Oecologia
DOI 10.1007/s00442-007-0669-3

POPULATION ECOLOGY

Hydrogen isotopic variation in migratory bird tissues of known
origin: implications for geographic assignment
Kathryn M. Langin Æ Matthew W. Reudink Æ
Peter P. Marra Æ D. Ryan Norris Æ T. Kurt Kyser Æ
Laurene M. Ratcliffe

Received: 27 September 2006 / Accepted: 23 January 2007

 Springer-Verlag 2007

Abstract Continent-wide variation in hydrogen isotopic
composition of precipitation is incorporated into animal
diets, providing an intrinsic marker of geographic location
at the time of tissue growth. Feathers from migratory birds
are now frequently analyzed for stable-hydrogen isotopes
(dD) to estimate the location of individuals during a preceding molt. Using known-origin birds, we tested several
assumptions associated with this emerging technique. We
examined hydrogen isotopic variation as a function of age,
sex, feather type and the timing of molt in a marked population of American redstarts (Setophaga ruticilla) breeding in southeastern Ontario. We measured dD in feathers
and blood from individuals that bred or hatched at our
study site during the year in which those tissues were
grown. Juvenile tissues from 5- to 10-day-old birds had
more negative dD values than those from adults, which
most likely reflected age-related differences in diet. Within
adults, primary feathers had more negative dD values than
contour feathers. The mean dD value in adult primary
Communicated by Mark Chappell.
K. M. Langin (&) 
 M. W. Reudink 
 L. M. Ratcliffe
Department of Biology, Queen’s University,
K7L 3N6 Kingston, ON, Canada
e-mail: langink@biology.queensu.ca
P. P. Marra
Smithsonian Migratory Bird Center, National Zoological Park,
Washington, DC 20008, USA
D. R. Norris
Department of Integrative Biology, University of Guelph,
N1G 2W1 Guelph, ON, Canada
T. K. Kyser
Department of Geological Sciences and Geological Engineering,
Queen’s University, K7L 3N6 Kingston, ON, Canada

feathers was relatively consistent among years and with the
value expected for our study population. However, amongindividual variation in dD corresponded to an estimated
latitudinal range of 6–8 (650–900 km). We conclude that
feathers sampled from recently hatched juveniles may not
provide a reliable estimate of expected local isotopic signatures for comparison with adult feathers of unknown
origin. Furthermore, we urge researchers to use caution
when using dD values in feathers to infer geographic origin, and suggest that the best approach is to assign individuals to broad geographic zones within a species’
potential molting range.
Keywords American redstart 
 Migration 
 Molt 

Passerine bird 
 Stable isotopes

Introduction
Tracking migratory animals between periods of the annual
cycle has been a challenging task because many species
travel thousands of kilometers between their breeding and
wintering areas (Greenberg and Marra 2005). Intrinsic
markers, such as stable isotopes, have proven useful for
tracking animals to study long-distance migration (Hobson
1999, 2005a; Rubenstein and Hobson 2004) and dispersal
(Hobson 2005b). The primary advantage of using isotopes
over mark–recapture techniques is that individuals only
have to be captured once to estimate their geographic
origin from a previous season. Stable-hydrogen isotopes
(dD), in particular, are a useful intrinsic marker in regions
where large-scale meteorological patterns create spatial
gradients in isotopic signatures (Bowen et al. 2005).
Hydrogen in precipitation is incorporated into plant tissues
and then transferred to higher trophic levels (Lajtha and

123

Oecologia

Michener 1994). Values of dD in animal tissues (dDt) can,
therefore, reflect the isotopic composition of the local food
web. The expected dDt value of a locally grown tissue is
commonly derived using the average dD value of growing
season precipitation (dDp), while taking into account the
tissue-specific discrimination factor that describes the offset between dDp and dDt.
For birds, stable-hydrogen isotope values in feathers
(dDf) can serve as a geographic marker for the location of
molt. Feathers are useful tissues for tracking individuals
because they retain a constant isotopic signature after they
are grown, unlike metabolically active tissues such as blood
and claws (Mazerolle and Hobson 2005). The application of
dDf for inferring geographic origins relies upon a priori
knowledge of variation in dDp within a species’ potential
molting range. Isotopic base maps of growing season precipitation have been created for North America (Meehan
et al. 2004; Bowen et al. 2005) and can be adjusted to
correct for isotopic discrimination from precipitation to
feathers. Birds of unknown origin can therefore be assigned
to a region of molt (often a latitudinal band) by interpolation
of the established isotopic base maps.
Several assumptions underlie the ability to use dDf as an
effective geographic marker (Hobson 2005a; Norris et al.
2006a). First, the discrimination factor linking dDp and dDf
is assumed to be constant among species (e.g., Hobson and
Wassenaar 1997; Kelly 2006) and across populations (e.g.,
Rubenstein et al. 2002; Norris et al. 2006b). Second, birds
molting at the same location are assumed to incorporate
similar isotopic signatures into their feathers, regardless of
age, sex or the type of feather being produced (e.g., Hobson
et al. 2004; Norris et al. 2004). Lastly, inter-annual and
seasonal variation in dDf is assumed to be negligible within
any given molt location (e.g., Rubenstein et al. 2002; Boulet et al. 2006). Recent studies on passerines (Powell and
Hobson 2006), shorebirds (Wunder et al. 2005; Rocque
et al. 2006) and raptors (Meehan et al. 2003; Smith and
Dufty 2005) have called these assumptions into question.
For example, Powell and Hobson (2006) reported that
feathers from wood thrush (Hylocichla mustelina) breeding
in the southern United States were more enriched in deuterium (i.e., had more positive dDf values) than expected
based on dDp. Another study sampled locally grown contour feathers from adults of two Pluvialis shorebird species
breeding within a narrow altitudinal range in Alaska
(Rocque et al. 2006); variation in dDf within one species
(113& in P. dominica) was substantially larger than the
other (20& in P. fulva). Finally, Meehan et al. (2003)
found that primary feathers grown by adult Cooper’s hawks
(Accipiter cooperii) were enriched in deuterium relative to
nestling feathers sampled from the same breeding territory.
Here, we examine variation in dDf as a function of age,
sex, feather type and the timing of molt using American

