Variation in Stable Hydrogen Isotope Values in a Wetland-Associated Songbird

Abstract

Stable isotope analysis is often used to identify the geographic origins of migratory bird populations. While this method can accurately predict the provenance of migratory species, stable hydrogen isotope values measured in feathers (δ²Hf) can be variable within a site and may be influenced by differences among age class (second year vs. after second year), type of feather (primary vs. rectrix), year of sampling, species, and local hydrology. In this study, sources of variation in δ²Hf values were assessed in a wetland-associated Neotropical migratory bird, the Prothonotary Warbler (Protonotaria citrea), by comparing δ²Hf values among age classes, sexes, years and feather types in individuals breeding in eastern Virginia, USA. Age and year were found to influence δ²Hf values, with individuals in their second year having more depleted δ²Hf values (-62.43 ± 9.56‰) than individuals older than 2 years (-53.73 ± 9.04‰). Differences between primaries and rectrices were within the range of sampling error (-2.98‰), and there was no effect of sex. For wetland-associated songbirds, age-related differences in the dietary proportion of aquatic vs. terrestrial prey may provide an additional mechanism for differences observed in δ²Hf values between age classes. In studies that use stable isotopes to assign geographic feather origin, researchers should attempt to account for and propagate known variation in δ²Hf values in assignment models whenever possible.

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Variation in Stable Hydrogen Isotope Values in a Wetland-
Associated Songbird
Author(s): Jessica A. Reese, Christopher Tonra, Catherine Viverette, Peter P.
Marra and Lesley P. Bulluck
Source: Waterbirds, 41(3):247-256.
Published By: The Waterbird Society
https://doi.org/10.1675/063.041.0304
URL: http://www.bioone.org/doi/full/10.1675/063.041.0304
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247
Variation in Stable Hydrogen Isotope
Values in a Wetland-associated Songbird
Jessica a. Reese1,*, chRistopheR tonRa2, catheRine ViVeRette3, peteR p. MaRRa4
and LesLey p. BuLLuck1,3
1Department of Biology, Virginia Commonwealth University, 1000 West Cary Street, Richmond, Virginia, 23284, USA
2School of Environmental and Natural Resources, The Ohio State University, 2021 Coffey Road,
Columbus, Ohio, 43210, USA
3Center for Environmental Studies, Virginia Commonwealth University, 1000 West Cary Street,
Richmond, Virginia, 23284, USA
4Migratory Bird Center, Smithsonian Conservation Biology Institute, P.O. Box 37012 MRC 5503,
Washington, D.C., 20013, USA
*Corresponding author; Email: jessica.a.reese@gmail.com
Abstract.—Stable isotope analysis is often used to identify the geographic origins of migratory bird populations.
While this method can accurately predict the provenance of migratory species, stable hydrogen isotope values mea-
sured in feathers (δ2Hf) can be variable within a site and may be influenced by differences among age class (second
year vs. after second year), type of feather (primary vs. rectrix), year of sampling, species, and local hydrology. In
this study, sources of variation in δ2Hfvalues were assessed in a wetland-associated Neotropical migratory bird, the
Prothonotary Warbler (Protonotaria citrea), by comparing δ2Hf values among age classes, sexes, years and feather
types in individuals breeding in eastern Virginia, USA. Age and year were found to influence δ2Hf values, with indi-
viduals in their second year having more depleted δ2Hf values (-62.43 ± 9.56‰) than individuals older than 2 years
(-53.73 ± 9.04‰). Differences between primaries and rectrices were within the range of sampling error (-2.98‰),
and there was no effect of sex. For wetland-associated songbirds, age-related differences in the dietary proportion
of aquatic vs. terrestrial prey may provide an additional mechanism for differences observed in δ2Hf values between
age classes. In studies that use stable isotopes to assign geographic feather origin, researchers should attempt to ac-
count for and propagate known variation in δ2Hf values in assignment models whenever possible. Received 30 October
2017, accepted 30 December 2017.
Key words.—forested wetlands, hydrogen isotopes, migratory connectivity, migratory songbird, Prothonotary
Warbler, Protonotaria citrea, stable isotope analysis.
