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The Auk 117(4):1051?1056, 2000
Identi?cation of the Extinct Hawaiian Eagle (Haliaeetus) by mtDNA
Sequence Analysis
ROBERT C. FLEISCHER,1,2,5 STORRS L. OLSON,3 HELEN F. JAMES,3 AND ALAN C. COOPER2,4
1Department of Biological Sciences, University of Durham, Durham DH1 3LE, United Kingdom;
2Molecular Genetics Laboratory, National Zoological Park, Smithsonian Institution, Washington, D.C. 20008, USA;
3Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution,
Washington, D.C. 20560, USA; and
4Departments of Zoology and Biological Anthropology, University of Oxford, Oxford, OX2 6QS, United Kingdom
Among the many bones of extinct birds discovered
in the ?rst extensive deposits studied in the Hawai-
ian Islands were a few from Molokai and Oahu that
clearly belonged to a large sea-eagle of the genus
Haliaeetus, as shown by their large size and the fusion
of the phalanges of the inner toe (Olson 1982, Olson
and James 1982). Even with the discovery of a nearly
complete skeleton of this eagle on Maui, its speci?c
identi?cation could not be entirely resolved. Size and
other characters eliminated most species of the ge-
nus, but no qualitative osteological characters could
be discerned between the Hawaiian fossils and avail-
able skeletons of the Old World White-tailed Eagle
(H. albicilla) and the New World Bald Eagle (H. leu-
cocephalus; Olson and James 1991). Unable to make a
morphological resolution, Olson and James (1991:62)
were constrained to list this interesting specimen as
``Haliaeetus sp., aff. H. leucocephalus/H. albicilla.'' To
identify the Hawaiian eagle more satisfactorily, we
5 Address correspondence to Molecular Genetics
Laboratory, National Zoological Park, Smithsonian
Institution, Washington, D.C. 20008, USA. E-mail:
r?eischer@nzp.si.edu
turned to ``ancient'' DNA methods to obtain mito-
chondrial DNA (mtDNA) sequences from its bones.
The bones used in ancient DNA analysis were ob-
tained from a nearly complete skeleton found on 4
April 1988 in a good state of preservation in Puu
Makua Cave, a lava tube at 1,463 m elevation on the
south slope of Mt. Haleakala, Maui (Olson and James
1991). The eagle had died in a cave that served as a
natural trap for ?ightless species such as moa-nalos,
rails, and ibises. The only entrance to the cave is a
skylight of approximately 5 m diameter above a ver-
tical drop of approximately 22 m. The eagle may
have ?own into the cave to scavenge ?ightless birds
that had fallen in beforehand. The eagle presumably
became trapped because the entrance was too small
for such a large bird to be able to ?y back out.
Previous analyses showed that the skeleton con-
tained 4.1% nitrogen and amino acids in proportions
only slightly different from those of modern cow
bone (T. Stafford, Jr. pers. comm.). An AMS radio-
carbon date on the skeleton gave an age of 3,300 6
60 years (two-sigma calibrated range 3,389 to 3,689
years; see James 1995). Because the nitrogen and ami-
no acid content suggested that organic compounds
1052 [Auk, Vol. 117Short Communications
were well preserved in the skeleton, and despite its
antiquity, we chose this specimen for ancient DNA
analysis.
We isolated DNA from two bones from the skele-
ton in two separate laboratories dedicated to ancient
DNA analysis. DNA was ampli?ed from the Hawai-
ian eagle bones for three different gene regions of
mtDNA. We compared sequence from part of the cy-
tochrome-b (cyt b) gene with cyt-b sequences ob-
tained from GenBank for seven of the eight species
of Haliaeetus known worldwide (Seibold and Helbig
1996). We also obtained sequences of the ATP sub-
unit 8 gene (ATPase8) and part of the 12S ribosomal
RNA gene (12S rRNA) and compared them with se-
quences we obtained from individuals of H. leucoce-
phalus, H. albicilla, and H. leucogaster.