123

redstarts (Setophaga ruticilla) of known origin. Redstarts
are abundant, widely distributed songbirds that breed
throughout North America and over-winter in the Caribbean, Central America and northern South America (Sherry
and Holmes 1997). Like most Nearctic–Neotropical
migratory songbirds, they are thought to undergo a postbreeding molt in the vicinity of their breeding or natal
territory (Sherry and Holmes 1997; but see Norris et al.
2004). Using feathers of known origin from individually
marked breeding adults and young, our goals were to: (1)
quantify variation in dDf within a breeding population of
redstarts, (2) compare dDf values with the expected local
value based on dDp, (3) examine variation in dDf as a
function of age, sex and feather type, and (4) test for interannual and seasonal variation in isotopic values.

Materials and methods
Field methods
Fieldwork was conducted during the 2003 to 2006 breeding
seasons (May–August) at the Queen’s University Biological Station, Ontario, Canada. Our 60-ha study area is a
maple-dominated, mixed-deciduous forest located alongside a 787-ha lake, with marshes, small lakes and streams
interspersed throughout (4433¢N, 7622¢W, elevation
120–140 m). Previous research on the same population
suggests that some adults molt their tail feathers south of
the breeding grounds (Norris et al. 2004). In light of this,
we sampled the first primary (P1), which we hypothesized
to be grown on or near the breeding site because adults
drop and commence regrowth of their P1 feather first in the
molt sequence (Pyle 1997) and have been observed doing
so while feeding dependent fledglings (Sherry and Holmes
1997; K. M. L., M. W. R., D. R. N., personal observation).
We therefore sampled the tip of P1 in year x + 1 from
adults (n = 30 males, 12 females) known to have bred at
our study site in year x (the same approach as in Norris
et al. 2004). Some adults were sampled in multiple years
(n = 8 in 2 years, n = 1 in 3 years). We also collected recently molted contour feathers (as indicated by the presence of a sheath) from adults during the post-breeding molt
period (n = 13). Contour feathers were sampled from
juveniles (n = 34, each individual from a different nest)
when they were either nestlings (age 5–6 days) or fledglings (age 9–10 days). Five juveniles were sampled during
the nestling and fledgling stages. We also sampled the tip
of P1 and contour feathers in year x + 1 from second-year
birds (in their first breeding season; n = 4, all males)
known to have hatched at our study site in year x. Juveniles
begin growing their primary feathers as nestlings and do
not replace them until the end of the next breeding season;

Oecologia

they do, however, drop and re-grow contour feathers after
they leave the nest (Pyle 1997). The contour feathers
sampled from second-year birds (in year x + 1) were the
re-grown feathers, so represent a distinct group from the
contour feathers sampled from juveniles (in year x).
To test for age-related variation in dD within a metabolically active tissue, we sampled blood from parents
(n = 7 females and 3 males, each from a different territory)
and one of their offspring on the same day in 2004. Most
offspring were sampled on the day of fledging (one was
still a nestling). Blood samples (10–50 ll from the brachial
vein) were collected in heparinized capillary tubes and
stored in a cooler until they could be centrifuged (within
8 h) for 8 min at 14,000 r.p.m. (protocol approved by the
Queen’s University Animal Care Committee). The plasma
component was removed using a Hamilton syringe before
transferring the hematocrit to separate microcentrifuge
tubes, which were then stored at –20C until isotopic
analysis.
Because the hydrogen in feathers and blood originates
from both dietary and water sources (Hobson et al. 1999),
we sampled redstart prey (insects), rainwater and standing
water in 2005. Insects were sampled using Malaise traps
(Diptera) and taken directly from vegetation (Lepidopteran
larvae) in mid June, then frozen at –20C until isotopic
analysis. Precipitation and standing water were sampled
from 14 June to 18 August. For precipitation, open jars
(n = 9) were located throughout the study site and a thin
layer of mineral oil was placed in them to prevent subsequent evaporation. The water was collected using a
syringe on three separate occasions, each sample representing precipitation over the previous 3 weeks. Standing
water was sampled 4 times at seven sites along marshes,
lakes and streams. Mean dD values at each site were used
for analysis.
Isotopic analysis
Stable-isotope analyses were conducted at the Queen’s
Facility for Isotope Research in Kingston, Ontario. Feathers were washed of surface oils and debris using a 2:1
chloroform:methanol solution and air dried under a fume
hood for 72 h, allowing them to equilibrate with the local
atmosphere. Blood samples and insect specimens were
freeze-dried and powdered prior to analysis (except for
Diptera, which were freeze-dried and analyzed whole).
Organic samples were loaded (0.10–0.15 mg) into silver
capsules and placed in an oven at 100C for either 24 h
(feathers) or 1 h (blood and insects) to remove any surface
water. The capsules were then crushed, loaded into a
reduction furnace (Finnigan TC/EA) at 1,450C and
introduced online into an isotope ratio mass spectrometer
(Finnigan MAT Delta Plus XL). For water samples, 1 ll