Waterbirds 41(3): 247-256, 2018
Migratory birds can be traced across their
annual cycle using intrinsic markers such as
stable isotopes, genetic markers, or trace
elements (Webster et al. 2002; Hobson and
Wassenaar 2008). Stable isotope ratios are
often used for provenance studies because
they exhibit predictable geographic varia-
tion, can be measured from a single capture,
and are relatively inexpensive to measure
(Hobson 1999). Feathers assimilate the iso-
tope ratio of the local environment and are
often retained for an entire migration cycle
(Hobson and Wassenaar 2008); additionally,
the location of feather growth is known for
many species of migratory birds (Pyle 1997;
but see Leu and Thompson 2002; Nordell et
al. 2016). Stable isotopes, especially stable
hydrogen, have been widely used to assign
migratory birds captured on the nonbreed-
ing grounds to their location of feather
growth on the breeding grounds (Chamber-
lain et al. 1997; Hobson and Wassenaar 1997;
Rushing et al. 2014; Gonzalez-Prieto et al.
2017) and to quantify natal dispersal move-
ments (Studds et al. 2012; Haché et al. 2014).
This allows the degree of migratory connec-
tivity, or the degree to which breeding popu-
lations mix on the wintering grounds, to be
measured (Webster et al. 2002), and allows
demographic units to be delineated (Haché
et al. 2014).
Stable hydrogen isotopes are useful for
migration and dispersal studies because pat-
terns of atmospheric circulation and tem-
perature create a latitudinal gradient of sta-
ble hydrogen values in precipitation (δ2Hp)
across North America (Fry 2006; Hobson
and Wassenaar 2008). Studies typically take
advantage of the strong correlation between
δ2Hp and stable hydrogen values measured

248 WateRBiRds
in bird feathers (δ2Hf) by correcting an in-
terpolated δ2Hp map (isoscape) using the
regression equation between the two values
and assigning individuals of unknown origin
to this surface in a spatially explicit manner
(Bowen et al. 2005; West et al. 2010). How-
ever, δ2Hf is often variable among (Powell
and Hobson 2006; Oppel et al. 2011) and
within (Tonra et al. 2015) individuals from
a single sampling location. Thus, large-scale
processes like continental precipitation pat-
terns do not fully describe the mechanisms
that result in a given individual’s δ2Hf value
in a given year.
For studies using precipitation-depen-
dent stable isotope patterns to measure mi-
gratory connectivity accurately, Wunder et al.
(2005) suggested that δ2Hf and δ2Hp should
have a linear relationship that does not
depend on species, age, location, or time,
and called for more direct tests of these as-
sumptions. Langin et al. (2007) further high-
lighted several assumptions of such studies,
including that all individuals at a given loca-
tion should have similar δ2Hf, regardless of
sex, and that a given location should have
a similar δ2Hf within and between breed-
ing seasons. However, previous studies have
found that δ2Hf can vary within a site (Table
1). Specifically, δ2Hf can vary among indi-
viduals of different ages (Meehan et al. 2003;
Langin et al. 2007; Gow et al. 2012; Haché et
al. 2012), and between years at the same site
(Haché et al. 2012; Van Dijk et al. 2014; Tonra
et al. 2015). An interaction between year and
other sources of variation has been found in
some studies, with species-specific (Nordell
et al. 2016) and age (Gow et al. 2012; Haché
et al. 2012) effects being different among
years. Most studies have not investigated dif-
ferences between sexes, but those that did
have been inconclusive (Langin et al. 2007)
or found no effect (Nordell et al. 2016). A
further assumption is that δ2Hf values should
not vary between feather types in the same
individual, but small differences have been
found between primary and rectrix feath-
ers in a songbird (Haché et al. 2012) and
large differences were seen between various
primary feathers in a raptor (Meehan et al.
2003). δ2Hf values can also vary seasonally
Table 1. Comparison of the effect of age, sex, year, and feather type on stable hydrogen isotope variation as reported in six studies. Age is denoted as nestling, HY (hatch year,
after fledging), SY (second year), or ASY (after second year). Feather type is denoted as P (primary) or R (rectrix). The direction of the effect is reported if known, otherwise it
is reported as present or absent. N/A denotes comparisons that were not performed in that study.
