3 375 13.91 2 –28.3 ± 2.1 2.7 ± 1.2 8.1 ± 1.0
Green chard 125 3 375 13.91 2 –29.9 ± 2.1 1.7 ± 2.6 9.2 ± 2.2
Mustard greens 125 3 375 13.91 2 –30.5 ± 1.0 2.1 ± 1.9 7.6 ± 1.9
Bok choy 125 2 250 9.28 1 –28.5 ± 0.0 –0.7 ± 0.0 9.4 ± 0.0
Escarole 125 2 250 9.28 1 –27.6 ± 0.0 –2.4 ± 0.0 12.0 ± 0.0
Kale 125 2 250 9.28 1 –31.0 ± 0.0 2.8 ± 0.0 11.6 ± 0.0
Root vegetables
a
15 5 75 2.78 4,4,4
a
–27.2 ± 1.8 2.5 ± 0.7 31.8 ± 9.3
Fruit
b
15 3 45 1.67 4,2,5
b
–26.0 ± 0.6 1.7 ± 0.4 52.6 ± 37.8
Zucchini 15 3 45 1.67 2 –26.1 ± 1.1 1.3 ± 0.2 11.73 ± 2.1
Green beans 15 2 30 1.11 1 –25.6 ± 0.0 2.8 ± 0.0 14.4 ± 0.0
Total weekly diet 2695
Overall diet –29.0 ± 0.5 1.4 ± 0.6
a
Root vegetables: carrot, turnip, yam.
b
Fruit: apple, honeydew, papaya.
Stable C and N isotope discrimination factors for reptiles
Rapid Commun. Mass Spectrom. 2016,30,9–21 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm
13
in sterile scintillation vials in a drying oven at 32 °C for
48 h.
[45]
The blood samples were homogenized by hand using
a mortar and pestle or metal spatula within a cryovial, and
~0.5 to 1.0 mg of these processed samples were weighed into
5 × 9 mm tin capsules. We did not extract lipids from blood as
whole blood has a very low lipid content;
[46]
this was
supported by the low (<4) C:N ratios observed from our
stable isotope data.
[47]
The scat and skin samples were freeze-dried for 48 h, and
the scat samples were further dried at 120 °C for 48 h to kill
any potentially remaining bacteria. We removed all
undigested plant matter from the scats and sampled the
remaining fecal matrix material by first dividing it into two
subsamples. The samples targeted for δ
13
C analysis were
agitated with 0.5 M HCl for 3 h to remove any potential
inorganic carbon, as we were interested in measuring the
δ
13
C values from the organic components only, then dried at
32 °C for 48 h. To test for the effects of HCl on the δ
13
C and
δ
15
N values from scat, subsamples of untreated scat material
were also analyzed. We homogenized all scat samples by
hand using either a mortar and pestle or metal spatula within
a cryovial, and packaged ~5 mg of each scat sample into
5 × 9 mm tin capsules.
The skin samples were cut into small pieces
(<0.5 × 0.5 mm) using surgical scissors, then each sample
was divided into two subsamples. Lipid extraction was
performed on the samples targeted for δ
13
C analysis via a
method modified from Folch et al.
[47–49]
We placed skin
samples in 15-mL glass centrifuge tubes, added 10 ml of
petroleum ether,
[50]
capped the vials with perforated lids,
and sonicated them for 10 min at 40 kHz in a 60 °C water
bath. We then centrifuged the samples at 12,000 gfor 5 min,
pipetted off the petroleum ether, rinsed each sample with
ultra-pure water, centrifuged them again at 12,000 gfor
5 min, and removed the excess water with a pipette. All the
samples were then dried in a drying oven at 45 °C for 72 h.
The samples were re-homogenized before analysis.
Subsamples from all skin samples were analyzed with their
lipids intact to test for the effects of lipid extraction on the
stable isotope values, and ~0.5 to 1.0 mg of all skin samples
were packaged into 5 × 9 mm tin capsules.
Throughout the sampling periods, we collected all diet
samples (Table 3), which were hand-washed, frozen at –20 °C
for at least 48 h, then freeze-dried for 48 h. The samples were
homogenized by hand and ~3 mg was packed into 5 × 9 mm
tin capsules.