Methods.?Two bones were selected for analysis
from the nearly complete skeleton found in Puu
Makua Cave (USNM 431238): a thoracic rib fragment
and a pedal phalanx. We obtained two tissue sam-
ples of H. albicilla from the Swedish Museum of Nat-
ural History (nos. 501 and 502) and feathers of H. al-
bicilla and H. leucogaster (White-bellied Fish-Eagle)
from captive birds in the United States. DNA sam-
ples of H. leucocephalus were obtained from W. Piper.
All pre-PCR protocols were conducted in isolated
ancient DNA laboratories located in buildings dif-
ferent from the ones containing the primary DNA
laboratories. DNA isolations and PCRs for ATPase8
and 12S rRNA segments were conducted at the Na-
tional Zoological Park (NZP) by A. C. Cooper, and
DNA isolations and PCRs for cyt b and 12S rRNA
were conducted at the University of Durham by R.
Fleischer. Methods to reduce the probability of con-
tamination followed Handt et al. (1994) and included
the use of barrier pipet tips; sterile solutions; DNA
destruction with UV illumination; liberal washing of
equipment and bench surfaces with bleach solutions;
and the use of extract, PCR, and ``large DNA'' con-
trols. In addition, equipment and supplies were ded-
icated to the ancient DNA laboratories, and work was
not conducted in an ancient DNA laboratory after
working in the main laboratory on the same day.
Isolation protocols in the two laboratories were
very similar. A small piece of each Haliaeetus bone
was cut away and cleaned in sterile water. This was
followed by a wash with rotation at room tempera-
ture in sterile 0.5 M EDTA, after which the EDTA was
discarded. At Durham, the bone was wrapped in
heavy aluminum foil (which had been decontami-
nated of DNA by UV illumination for 20 min) and
crushed in a mortar and pestle (decontaminated by
soaking in 50% bleach solution). At the NZP, a
bleach-sterilized coffee grinder was used to pulver-
ize the bone sample. The crushed bone was placed in
5 mL of sterile UV-treated lysis buffer (0.01 M Tris,
ph 8.0; 0.002 M EDTA; 0.01 M NaCl; 10 mg/mL DTT;
1% SDS; and 1 mg/mL Proteinase K) and rotated
overnight at 608C. The sample was then extracted
twice with 5 mL of phenol and once with 5 mL of
chloroform. The supernatant was placed in 1 mL of
sterile water in a Centricon 30 microconcentrator and
centrifuged until about 50 to 100 mL covered the
membrane. A second 1 mL of sterile water was added
and the centrifugation repeated. The remaining sam-
ple was collected from the membrane by a short cen-
trifugation and diluted to 200 mL total volume. From
2 to 4 mL of this were used in subsequent PCR ex-
periments.
We attempted PCR ampli?cations with primers
that amplify different-sized segments of the three
mtDNA genes: 12S rRNA, ATPase8, and cyt b. We de-
signed a set of primers (AncEag. 2: forward, 59-AG-
GAATCTGCCTACTAACACA-39, 39 is base 15,026 of
Gallus gallus sequence; Desjardins and Morais 1990;
reverse, 59-TTCGACATGTATGGGCTACG-39, 39 is
base 15,090) from Haliaeetus eagle cyt-b sequence
(Seibold and Helbig 1996) such that they ?anked a
small (63 base pairs [bp]) region that contained four
transitional substitutions between H. albicilla and H.
leucocephalus and also differed by at least this much
from the other ?ve Haliaeetus species. The AncEag. 2
product size was 104 bp.
Primers cyt-b 2-rc (59-TGAGGACAAATATCC
TTCTGAGG-39, 15,320 of Gallus, reverse comple-
ment of cyt b 2; Kocher et al. 1989) and cyt-b ack (59-
CCTCCTCAGGCTCATTCTAC-39, 15,376 of Gallus)
ampli?ed a 98-bp segment of cyt b disjunct from
AncEag. 2. The 12S rRNA segments were ampli?ed
and sequenced using primers 12SA, 12SH, 12SJ, and
12SB2 (Cooper 1994), producing 392 bp of sequence.
