A test of comparative equilibration for determining non-exchangeable stable hydrogen isotope values in complex organic materials

Article (PDF Available)inRapid Communications in Mass Spectrometry 23(15):2316-20 · August 2009with19 Reads
DOI: 10.1002/rcm.4150 · Source: PubMed
Abstract
Comparative equilibration has been proposed as a methodological approach for determining the hydrogen isotopic composition (deltaD) of non-exchangeable hydrogen in complex organic materials, from feathers to blood and soils. This method depends on using homogenized standards that have been previously calibrated for their deltaD values of non-exchangeable H, that are compositionally similar to unknown samples, and that span an appropriate isotopic range. Currently no certified organic reference materials with exchangeable H exist, and so isotope laboratories have been required to develop provisional internal calibration standards, such as the keratin standards currently used in animal migration studies. Unfortunately, the isotope ratios of some samples fall outside the range of keratin standards currently used for comparative equilibration. Here we tested a set of five homogenized keratin powders as well as feathers from Painted Buntings and Dark-eyed Juncos to determine the effects of extrapolating comparative equilibration normalization equations outside the isotopic range of keratin standards. We found that (1) comparative equilibration gave precise results within the range of the calibration standards; (2) linear extrapolation of normalization equations produced accurate deltaD results to approximately 40 per thousand outside the range of the keratins standards used (-187 to -108); and (3) for both homogenized keratin powders and heterogeneous unknown samples there was no difference in variance between samples within and outside the range of keratin standards. This suggested that comparative equilibration is a robust and practical method for determining the deltaD of complex organic matrices, although caution is required for samples that fall far outside the calibration range.
A test of comparative equilibration for determining
non-exchangeable stable hydrogen isotope values in
complex organic materials
Jeffrey F. Kelly
1
*
, Eli S. Bridge
1
, Adam M. Fudickar
2
and Leonard I. Wassenaar
3
1
Oklahoma Biological Survey and Department of Zoology, 111 East Chesapeake St., University of Oklahoma, Norman, OK, USA
2
Max Planck Institute for Ornithology, Department of Migration and Immuno-ecology, Vogelwarte Radolfzell, Schlossallee 2, D-78315 Radolfzell,
Germany
3
Environment Canada, 11 Innovation Boulevard, Saskatoon, Saskatchewan, S7N 3H5 Canada
Received 29 April 2009; Revised 3 June 2009; Accepted 10 June 2009
Comparative equilibration has been proposed as a methodological approach for determining the
hydrogen isotopic composition (dD) of non-exchangeable hydrogen in complex organic materials,
from feathers to blood and soils. This method depends on using homogenized standards that have
been previously calibrated for their dD values of non-exchangeable H, that are compositionally
similar to unknown samples, and that span an appropriate isotopic range. Currently no certified
organic reference materials with exchangeable H exist, and so isotope laboratories have been
required to develop provisional internal calibration standards, such as the keratin standards
currently used in animal migration studies. Unfortunately, the isotope ratios of some samples fall
outside the range of keratin standards currently used for comparative equilibration. Here we tested a
set of five homogenized keratin powders as well as feathers from Painted Buntings and Dark-eyed
Juncos to determine the effects of extrapolating comparative equilibration normalization equations
outside the isotopic range of keratin standards. We found that (1) comparative equilibration gave
precise results within the range of the calibration standards; (2) linear extrapolation of normalization
equations produced accurate dD results to 40% outside the range of the keratins standards used
(187 to 108); and (3) for both homogenized keratin powders and heterogeneous unknown samples
there was no difference in variance between samples within and outside the range of keratin
standards. This suggested that comparative equilibration is a robust and practical method for
determining the dD of complex organic matrices, although caution is required for samples that fall
far outside the calibration range. Copyright # 2009 John Wiley & Sons, Ltd.
Hydrogen stable isotope ratios (dD) of many complex organic
materials (collagen, chitin, soil, plants, feathers, etc.) are
controlled by the dD of precipitation at locations where they
are grown and therefore are useful for inferring both origins
and paleo-climatic trends.
1,2
Globally, dD values of precipi-
tation are spatially and temporally predictable.
3
Most
notably there are strong long-term latitudinal gradients
in the dD values of precipitation, and this makes keratin
(e.g. feathers) dD values useful for tracing the origins of long-
distance avian migrants that move across large latitudinal
dD gradients.
