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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
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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:
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.
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).
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 ±
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).
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
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.
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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... One outermost rectrix or one innermost primary was sampled from each individual. δ 2 H f values between these feather types are correlated (with a slope close to 1) within the same individual, and the offset between the 2 feather types is close to within measurement error (Reese et al. 2018). Both rectrices and primaries are retained for 1 yr after the definitive prebasic molt in this species (Pyle 1997), which likely occurs on or near the breeding grounds; Prothonotary Warblers exhibit higher than average predicted probability of molting at their location of breeding, and there is no evidence that they engage in molt-migration (Pyle et al. 2018). ...
... One explanation may be the influence of yearly variation in δ 2 H p ; however, when we modeled δ 2 H f values as a function of the interactive effect of δ 2 H p and sampling year, there was no support for an effect of year (all P values >0.55). Nonetheless, differences in δ 2 H p can occur between years at a given site ) and year of sampling has been shown to be an important predictor of variation in δ 2 H f (Haché et al. 2012, Van Dijk et al. 2014, Reese et al. 2018, so the influence of year should not be discounted, especially for studies covering a broad range of sampling years. Vander Zanden et al. (2014) and Tonra et al. (2015) attempted to account for yearly variation in δ 2 H f by developing year-specific isoscapes using the IsoMap tool, but found assignment accuracy and precision were not improved. ...
... Sampling sites under the influence of the Mississippi River may have a more negative δ 2 H f value due to the influx of isotopically depleted water associated with the north-south flow of this river (Bowen et al. 2011). A study in Prothonotary Warblers breeding at a tidal freshwater river in Virginia showed that age and year influence δ 2 H f values (Reese et al. 2018) in a similar manner as reported in terrestrial species (Meehan et al. 2003, Langin et al. 2007, Gow et al. 2012, Haché et al. 2012). To our knowledge, no studies have directly examined avian δ 2 H f values among different hydrologic regimes. ...
Full-text available
Estimates of migratory connectivity are needed for full annual cycle population models of migratory bird species experiencing rapid declines in abundance. One technique to determine migratory connectivity is through stable isotope analysis. This low-resolution method may be influenced by how data are calibrated between isotopes measured in precipitation and those measured in feathers, and can be informed by incorporating relative abundance into the assignment model. eBird abundance maps are a new tool combining citizen science data into a predictive species distribution model. In the Prothonotary Warbler (Protonotaria citrea), a wetland-associated songbird with a patchy breeding distribution, we sought to use stable-hydrogen isotope analysis informed by a species-specific calibration equation and eBird abundance data to determine the strength of migratory connectivity. We developed a species-specific calibration equation using known-origin samples from the breeding grounds and found that stable-hydrogen isotope values measured in precipitation explained 50% of the variation in stable-hydrogen isotope values among feathers. We found that the assignment model incorporating eBird abundance data correctly identified the true origins of 66% of individuals, and that the average assignment area (as a measure of precision) was 64% of the breeding distribution. These results represented a 7% increase in precision and a 3% decrease in accuracy when compared to a model that was not informed by abundance. Based on these models, wintering populations from 6 countries represented a mix of likely breeding origins, suggesting low migratory connectivity for Prothonotary Warblers. We found evidence that wintering latitude was related to likely breeding origin, with individuals at western wintering locations more likely to have southern breeding origins, but this relationship was weak. These results corroborate studies using archival light-level geolocators and high-resolution genetic markers, which also demonstrated weak migratory connectivity in this species. For patchily distributed species, eBird abundance data may not provide a useful increase in precision and accuracy for isotope assignments.