The ATPase8 segment included 35 bp of tRNA-lysine
and was ampli?ed and sequenced with ``Birds-R-Us''
(59-TGGTCGAAGAAGCTTAGGTTCA-39, 9,241 of
Gallus) and t-lys (59-CACCAGCACTAGCCTTTT
AAG-39, 9,051 of Gallus).
Final PCR components in a 50-mL reaction volume
were 1X taq buffer (Perkin Elmer), 1.25 units of Taq-
gold polymerase, 0.2 mM each dNTP, 2.0 mM MgCl2,
3 mg/mL non-acetylated BSA, and 1 mM of each
primer. PCR was conducted for 45 cycles beginning
with a ``hot-start'' for 10 min at 948C and ending
with a 6-min soak at 728C. Conditions per cycle were
928C denaturation for 40 s, 508C annealing for 1 min,
and 728C extension for 1 min.
Successful ampli?cation was determined by visu-
alizing PCR products on a 2% agarose gel. Single-
banded products were cleaned by centrifugation us-
ing a Qiagen kit following the manufacturer's in-
structions. About 100 ng of product were used for cy-
cle sequencing of both strands using dye-labeled
dideoxynucleotide terminators; resulting sequence
reactions were run on an ABI 377 sequencer. We as-
sessed chromatograms for accuracy and aligned se-
quences using Sequencher 3.0.
We compared sequences from the Hawaiian eagle
bones directly with those of other Haliaeetus species.
We also conducted a total-evidence phylogenetic
October 2000] 1053Short Communications
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analysis of the sequences of the Hawaiian bone and
composite sequences from three outgroup Haliaeetus
taxa. Replicate sequences for H. leucocephalus and H.
albicilla for ATPase8, and for H. leucocephalus for
12srRNA, were identical; thus, we used only one se-
quence from these to represent each taxon in the
analysis. The cyt-b sequences of H. albicilla, H. leu-
cocephalus, and H. leucogaster were from a different
study and therefore arise from different individuals
than the sequences obtained for these same species
in this study. Nonetheless, we combined the gene re-
gions into the full 704 bp of sequence (shown
abridged in Table 1 as variable sites only) and con-
ducted an exhaustive search, with no character
weighting, in PAUP* (Swofford 1999). We also ran a
bootstrap (1,000 replications) using a branch-and-
bound search algorithm to determine the relative
support for different clades. In addition, we con-
strained the Hawaiian bone to be a sister taxon to H.
albicilla in one tree and H. leucocephalus in another
and then used a Kishino-Hasegawa test (Kishino and
Hasegawa 1989) to determine whether the alterna-
tive trees differed signi?cantly in length.
Results.?We obtained a total of 704 nucleotide
sites of sequence from the Hawaiian eagle bones: 392
nucleotide sites of 12S rRNA, 118 bp of cyt b, and 194
bp of ATPase8 (Table 1). We compared the cyt-b se-
quences with those obtained by Seibold and Helbig
(1996) for seven species of Haliaeetus, and the 12S and
ATPase8 sequences with ones we obtained from H.
albicilla, H. leucocephalus, and H. leucogaster (Table 1).
The cyt-b sequence of the Hawaiian Haliaeetus was
identical to that of H. albicilla and differed by 5 to 17
bp from all other Haliaeetus taxa, including H. leu-
cocephalus. The more-conserved 12S sequence was
identical to that of H. albicilla but differed by two
transitions from the sequence for H. leucocephalus and
by 24 substitutions from H. leucogaster. The Hawaiian
eagle bone ATPase8 sequence differed at only 3 po-
sitions from H. albicilla, but differed at 6 positions
from H. leucocephalus and at 24 positions from H. leu-
cogaster.