4–6
This utility has led to a huge increase in
application of hydrogen stable isotopes to study movement
of migrant species over the past decade.
7–9
A challenge for
the future of H isotope applications in migratory ecology is to
better understand the source and meaning of variance in
dD isotope data obtained from various species.
10–12
One of the first challenges encountered is that complex
organic substrates like most biological tissues contain
exchangeable H.
13
Left untreated, tissues dynamically
exchange a proportion of their H atoms with ambient
moisture, which leads to dD results that cannot be compared
among laboratories. Some biological substrates (e.g. chitin)
are amenable to nitration to remove exchangeable H, but
most biological tissues are not. A corresponding challenge
was the need for rapid and reproducible high sample
throughput with comparably precise results among labora-
tories. The key innovation to address this challenge was the
development of the ’Comparative Equilibration’ method.
14
Comparative equilibration uses homogenized powdered
complex organic standards that have previously been
calibrated offline for their non-exchangeable dD. These
standards are then analyzed with ’like’ unknown samples
and are used to correct measured dD values of bulk tissues
RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2009; 23: 2316–2320
Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4150
*Correspondence to: J. F. Kelly, Oklahoma Biological Survey and
Department of Zoology, 111 East Chesapeake St., University of
Oklahoma, Norman, OK, USA.
E-mail: jeffrey.f.kelly@gmail.com
Contract/grant sponsor: US National Science Foundation; con-
tract/grant number: 0541740.
Contract/grant sponsor: US Environmental Protection Agency;
contract/grant number: R 833778.
Copyright # 2009 John Wiley & Sons, Ltd.
(e.g. feathers) to determine the dD value of the non-
exchangeable fraction of the feather H.
14,15
This method
allows for rapid sample throughput and makes it possible to
compare measurements within and among different
sampling periods within and among laboratories.
2
Comparative equilibration is typically implemented by
measuring unknown complex organic samples and chemi-
cally similar calibration standards within the same analysis
runs (e.g. keratin standards with keratinous feathers, hair,
etc.). A regression equation (corrected for instrumental drift)
is then fitted using the measured values of standards as the
independent variable and accepted values of the non-
exchangeable fraction of the H isotopes in the standards
as the dependent variable. This best-fit regression equation is
then used to estimate dD values of the non-exchangeable
fraction of H of the unknown samples.
14
This method requires
an appropriate isotopic range of homogenous powdered
organic matrices that have accepted dD values for the non-
exchangeable fraction of H. These homogenous complex
organic standard powders are time consuming and costly to
develop and to calibrate; although recent efforts have
reduced these difficulties somewhat.
16
At present, for
keratins used in animal studies, the most widely used
provisional dD standards are chicken feathers (CFS), cow
hooves (CHS) and bowhead whale baleen (BWB), which
span about 80% (187% to 108%).
14
Many animals grow keratins that fall within the range of
these standards, but a significant proportion of these keratins
and other tissues do not. When unknown samples fall
outside the range of the standards it is often assumed that
the comparative equilibration regression equation can be
extrapolated to these values; that is, analytical linearity is
assumed. This assumption is not unique to measurement of
H isotope ratios in organic materials and has a long history in
stable isotope applications.
17
Nonetheless, we are not aware
of any specific attempts to test the validity of comparative
equilibration when extrapolating beyond the range of
the standards, especially with keratin. Recent studies have
criticized this practice, despite a weak empirical basis for
doing so.
18
We tested the assumption of linearity and its
effect on the mean and variation in powders and hetero-
geneous samples measured outside the calibration range of
known standards.
We used three keratin standards (BWB, CFS, CHS) and two
additional homogenous keratin powders that had more
positive dD values than the accepted keratin standards to test
the assumption of linearity and to compare the variance
among raw values and normalized values within and outside
the range of standards. We then examined the variance in
two sets of non-homogenized feather samples to examine
effects of extrapolation.