... However, the time-integrated nature of isotopic incorporation in animal tissues and food webs is influenced by various biotic factors, e.g., trophic position, metabolism, tissue type (Vander Zanden et al. 2015;van Wijke et al. 2021). Descriptions of the relationship among the three components (i.e., ambient water, prey bound to the aquatic environment, and consumers) are limited to a few studies with most considering the connectivity between ambient water and consumers (e.g., Reese et al. 2018;Sturgis et al. 2019;Genier et al. 2021). Vander Zanden et al. (2016 highlighted data gaps associated with the utility of δ 2 H tracers in freshwater food webs, which consider aquatic prey items and multiple hydrologic regimes. ...
... Our predictions relied on previous investigations of the dynamics of δ 2 H in surface waters of shallow prairie ponds and corresponding values in consumers (e.g., Coulton et al. 2009;Bortolotti et al. 2013;Reese et al. 2018;Sturgis et al. 2019). We predicted ambient water δ 2 H values in boreal lakes and prairie wetlands would gradually increase over the spring to summer sampling period each year due to net water volume loss from evaporative processes, especially in seasonal wetlands, and ambient water δ 2 H values would be significantly influenced by local-scale hydrologic variables. ...
We investigated variability in δ2H values for two aquatic food webs involving avian consumers (lesser scaup, Aythya affinis Eyton and tree swallow, Tachycineta bicolor Vieillot) foraging from boreal lakes in the Yukon Flats (Alaska, United States) and wetlands in the Prairie Pothole Region (Saskatchewan, Canada), respectively. Generalized linear mixed-effects models were used to decompose sources of variation in water and tissue δ2H. We found inter-annual variation in boreal lakes δ2HW influenced by surface water connectivity (e.g., open vs. closed basins) and distance to the nearest river. Prairie pothole wetland δ2HW displayed intra-annual variability dictated by wetland type (e.g., semipermanent and seasonal) with a range greater than 60‰, which incrementally increased over the summer months. Variance in invertebrate δ2HI was explained by taxonomic category, but factors such as the relative distance to the nearest river (boreal model) and wetland type (prairie model) should be explored in future studies. Lesser scaup duckling feather δ2HF displayed inter-annual and spatial variability with the top model explaining 86% of the overall variation, including the following fixed effects: basin type (open/closed), year, and calendar date of sampling. Similar spatial patterns with known lesser scaup prey items, such as relative site distance to the nearest river, most closely aligned with Amphipoda δ2HI. Variation in tree swallow nestling δ2HF was attributed to sampling date with the top model explaining 38% of overall variation, while sampled prey items (e.g., Chironomidae) did not closely follow this pattern. Our findings quantify the extreme temporal and spatial δ2H variability in food webs fundamentally linked to seasonal evaporative effects in shallow lentic aquatic ecosystems.
... A key premise underpinning the utilization of isotope-based methods to infer origin and migration is that tissue grown at the same site are isotopically similar. However, isotopic data from known-origin samples commonly show high levels of variation even among conspecific individuals living at a common site (e.g., Kelly et al., 2002;Meehan et al., 2003;Smith and Dufty, 2005;Wunder et al., 2005;Rocque et al., 2006;Langin et al., 2007;Gow et al., 2012;Haché et al., 2012;van Dijk et al., 2014;Reese et al., 2018). Such variability can often be quantified and incorporated in geographic assignment tests (Hobson et al., , 2014 but may limit the utility of such tests. ...
... Although we have a solid understanding of the processes controlling broad-scale isotope patterns (reviewed by Bowen, 2010), a more fundamental understanding of the underlying mechanisms of small-scale isotope variance is needed to improve estimates of such variance and assess the potential utility of isotope-based methods for specific groups or environments. Several studies have investigated the causes of within-site amongindividual isotopic variance through exploratory approaches such as correlation analysis and model selection (e.g., Clegg et al., 2003;Meehan et al., 2003;Smith and Dufty, 2005;Langin et al., 2007;Reese et al., 2018; see also Hobson et al., 2012;Nordell et al., 2016). Relationships between feather δ 2 H values and age have been observed for several taxa (e.g., Meehan et al., 2003;Smith and Dufty, 2005;Langin et al., 2007;Haché et al., 2012;Studds et al., 2012;Gomez et al., 2019) and attributed to age-related differences in diet, access to water, metabolism (Meehan et al., 2003;Langin et al., 2007;Storm-Suke et al., 2012), and/or evaporative water loss (Wolf and Martinez del Rio, 2000;Meehan et al., 2003, McKechnie et al., 2004Smith and Dufty, 2005; but see Kirkley and Gessaman, 1990;Marder et al., 2003). ...