was injected into an H/Device coupled to an isotope ratio
mass spectrometer (Finnigan MAT 252). One in-house
standard was run for every five unknowns. Stable-hydrogen
isotope values are reported in per mil notation (&) relative
to Vienna standard mean ocean water according to the
formula:
dD =

 2

H/1 Hsample /2 H/1 Hstandard





1  1,000

Measurements on the same feather were repeatable to
within 2 ± 2& (mean ± SD, n = 12). Similarly, SDs for
repeated measurements of mineral standards were between
2 and 3& (kaolinite n = 16; brucite n = 13).
The hydrogen isotope values reported here include a
combination of exchangeable and non-exchangeable
hydrogen. To control for potential seasonal variation in dD
in the ambient water vapor of the laboratory—and hence in
the exchangeable hydrogen portion of the feathers—we
analyzed the majority of samples (n = 95) within a span of
2 months in 2006. Excluding samples analyzed outside of
this period (n = 17) did not change the results of most
analyses, with the exception of sex-related variation in dDf.
There was no isotopic difference between the sexes when
all of the feathers were included in the analysis (see results); however, with the reduced sample size primary
feathers from males had more positive dDf values than
those from females (t-test, t = 3.6, P = 0.001, df = 27).
Further sampling is needed to determine if there is sexrelated variation in dDf in American redstarts.
Data analysis
The interpolated growing season mean dDp value at our study
site is –58&, based on isotopic data from the International
Atomic Energy Agency (IAEA) and the interpolation method
described in Bowen et al. (2005). Confidence intervals are
not available for interpolations of growing season precipitation (Bowen et al. 2005) but are for annual precipitation
(Bowen and Revenaugh 2003). At our study site the 95%
confidence interval for the interpolated annual mean dDp
value is relatively low (4&) due to the close proximity of the
nearest IAEA sampling site (within 100 km). Two different
discrimination factors are frequently used to estimate the
origin of passerine birds in North America, one based on
measures of non-exchangeable hydrogen in feathers from
central portions of the continent (–25&; Wassenaar and
Hobson 2001), and the other based on measures of
exchangeable and non-exchangeable hydrogen in feathers
from across the continent (–19&; Bowen et al. 2005).
According to these estimates, the predicted dDf value for our
study population is between –83 and –77&.
When individuals were sampled on multiple occasions,
we only included the first feather in our analyses. Contour

123

Oecologia

feathers sampled from nestlings [–92 ± 8& (mean ± SD),
n = 20] and fledglings (–90 ± 5&, n = 14) did not differ
significantly in isotopic composition ( test, t = 0.74, P = 0.47,
df = 32), so these groups were pooled for all analyses and
are hereafter referred to as ‘‘juvenile contour feathers’’.
Multiple comparison procedures were used to test for
variation in dDf as a function of sex (t-test), feather type
(one-way ANOVA) and growth year (two-way ANOVA,
with growth year and feather type as model factors). Raw
data were converted to ranks before performing both
ANOVA procedures because of unequal variance among
groups. For the two-way ANOVA, there was no interaction
between growth year and feather type (P = 0.08). We
tested for seasonal variation in dDf using hatch date as a
proxy for the start of feather growth in juveniles and offspring fledge date as a proxy for the start of molt in adults.
For the latter analysis, we only included adults that raised a
brood to fledging during the year of feather growth
(n = 15). Data were analyzed using JMP (SAS Institute
2006).