EXPERIMENTAL
We analyzed samples in batch sequences of 49 samples and
references that we refer to as autoruns. Each autorun
contained three replicates of five homogenized keratin
powders. Of these keratin powders, three were supplied
by one of the authors (LIW) and were documented by
Wassenaar and Hobson;
14,15
chicken feathers (CFS), cow
hooves (CHS) and bowhead whale baleen (BWB). The
remaining two were prepared at the University of Oklahoma;
human hair from one of the authors – JFK (HOJ), and Brown
headed cowbird feathers (BHCO; Table 1). Powders
prepared at the University of Oklahoma were cut into small
pieces and cryoground in liquid nitrogen (Spex Certiprep
6750 freezer mill; Metuchen, NJ, USA). Each subsample was
cryoground for three cycles of 3 min with 1 min between
cycles. The resulting powder was sieved to remove particles
larger than 63 mm and then blended to ensure isotopic
homogeneity.
The five keratin powders were analyzed in sequence
positions 1–5, 22–26, and 45–49 in each autorun, as is
typically done in laboratories. Samples 5–21 and 27–44 were
the feathers of unknown dD value. Many of the unknown
feather samples in these autoruns were from Painted
Buntings (Passerina ciris,n¼ 80) captured at Fort Sill, OK,
USA, in the summer of 2007 and 2008 or Dark-eyed Juncos
(Junco hyemalis,n¼ 72) captured in Norman, OK, USA, in
the winter of 2009. Because neither of these species molt at
the sampling location we made no assumption that these
unknown samples represent a homogeneous grouping. We
also had no a priori expectation that each feather would
be isotopically homogenous.
All feather sample materials were cleaned with dilute
detergent and then 2:1 chloroform/methanol following the
method of Parrite and Kelly.
19
We packed 140 to 160 mgof
each sample into a 3.5 mm 5 mm silver capsule. A tight
allowable range of 10 mg was required to avoid variance in
dD values due to variable H yields on the mass spec-
trometer.
2
All isotope ratio data were collected at the
University of Oklahoma with a ThermoFinnigan Delta V
isotope ratio mass spectrometer connected to a high-
temperature pyrolysis elemental analyzer (TC/EA, Thermo-
Finnigan, Bremen, Germany) through an open split valve
(Conflo III, ThermoFinnigan). The trap and box currents of
the isotope ratio mass spectrometer were 0.8 mA and 0.7 mA,
respectively. The TC/EA reactor was operated at 14508C,
and contained glassy C, quartz wool and silver wool
according to ThermoFinnigan specifications.
20
The crucible
in the reactor was changed every 100 samples and the reactor
Table 1. Measured and equilibrated H stable isotope ratios of
five keratin powders and feathers from two species of birds.
For each equilibrated value the standards used to generate
the equilibration equations are provided. Equilibrated values
were used to test for effects on linearity and variance. All
values are means of 13 autoruns reported as dD SD per mil
VSMOW except where noted
Sample
(accepted) Raw Equilibrated Standards used Test
CHS (187)
175.8 (3.6)
None
CFS (147.4)
145.8 (3.5) 146.8 (3.5)
CHS, BWB Variance
BWB (108)
116.9 (4.2) 109.7
a
(7.6)
CHS, CFS Linearity
HOJ
90.5 (4.2) 73.0 (5.0)
CHS, BWB Variance
BHCO
76.2 (5.0) 54.0 (4.3)
CHS, BWB Variance
Juncos
148.6 (11.8) 151.8 (14.0)
CHS, CFS, BWB Variance
Buntings
78.4 (12.9) 62.0 (16.1)
CHS, CFS, BWB Variance
a
n ¼ 39.
Copyright # 2009 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2009; 23: 2316–2320
DOI: 10.1002/rcm
Stable hydrogen isotope values in complex organic materials 2317
contents were re-packed every <200 samples. The He flow
rate was 100 mL/min maintained at a pressure of 21 psi, and
the gas chromatography (GC) column was held at 1008C. H
3þ
corrections were carried out when the column was repacked
and the H
3þ
signal ranged from 4.48 to 4.53 ppm/nA.
For each sample run, two reference pulses of ultra-high
purity H
2
gas (99.999%, Air Gas) were injected into the ion
source; the first at 40 s and the second at 90 s after the start of
acquisition. Each pulse lasted 24 s with an intensity of
3000 mV at mass 2. The second reference peak was used in
calculation of the sample dD value. Based on the dD values of
standard materials the approximate dD value of the reference
gas was 320%. We used this value of the reference gas to
calculate the raw dD values of the samples. Samples were
automatically dropped from a 50-position zero-blank auto-
sampler with a 1 s drop time at 110 s after the beginning of
acquisition. The H
2
gas from the sample was chromato-
graphically separated from N
2
and CO using a 5 A
˚
packed
molecular sieve. The sample peak was detected at about 135 s
after the beginning of acquisition. The total analysis time of
300 s per sample allowed complete elution of CO and N
2
prior to the next sample acquisition.