In summary, the Hawaiian Haliaeetus bone se-
quences differed at only 3 of 704 bp (0.4 %) from the
White-tailed Eagle, but by 13 of 704 (1.8 %) from the
Bald Eagle. Divergence of the Hawaiian eagle bone
sequence from the Bald Eagle sequence was signi?-
cantly greater than its divergence from the White-
tailed Eagle sequence (Fisher exact test, P 5 0.021).
Divergence of the Hawaiian bone sequence from the
White-tailed Eagle sequence was not signi?cantly
different from zero (Fisher exact test, P 5 0.250).
The three ATPase8 sequence differences between
the Hawaiian eagle skeleton and White-tailed Eagle
sequences included two A-G transitions and one A-
T transversion (Table 1). Because of the rarity of A-T
transversions in mtDNA at this level of divergence,
and the fact that the T site of the latter was unique to
the Hawaiian eagle bone, it is possible that it repre-
1054 [Auk, Vol. 117Short Communications
FIG. 1. Total-evidence parsimony phylogram showing relationships among mtDNA sequences from Ha-
waiian bone and from three species of Haliaeetus (see Table 1). Note that the outgroup sequences are chimeric
for individuals: ATPase8 and 12S rRNA were obtained from individuals sequenced as part of this study, and
cyt-b sequences are from different individuals described in Seibold and Helbig (1996). The 98% bootstrap
value for the Hawaiian bone and H. albicilla clade is on the clade's node.
sents a postmortem artifact rather than a real sub-
stitution. Other than this single change, which does
not modify our conclusions, no other differences are
suggestive of artifacts owing to environmental mod-
i?cation.
The reconstructed phylogeny (Fig. 1) shows the
close relationship of the Hawaiian bone sequences to
those of H. albicilla and their more-distant relation-
ship to H. leucocephalus and the outgroup taxon H.
leucogaster. Bootstrap support for a Hawaiian bone/
H. albicilla clade was very high (98%). Constraining
the Hawaiian bone sequences to be sister to H. leu-
cocephalus increased the tree length by ?ve steps, but
the Kishino-Hasegawa test was not quite signi?cant
in supporting the Hawaiian bone/H. albicilla clade
over the Hawaiian bone/H. leucocephalus clade (t 5
1.9, P 5 0.058).
Discussion.?We obtained mtDNA sequences from
two subfossil Hawaiian eagle bones that are nearly
identical to ones from Haliaeetus albicilla. Our anal-
yses were conducted in two separate laboratories us-
ing independently purchased supplies, solutions,
and synthesized primers. No outgroup samples of
Haliaeetus were present in the laboratories used at
Durham University (where the cyt-b and some
12SrRNA sequences were obtained). In the NZP lab-
oratory (where the ATPase8 and 12SrRNA sequences
were obtained), all outgroups were analyzed after
DNA isolation from the subfossil. Thus, it is extreme-
ly unlikely that these sequences are the result of con-
tamination by external sources of DNA.
It is remarkable that DNA and amino acids are well
preserved in this skeleton that has lain in a cave in a
subtropical climate for perhaps 3,500 years, espe-
cially when considering that the eagle skeleton was
exposed to dripping water in the cave and was moist
when collected. Factors that may have favored pres-
ervation of organic compounds include the relatively
constant, cool temperatures of this high-elevation
cave, and the fact that the eagle corpse was always
protected from UV radiation, being preserved in
darkness.
Our results show that DNA sequences of the Ha-
waiian Haliaeetus are more similar to sequences of
the White-tailed Eagle than they are to those of the
Bald Eagle. They do not support a hypothesis that
the Hawaiian eagle skeleton is conspeci?c with the
Bald Eagle. The protein-coding sequence divergence
(i.e. cyt b and ATPase8, combined) between the two
(3.5%) is higher than might be expected for a within-
species level divergence in birds and more typical of
divergence between congeneric species (Klicka and
Zink 1997, Avise and Walker 1998). On the other
hand, the divergence between the White-tailed Eagle
and Hawaiian bone protein-coding sequences (1.0%)
is much lower and is within the typical range of
within-species variation. Our results do not allow us
to reject the hypothesis that the Hawaiian skeleton is
that of a White-tailed Eagle, or its very close relative.