Full-text available
Variations in stable hydrogen and oxygen isotope ratios in terrestrial animal tissues are used to reconstruct origin and movement. An underlying assumption of these applications is that tissues grown at the same site share a similar isotopic signal, representative of the location of their origin. However, large variations in tissue isotopic compositions often exist even among conspecific individuals within local populations, which complicates origin and migration inferences. Field-data and correlation analyses have provided hints about the underlying mechanisms of within-site among-individual isotopic variance, but a theory explaining the causes and magnitude of such variance has not been established. Here we develop a mechanistic modeling framework that provides explicit predictions of the magnitude, patterns, and drivers of isotopic variation among individuals living in a common but environmentally heterogeneous habitat. The model toolbox includes isoscape models of environmental isotopic variability, an agent-based model of behavior and movement, and a physiology-biochemistry model of isotopic incorporation into tissues. We compare model predictions against observed variation in hatch-year individuals of the songbird Spotted Towhee ( Pipilo maculatus ) in Red Butte Canyon, Utah, and evaluate the ability of the model to reproduce this variation under different sets of assumptions. Only models that account for environmental isotopic variability predict a similar magnitude of isotopic variation as observed. Within the modeling framework, behavioral rules and properties govern how animals nesting in different locations acquire resources from different habitats, and birds nesting in or near riparian habitat preferentially access isotopically lighter resources than those associated with the meadow and slope habitats, which results in more negative body water and tissue isotope values. Riparian nesters also have faster body water turnover and acquire more water from drinking (vs. from food), which exerts a secondary influence on their isotope ratios. Thus, the model predicts that local among-individual isotopic variance is linked first to isotopic heterogeneity in the local habitat, and second to how animals sample this habitat during foraging. Model predictions provide insight into the fundamental mechanisms of small-scale isotopic variance and can be used to predict the utility of isotope-based methods for specific groups or environments in ecological and forensic research.
... We washed all feathers in a 2:1 choloroform:methanol solution to remove excess dirt and oils before drying them at 60°C for 48 h (Reese et al., 2018;Reynolds et al., 2019;Tollington et al., 2019). Small fragments of feather vane were cut from the distal end and weighted into tin capsules (1-2 mg) for SIA (δ 13 C and δ 15 N). ...
Theory suggests that overcrowding and increased competition in urban environments might be detrimental to individual condition in avian populations. Unfavourable conditions could be compounded by changes in dietary niche with additional consequences for individual quality of urban birds. We analysed the isotopic signatures, signal coloration, body condition, parasitic loads (feather mites and coccidia), and immune responsiveness of 191 adult common (Indian) mynas (Acridotheres tristis) captured in 19 localities with differing levels of urbanization. The isotopic signature of myna feathers differed across low and high urbanized habitats, with a reduced isotopic niche breadth found in highly urbanized birds. This suggests that birds in high urban environments may occupy a smaller foraging niche to the one of less urbanized birds. In addition, higher degrees of urbanization were associated with a decrease in carotenoid-based coloration, higher ectoparasite loads and higher immune responsiveness. This pattern of results suggests that the health status of mynas from more urbanized environments was poorer than mynas from less modified habitats. Our findings are consistent with the theory that large proportions of individual birds that would otherwise die under natural conditions survive due to prevailing top-down and bottom-up ecological processes in cities. Detrimental urban ecological conditions and search for more favourable, less crowded habitats offers the first reasonable explanation for why an ecological invader like the common myna continues to spread within its global invasive range.