All stable isotope analyses were performed with a Carlo Erba
CE1108 elemental analyzer (CE Elantech, Lakewood, NJ, USA)
interfaced via a ConFlo III device (Thermo Fisher Scientific, San
Jose, CA, USA) to a Delta Plus XP isotope ratio mass
spectrometer (Thermo Fisher Scientific) at the Department of
Earth and Marine Sciences, University of California, Santa
Cruz. The average precision of these data was calculated as
the standard deviation (SD) of the δ
13
Candδ
15
Nvaluesfrom
a set of standards (acetanilide), and it was 0.1‰for both.
Statistical analysis
All individuals were kept on a weekly diet schedule (Table 3)
for which the weighted percentage of each diet item was
calculated by comparing the weekly total weight of each diet
item with the weekly total weight of all food consumed by
each individual. Using this weighted percentage and the
δ
13
C and δ
15
N values of each dietary item and the iguana
tissues, the discrimination factors were calculated using the
following equation:
ΔX‰ðÞ¼δXconsumer
ðÞ–h%source 1δXsource 1
ðÞ
þ%source 2δXsource 2
ðÞþ…i;
where ΔX(‰) is the C or N isotope discrimination factor
(Δ
13
CorΔ
15
N, respectively), δX
consumer
is the δ
13
Corδ
15
N
value of the tissues from the iguanas, %
source
is the
contribution of a specific diet item to the animals’total,
aggregate diet, and δX
source
is the δ
13
Corδ
15
N value of that
particular diet or source item.
[15,16]
To determine the variance around the weighted average
source δ
13
C and δ
15
N values, we squared the SD from the
mean δ
13
C and δ
15
N values for each source item. We then
divided the % source value by 100, squared each of those
values, then multiplied these by the squared SD for each of
the mean δ
13
C and δ
15
N values from the sources. We added
these numbers to arrive at a weighted sum of variances, then
took the square root of that to obtain the SD around the
weighted mean δ
13
C and δ
15
N values for the overall diet
sources. We used the weighted sum of variances to calculate
a potential range of low to high δ
13
C and δ
15
N values for
the diet source and used those to calculate a range of potential
discrimination factors. The mean C and N discrimination
factor ± SD for each tissue type was calculated using the mean
source isotope values in the equation above, and the mean
range of potential discrimination factors was calculated by
subtracting the lowest and highest potential source stable
isotope values from the stable isotope values from the iguana
tissues and taking the averages of those values.
All statistical tests were performed with R,
[51]
using
parametric methods as all the data met the assumptions for
parametric tests. Paired t-tests were used to evaluate the
effects of acid treatment and lipid extraction on the isotope
values from scat and skin samples, respectively, and to test
for differences in isotope values between the two collection
periods. We used t-tests to determine the effects of sex, age-
group, and sampling period on the stable isotope values from
scat and skin, and of sex and age-group on the isotope values
from blood samples.
To test for relationships between the stable isotope values
from all tissues and iguana age, size (SVL; mm), weight (g),
and the relationship between size and weight, linear
regressions were conducted for all tissues using individuals
for which age, SVL, and/or weight data were available for
each respective analysis. For some, these measurements were
not available and they were omitted from the regression
analysis. Results were included from regression analyses for
age, size, weight (although they are all related), so that the
results will be useable by researchers with access to any of
those variables as they work to best determine which Δ
13
C
or Δ
15
N values would be most applicable to the reptile of
interest. To test for differences in the stable isotope values
among species for all tissues collected and among tissues for
each species, we conducted analysis of variance (ANOVA)
tests followed by Tukey’s pairwise comparisons.
[51]
The
values reported are means ± SD and significance was tested
at the α= 0.05 level.
R. Steinitz et al.
wileyonlinelibrary.com/journal/rcm Copyright © 2015 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016,30,9–21
14
RESULTS
The δ
13
C and δ
15
N values from all tissues and dietary items
collected during the two sampling periods were not different
(paired t-tests, all p ≥0.07; see Supplementary Table S1,
Supporting Information). Therefore, as we had slightly more
data from the first sampling period, we used the isotope values
collected during this period (February 2013) for the analysis of
diet items, scat, and skin. As blood samples were only
available from the second sampling period (August 2013),
these were used for the isotope analysis of blood. The δ
13
C
and δ
15
N values were the same between sexes within each
tissue type (t-tests, all p ≥0.25; see Supplementary Table S1,
Supporting Information), so females and males were grouped
together for all comparisons.