Therefore, we suggest that the Hawaiian eagle is con-
speci?c with and represents a disjunct Hawaiian
population of the White-tailed Eagle.
The similarity of the sampled sequence from the
Hawaiian eagle to that of the White-tailed Eagle also
suggests that this species colonized the archipelago
at a relatively late date, although one that apparently
October 2000] 1055Short Communications
preceded the arrival of humans (James 1995). The
relative recency of colonization is also supported by
the absence of eagle fossils in the sediments of a
Pleistocene lake at Ulupau Head, Oahu (James 1987)
and the lack of skeletal differences between the Ha-
waiian eagle and H. albicilla (Olson and James 1991).
The Hawaiian eagle and the Black-crowned Night-
Heron (Nycticorax nycticorax) are the only lineages of
Hawaiian birds that successfully colonized the ar-
chipelago without the aid of humans, but that have
not differentiated at least to the level of subspecies.
This, and other evidence (e.g. Olson and James 1991,
Fleischer and McIntosh 2000) suggest that coloniza-
tion of the Hawaiian Islands is an ongoing process
and that ``equilibrium'' diversities predicted by the-
ories of island biogeography have not yet been
reached.
Although the eagle appears to have been the only
indigenous predator capable of depredating the
large ?ightless waterfowl of the islands (see Olson
and James 1991), genetic and other data indicate that
these waterfowl existed in the islands well before the
arrival of H. albicilla (Paxinos 1998, Sorenson et al.
1999).
Our study demonstrates the utility of analyzing
fossil or ancient DNA for identifying species when
other approaches will not work. The identi?cation of
the Hawaiian eagle adds another Palearctic element
to the sources of colonization of the Hawaiian avi-
fauna. Most avian lineages in Hawaii appear to have
colonized from the New World, particularly North
America, and very few from the Palearctic (Mayr
1943, Fleischer and McIntosh 2000). Other lineages of
Palearctic origin include the Laysan Rail (Porzana pal-
meri; Olson 1973a,b; Fleischer and McIntosh 2000)
and possibly the Acrocephalus warblers and the dre-
panidines (Groth 1994, Fleischer et al. 1998). Histor-
ical records of eagles in the Hawaiian chain include
a Golden Eagle (Aquila chrysaetos) resident on Kauai
for 17 years (Berger 1981, Pyle 1997) that may have
originated in either North America or Asia, and the
Asian Steller's Sea-Eagle (H. pelagicus) that was re-
corded from the Northwestern Hawaiian Islands of
Kure and Midway (Pyle 1997). Haliaeetus albicilla is a
latitudinal migrant in Eurasia, especially in the east-
ern part of its range (del Hoyo et al. 1994), and some-
times is a vagrant and thus could be expected to have
reached the islands.
Acknowledgments.?We thank R. Michael Severns
and Pauline Fiene-Severns for guiding us to Puu
Makua Cave and helping to collect the eagle skele-
ton. C. Pickett (National Zoological Park), P. Erickson
(Swedish Museum of Natural History), and W. Piper
generously contributed samples of outgroup Haliaee-
tus, and R. Hoelzel and C. Shaw provided lab facili-
ties in Durham. We also thank Thomas Stafford, Jr.,
for helping to collect the skeleton and for providing
results of his CHN, amino acid, and radiocarbon
analyses. We thank R. Chandler, R. Prum, and an
anonymous reviewer for comments on the manu-
script, including the request for phylogenetic anal-
ysis. The research was supported by the National
Geographic Society; Friends of the National Zoo; and
the Wetmore, Walcott, and Scholarly Studies Funds
of the Smithsonian Institution.
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logeographic effects on avian populations and
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BERGER, A. J. 1981. Hawaiian birdlife, 2nd ed. Uni-
versity of Hawaii Press, Honolulu.