Full-text available
In birds, patterns of migratory connectivity are influenced by landscape structure experienced throughout the annual cycle. Assessing how landscape patterns drive the movements and distributions of migratory species is important for understanding the evolution of migration and determining conservation priorities. We determined migratory connectivity in the Canada Warbler (Cardellina canadensis), a declining Neotropical migratory bird, and evaluated the degree to which the Andean mountains influence winter population distribution in this montane forest specialist.
Full-text available
Understanding bird migration and dispersal is important to inform full life-cycle conservation planning. Stable hydrogen isotope ratios from feathers (δ²Hf) can be linked to amount-weighted long-term, growing season precipitation δ²H (δ²Hp) surfaces to create δ²Hf isoscapes for assignment to molt origin. However, transfer functions linking δ²Hp with δ²Hf are influenced by physiological and environmental processes. A better understanding of the causes and consequences of variation in δ²Hf values among individuals and species will improve the predictive ability of geographic assignment tests. We tested for effects of species, land cover, forage substrate, nest substrate, diet composition, body mass, sex, and phylogenetic relatedness on δ²Hf from individuals at least two years old of 21 songbird species captured during the same breeding season at a site in northeastern Alberta, Canada. For four species, we also tested for a year × species interaction effect on δ²Hf. A model including species as single predictor received the most support (AIC weight = 0.74) in explaining variation in δ²Hf. A species-specific variance parameter was part of all best-ranked models, suggesting variation in δ²Hf was not consistent among species. The second best-ranked model included a forage substrate × diet interaction term (AIC weight = 0.16). There was a significant year × species interaction effect on δ²Hf suggesting that interspecific differences in δ²Hf can differ among years. Our results suggest that within- and among-year interspecific variation in δ²Hf is the most important source of variance typically not being explicitly quantified in geographic assignment tests using non-specific transfer functions to convert δ²Hp into δ²Hf. However, this source of variation is consistent with the range of variation from the transfer functions most commonly being propagated in assignment tests of geographic origins for passerines breeding in North America.
Full-text available
Aquatic prey subsidies entering terrestrial habitats are well documented, but little is known about the degree to which these resources provide fitness benefits to riparian consumers. Riparian species take advantage of seasonal pulses of both terrestrial and aquatic prey, although aquatic resources are often overlooked in studies of how diet influences the reproductive ecology of these organisms. Ideally, the timing of resource pulses should occur at the time of highest reproductive demand. This study investigates the availability of aquatic (mayfly) and terrestrial (caterpillar) prey resources as well as the nestling diet of the prothonotary warbler (Protonotaria citrea) at two sites along the lower James River in Virginia during the 2014 breeding season. We found large differences in availability of prey items between the two sites, with one having significantly higher mayfly availability. Nestling diet was generally reflective of prey availability, and nestlings had faster mean growth rates at the site with higher aquatic prey availability. Terrestrial prey were fed more readily at the site with lower aquatic prey availability, and at this site, nestlings fed mayflies had higher mean growth rates than nestlings fed only terrestrial prey. Our results suggest that aquatic subsidies are an important resource for nestling birds and are crucial to understanding the breeding ecology of riparian species.