The δ
13
C and δ
15
N values were different among tissue
types (scat, blood, and skin) for each species (ANOVA, all
p≤0.01) and Tukey’s post hoc tests confirmed that the isotope
values from all tissues within each species were different
except for the δ
13
C values from blood and skin from C. lewisi
(p = 0.26), and the δ
15
N values from blood and skin for
C. pinguis (p = 0.22) and from blood and scat for all three
species (p = 0.10 to 0.56) (see Table 4 and Supplementary
Table S1, Supporting Information, and Fig. 1). The Δ
13
C and
Δ
15
N values from all tissue types and across all species ranged
from –2.5 to +6.5‰and from +2.2 to +7.5‰, respectively, for
adults, and from –1.6 to +2.8‰and from +0.8 to +5.3‰,
respectively, for juveniles (Table 4). We found that the
relationship between weight (g) and size (SVL, mm) for
all individuals (adults and juveniles) was curvilinear
(W = (1.2274 × 10
–5
)×S
3.216
; log-transformed for regression:
R
2
adj
= 1.0, F
1,24
= 5301.0, p <<0.0001), and juveniles had
higher growth rates than the adults (Supplementary Table
S1 and Fig. S1, Supporting Information).
Scat
There were no differences in the δ
13
C values from scat samples
processed with and without the HCl agitation to potentially
remove inorganic carbon prior to isotope analysis (paired t-test,
t=–1.0, df = 37.0, p = 0.4) (Supplementary Table S1, Supporting
Information). We expected the acid treatment to have no effect
on the δ
15
N values; however, we found that scat samples
processed with HCl exhibited slightly higher δ
15
N values than
those processed without HCl (5.3 ± 1.1‰vs 4.8 ± 0.7‰,
respectively; paired t-test, t = 3.5, df =9.0, p <0.01) (Supplementary
Table S1, Supporting Information). Thus, we used the δ
13
Cand
δ
15
N values from the untreated scat samples for our analyses.
The Δ
13
C and Δ
15
N values from scats from all of the species
had high degrees of variability, ranging from –2.5 to 0.7‰and
from 2.2‰to 4.2 ± 0.6‰, respectively, but none of these
values were statistically different among species (ANOVA,
F
2,8
= 3.4, p = 0.09 and F
2,8
= 1.5, p = 0.28, respectively; Table 4
and Supplementary Table S1, Supporting Information, and
Fig. 1). There were no differences in the Δ
13
C and Δ
15
N values
from scat between the two age groups sampled from C. lewisi
(adults vs juveniles; t-tests, δ
13
C: t = 0.2, df = 4.2, p = 0.85; δ
15
N:
t = 0.4, df = 6.6, p = 0.69) (Table 4 and Supplementary Table S1,
Supporting Information, and Fig. 1). Linear regression
analysis demonstrated no correlation between age as a
continuous variable and the Δ
13
C and Δ
15
N values from scat
samples (δ
13
C: R
2
adj
= 0.1, F
1,9
= 2.2, p = 0.17; δ
15
N: R
2
adj
=–0.1,
F
1,9
= 0.1, p = 0.82; Table 4 and Supplementary Table S1 and
Figs. S2(a) and S2(b), Supporting Information). However,
linear regression analysis exhibited a negative correlation
between the Δ
13
C values from scats and weight (R
2
adj
= 0.6,
F
1,5
= 9.1, p = 0.03; Supplementary Table S1, Supporting
Information, and Fig. 2(a)) and body size (SVL, R
2
adj
= 0.8,
F
1,5
= 20.6, p = 0.01; Supplementary Table S1 and Fig. S3(a),
Supporting Information) for adults of all three species. There
were no significant relationships between weight, age, or size
and the Δ
15
N values from scats (Supplementary Table S1 and
Fig. 2, and Supplementary Figs. S2(b) and S3(b) (Supporting
Information)).
Blood
The Δ
13
C values from blood differed among all three species
(ANOVA, F
2,11
= 22.7, p <0.01), but the Δ
15
N values were
not different (F
2,11
= 1.8, p = 0.21) (Table 4 and Supplementary
Table S1, Supporting Information, and Fig. 1). Post hoc Tukey
pairwise comparisons revealed that all three species had
different mean Δ
13
C values (all p ≤0.02), with C. lewisi as the
lowest (1.5 ± 0.3‰), then C. collei (2.1 ± 0.4‰), and C. pinguis
(2.8 ± 0.2‰) (Table 4 and Supplementary Table S1,
Supporting Information, and Fig. 1). In addition, the mean
Δ
15
N values from C. lewisi juveniles were significantly lower
(2.9 ± 0.6‰) than those from the adults (4.1 ± 0.4‰; t-test,
t = 2.9, df = 7.4, p = 0.02), but the Δ
13
C values (1.4–3.5‰) did
not differ between age groups (t-test, t = 1.0, df = 6.3,
p = 0.38) (Table 4 and Supplementary Table S1, Supporting
Information, and Fig. 1).