Results
Based on the dDp value for local growing season precipitation, the predicted isotopic composition of feathers grown
at our study site was between –83 and –77&. On average,
dDf values in American redstart feathers were slightly more
negative than predicted [–85 ± 8& (mean ± SD), n = 97]
and ranged from –115 to –70&. Age accounted for much
of this variation. Hydrogen isotope values in adult primary
feathers corresponded more closely with the predicted
values and were not as variable (–82 ± 4&, n = 42; ranged
from –92 to –70&). Half of those values were within ±2&
of the mean and 80% were within ±6&. Based on the
isotopic base map proposed by Meehan et al. (2004) for
growing season precipitation, the total variation in dDf we
measured among adult primary feathers corresponds to an
estimated latitudinal range of 6–8 (650–900 km) around
our study site (see Fig. 1).
Primary feathers from males (–81 ± 4&, n = 30) and
females (–84 ± 5&, n = 12) had similar dDf values (t-test,
t = 1.8, P = 0.08, df = 40), so adults were pooled for all
subsequent analyses. Juvenile contour feathers were more
depleted in deuterium than both adult feather types
[ANOVA on ranked data, F2,88 = 51.6, P < 0.0001, Tukey’s honestly significant difference (HSD) test; Fig. 2a].
Within adults, contour feathers had more positive dDf
values than primary feathers (Tukey’s HSD test). Only a
small fraction of birds banded as nestlings returned the
following year (n = 4), so they could not be included in the
above analysis. Nevertheless, values of dDf in feathers
from second-year birds (nestlings returning in year x + 1)

123

appear more similar to those of adult feathers than to those
of juvenile contour feathers (Fig. 2a).
Temporal variation in dDf was examined using adult
primary feathers and juvenile contour feathers, the groups
with the most samples. Values of dDf did not vary among
years when controlling for feather type (two-way ANOVA
on ranked data, F2,75 = 1.8, P = 0.18; Fig. 2b). However,
when juveniles were analyzed separately feathers grown in
2005 had more negative dDf values than those grown in
2004 (ANOVA, F2,33 = 6.9, P = 0.003, Tukey’s HSD test).
We did not detect seasonal variation in the isotopic composition of juvenile contour feathers (ordinary least squares
r2 = 0.01, P = 0.54, n = 34) or adult primary feathers
(r2 = 0.04, P = 0.49, n = 15; Fig. 2c).
For birds that were sampled on multiple occasions,
within-individual variation in dDf was highly variable (see
Fig. 3). Hydrogen isotope values were more variable
within juveniles sampled twice in 4–5 days than within
adults sampled in consecutive years. The SD for any given
individual was also largest when its mean dDf value was
furthest removed from the population mean (controlling for
age). Thus, individuals that formed a feather with a dDf
value more positive (or negative) than expected did not
consistently do so.

Fig. 1 Inset Histogram of stable-hydrogen isotope values in primary
feathers (dDf) (n = 42) sampled in year x + 1 from adult American
redstarts (Setophaga ruticilla) known to have bred at the study site
(Queen’s University Biological Station, Ontario, Canada; dark filled
circle) in year x. Feathers were grown during the post-breeding molt
period in 2003, 2004 and 2005. Contour lines represent mean dDp
values for growing season precipitation across eastern North America
(approximated from Meehan et al. 2004). Feathers grown in these
locations are assumed to reflect the local dDp value with an offset in
the order of –19 (Bowen et al. 2005) to –25& (Wassenaar and
Hobson 2001 )

Oecologia

Age-related variation in dD was also detected in blood, a
metabolically active tissue. Controlling for location within
the study site (adults and offspring were from the same
territory) and time of season, hematocrit sampled from
parental redstarts was more enriched in deuterium than
hematocrit sampled from their offspring on the same day
(paired t-test, t = 2.8, P = 0.02, df = 9; Table 1).
Potential sources of hydrogen for redstarts in this population—insects and drinking water—differed markedly in
isotopic composition. Hydrogen isotope values in insects
ranged from –170 to –97&, with Diptera samples having
more negative dD values than Lepidopteran larvae
(Table 1). Relative to insects, all potential sources of
drinking water were enriched in deuterium (dD ranged
from –71 to –38&). Hydrogen isotope values were similar
in precipitation and standing water (Table 1).

Discussion
Stable-hydrogen isotopes are increasingly used to address
fundamental questions about the ecology and life history of
mobile organisms. Yet, few researchers have adequately
tested critical assumptions of this method by analyzing
tissues of known origin. Most such studies on passerine
birds (Hobson and Wassenaar 1997; Kelly et al. 2002;
Hobson et al. 2004) have used feathers collected from

Fig. 2 dDf in American redstart feathers grown in southeastern
Ontario as a function of a bird age (adult, juvenile, second-year) and
feather type (primary, contour), b growth year (2003, 2004, 2005) and
c relative timing of molt. Age designations are used to describe the
birds’ age at the time of feather sampling. Feathers collected in the
same year they were grown are labelled as (x) and those collected in
the following year are labelled as (x + 1). a and b Box plots show 10,
25, 50, 75 and 90th percentiles as horizontal lines and outlying values
as circles. Sample sizes are noted below each box. The data for
second-year birds are represented as individual circles, rather than as
boxes, because of low sample size (n = 4). c The date an adult’s
offspring left the nest in year x was used as a proxy for the timing of
molt (n = 15), while for juveniles hatch date was used as a proxy
(n = 34)