For each autorun we corrected all measurements for
instrumental drift between the first and last sample.
Instrumental drift corrections were based on the slopes of
best-fit lines for dD values regressed against analysis time of
references within each autorun. A slope was calculated for
the five powders in the run and these five slopes were
averaged to achieve the drift correction coefficient. All data
are reported in per mil notation (%) relative to VSMOW
(Vienna Standard Mean Ocean Water).
We used results from the three known keratin standards
(BWB, CFS and CHS) to determine if it was reasonable to
assume analytical linearity outside the range of known
standards. For this analysis we treated CHS and CFS
standards as known samples and treated the BWB standard
as an unknown. For each of the 13 autoruns we calculated a
comparative equilibration equation based on CHS and CFS
samples and then used this equation to correct the
measurements of BWB. We use a one-sample t-test to
determine if the estimated value of BWB was different from
the accepted provisional value (108). Because unknowns
are typically analyzed as single samples, we did not average
our BWB replicates in each run for this analysis (i.e., n ¼ 39
for BWB).
In our next analysis, we focused on variability that may
result from comparative equilibration for samples within
and outside the range of the known standards. For this
analysis we used the same data described above from three
keratin standards powders, but in this analysis we treated the
CFS samples as unknown along with two additional keratin
powders: human hair (HOJ) and Brown-headed Cowbird
feathers (BHCO). We calculated comparative equilibration
normalization equations for each of the 13 autoruns from
CHS and BWB samples. We then used a Levene’s test to
check for homogeneity of variances among the raw
measurements of the five powders and the normalized
values of these powders. We note that the raw values of
individual samples are not meaningful, but the variation
among replicate measurements is informative. Because all
the keratin powders were homogenized, we expected them
to produce equally variable raw measurements. Therefore, if
a Levene’s test on the corrected values indicated significant
heterogeneity in variance among the powders, we attributed
this variation to the comparative equilibration method.
Because it has the most positive dD value, we expected
variance to be greatest in the corrected BHCO samples
followed by HOJ with CFS having the least variation.
In a similar manner we used raw data values and
comparative equilibration normalized values of Dark-eyed
Junco and Painted Bunting feathers to infer the likely impact
of comparative equilibration on sample variation.
Because the Junco samples tended to be within the range
of the standards we expected the variation in this sample to
be unaffected by comparative equilibration. In contrast, the
dD values of Painted Buntings tend to be more positive than
the range of keratin standards. Therefore, we expected to see
an increase un the variance among these samples. We again
used Levene’s test to compare the variation between Junco
and Bunting feathers both before and after equilibration.
RESULTS
We failed to reject the null hypothesis of linearity outside the
range of standard values. We calculated a grand mean of
109.7% for BWB based on the CFS and CHS samples
(Table 1, Fig. 1). This value does not significantly differ from
the accepted one of 108% (t
1,38
¼ 1.35, p ¼ 0.19). More
importantly, the total effect size of 1.7% across a spread of
40% was small relative to other sources of error, and is
acceptable for dD measurements. Generally, 2% is
considered to be an acceptable level of precision for
dD measurements.
When using CFS, BWB and CHS as standards, our 13
calibration slopes ranged from 1.24 to 1.47 with intercepts
that ranged from 32.8 to 70.0. Using these corrections we
Figure 1. Solid circles are measured mean values (n ¼ 13
autoruns) for CHS and CFS plotted against their accepted
values (CHS ¼187; CFS ¼147.4); comparative equi-
librium normalizations were based on these data. Open cir-
cles are corrected values of BWB measurements based on
the data from the solid circles (n ¼ 39, x axis) plotted against
the accepted value for BWB (108). A one-sample t-test
indicates no dif ference between the normalized value for
BWB and the accepted value. The insert is a histogram of
BWB values.