We found positive correlations between age as a
continuous variable and both the Δ
13
C and the Δ
15
N values
from blood (R
2
adj
= 0.7, F
1,12
= 26.8, p <0.01 and R
2
adj
= 0.3,
F
1,12
= 7.8, p = 0.02, respectively; Supplementary Table S1
and Figs. S2(a) and S2(b), Supporting Information). The
Δ
13
C and Δ
15
N values from blood were also significantly,
positively correlated with weight (R
2
adj
= 0.6, F
1,11
= 17.5,
p<0.01 and R
2
adj
= 0.6, F
1,11
= 19.1, p <0.01, respectively;
Supplementary Table S1, Supporting Information, and Fig. 2)
and size (R
2
adj
= 0.3, F
1,11
= 6.6, p = 0.03 and R
2
adj
= 0.7,
F
1,11
= 32.0, p <0.01, respectively; Supplementary Table S1
and Figs. S3(a) and S3(b), Supporting Information).
Skin
There were no differences in the δ
13
C and δ
15
N values from
skin that had been lipid extracted or left intact (paired t-tests,
t=–0.1, df = 7.0, p = 0.90 and t = 0.6, df = 9.0, p = 0.56,
respectively). Therefore, we used isotope values from
samples that were not lipid extracted to calculate the
discrimination factors for skin. The Δ
13
C values from skin
varied among species (ANOVA, F
2,14
= 8.5, p <0.01). The
order of Δ
13
C values from skin was the same as for blood:
C. lewisi (2.4 ± 0.1‰), C. collei (3.7 ± 1.2‰), and C. pinguis
(5.2 ± 0.9‰); however, Tukey’s tests demonstrated that the
δ
13
C values from skin were not significantly different between
C. lewisi and C. collei (p = 0.19). All other comparisons were
significantly different (p ≤0.04; Table 4 and Supplementary
Table S1, Supporting Information, and Fig. 1). The Δ
15
N
values from skin did not differ among species (ANOVA,
F
2,14
= 0.6, p = 0.6, Table 4 and Supplementary Table S1,
Supporting Information, and Fig. 1). C. lewisi juveniles
Stable C and N isotope discrimination factors for reptiles
Rapid Commun. Mass Spectrom. 2016,30,9–21 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm
15
Table 4. Species, mean (±SD) stable isotope ratios and stable isotope discrimination values (‰), range of discrimination factors, and C:N ratios for tissues and diet items
collected in 2013 for all Cyclura species in this study. All animals were adults except where otherwise noted. Stable isotope values from males and females were grouped for
each tissue type within each species as there were no differences in isotope values between sexes. See Supplementary Table S1 (Supporting Information) for more details
Species Tissue N
Animal Diet
Mean discrimination
factors
Range of discrimination
factors
δ
13
Cδ
15
Nδ
13
Cδ
15
NΔ
13
CΔ
15
NΔ
13
CΔ
15
N C:N
C. lewisi
a
Scat 4 –29.5 ± 0.9 4.3 ± 0.8 –29.0 ± 0.5 1.4 ± 0.6 –0.5 ± 0.9 2.9 ± 0.8 –1.5 to 0.7 2.2 to 3.8 9.6 ± 1.2
Blood 5 –27.1 ± 0.3* 5.2 ± 0.6* –29.0 ± 0.5 1.4 ± 0.6 1.9 ± 0.3* 3.8 ± 0.6* 1.5 to 2.1 3.2 to 4.6 3.6 ± 0.3
Skin 3 –26.2 ± 0.1* 7.4 ± 0.7* –29.0 ± 0.5 1.4 ± 0.6 2.8 ± 0.1* 6.0 ± 0.7* 2.7 to 2.8 5.4 to 6.8 3.5 ± 0.4