Species Sex Age Year Age*Year Feather Type Study
Prothonotary Warbler (Protonotaria citrea) no SY < ASY present absent P slightly < R This study
Ovenbird (Seiurus aurocapillus) N/A nestling < ASY present present P slightly < R Haché et al. 2012
Wood Thrush (Hylocichla mustelina) N/A present absent present absent Gow et al. 2012
American Redstart (Setophaga ruticilla) absent nestling < ASY absent N/A P < contour Langin et al. 2007
Cooper’s Hawk (Accipiter cooperii) N/A nestling < adult N/A N/A P ≠ P Meehan et al. 2003
Mallard (Anas platyrhynchos) N/A HY < adult absent N/A N/A Van Dijk et al. 2014

songBiRd isotope VaRiation 249
over the course of the breeding season (Bor-
tolotti et al. 2013).
Differences in habitat, migration dis-
tance, and foraging pattern have also been
considered as sources of variation in δ2Hf
values across species (Hobson et al. 2012;
Nordell et al. 2016). For example, Hobson et
al. (2012) found that migration distance and
foraging guild influence δ2Hf values and the
relationship between δ2Hf and δ2Hp. Many
studies now use guild-specific transfer func-
tions to relate δ2Hf and δ2Hp, which classify
species as short-distance, long-distance, or
non-migratory and whether they forage on
the ground or in the canopy (Hobson et al.
2014; Pillar et al. 2015; Gonzalez-Prieto et al.
2017). While Hobson et al. (2012) did not
find support for an effect of aquatic vs. up-
land habitat use on δ2Hf values, hydrologic
processes occurring within aquatic habitats
may distort the relationship between δ2Hp
and δ2Hf (Hobson et al. 2004; Clark et al.
2006; Coulton et al. 2009; but see Hebert
and Wassenaar 2005). Bowen et al. (2011)
found that surface water δ2H values may be
between 30‰ more depleted to 20‰ more
enriched relative to local precipitation, dif-
ferences that likely propagate through the
trophic web to influence δ2Hf values of in-
dividuals in these ecosystems. Some studies
have excluded aquatic-associated species
from analyses (Hobson et al. 2004) or justi-
fied the exclusion of outliers for this reason
(Hobson et al. 2012), but few studies have
explicitly investigated sources of variation in
δ2Hf values of wetland-associated birds (but
see Betini et al. 2009; Bortolotti et al. 2013).
There has recently been considerable in-
terest in using stable isotope methods to de-
termine migratory connectivity for the many
species of long-distance migratory birds that
rely on wetlands (Hobson et al. 2006; Pérez et
al. 2010; Bridge et al. 2015). Thus, there is a
need to explicitly assess both how much vari-
ation in δ2Hf values occurs in these wetland-
associated species relative to terrestrial spe-
cies (Table 1), and how much variation can
be identified and accounted for when using
stable isotope analysis to investigate disper-
sal or migratory movements. In this study,
we measured δ2Hf values in a population of
Prothonotary Warblers (Protonotaria citrea)
breeding in eastern Virginia, USA. Our goal
was to assess variation in δ2H values in Pro-
thonotary Warbler feathers as a function of
age, sex, year, and feather type.
Methods
Study Area
We collected feather samples from adult male (n =
33) and female (n = 63) Prothonotary Warblers during
the 2013-2016 field seasons at long-term study sites in
the Lower James River Important Bird Area (Blem and
Blem 1994; Bulluck et al. 2013; Table 2). The majority
of samples used in this study (n = 89) were from Deep
Bottom Park (37° 24ʹ N, 77° 18ʹ W), with additional
samples (n = 7) from Presquile National Wildlife Ref-
uge (37° 21ʹ N, 77° 15ʹ W), both in Henrico County,
Virginia, USA. At both sites, we obtained samples from
individuals attending nest boxes positioned over water
near the shore of tidal freshwater creeks and the main
stem of the James River.
Study Species
The Prothonotary Warbler is a Neotropical migra-
tory songbird and a habitat specialist of forested wet-
lands such as bald cypress-tupelo swamps, riparian cor-
ridors, and flooded bottomland hardwood forests (Petit
1999). Their breeding range encompasses the south-
eastern United States, extending through the Atlantic
Coastal Plain and the Mississippi Alluvial Valley north
to Ontario, Canada, and they winter in mangroves and
Table 2. Sample sizes of feathers in which δ2H was measured from Prothonotary Warblers from Henrico County,
Virginia, USA. Age is reported as second-year (SY) or after second-year (ASY).