Fig. 3 Within-individual variation in dDf for American redstarts
sampled on multiple occasions. Adults (n = 9; dark filled circle) were
sampled for primary feathers in 2 consecutive years (one in all
3 years), while juveniles (n = 5; light filled circle) were sampled for
contour feathers twice within the first 10 days of hatching in 2004
(mean ± SD for each individual). Horizontal lines denote the mean
dDf value for adult primary feathers in all years and for juvenile
contour feathers in 2004. The analytical error includes the mean
difference between repeated dDf measurements of the same feather
(solid line) and the SD of those measurements (dotted line; n = 12)

123

Oecologia
Table 1 Stable-hydrogen isotope values in American redstart (Setophaga ruticilla) feathers and blood hematocrit, as well as in potential sources of dietary hydrogen (insects) and drinking water
Mean ± SD (n)
Feathersa
Adult (primary)

–82 ± 4 (42)

Juvenile (contour)

–91 ± 7 (34)

Blood

b

Adult

–84 ± 9 (10)

Juvenile

–92 ± 9 (10)

Insectsc
Diptera

–143 ± 27 (5)

Lepidoptera
Drinking water

–116 ± 22 (5)
d

Precipitation

–54 ± 3 (9)

Standing water

–57 ± 13 (7)

a

Grown in 2003, 2004 and 2005

b

Sampled in June and July 2004

c

Sampled in June 2005

d

Sampled in June, July and August 2005

breeding areas and have assumed that those individuals
bred and molted in the same location the previous year
(i.e., no dispersal or molt migration; but see Powell and
Hobson 2006). Our study analyzed isotopic signatures of
individually marked birds over multiple years and could
therefore limit feather collection to birds known to have
hatched or bred at our study site during the year in which
those feathers were grown. Furthermore, by sampling the
first primary feather (sampling tail feathers is a more
common approach in passerines; Smith et al. 2003) we
increased the likelihood that adults completed feather
growth prior to their departure for fall migration. It does,
however, remain possible that some primary feathers were
replaced south of the study site, as we could not track the
post-breeding movements of adults. Had that been the case,
we would have expected the distribution to be skewed, with
the tail pointing towards more positive dDf values (more
enriched in deuterium than expected). Instead, outliers
were dispersed evenly around the mean (i.e., both positive
and negative; see Fig. 1).
Assigning breeding origin of migratory birds using
geographic patterns in dDp depends on an accurate estimate
for the discrimination factor linking dDp and dDf. Across
North America, a strong relationship exists between dDp
and dDf (Hobson and Wassenaar 1997; Lott and Smith
2006); however, the discrimination factor linking them has
been found to vary among taxonomic groups (songbirds
–25 to –19&, Wassenaar and Hobson 2001; Bowen et al.
2005; raptors –5.6&, Lott and Smith 2006) and, in the case
of raptors, at a regional level (Lott and Smith 2006). The
accurate assignment of migratory birds is further compli-

123

cated by potential inconsistency in the measurement of dDf
values. As mentioned previously, a small portion of the
hydrogen in feathers can exchange with the hydrogen in
ambient water vapor, the isotopic signature of which may
vary seasonally or among laboratories. Despite the potential influence of exchangeable hydrogen, the mean dDf
value we measured in adult primary feathers (–82&) corresponded closely with the predicted value (–83&) based
on the discrimination factor for non-exchangeable hydrogen (Wassenaar and Hobson 2001). The isotopic signature
of adult feathers was also remarkably consistent across all
3 years, with the mean varying by less than 2&.
Recent studies on shorebirds have reported substantial
within-population variation in dDf, questioning the precision of this intrinsic geographic marker. Wunder et al.
(2005) sampled contour feathers from mountain plover
(Charadrius montanus) chicks at multiple breeding sites
ranging from Colorado to Montana and found that, within a
given site, the isotopic signature of feathers grown in the
same year varied by upwards of 60&. The use of juvenile
contour feathers—which, in redstarts at least, are more
isotopically variable than adult feathers—may have contributed to the large within-site variation. However, Rocque et al. (2006) reported an even larger isotopic range for
adult American golden plovers (Pluvialis dominica)
breeding in Alaska, with dDf ranging from –175 to –62&.
For American redstarts in our study population, in contrast,
the range of dDf values among adult primary feathers in a
given year was as high as 20& in 2003 and as low as 12&
in 2004. Furthermore, half of the values were within ±2&
of the mean and 80% were within ±6&. These results
suggest that dDf may serve as a more precise estimate of
geographic origin in passerines than in shorebirds.
We also found that adult tissues were more enriched in
deuterium than juvenile tissues from 5- to 10-day-old birds.
Similar results have been previously reported for feathers
of Cooper’s hawks (Meehan et al. 2003) and northern
goshawks (Accipiter gentilis; Smith and Dufty 2005).
Adults of those species, however, begin molt during the
incubation phase, so the authors presumed their findings
would not apply for passerines that undergo a post-breeding molt. In redstarts, age-related variation in dD appears to
be limited to recently hatched juveniles, as feathers from
second-year birds (nestlings returning in year x + 1) had
isotopic signatures that were consistent with adult feathers
of the same type (Fig. 2a). Primary feathers begin growing
when juveniles are in the nest, but they do not become inert
until the feather barbs emerge and the follicles reabsorb the
blood vessels (Gill 1989). The isotopic signature of flight
feathers could therefore be altered later during the fledgling
phase. In the case of contour feathers, they are re-molted
after the juveniles fledge (Pyle 1997), so a distinct isotopic
signature may be incorporated into the re-grown feathers