C. lewisi juvenile Scat 10 –29.6 ± 0.7 4.1 ± 1.0 –29.0 ± 0.5 1.4 ± 0.6 –0.6 ± 0.7 2.7 ± 1.0 1.0 to -1.6 0.8 to 3.9 9.1 ± 0.8
Blood 13 –27.3 ± 0.2 4.4 ± 0.6* –29.0 ± 0.5 1.4 ± 0.6 1.7 ± 0.2 2.9 ± 0.6* 1.4 to 2.2 2.3 to 4.0 3.4 ± 0.1
Skin 9 –26.8 ± 0.3* 5.6 ± 0.8* –29.0 ± 0.5 1.4 ± 0.6 2.3 ± 0.3* 4.2 ± 0.8* 1.8 to 2.8 2.8 to 5.3 3.3 ± 0.1
C. collei
b
Scat 4 –31.0 ± 0.4 5.3 ± 0.3 –29.0 ± 0.5 1.4 ± 0.6 –2.0 ± 0.4 3.4 ± 0.3 –2.5 to -1.7 3.6 to 4.2 9.7 ±1.4
Blood 4 –26.5 ± 0.4* 5.6 ± 0.2 –29.0 ± 0.5 1.4 ± 0.6 2.1 ± 0.4* 3.9 ± 0.3 2.2 to 3.0 4.0 to 4.5 4.3 ± 0.8
Skin 7 –24.9 ± 1.2* 7.6 ± 0.8 –29.0 ± 0.5 1.4 ± 0.6 4.1 ± 1.2* 6.2 ± 0.8 2.8 to 6.5 5.0 to 7.5 3.0 ± 0.3
C. pinguis
c
Scat 3 –30.1 ± 1.2 3.6 ± 2.3 –29.0 ± 0.5 1.4 ± 0.6 –1.6 ± 1.1 3.5 ± 0.6 –2.4 to -0.9 3.1 to 3.9 6.4 ±4.7
Blood 5 –25.9 ± 0.2* 5.7 ± 0.3 –29.0 ± 0.5 1.4 ± 0.6 3.1 ± 0.2* 4.3 ± 0.3 2.9 to 3.5 4.0 to 4.7 3.4 ± 0.1
Skin 7 –23.5 ± 0.9* 7.2 ± 0.4 –29.0 ± 0.5 1.4 ± 0.6 5.5 ± 0.9* 5.8 ± 0.4 4.1 to 6.5 5.4 to 6.5 3.3 ± 0.1
Overall
Scat
Δ
13
CΔ
15
N
–1.3 ± 1.0 3.4 ± 0.7
Blood 2.5 ± 0.6 4.1 ± 0.4
Skin 4.5 ± 1.4 6.0 ± 0.6
*Significant differences in stable isotope values and discrimination factors among species comparisons for each tissue type (ANOVAs, Tukey’s pairwise comparisons). All
statistical details are provided in Supplementary Table S1 (Supporting Information).
All among-tissue comparisons within species were statistically different (all p ≤0.02), except:
a
blood vs. skin Δ
13
C values, p = 0.26; blood vs scat Δ
15
N values, p = 0.21
b
blood vs. scat Δ
15
N values, p = 0.56
c
blood vs. scat Δ
13
C values, p = 0.10; blood vs skin Δ
15
N values, p = 0.20
R. Steinitz et al.
wileyonlinelibrary.com/journal/rcm Copyright © 2015 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016,30,9–21
16
exhibited lower Δ
13
C and Δ
15
N values from skin (2.3 ± 0.3‰
and 4.2 ± 0.8‰, respectively) than C. lewisi adults (4.5 ± 0.1‰
and 6.0 ± 0.6‰, respectively) (t-test; Δ
13
C: t = 4.6, df = 9.9,
p<0.01; Δ
15
N: t = 3.6, df = 3.6, p = 0.03) (Table 4 and
Supplementary Table S1, Supporting Information, and Fig. 1).
The Δ
13
C values from skin collected from adults of all
species were positively correlated with age as a continuous
variable (R
2
adj
= 0.8, F
1,15
= 53.9, p <<0.01) (Supplementary
Fig. S2(a), Supporting Information), but the Δ
15
N values were
not (R
2
adj
= 0.0, F
1,15
= 0.8, p = 0.37) (Supplementary Table S1
and Fig. S2(b), Supporting Information). In addition, weight
and size were positively correlated with the Δ
13
C values from
skin from adults (R
2
adj
= 0.7, F
1,9
= 27.4, p <0.01 and R
2
adj
= 0.5,
F
1,9
= 10.2, p = 0.01, respectively; Supplementary Figs. S2(a)
and S3(a), Supporting Information), whereas there were no
relationships between weight and size and the Δ
15
N values
from skin (R
2
adj
=–0.1, F
1,9
= 0.1, p = 0.80 and R
2
adj
=–0.1,
F
1,9
= 0.1, p = 0.79, respectively; Supplementary Table S1 and
Figs. S2(b) and S3(b), Supporting Information).