Year
Age Sex Feather Type
TotalSY ASY Female Male Primary Rectrix
2013 8 14 16 6 0 22 22
2014 10 15 16 9 0 25 25
2015 6 12 13 5 18 0 18
2016 14 17 18 13 30 16 46
Total 38 58 63 33 48 63 111

250 WateRBiRds
other forested wetlands in Central and South America
(Petit 1999). As a riparian- and wetland-associated spe-
cies, they consume both terrestrial and aquatic prey
throughout the nesting season (Petit 1999; Dodson et
al. 2016).
Data Collection
We captured males by playing conspecific songs and
calls near a mist net with a decoy. We captured females
using a hand held net placed over the cavity entrance
hole during incubation. We determined age for each
bird as after second-year (ASY, n = 58) or second-year
(SY, n = 38) following molt criteria outlined in Pyle
(1997), and we determined sex using plumage charac-
teristics and presence of breeding condition (i.e., brood
patch or cloacal protuberance). Feathers sampled from
all individuals were grown the previous summer; SY in-
dividuals would have molted their flight feathers while
nestlings, and ASY individuals would have molted their
flight feathers following breeding. Seventy-one percent
of individuals had been banded at the study site pre-
viously. We collected one outermost rectrix (R6) in all
years, and in 2015 and 2016, we also collected one in-
nermost primary (P1). As a result, we used a mixture of
primary and rectrix feathers for comparisons between
age classes and sexes, and all samples used in that analy-
sis from 2013 and 2014 were rectrices, while those from
2015 and 2016 were primaries. We also compared δ2Hf
between primary and rectrices collected in 2016.
Stable Isotope Analysis
We conducted stable isotope analysis in August 2016.
We cleaned all feathers in a 2:1 chloroform:methanol
solution to remove oil and debris, then dried the feath-
ers in a fume hood for 48 hr. Feather samples were
equilibrated in the laboratory where analysis was con-
ducted for ~72 hr (Wassenaar and Hobson 2003), then
subsamples of feather vane from the distal end (0.3-0.4
mg) were analyzed via high-temperature combustion in
an elemental analyzer (Thermo TC/EA; Thermo Scien-
tific) and an isotope ratio mass spectrometer (Thermo
Scientific Delta V Advantage). We present stable isotope
values in units of per mil (‰) with non-exchangeable
hydrogen reported in relation to the Vienna Standard
Mean Ocean Water-Standard Light Antarctic Precipi-
tation standard scale (Coplen 2011). We corrected
measurements of stable hydrogen for exchangeable
atmospheric hydrogen via the comparative equilibrium
method (Wassenaar and Hobson 2003) using three in-
house keratin reference standards (USGS42 [Tibetan
Human Hair]: -78.5‰, CBS [Caribou Hoof Standard]:
-197‰, KHS [Kudu Horn Standard]: -54.1‰). Analyti-
cal error of laboratory measurements was ± 2‰.
Statistical Analysis
We used a paired t-test and linear regression to
compare within-individual differences in δ2Hf between
15 paired rectrix and primary feathers sampled in
2016 from adult (10 ASY and 5 SY) female Protho-
notary Warblers. We performed a two-way ANOVA to
look for differences in δ2Hf between age class, year,
and an interaction between age class and year. Pairwise
comparisons between these categories were made us-
ing Tukey’s honestly significant difference (HSD) test.
We used a generalized linear model with a Gaussian
distribution to examine differences in δ2Hf between
the sexes because a Levene’s test indicated unequal
variances between male and female individuals (F94, 1
= 4.68; P = 0.03). To examine the percentage of varia-
tion explained by each mechanism of interest, we per-
formed a multiple regression using age class, sex, year,
feather type, and an interaction between age and year
as predictors. We also performed each analysis using
only individuals that were known to have been present
on the study site previously. All individuals used in the
comparison between primaries and rectices had been
encountered at the site previously. We conducted all
statistical analyses in statistical program R (R Develop-
ment Core Team 2016) using an alpha value of 0.05
when applicable, and we report all results as mean ±
SD.