Oecologia

compared to those grown in the nest. We suggest that
during the early stages of growth, juvenile tissues are depleted in deuterium relative to adults and even relative to
juveniles in the later stages of growth.
In raptors, evaporative water loss was proposed as a
possible explanation for elevated dDf values in adults relative to nestlings (Meehan et al. 2003; Smith and Dufty
2005). Birds release heat through the evaporation of water
along cutaneous and respiratory surfaces (Wolf and
Walsberg 1996). Water containing protium is preferentially
evaporated during this process, leaving the body water pool
enriched in the heavier hydrogen isotope (McKechnie et al.
2004). Raptors combine molt with reproductive activities;
thus it was suggested that they may rely on evaporation
during the period of feather growth more so than in
passerines. We detected an isotopic difference between
adults and nestlings that was in the same direction, but of a
lower magnitude, than that reported in raptors (difference
of 30–60&, on average). It is possible that adults have
higher rates of evaporative water loss than nestlings,
regardless of the timing of molt. However, there is some
evidence that the reverse may actually be the case in
temperate-breeding species (Kirkley and Gessaman 1990;
Marder et al. 2003).
Instead of a physiological mechanism, we suggest that
variation in diet is a more parsimonious explanation for the
age-related variation in dD. Hydrogen incorporated into
feathers and blood can be derived from food items and
drinking water (Hobson et al. 1999), sources that may
differ in isotopic composition. Plants discriminate against
the heavier hydrogen isotope during the synthesis of organic compounds, producing tissues with more negative
dD values than local waters (Smith and Epstein 1970;
Epstein et al. 1976). Isotopic fractionation of hydrogen is
thought to be minimal during the formation of animal tissues (Estep and Dabrowski 1980; Hobson and Wassenaar
1997). Even so, a recent study found that animals at higher
trophic levels tend to have more positive dD values
(Birchall et al. 2005), a pattern that may reflect the incorporation of hydrogen derived from drinking water. We
sampled a variety of local waters in 2005, all of which had
more positive dD values than those in insects (Diptera and
Lepidopteran larvae) sampled at the same time (Table 1).
Adults in our study population have access to both of these
sources of hydrogen whereas nestlings only have access to
food items (assuming precipitation plays a negligible role).
All else being equal, if adults incorporate more water-derived hydrogen into their tissues they would have more
positive dD values than nestlings, which is consistent with
our observations. Once the nestlings fledge their access to
drinking water may increase, thus altering the isotopic
composition of their tissues. Given the wide range in dD
values of insects, the exclusive use of food items by nes-

tlings may have also contributed to the substantial variance
in dDf among juvenile contour feathers.
Regardless of the underlying mechanism, deuterium
enrichment in adults relative to nestlings appears to be
present in multiple tissue types (feathers, blood) and across
diverse avian taxa (raptors, passerines). Based on these
findings, we caution against using feathers sampled during
the nestling phase or shortly thereafter to estimate expected
local isotopic signatures for comparison with adult feathers
of unknown origin (e.g., Hobson and Wassenaar 2001;
Norris et al. 2004). Feathers from fully grown hatch-year
or second-year birds, however, appear to reflect the same
local isotopic signature as adults. A similar discrimination
factor should therefore apply when estimating their geographic origin.
Implications for future research
Feathers from migratory birds are more and more frequently being analyzed for stable-hydrogen isotopes to link
the location of molt (as inferred using dDf) with the location of feather collection. One of the most useful applications of this technique is to document patterns of migratory
connectivity, which is the degree to which breeding, wintering and migratory stopover areas are geographically
linked (Webster et al. 2002; Marra et al. 2006). Hydrogen
isotope values in feathers have also been used to study
long-distance dispersal, an application that applies to both
resident and migratory species (Hobson 2005b). The ability
to infer the mean dDp value in the source region based on
the mean dDf value for a group of birds has been reported
to be as precise as ±3& (Meehan et al. 2001). Our findings
are consistent with that level of resolution. Furthermore, at
the individual level more than half of the adults sampled
for primaries had dDf values within ±3& of the mean. Yet,
a few individuals known to be local breeders had isotopic
signatures that were indicative of much more northerly or
southerly origins (see Fig. 1). Our results therefore suggest
that the origin of individual redstarts, and possibly other
songbirds, cannot be accurately estimated within a narrow
band of latitude using dDf values alone.
When used cautiously, we believe stable-hydrogen isotopes remain a valuable tool with which to study movement
ecology. Instead of treating dDf values as pinpoint estimates of molting latitude, we recommend that individuals
should be assigned to a broad geographic zone within a
species’ potential molting range (e.g., Norris et al. 2006b).
The recent development of likelihood-based assignment
tests has provided more rigorous statistical techniques for
assigning birds to geographic zones of origin (Royle and
Rubenstein 2004; Wunder et al. 2005; Marra et al. 2006).
Multivariate approaches, which combine dDf with the use
of additional markers such as trace elements (e.g., iron,