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Associate Editor: R. O. Prum
The Auk 117(4):1056?1060, 2000
Are Nest Predation and Brood Parasitism Correlated in Yellow Warblers? A Test of
the Cowbird Predation Hypothesis
CELIA M. MCLAREN AND SPENCER G. SEALY1
Department of Zoology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
Brown-headed Cowbirds (Molothrus ater) are gen-
eralist brood parasites that reduce the productivity
of many of their passerine hosts through egg removal
and competition between cowbird and host nestlings
(Lowther 1993, Payne 1998, Lorenzana and Sealy
1999). Recently, it has been suggested that cowbirds
exert a previously unappreciated in?uence on the de-
mography of their hosts through predation (Arcese
et al. 1992, 1996; Smith and Arcese 1994; Arcese and
Smith 1999). The cowbird predation hypothesis (Ar-
cese et al. 1992) was suggested to explain the fre-
quently observed link between nest predation and
interspeci?c brood parasitism in many passerines
(e.g. Wolf 1987, Payne and Payne 1998). Speci?cally,
the hypothesized link is a direct result of cowbird
1 Address correspondence to this author. E-mail:
sgsealy@cc.umanitoba.ca
predation of host nests that are discovered too late in
the host's nesting cycle for successful parasitism.
This creates future opportunities for parasitism by
causing hosts to renest (Arcese et al. 1996). Alter-
natively, a link between predation and parasitism
may result if cowbird parasitism facilitates preda-
tion, or if both factors are correlated with a third var-
iable such as nest vulnerability (Arcese and Smith
1999). Determining the independent effects of brood
parasitism and nest predation on host populations is
dif?cult because of this potential functional depen-
dence (Pease and Grzybowski 1995, Arcese and
Smith 1999).
In a resident population of Song Sparrows (Melos-
piza melodia) on Mandarte Island, British Columbia,
the frequency of nest failure was positively related to
the frequency of cowbird parasitism (Arcese et al.
1996). In 5 of 19 years when cowbirds were rare, ab-
sent, or removed on the island, nest failure was lower
October 2000] 1057Short Communications
than when one or more cowbirds were present on a
consistent basis. To explain these trends, Arcese et al.
(1992) proposed the cowbird predation hypothesis,
which they subsequently tested directly (Arcese et
al. 1996). A critical prediction of the hypothesis was
supported when Arcese et al. (1996) found that par-
asitized nests failed less often than unparasitized
nests when female cowbirds maintained exclusive
territories, because cowbirds should regularly dep-
redate unparasitized nests but not nests they have al-
ready parasitized.
To assess whether results from this island popu-
lation were unique, J. N. M. Smith and M. J. Taitt (un-
publ. data) and Rogers et al. (1997) analyzed com-
parable data from Song Sparrows on Westham Is-
land, near mainland British Columbia. In both stud-
ies, lower cowbird numbers were associated with
higher host nesting success, as predicted. However,
Rogers et al. (1997) found no difference in the fre-
quency of nest failure in parasitized and unparasit-
ized nests, contrary to the key prediction of the hy-
pothesis. Published analyses of potential correlates
of cowbird predation behavior are limited to these
populations of Song Sparrows. Arcese et al. (1996)
cited several anecdotal accounts of cowbirds de-
stroying passerine nests and also called for addition-
al independent tests of the cowbird predation hy-
pothesis (see also Arcese and Smith 1999).
We compared the frequency of failure of parasit-
ized versus unparasitized nests in a population of
Yellow Warblers (Dendroica petechia) in the Delta
Marsh region of southern Manitoba, using data col-
lected from 1974 to 1976. The cowbird predation hy-
pothesis predicts that unparasitized nests should fail
more often than parasitized nests when cowbird
egg-laying ranges do not overlap. In contrast, para-
sitized nests may fail more often than unparasitized
nests if cowbird egg-laying ranges overlap, or if cow-
birds facilitate rather than cause predation. We also
examined the evidence in support of the prediction
that unparasitized hosts whose nests are depredated
should have a high probability of their replacement
nests being parasitized. We further tested the general
hypothesis that nest predation is linked to brood par-
asitism, either directly or through facilitation, by ex-
amining the relationship between frequency of nest
predation and parasitism in seven Yellow Warbler
populations across North America (cf. Arcese and
Smith 1999).