Copyright # 2009 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2009; 23: 2316–2320
DOI: 10.1002/rcm
2318 J. F. Kelly et al.
failed to reject the hypothesis of equal variances across the
raw measured values and the corrected values of the keratin
powders (Fig. 2) suggesting that extrapolation to values
outside the range of standards does not result in an inflation
of the variance in measurements. All the Dark-eyed Junco
feathers that we measured were within the range of our three
keratin standards, while all the Painted Buntings were
outside this range (Table 1). Comparison of the raw
measured values of these birds showed that there was no
significant difference in the variance between Junco and
Bunting samples prior to or after comparative equilibration
normalization corrections (Fig. 3).
DISCUSSION
Overall when comparative equilibration was used as
designed, that is samples are within the range of the
standards, the method was highly effective at correcting
measured values to the accepted value (CFS data, Table 1).
Further, our results supported the assumption that equi-
librium H exchange is a linear process, at least over the range
from 187 to 108%. The variation in slopes and intercepts
of our calibration lines suggested that routine measurement
of multiple standards with samples was necessary for
adequate correction. It is unlikely that single-point or offset
calibration approaches would produce similar results. There
was minimal difference between the grand mean of our
comparatively equilibrated samples of BWB (109.7) and the
accepted value derived through offline steam equilibration
(108) by Wassenaar and Hobson.
14
This finding suggested
that it was possible to infer the values of samples outside the
range of known standards and it supported the validity of
this approach. Further testing is needed to validate this
method with samples that greatly exceed the range of the
standards. Nonetheless, our data indicate that comparative
equilibration provides a viable approach for arriving at
precise values for new standards without having to resort to
offline steam equilibration. Wunder et al. also employed
this approach effectively to validate a working standard at
57% that was used in their comparative equilibrium
corrections.
21
Our results also indicated no detectable inflation in
variation in dD values as the distance between the standards
and unknowns increased. The absence of this pattern was
surprising, and probably indicated that within our range of
samples comparative equilibration was a minimal source of
error. Even for our unknown feather samples the standard
deviations of the population of samples were not signifi-
cantly inflated by comparative equilibration (Table 1).
We urge researchers to continue to apply and test the
comparative equilibration method when measuring stable H
isotope ratios of feathers and other biological substrates.
While we are hesitant to dismiss the possibility of inflated
variation well outside the range of lab standards based on
our data alone, we think that within the range of our
measured values this problem is not a primary concern.
However, researchers should be aware of this possibility (as
well as potential intra-sample isotopic heterogeneity) when
unknown samples are far more enriched (or depleted)
relative to the standards used to develop comparative
equilibration correction equations. Finally, development of
large batches of complex organic substrates that span the
range of normally encountered keratins and other biological
substrates is a pressing need for laboratory standardization
in this field.
Figure 2. Deviations from the grand mean for measured
(top) and equilibrated (bottom) values for five keratin powders.
Solid symbols indicate powders that were treated as lab
standards and used to generate comparative equilibration
normalization equations. Open symbols were corrected with
these equations. Levene’s tests indicate that the measured
and corrected values of the powders were equally variable.
Figure 3. Deviations from the grand mean for samples of
non-homogenized 80 Dark-eyed Junco and 70 Painted Bunt-
ing feathers. There was no difference in the variation between
measured and corrected values of juncos or buntings.
Copyright # 2009 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2009; 23: 2316–2320
DOI: 10.1002/rcm
Stable hydrogen isotope values in complex organic materials 2319
Acknowledgements
We thank R. Maynard and M. Engel for assistance with
analysis. This research was supported by the US National
Science Foundation (IOS: 0541740) and the US Environmen-
tal Protection Agency (R 833778). Although the research
described in this article has been funded wholly or partially
by the United States Environmental Protection Agency
through grant/cooperative agreement R 833778, it has not
been subjected to the Agency’s required peer and policy
review and therefore does not necessarily reflect the views of
the Agency and no official endorsement should be inferred.
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2320 J. F. Kelly et al.