123

Oecologia

mercury), other stable isotopes (e.g., d13C, d15N) and genetic markers, can also be used to provide more precise
estimates of geographic origin, particularly when the
additional markers vary with longitude (Kelly et al. 2005;
Boulet et al. 2006).
Acknowledgements Funding was provided by the Natural Sciences
and Engineering Research Council of Canada (L. M. R., T. K. K.,
K. M. L., D. R. N.), the National Science Foundation (0089565;
P. P. M.), the Canadian Foundation for Innovation (T. K. K.,
L. M. R.), the Wilson Ornithological Society (D. R. N.), Sigma Xi
(M. W. R.), the American Ornithologists’ Union (M. W. R.,
D. R. N.), the Society of Canadian Ornithologists (M. W. R.,
D. R. N.), the Ontario Government (K. M. L.), Queen’s University
(M. W. R., D. R. N.) and the Smithsonian Institution (P. P. M.).
Thanks to C. Studds, S. McWilliams, J. Kelly and one anonymous
reviewer for helpful comments, to F. Phelan and F. Connor for
logistical support at the Queen’s University Biological Station, to K.
Klassen and A. Vuletich for assistance with isotope analysis, to K.
Shorter and R. Zavitz for collecting and analyzing water samples, to
C. Hasler for GIS help, and to our many excellent field crews for their
tireless efforts.

References
Birchall J, O’Connell TC, Heaton THE, Hedges REM (2005)
Hydrogen isotope ratios in animal body protein reflect trophic
level. J Anim Ecol 74:877–881
Boulet M, Gibbs HL, Hobson KA (2006) Integrated analysis of
genetic, stable isotope, and banding data reveal migratory
connectivity and flyways in the yellow warbler (Dendroica
petechia). In: Boulet M, Norris DR (eds) Migratory connectivity
of two species of Neotropical–Nearctic migratory songbirds. Orn
Monogr 61
Bowen GJ, Revenaugh J (2003) Interpolating the isotopic composition of modern meteoric precipitation. Water Resour Res
39:1299
Bowen GJ, Wassenaar LI, Hobson KA (2005) Global application of
stable hydrogen and oxygen isotopes to wildlife forensics.
Oecologia 143:337–348
Epstein S, Yapp CJ, Hall JH (1976) The determination of the D/H
ratio of non-exchangeable hydrogen in cellulose extracted from
aquatic and land plants. Earth Planet Sci Lett 30:241–251
Estep MF, Dabrowski H (1980) Tracing food webs with stable
hydrogen isotopes. Science 209:1537–1538
Gill FB (1989) Ornithology. Freeman, New York
Greenberg R, Marra PP (2005) Birds of two worlds: the ecology and
evolution of migration. Johns Hopkins University Press, Baltimore, Md.
Hobson KA (1999) Tracing origins and migration of wildlife using
stable isotopes: a review. Oecologia 120:314–326
Hobson KA (2005a) Stable isotopes and the determination of avian
migratory connectivity and seasonal interactions. Auk
122:1037–1048
Hobson KA (2005b) Using stable isotopes to trace long-distance
dispersal in birds and other taxa. Divers Distrib 11:157–164
Hobson KA, Wassenaar LI (1997) Linking breeding and wintering
grounds of neotropicalmigrant songbirds using stable hydrogen
isotopic analysis of feathers. Oecologia 109:142–148
Hobson KA, Wassenaar LI (2001) Isotopic delineation of North
American migratory wildlife populations: loggerhead shrikes.
Ecol Appl 11:1545–1553