Yellow Warblers are a common host of the cowbird
at Delta Marsh (19.1% of 2,163 nests parasitized;
Neudorf and Sealy 1994) and elsewhere. They typi-
cally accept cowbird eggs laid after the midpoint of
the laying stage but respond to eggs laid earlier ei-
ther by burial of the cowbird egg (and any host eggs)
under a new nest, or by desertion followed by re-
nesting at a different site (Clark and Robertson 1981,
Sealy 1995).
Methods.?During 1974 to 1976, Yellow Warblers at
Delta Marsh suffered high cowbird parasitism and
high predation. Further details of the study site, spe-
cies, and methods used to ?nd and follow nests are
given in Goossen (1978) and Sealy (1995). We ex-
amined data for 331 Yellow Warbler nesting at-
tempts. Parasitism frequency (proportion of all nests
that received one or more cowbird eggs) was 26%.
We calculated the frequency of failure of parasitized
and unparasitized nests in the subset of nests sur-
viving at least to the second day of incubation to
eliminate the bias of unequal exposure to parasitism.
Nest-failure frequency is de?ned as the proportion of
all nests that ?edged no young. A parasitized nest in
which the cowbird egg (and Yellow Warbler eggs, if
present) was buried and a new clutch was laid was
considered a single parasitized nesting attempt.
Where possible, we identi?ed cause of nest failure for
each unsuccessful nesting attempt. For example,
weather or predation could be implicated con?dently
in many cases. Only nests that clearly failed as a re-
sult of loss of most or all of the eggs or nestlings were
considered to have been depredated.
Nesting stage at which failure occurred was as-
signed on the basis of laying date of the ?rst egg for
nests found during building or laying or by back-
dating from the ?rst day of incubation (i.e. the day
after the last warbler egg is laid) or from the date of
hatching for nests found later. For nests that were
partially depredated over several days, the stage at
the ?rst day of egg or nestling loss was considered
the stage of predation. Common nest predators at
Delta Marsh include red squirrels (Tamiasciurus hud-
sonicus), Common Grackles (Quiscalus quiscala), Yel-
low-headed Blackbirds (Xanthocephalus xanthocephal-
us), Red-winged Blackbirds (Agelaius phoeniceus),
Baltimore Orioles (Icterus galbula), and Gray Cat-
birds (Dumetella carolinensis; see Sealy 1994).
We used corrected x2 tests to compare the frequen-
cy of failure of parasitized and unparasitized nests,
combining data for all years. We also performed an
additional test con?ned to nests for which failure
could be con?dently attributed to predation. We ex-
amined the relationship between parasitism and pre-
dation in seven populations of Yellow Warblers from
Ontario (S. Yezerinac unpubl. data), Michigan (Batts
1958, DellaSala 1985), Colorado (C. Ortega and J. Or-
tega unpubl. data, Howe and Knopf 2000), Montana
(J. Tewksbury unpubl. data) and Manitoba (this
study). We used Spearman rank correlation, weight-
ed by sample size, to determine whether the per-
centage of nests depredated was related to the per-
centage of nests parasitized.
Results and Discussion.?The frequencies of failure
of parasitized and unparasitized nests were opposite
to predictions of the cowbird predation hypothesis:
parasitized nests failed more often (52% of 64 nests)
than unparasitized nests (37% of 214 nests; x2 5 3.81,
P 5 0.05). To assess whether predation and parasit-
ism are linked, we compared the frequency of failure
1058 [Auk, Vol. 117Short Communications
of parasitized and unparasitized nests for failures
that we could con?dently attribute to predation.
Again, the trend was opposite to predictions, al-
though the difference was not statistically signi?cant
(40% of 52 parasitized nests vs. 30% of 192 unpara-
sitized nests depredated; x2 5 1.69, P 5 0.19). These
results did not support the cowbird predation hy-
pothesis.