DISCUSSION
The range of Δ
13
C and Δ
15
N values from scat, blood, and skin
from the captive rock iguanas was from –2.5 to +6.5‰and
from +2.2 to +7.5‰, respectively, for adults, and from –1.6 to
2.8‰and from +0.8 to 5.3‰, respectively, for juveniles. These
values are similar to those found in previous studies
examining isotope discrimination factors in captive reptiles,
although both Δ
13
C and Δ
15
N values from skin from adult
Rock Iguanas are generally higher than those reported for skin
from other adult reptiles, while values from skin from juvenile
Rock Iguanas are similar to those from other species (Table 1).
Of 24 comparisons of the Δ
13
C and Δ
15
N values among tissues
for all species (blood vs skin vs scat for the adults of three
species and for juvenile C. lewisi), only six were not different:
the Δ
13
C values from blood and skin in C. lewisi adults, the
Δ
15
N values from blood and skin in C. pinguis adults, and
the Δ
15
N values from blood and scat from adults of all three
species and juvenile C. lewisi (Table 4 and Supplementary
Table S1, Supporting Information, and Fig. 1). Different tissues
are composed of different amino acids, which vary in their
δ
13
C and δ
15
N values.
[52,53]
Therefore, it is expected that
different tissues from the same animal held on a constant diet
could exhibit varying stable isotope values.
[12]
The δ
15
N (and Δ
15
N) values from blood and skin among
species were the same (Table 4, Fig. 1); however, they were
significantly lower for juvenile C. lewisi than for the adult
C. lewisi. In addition, all among-species comparisons for
Δ
13
C values from blood and skin were significantly different
(Table 4 and Supplementary Table S1, Supporting Information).
The Δ
15
N values of tissues from vertebrates with higher growth
rates, such as more rapidly growing hatchlings or juveniles,
have been shown to be lower than those from animals
that are not growing or have slowed growth.
[12,39,54,55]
This
is because growing animals retain more
14
N via tissue
deposition than they lose via excretion of waste than an
animal that is not growing (but see
[56]
). This could explain
the lower Δ
15
N values that we observed in the juvenile
C. lewisi than from the adult C. lewisi.
To our knowledge, this same phenomenon has not been
shown to occur for δ
13
C (and thus Δ
13
C) values and, in fact,
rapidly growing hatchling and juvenile sea turtles
Figure 1. Mean (±SD) stable carbon (Δ
13
C) and nitrogen
(Δ
15
N) isotope discrimination factors between diets and
tissues (skin, blood and scat) from adult and juvenile
captive Rock Iguanas (Cyclura spp.). See Table 2 for specific
stable isotope and discrimination factor values, and
Supplementary Table S1 (Supporting Information) for tests
for differences among tissues, species and age groups.
Figure 2. Linear relationships between (a) the stable carbon
(Δ
13
C) and (b) the nitrogen (Δ
15
N) isotope discrimination
factors and weight (g) from adult, captive Rock Iguanas
(Cyclura spp.). There were significant linear relationships
between the Δ
13
C values from scat, blood, and skin and
between the Δ
15
N values from blood and weight from the
three iguana species sampled. No other linear relationships
were significant. See Supplementary Table S1 (Supporting
Information) for all results and Supplementary Figs. S2 and
S3 for linear regressions for age and size.
Stable C and N isotope discrimination factors for reptiles
Rapid Commun. Mass Spectrom. 2016,30,9–21 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm
17
(loggerheads, Caretta caretta) demonstrated δ
13
C values in line
with other published values from adults.
[39]
However, the
Δ
13
C values from blood and skin from the adults in this study
increased with increasing size and age (Tables 2, 4 and
Supplementary Table S1, and Figs. S2(a) and S3(a),
Supporting Information), which may be attributed to
differential growth rates at different ages for reptiles.
Supplementary Fig. S1 (Supporting Information) illustrates
an increased growth rate for juveniles over adults in this
study and the youngest, and physically smallest, species
(C. lewisi) had the lowest δ
13
C values, followed by the
mid-size C. collei, then the oldest, largest C. pinguis. While
little data exists on Cyclura growth rates, they are known
to grow continuously throughout their lives,
[57]
but exhibit
different growth rates at different life stages, including
higher growth and metabolic rates at younger ages.