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The measurement of stable carbon (δ13C) and nitrogen (δ15N) isotopes in tissues of organisms has formed the foundation of isotopic food web reconstructions, as these values directly reflect assimilated diet. In contrast, stable hydrogen (δ2H) and oxygen (δ18O) isotope measurements have typically been reserved for studies of migratory origin and paleoclimate reconstruction based on systematic relationships between organismal tissue and local environmental water. Recently, innovative applications using δ2H and, to a lesser extent, δ18O values have demonstrated potential for these elements to provide novel insights in modern food web studies. We explore the advantages and challenges associated with three applications of δ2H and δ18O values in food web studies. First, large δ2H differences between aquatic and terrestrial ecosystem end members can permit the quantification of energy inputs and nutrient fluxes between these two sources, with potential applications for determining allochthonous vs. autochthonous nutrient sources in freshwater systems and relative aquatic habitat utilization by terrestrial organisms. Next, some studies have identified a relationship between δ2H values and trophic position, which suggests that this marker may serve as a trophic indicator, in addition to the more commonly used δ15N values. Finally, coupled measurements of δ2H and δ18O values are increasing as a result of reduced analytical challenges to measure both simultaneously and may provide additional ecological information over single element measurements. In some organisms, the isotopic ratios of these two elements are tightly coupled, whereas the isotopic disequilibrium in other organisms may offer insight into the diet and physiology of individuals. Although a coherent framework for interpreting δ2H and δ18O data in the context of food web studies is emerging, many fundamental uncertainties remain. We highlight directions for targeted research that will increase our understanding of how these markers move through food webs and reflect ecological processes.
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In contrast to the majority of migratory songbirds in North America, which moult on or near their breeding grounds, the Bullock’s oriole (Icterus bullockii) is reported to stop during fall migration to moult en route to the wintering grounds. These birds seem to take advantage of food resources during the Mexican monsoon season in the Southwestern USA and Northwestern Mexico. We studied a population of Bullock’s orioles at the northern limit of their breeding range in Kamloops, British Columbia, Canada, using a combination of light-level geolocators and stable hydrogen isotope analysis. We found evidence that supports the existence of moult migration in this species, with geolocators indicating that all birds appeared to stay in the Mexican monsoon region for moult in an extended stopover period during fall migration. Feathers were isotopically enriched with deuterium compared to predicted breeding isotope values and were significantly more negative than winter-grown claws, confirming that moult occurred somewhere between the breeding and wintering grounds. Stable isotope data were consistent with complete prebasic stopover moult in adults and complete contour feather and variable tail feather moult in first-year orioles. Our results confirm that this northern population of Bullock’s orioles employs a moult migration strategy and highlight the usefulness of combining geolocator and stable isotope studies.
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Population connectivity is an important consideration in studies of disease transmission and biological conservation, especially with regard to migratory species. Determining how and when different subpopulations intermingle during different phases of the annual cycle can help identify important geographical regions or features as targets for conservation efforts and can help inform our understanding of continental-scale disease transmission. In this study, stable isotopes of hydrogen and carbon in contour feathers were used to assess the degree of molt-site fidelity among Bar-headed Geese (Anser indicus) captured in north-central Mongolia. Samples were collected from actively molting Bar-headed Geese (n = 61), and some individual samples included both a newly grown feather (still in sheath) and an old, worn feather from the bird's previous molt (n = 21). Although there was no difference in mean hydrogen isotope ratios for the old and new feathers, the isotopic variance in old feathers was approximately three times higher than that of the new feathers, which suggests that these birds use different and geographically distant molting locations from year to year. To further test this conclusion, online data and modeling tools from the isoMAP website were used to generate probability landscapes for the origin of each feather. Likely molting locations were much more widespread for old feathers than for new feathers, which supports the prospect of low molt-site fidelity. This finding indicates that population connectivity would be greater than expected based on data from a single annual cycle, and that disease spread can be rapid even in areas like Mongolia where Bar-headed Geese generally breed in small isolated groups.