    • "Approaches for controlled and repeatable accounting of the exchangeable-H in organic samples have varied (Wassenaar and Hobson, 2003; Bowen et al., 2005a; Kelly et al., 2009; MeierAugenstein et al., 2011; Coplen and Qi, 2012 ). Some of the recommended methods are described in more detail by Augenstein et al. (2013) and include a two-stage exchange equilibration process (Bowen et al., 2005a), a comparative two-point end-member equilibration (Wassenaar and Hobson, 2000; Kelly et al., 2009 ), online comparative twopoint end-member equilibration with an autosampler that will allow steam equilibration (EuroVector 1 ), or an online vacuum equilibration system (Wassenaar et al., 2015). Regardless of the method used, we emphasize that the methods must be reliable and reproducible, and reported with sufficient detail in publications. "
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Full-text · Article · Mar 2016
    • "Measurement precision for d 13 C and d 15 N were ±0.1 and ±0.2 %, respectively, based on the analyses of internal laboratory standards (i.e. powdered Brown-headed Cowbird feathers; Kelly et al. 2009) and two National Institute of Standards and Technologies (Gaithersburg, MD) reference materials (USGS 40 and USGS 41). We only sampled females at least 15 days into the breeding season and which were nesting for at least 10 days (nest-building period included) to ensure that the isotope ratios measured in their whole blood samples reflected those of their local breeding grounds. "
    [Show abstract] [Hide abstract] ABSTRACT: Throughout their annual cycle, migrants often adopt different foraging and microhabitat usage strategies. Previous studies treat migrants as niche-trackers/niche-followers, i.e., they track similar niches along their annual cycle, almost exclusively based on food resource availability, which is inferred based on the climate at either the wintering or breeding grounds. An alternative approach is the use of such techniques as stable isotope analyses that allow researchers to more directly infer a migrant’s niche across seasons. While the use of carbon isotopes enables an assessment of microhabitat traits, that of nitrogen isotopes provides information on a bird’s trophic level. In the study reported here, we performed comparative analyses of stable carbon and nitrogen isotope ratios in tissues of the resident Plain-crested Elaenia and the intratropical migrant Lesser Elaenia to evaluate their year-round ecological niches. Our data suggest that both residents and migrants were consistent in their use of similar microhabitats throughout the year, which indicates a niche-tracking behavior on the part of migratory individuals. Migrants often fed at higher trophic levels than residents, but both species exhibited similar trophic level shifts through the year, feeding on higher trophic levels during breeding and on the lowest ones while wintering. The observed patterns could be due to several factors, including differential energetic demand needed for the migratory journey, species-specific nutritional needs during each stage of the year, and/or the use of multiple wintering grounds by migrants.
    Full-text · Article · Feb 2016
    • "The long-term analytical precision (1σ) of these standards at CASIF is 2.1‰ and 2.0‰, respectively. Our δ 2 H hair values span a larger range (−122.7‰ to −8.8‰) than do USGS42 and USGS43, but prior studies suggest that linear extrapolation of normalization relationships for δ 2 H values has minimal influence on values within ~100‰ of the range of the standards used for normalization, provided that at least two standards are analyzed (Kelly et al. 2009, Wiley et al. 2012). The long-term accepted δ 2 H value of the internal keratin standard at CASIF is −59.5‰ ± 2.3‰. "
    [Show abstract] [Hide abstract] ABSTRACT: An unanticipated impact of wind-energy development has been large-scale mortality of insectivorous bats. In eastern North America, where mortality rates are among the highest in the world, the hoary bat (Lasiurus cinereus) and the eastern red bat (L. borealis) comprise the majority of turbine-associated bat mortality. Both species are migratory tree bats with widespread distributions; however, little is known regarding the geographic origins of bats killed at wind-energy facilities or the diversity and population structure of affected species. We addressed these unknowns by measuring stable hydrogen isotope ratios (δ2H) and conducting population genetic analyses of bats killed at wind-energy facilities in the central Appalachian Mountains (USA) to determine the summering origins, effective size, structure, and temporal stability of populations. Our results indicate that ∼1% of hoary bat mortalities and ∼57% of red bat mortalities derive from non-local sources, with no relationship between the proportion of non-local bats and sex, location of mortality, or month of mortality. Additionally, our data indicate that hoary bats in our sample consist of an unstructured population with a small effective size (Ne) and either a stable or declining history. Red bats also showed no evidence of population genetic structure, but in contrast to hoary bats, the diversity contained in our red bat samples is consistent with a much larger Ne that reflects a demographic expansion after a bottleneck. These results suggest that the impacts of mortality associated with intensive wind-energy development may affect bat species dissimilarly, with red bats potentially better able to absorb sustained mortality than hoary bats because of their larger Ne. Our results provide important baseline data and also illustrate the utility of stable isotopes and population genetics for monitoring bat populations affected by wind-energy development.
    Full-text · Article · Feb 2016
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