ResuLts
The mean δ2Hf value for the study area
across ages, sexes, years, and feather types
was -57.17 ± 10.12‰ (n = 96; Range = -85.27
to -38.09‰; 95% CI = -59.2; -55.14‰). The
predicted δ2Hf value for our study site based
on the growing-season δ2Hp isoscape of Bow-
en et al. (2005) calibrated using the Hob-
son et al. (2012) equation for long-distance
non-ground foraging migratory songbirds
was -60.13‰. Primary feathers (-57.89 ±
10.91‰) had more negative δ2Hf values than
rectrices (-54.914 ± 10.12‰) in paired sam-
ples (t14 = -2.66; P = 0.02; Fig. 1), but their val-
ues were also positively correlated (R2 = 0.84;
F = 69.7; P < 0.001; Fig. 1). Age (F1,88 = 23.6; P
< 0.001) and year (F3,88 = 6.39; P < 0.001) had
an effect on δ2Hf values, but an interaction
between age and year was not found (F3,88 =
0.66; P = 0.58; Fig. 2). SY individuals (-62.43
± 9.56‰, n = 38) had more negative δ2Hf val-
ues than ASY individuals (-53.73 ± 9.04‰,
n = 58, P < 0.001), and feathers sampled in
2015 (-64.34 ± 8.32‰; n = 18) had more
negative δ2Hf values than feathers sampled
in any other year (2013: -56.81 ± 12.01‰, n
= 22; P = 0.02; 2014: -54.55 ± 7.40‰, n = 25;
P < 0.001; 2016: -55.38 ± 10.11‰, n = 31; P =
0.001; Fig. 2). δ2Hf values were not different
in male and female individuals (t94 = 1.77;
P = 0.08). When we repeated these analyses
using only individuals known to have been

songBiRd isotope VaRiation 251
present on the study sites previously, the re-
sults did not change (age: P < 0.001; year: P
= 0.002 [2013], P = 0.001 [2014], P = 0.003
[2016]; sex: t66 = 0.89, P = 0.377). For all indi-
viduals, age explained 17.8% of the variation
in δ2Hf values, year explained 14.2% of the
variation, and all other variables (sex, feath-
er type, and an interaction between age and
year) each explained < 1.5% of the variation
(P < 0.001; R2 = 0.23). For individuals known
to have been present previously at the study
sites, age explained 15.3% of the variation,
year explained 21.8% of the variation, and
all other variables each explained < 2% of
the variation (P < 0.001; R2 = 0.30).
discussion
In this study, we sought to explore differ-
ent sources of variation in δ2Hf values in a
wetland-associated songbird, the Prothono-
tary Warbler, in southeastern Virginia, USA.
We found strong support for age and year ef-
fects on δ2Hf values, limited support for a dif-
ference between feather types, and no differ-
ence between the sexes. We found that the
mean δ2Hf value for our study site was similar
to the value predicted for this location based
on growing-season δ2Hp values. The range
of δ2Hf values that we observed was similar
to that of other studies that have success-
fully measured the degree of migratory con-
nectivity using stable isotopes (Paxton et al.
2007; Tonra et al. 2015). Of the individuals
used in this study, 58% were known to have
been present on the study site the previous
year (i.e., the year the feather was grown)
and 71% were known to have been present
at the study site in at least one of the pre-
vious sampling years. However, the results
did not change when we analyzed these in-
dividuals separately, which suggests that the
observed variation is not a result of disper-
sal from other breeding areas into our study
sites. Our results corroborate those found
in non-wetland-associated bird species, sug-
gesting that differences in δ2Hf values among
age classes and years are important sources
of variation regardless of the hydrological
regime of a given site.
Primary feathers were found to have de-
pleted δ2Hf values relative to rectrices for
paired samples; however, the average differ-
ence between the two feather types (-2.98‰)
was similar to the analytical error for the lab-
oratory analysis (± 2‰). Haché et al. (2012)
also found that primaries were on average
slightly depleted relative to rectrices (-1.9‰)
in nestling Ovenbirds (Seiurus aurocapilla).