[27,34,41,43]
Each species in this study includes individuals of
significantly different mean ages (C. pinguis: 23.0 ± 6.7 years,
C. collei: 12.1 ± 6.4 years, and C. lewisi: 2.2 ± 2.9 years (adults
and juveniles); p ≤0.001; Table 2) and they may be
experiencing different growth and metabolic rates specific
to these different ages (Supplementary Fig. S1, Supporting
Information). Interestingly, only the Δ
13
C values from skin
(and not those from blood) were lower for the juveniles than
the adult C. lewisi. At present, we do not know why the Δ
13
C
values correlated so strongly with age/size in the adults,
whereas the Δ
15
N values did not, and the potential role of
differential sizes and growth rates in determining Δ
13
C and
Δ
15
N values deserves further study in reptiles and other taxa.
The Δ
13
C values from scats were all negative (excluding one
adult with a Δ
13
C value of +0.7‰). Consumer tissues
generally contain more of the heavy isotopes of C and N than
their food sources, thus leading to positive discrimination
factors.
[7,13,58]
However, excreta generally have lower δ
13
C
values than their diets and their other tissues.
[30,59,60]
It is not
known exactly why this happens (see
[60]
), but one hypothesis
is that it is because the lighter isotope of carbon reacts more
quickly than the heavier isotope, becoming more readily
incorporated into waste products. This fractionation effect
contributes to a predictable increase in both δ
13
C and δ
15
N
values between trophic levels in a food web.
[7,13,59]
Thus, an
accumulation of more of the light isotope of carbon in excreta
could explain the negative Δ
13
C values, and the somewhat
lower Δ
15
N values observed for iguana scats than for the other
tissues. Finally, the Δ
13
C values from scats were negatively
correlated with iguana age, size, and weight. As growing
animals tend to retain more of the lighter isotopes, it follows
that their excreta would contain higher ratios of heavier to
lighter isotopes than slower growing adults, leading to higher
δ
13
C (and thus Δ
13
C) values for their excreta. We might expect
a similar pattern for the stable nitrogen isotope ratio values
from scats, but there were no relationships between the Δ
15
N
values from iguana scats and their age, size, or weight.
In studies of wild reptile populations (Green Sea Turtles,
Chelonia mydas, Loggerhead Turtles, Caretta caretta, and
Leatherback Sea Turtles, Dermochelys coriacea), correlations of
δ
13
C and δ
15
N values with body size, weight, or age are
typically attributed to ontogenetic, or size- and age-related,
shifts in diet preferences.
[61–63]
For example, such changes
can occur as animals grow larger and are able to acquire larger
prey. However, the San Diego Zoo ICR held individuals of all
ages and species on a steady, identical diet throughout our
study (12+ months). Therefore, diet shifts with increasing
age or size would not account for the observed isotopic
changes and this should be considered when examining stable
isotope values from different age and size-class individuals in
the wild. In addition, it is frequently difficult to obtain Δ
13
C
and Δ
15
N values from captive animals for use in studies
estimating wild animal foraging ecology. Our data indicate
linear relationships between the Δ
13
C values from blood, skin,
and scat and iguana weight, size, and age, and the Δ
15
N values
from blood and iguana weight, size, and age. Therefore, it
may be possible for others to use our regressions to estimate
the best Δ
13
C and Δ
15
N values for these tissues for use in
interpreting wild iguana foraging ecology if the weights, sizes,
and/or ages of the iguana species of interest are known.
The captive populations at the San Diego Zoo ICR are kept
on a constant, healthy diet, and are thus neither malnourished
nor overweight.
[34,41,43]
Therefore, our weight-size curvilinear
relationship (Supplementary Fig. S1, Supporting Information)
represents that of healthy individuals. This model may be used
for comparison of wild individuals for estimations of physical
condition. This may be useful as an animal’sphysicalcondition
can affect its tissue stable isotope values. For example, when
resources are scarce, tissues undergo catabolism causing an
increase in their δ
15
Nvalues(
[64]
and see review in
[65]
), an
occurrence observed across taxa, including in reptiles.