123

Hobson KA, Atwell L, Wassenaar LI (1999) Influence of drinking
water and diet on the stable-hydrogen isotope ratios of animal
tissues. PNAS 96:8003–8006
Hobson KA, Wassenaar LI, Bayne E (2004) Using isotopic variance
to detect long-distance dispersal and philopatry in birds: an
example with ovenbirds and American redstarts. Condor
106:732–743
Kelly JF (2006) Stable isotope evidence links breeding geography and
migration timing in wood warblers (Parulidae). Auk 123:431–
437
Kelly JF, Atudorei V, Sharp ZD, Finch DM (2002) Insights into
Wilson’s Warbler migration from analyses of hydrogen stableisotope ratios. Oecologia 130:216–221
Kelly JF, Ruegg KC, Smith TB (2005) Combining isotopic and
genetic markers to identify breeding origins of migrant birds.
Ecol Appl 15:1487–1494
Kirkley JS, Gessaman JA (1990) Water economy of nestling
Swainson’s hawks. Condor 92:29–44
Lajtha K, Michener RH (1994) Stable isotopes in ecology and
environmental science. Blackwell, London
Lott CA, Smith JP (2006) A Geographic-Information-System
approach to estimating the origin of migratory raptors in North
America using stable hydrogen isotope ratios in feathers. Auk
123:822–835
Marder J, Withers PC, Philpot RG (2003) Patterns of cutaneous water
evaporation by Australian pigeons. Isr J Zool 49:111–129
Marra PP, Norris DR, Haig SM, Webster MS, Royle JA (2006)
Migratory connectivity. In: Crooks K, Sanjayan MA (eds)
Conservation connectivity. Cambridge University Press, New
York
Mazerolle DF, Hobson KA (2005) Estimating origins of shortdistance migrant songbirds in North America: contrasting
inferences from hydrogen isotope measurements of feathers,
claws, and blood. Condor 107:280–288
McKechnie AE, Wolf BO, Martinez del Rio C (2004) Deuterium
stable-isotope ratios as tracers of water use: an experimental test
with rock doves. Oecologia 140:191–200
Meehan TD, Lott CA, Sharp ZD, Smith RB, Rosenfield RN, Stewart
AC, Murphy RK (2001) Using hydrogen isotope geochemistry to
estimate the natal latitudes of immature Cooper’s hawks
migrating through the Florida Keys. Condor 103:11–20
Meehan TD, Rosenfield RN, Atudorei VN, Bielefeldt J, Rosenfield
LJ, Stewart AC, Stout WE, Bozek MA (2003) Variation in
hydrogen stable-isotope ratios between adult and nestling
Cooper’s hawks. Condor 105:567–572
Meehan TD, Giermakowski JT, Cryan PM (2004) GIS-based model
of stable hydrogen isotope ratios in North American growingseason precipitation for use in animal movement studies.
Isotopes Environ Heath Stud 40:291–300
Norris DR, Marra PP, Montgomerie R, Kyser TK, Ratcliffe LM
(2004) Reproductive effort, molting latitude, and feather color in
a migratory songbird. Science 306:2249–2250
Norris DR, Wunder MB, Boulet M (2006a) Perspectives in migratory
connectivity. In: Boulet M, Norris DR (eds) Migratory connectivity of two species of Neotropical–Nearctic migratory songbirds. Orn Monogr 61
Norris DR, Marra PP, Bowen GJ, Ratcliffe LM, Royle JA, Kyser TK
(2006b) Migratory connectivity of a widely distributed songbird,
the American Redstart (Setophaga ruticilla). In Boulet M, Norris
DR (eds) Migratory connectivity of two species of Neotropical–
Nearctic migratory songbirds. Orn Monogr 61
Powell LA, Hobson KA (2006) Enriched feather hydrogen isotope
values for Wood Thrushes sampled in Georgia, USA, during the
breeding season: implications for quantifying dispersal. Can J
Zool 84:1331–1338

Oecologia
Pyle P (1997) Identification guide to North American birds. Part I.
Slate Creek Press, Bolinas
Rocque DA, Ben-David M, Barry RP, Winker K (2006) Assigning
birds to wintering and breeding grounds using stable isotopes:
lessons from two feather generations among three intercontinental migrants. J Ornithol 147:395–404
Royle JA, Rubenstein DR (2004) The role of species abundance in
determining breeding origins of migratory birds with stable
isotopes. Ecol Appl 14:1780–1788
Rubenstein DR, Hobson KA (2004) From birds to butterflies: animal
movement patterns and stable isotopes. Trends Ecol Evol
19:256–263
Rubenstein DR, Chamberlain CP, Holmes RT, Ayres MP, Waldbauer
JR, Graves GR, Tuross NC (2002) Linking breeding and
wintering ranges of a migratory songbird using stable isotopes.
Science 295:1062–1065
SAS Institute (2006) JMP Statistical Discovery 6.0.2. Duxbury,
Pacific Grove, Calif.
Sherry TW, Holmes RT (1997) American redstart (Setophaga
ruticilla). In: Poole A, Gill FB (eds) The birds of North
America, no. 277. Academy of Natural Science, Philadelphia,
American Ornithological Union, Washington, D.C.

Smith AD, Dufty AM Jr (2005) Variation in the stable-hydrogen
isotope composition of Northern Goshawk feathers: relevance to
the study of migratory origins. Condor 107:547–558
Smith BN, Epstein S (1970) Biogeochemistry of the stable isotopes of
hydrogen and carbon in salt marsh biota. Plant Physiol 46:738–
742
Smith TB, Marra PP, Webster MS, Lovette I, Gibbs HL, Holmes RT,
Hobson KA, Rohwer S (2003) A call for feather sampling. Auk
120:218–221
Wassenaar LI, Hobson KA (2001) A stable-isotope approach to
delineate geographical catchment areas of avian migration
monitoring stations in North America. Environ Sci Technol
35:1845–1850
Webster MS, Marra PP, Haig SM, Bensch S, Holmes RT (2002)
Links between worlds: unraveling migratory connectivity.
Trends Ecol Evol 17:76–83
Wolf BO, Walsberg GE (1996) Respiratory and cutaneous evaporative water loss at high environmental temperatures in a small
bird. J Exp Biol 199:451–457
Wunder MB, Kester CL, Knopf FL, Rye RO (2005) A test of
geographic assignment using isotope tracers in feathers of known
origin. Oecologia 144:607–617

123