Figure 1. (A) δ2Hf values (‰) for primary and rectrix feathers from paired samples of 15 female Prothonotary
Warblers from Henrico County, Virginia, USA. (B) Correlation of δ2Hf values for primary and rectrix feathers. The
line of best fit (black) and a 1:1 line (gray) are shown.

252 WateRBiRds
In passerine birds, including Prothonotary
Warblers, the innermost primary is the first
primary to be molted, while the outermost
rectrix is the final rectrix to be molted (Pyle
1997). A less negative δ2Hf value in rectrices
could indicate that these feathers are molted
in an isotopically enriched region compared
to the breeding territory (e.g., areas further
south, downslope, or downriver), that a diet
shift occurs during the molting period, or
that the δ2H value of prey items changes
during this time. Because the difference in
δ2Hf values between the feather types was
neither large nor systematic, it does not pro-
vide evidence that Prothonotary Warblers
in our study area regularly engage in molt
migration, though more study is needed to
determine if and how movement during the
molt period may influence δ2Hf values for
some individuals. While we suggest that fu-
ture studies continue to test for differences
between feather types in other species when
planning to combine multiple feather types
for an analysis, we conclude that primary
and rectrix feathers in Prothonotary War-
blers do not differ enough to preclude their
simultaneous use in studies assessing migra-
tory connectivity.
Similar to several other studies, we found
that SY individuals had more depleted δ2Hf
Figure 2. δ2Hf values (‰) across year and age classes for 96 feather samples from Prothonotary Warblers collected
in Henrico County, Virginia, USA. Individuals in their first year of breeding are shown with open circles and individ-
uals in their second year of breeding or older are shown in closed circles. Error bars show 95% confidence intervals.

songBiRd isotope VaRiation 253
values compared to ASY individuals (Mee-
han et al. 2003; Langin et al. 2007; Gow et al.
2012; Haché et al. 2012). Explanations for
this variation include differences in physiolo-
gy and diet between the age classes (Hobson
et al. 1999; Meehan et al. 2003). Differences
in δ2H values of prey items and drinking wa-
ter and/or differences in the proportion of
those items consumed among age classes
may lead to systematic variation in δ2Hf val-
ues between younger and older individuals
(Langin et al. 2007; Betini et al. 2009). These
different sources of consumed hydrogen
may also lead to individual variation within
age classes because diet/resource availabil-
ity may vary among individuals. For wetland-
associated birds, the quantity of aquatic vs.
terrestrial prey consumed may be especially
important because these prey sources can
have different δ2H values (Vander Zanden et
al. 2016). Betini et al. (2009) suggested that
the contribution of aquatic insects in the
diet of box-nesting Tree Swallow (Tachycineta
bicolor) nestlings from a riparian site could
have led to depleted δ2H values measured
in nestling blood samples. In contrast, Bor-
tolotti et al. (2013) found that aquatic in-
sects had slightly more enriched δ2H values
compared to terrestrial insects. To date, no
studies have examined δ2H values in Protho-
notary Warbler food webs, but an analysis
of carbon and nitrogen stable isotope ratios
in Prothonotary Warbler nestling and adult
breast muscle indicated that adults likely
consume more terrestrial prey (L. Bulluck,
unpubl. data). The majority (92%) of our
feather samples came from Deep Bottom
Park, where aquatic food makes up at least
68% of nestlings’ diet (Dodson et al. 2016).
While it is possible that the larger propor-
tion of aquatic food in nestlings’ diet may be
responsible for the depleted δ2Hf values seen
in SY individuals, more study is needed to
understand the contributions of aquatic and
terrestrial prey to adult and nestling Protho-
notary Warbler diets as well as to explicitly
test how those prey items differ in isotopic
composition.
We examined δ2Hf values from 4 years of
data, and found that one year (2015) had a
more negative mean δ2Hf value compared
to all other years. While the samples from
2015 were primaries, which were slightly
depleted compared to rectrices, there is no
evidence that differences in δ2Hf values be-
tween feather types is the source of between-
year variation in this study, because the mag-
nitude of the difference between years was
greater than the difference between feather
types. The sampling year with the most dif-
ferent δ2Hf values (2015) was the driest year
of the four included in this study; eastern
Virginia received 400 mm of precipitation
from March through July compared with
an average of 520 mm. However, 2012 was
similarly dry (425 mm), and δ2Hf did not
show a corresponding change. Other stud-
ies have also found yearly differences both
in δ2Hf (Haché et al. 2012; Van Dijk et al.