[65]
The treatment of scat samples with HCl was done to
remove any potential inorganic carbon (Brown Reid,
personal communication, Washington University, St. Louis,
MO, USA) before stable isotope analysis. This is to ensure
that organic carbon is targeted for analysis, as the δ
13
C value
from organic carbon is what reflects animal diet. Although
we found that agitation of scat matrix in HCl did not
significantly affect the δ
13
C values in our samples, indicating
that no inorganic carbon was present, those that were treated
with HCl did exhibit higher δ
15
N values than those that were
not. The acidification process could cause leaching of organic
nitrogen compounds (i.e. proteins or amino acids),
[66]
which
could affect the δ
15
N values in the scat samples. As the
acidification treatment of scats did not affect their δ
13
C
values, but it did affect the δ
15
N values, we recommend
omitting this treatment from future protocols for preparing
scats from iguanas for stable isotope analysis unless the
samples in question clearly contain significant amounts of
materials with inorganic carbon. In addition, we recommend
that the use of stable isotope values from scat in future studies
be considered with caution as scat proved to be the least
reliable tissue in its consistency of stable isotope values across
species and age groups (see Table 4 and Supplementary Table
S1, Supporting Information, and Fig. 1). Studies using stable
isotope analysis are often performed for the purpose of
reconstructing diets of wild animals and scat would be a
relatively easy, accessible, and noninvasive tissue to use for
such analyses. However, Hwang et al.
[67]
found that the
δ
15
N and δ
13
C values from scat did not consistently reflect
the isotopic composition of the diet, and the Δ
15
N and Δ
13
C
values from scat offered evidence of great variation in a study
across several mammalian fore- and hindgut fermenters.
These findings are in line with our scat analyses results. There
may also be a representational bias as a scat sample will
contain the highest proportion of remains of the least
digestible dietary items, while the most digestible items will
be least represented.
R. Steinitz et al.
wileyonlinelibrary.com/journal/rcm Copyright © 2015 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016,30,9–21
18
The skin from the iguanas was treated with petroleum
ether to remove any lipids that might be present as reptile
skin can contain significant lipid concentrations that are
thought to contribute to decreased cutaneous water loss
[68]
and the presence of lipids in animal tissues reduces δ
13
C
values.
[47]
However, lipid extraction had no significant effect
on the δ
13
Corδ
15
N values from skin, indicating that it was
unnecessary. This is probably because the lipid content of
theiguanaskininourstudywasnothighenoughtoaffect
the stable isotope values andthisissupportedbytheC:N
ratios from the skin samples which were less than 4.0,
indicating a lipid content of less than 10%.
[47]
In order to provide overall stable isotope discrimination
factors across all three adult iguana species for use in studies
employing other species, we have reported the mean Δ
13
C
and Δ
15
N discrimination factors across all species for each
tissue in Table 4 (and data are presented in Fig. 1). They were
+4.5 ± 1.4‰and +6.0 ± 0.6‰, respectively, for skin; +2.5
±0.6‰and +4.1 ± 0.4‰, respectively, for blood; and –1.3
±1.0‰and +3.4 ± 0.7‰, respectively, for scat (Table 4 and
Supplementary Table S1, Supporting Information). However,
since we found relationships between discrimination factors
and size, we recommend using the Δ
13
CandΔ
15
N factors from
the species in this study that are closest in size to the wild
species of interest when applying stable isotope discrimination
factors to known-size iguanas in the wild. In addition, where
possible, we recommend utilizing our reported linear
regression equations to estimate the appropriate discrimination
factors for the size of the iguana species.
CONCLUSIONS
We found that age, size, and the growth rates associated with
specific life stages probably play a role in affecting the δ
13
C
and δ
15
N (and thus Δ
13
C and Δ
15
N) values in Rock Iguanas,
although the mechanisms causing these relationships require
further study, especially for stable carbon isotopes. Tissues of
varying molecular composition also varied isotopically, and
the δ
13
C values from scat exhibited inverse relationships with
age and size to those from other tissues, possibly due to the
digestion adaptations unique to hindgut fermenters.
Acknowledgements
The authors extend their thanks to Ron Swaisgood and the
San Diego Zoo –Institute for Conservation Research, Applied
Animal Ecology/Behavioral Ecology –for their collaboration;
UCSD, Heart di Vite and the UCSD Undergraduate Academic
Enrichment Program and David Marc Belkin Memorial
Research Scholarship for providing funding that permitted
this study; IUCN –Iguana Specialist Group for providing
funding to present this research at the IUCN –ISG conference
2013; The UCSC Stable Isotope Lab and Dyke Andreasen; we
thank Dr D. Holway, Dr E. Cleland, L. Bailey, K. Richardson,
C. Turner Tomaszewicz, E. Hetherington, V. Hanna, S. Urata,
C.Kelleherandmembers(two-andfour-legged)ofthe
Kurle Lab for laboratory help and comments that greatly
improved this manuscript; and we thank the three
anonymous reviewers for their comments that improved
this manuscript.
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SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publisher’s website.
Stable C and N isotope discrimination factors for reptiles
Rapid Commun. Mass Spectrom. 2016,30,9–21 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm
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