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Management decisions involving species at risk are typically made in the face of substantial biological uncertainty. Incorporating uncertainty into decision-making allows managers to perform risk/benefit analysis. Stable hydrogen isotope ratios (δD) provide a tool for studying largescale movements of migratory organisms and can contribute to the management of migratory species at risk. However, this analytical approach is based on predicted precipitation amount-weighted average deuterium (δDp) values, and individual years of interest can involve substantial departures from long-term averages. Local growing-season δDp at locations within the Great Plains of North America deviates from that described by GIS-based models of δDp derived from long-term estimates based on the Global Network of Isotopes in Precipitation (GNIP). We augmented the GNIP dataset with information from the US Network for Isotopes in Precipitation database for sites in the Great Plains, refining a previously published isoscape for inferring origins of migratory wildlife. Accounting for longitude and the coefficient of variation within precipitation in a single year at specific locations allowed us to determine year- and site-specific estimates of δDp and estimated rates of long-distance dispersal for 3 species of grassland songbirds based upon δD values of feathers (δDf). Using a likelihood-based approach to classify individuals as 'local' or 'immigrant,' we incorporated uncertainty in making these designations. This approach provides a convenient tool to effectively communicate research results to policy makers, who must make decisions at the level of risk which they are willing to assume when determining management strategies.
Stable isotope ratio variation in natural systems reflects the dynamics of Earth systems processes and imparts isotope labels to Earth materials. Carbon isotope ratios of atmospheric CO2 record exchange of carbon between the biosphere and the atmosphere; the incredible journeys of migrating monarchs is documented by hydrogen isotopes in their wings; and water carries an isotopic record of its source and history as it traverses the atmosphere and land surface. Through these and many other examples, improved understanding of spatio-temporal isotopic variation in Earth systems is leading to innovative new approaches to scientific problem-solving. This volume provides a comprehensive overview of the theory, methods, and applications that are enabling new disciplinary and cross-disciplinary advances through the study of "isoscapes": isotopic landscapes. "This impressive new volume shows scientists deciphering and using the natural isotope landscapes that subtly adorn our spaceship Earth." Brian Fry, Coastal Ecology Institute, Louisiana State University, USA "An excellent timely must read and must-have reference book for anybody interested or engaged in applying stable isotope signatures to questions in e.g. Anthropology, Biogeochemistry, Ecology, or Forensic Science regarding chronological and spatial movement, changes, or distribution relating to animals, humans, plants, or water." Wolfram Meier-Augenstein, Centre for Anatomy & Human Identification, University of Dundee, UK "Natural resources are being affected by global change, but exactly where, how, and at what pace? Isoscapes provide new and remarkably precise answers." John Hayes, Woods Hole Oceanographic Institution, USA "This exciting volume is shaping a new landscape in environmental sciences that is utilizing the remarkable advances in isotope research to enhance and extend the capabilities of the field." Dan Yakir, Weizmann Institute of Science, Israel.
The study of migratory connectivity is rapidly growing in ornithology, as is the technology used to measure it. While use of extrinsic markers, such as archival tags, is becoming more prevalent, for many small species the best tool available for tracking birds remains intrinsic markers, such as stable-hydrogen isotope ratios (δ2H). Many researchers have raised concerns that spatial and temporal environmental variation introduces a large amount of error into isotope-based assignments, limiting their utility. Here, using feathers, we sought to address these issues in developing δ2H base maps for assigning pied flycatchers Ficedula hypoleuca of known origin to 15 sites across the breeding range (approx. 4 020 800 km2). We evaluated the effects of including random site variation and year-specific precipitation δ2H (δ2HP) maps on assignments, compared to using mean annual growing season δ2Hp and no site effects. We found a positive correlation between feather δ2H (δ2HF) and mean annual δ2HP, resulting from large scale geographic variation. Repeatability of feather δ2H for individuals sampled in multiple years was strong overall, but variable among populations. Annual variation in δ2HP explained 21% of within individual variation in δ2HF. Neither year- nor site-specific methods improved assignment precision or accuracy. All three methods assigned flycatchers of unknown origin captured at an African overwintering site to similar breeding areas. However, methods using long-term means of δ2Hp assigned birds more precisely than year-specific methods. Our results suggest that annual variation in this system is primarily a result of food web or individual level processes and that random site effects are not strong enough to drastically impact accuracy. We conclude that improvements in isotope based geographic assignments will rely on the addition of prior information, such as relative abundance in a Bayesian framework, or additional intrinsic markers.