2014) and δ2Hp (Van Wilgenburg et al. 2012).
Yearly variation in δ2Hf values may be related
to large-scale variation in climate processes
such as the North Atlantic Oscillation or the
El Nino-Southern Oscillation, which cause
deviations from long-term averages in δ2Hp
(Hobson et al. 2012). Van Wilgenburg et al.
(2012) found that isoscapes based on the
long-term average of δ2Hp values were ac-
curate for predicting the mean δ2Hp across
all years at their study sites, but that yearly
variation in δ2Hp ranged as much as 40‰.
Locally, rates of evapotranspiration may vary
on a yearly or seasonal basis, which can lead
to surface waters enriched in the heavy iso-
tope of hydrogen relative to local δ2Hp values
(Craig and Gordon 1965; Gat 2010; Bowen
et al. 2011).
More study is needed to understand the
mechanisms leading to variation in δ2Hf val-
ues beyond those addressed here. For exam-
ple, we considered testing for within-season
variation in δ2Hf values, which has been dem-
onstrated by other studies (Bortolotti et al.
2013), but we were unable to test for this re-
lationship because our sample sizes were too
small among years. In particular, how local
precipitation and surface water δ2H values
interact with individual-specific differences
in diet and physiology and ultimately δ2Hf
values is poorly understood at present (Ton-
ra et al. 2015). δ2Hf values measured at our
study site, located along a tidal freshwater

254 WateRBiRds
river, were similar to predicted values, which
suggests that hydrological processes did not
have an acute influence on δ2Hf values. How-
ever, processes such as evapotranspiration
may exert a larger influence in habitats with
open or standing water, such as reservoirs
and swamps. Additionally, rivers fed by snow-
melt may be depleted in the heavy isotope
of hydrogen relative to local δ2Hp values (Fry
2006; Bowen et al. 2011), and large north-
south flowing rivers may transport depleted
δ2H values downstream. Future work should
characterize the influence of both aquatic
prey items and multiple hydrologic regimes
on δ2Hf values (Bowen et al. 2011; Vander
Zanden et al. 2016), which may help ex-
plain variation not accounted for by factors
measured in this study. Understanding the
sources of variation in δ2Hf values can help
researchers better account for this variation,
such as by limiting sampling collection to
a single age class and/or year. Researchers
should attempt to collect large enough sam-
ple sizes to adequately capture site-specific
variation when using known-origin samples,
which will allow this variation to be propa-
gated in assignment models.
acknoWLedgMents
We thank Christine France for assistance with iso-
tope analysis at the Smithsonian Institute Stable Isotope
Mass Spectrometry Laboratory in Suitland, Maryland,
USA. We thank Charles and Leanne Blem for initiat-
ing the long-term study of Prothonotary Warblers at
Virginia Commonwealth University (VCU) and the
many graduate and undergraduate students who have
collected feather samples over the years. We thank
Cyrus Brame and the staff of the Eastern Virginia Rivers
National Wildlife Refuge Complex for access to refuge
property as well as financial and facilities support over
30 years. Partial funding for this study was provided
by two grants from the U.S. Department of Defense
through a Cooperative Agreement (14-0077 and 15-
0059) with the Fort A.P. Hill Environmental Program
awarded to C. Viverette and L. Bulluck, a VCU Rice Riv-
ers Center Research Award to J. Reese, and the VCU
Center for Environmental Studies. J. Reese was support-
ed by a Career Development Grant from the American
Association of University Women during this study. All
applicable ethical guidelines for the use of birds in re-
search have been followed, including those presented
in the Ornithological Council’s “Guidelines to the Use
of Wild Birds in Research” (Fair et al. 2010). All feather
samples were obtained under appropriate Animal Care
and Use Committee (IACUC protocol # AM10230, Vir-
ginia Commonwealth University) and State and Federal
banding and scientific collection permits (U.S. Geologi-
cal Survey permit #23486 and Virginia Department of
Game and Inland Fisheries permit #053965). This is
VCU Rice Rivers Center Contribution Number 85.
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