In contrast to Arcese et al.'s (1996) results, unpar-
asitized nests were less likely to fail than were par-
asitized nests. This result was not unexpected be-
cause Yellow Warblers frequently respond to para-
sitism by nest desertion (Clark and Robertson 1981,
Burgham and Picman 1989, Sealy 1995). However,
because we considered only nests that failed after the
window for parasitism (after the second day of in-
cubation), any nests deserted in response to parasit-
ism within this window were not included. Similarly,
Payne and Payne (1998) found that parasitized In-
digo Bunting (Passerina cyanea) nests failed more of-
ten than unparasitized nests. This result may occur
if cowbirds facilitate predation by their activities
near host nests (Arcese and Smith 1999). However,
because the frequency of failure was not signi?cantly
higher in parasitized nests, we have no evidence to
support the cowbird facilitation hypothesis.
Identi?cation of nest predators is crucial to under-
standing the association between nest failure and
brood parasitism. Field signs after nest predation
only occasionally are useful in identifying nest pred-
ators (Arcese and Smith 1999, Thompson et al. 1999);
hence, we have no clear record of whether cowbirds
or some other predator were responsible for nesting
failures. However, many nest predators exist in the
study area, and of several observed predation events
at Yellow Warbler nests, none involved cowbirds
(Sealy 1994). In a similar habitat, Thompson et al.
(1999) documented only one case out of 25 predation
events recorded on video of nest predation by a cow-
bird. We conclude that cowbirds probably were not
responsible for many of the predation events at the
Yellow Warbler nests in our sample.
The critical prediction of the cowbird predation
hypothesis is that parasitized nests should fail less
often than unparasitized nests, but this prediction
would hold only when individual female cowbirds
have distinct egg-laying ranges, because a female
should destroy unparasitized nests but not nests she
has parasitized (Arcese et al. 1996). When female
cowbirds defend exclusive intraspeci?c nesting ter-
ritories, any parasitized nest a female encounters is
likely to contain her own egg. On the other hand, if
cowbird egg-laying ranges overlap, different females
would be likely to discover the same nests if some
nests are especially conspicuous to cowbirds in gen-
eral, and subsequent females may depredate a par-
asitized nest. Therefore, parasitized nests should fail
more often than unparasitized nests (Arcese et al.
1996).
One could argue that the trend we documented re-
sulted from overlap of egg-laying ranges among fe-
male cowbirds. However, recent DNA work has
shown that egg-laying ranges of female cowbirds do
not overlap at Delta Marsh (Alderson et al. 1999),
and we have no reason to believe that this was not
the case 25 years ago. Indeed, egg-laying ranges like-
ly overlapped less often then, because Yellow War-
bler nesting density was about twice what it is at pre-
sent, but cowbird densities were similar (S. Sealy un-
publ. data). If host nests are abundant, female cow-
birds should not need to search wide areas for host
nests. The low frequency of multiple parasitism in
the Yellow Warbler data set (8 of 85 parasitized nests
received more than one cowbird egg) also supports
the claim that egg-laying ranges overlapped little.
Yellow Warblers frequently respond to parasitism
by deserting (burying) the cowbird egg. In the pre-
sent sample, burial occurred at 39% of parasitized
nests. A nest at which burial has occurred and a new
clutch is laid may suffer increased exposure to pre-
dation compared with a nest that proceeds directly
to incubation. This difference would bias our results
against the prediction of higher frequency of failure
of unparasitized nests. When we excluded nests at
which burial had occurred, our results were un-
changed. In addition, the frequency of nesting failure
did not differ between parasitized nests in which
cowbird eggs were buried versus accepted (x2 5 3.02,
P 5 0.08), and the trend was opposite to expectation
because ``burial'' nests failed less often (36% of 25
nests) than ``acceptance'' nests (62% of 39 nests).
Predation of a parasitized nest in which the cow-
bird egg is buried is predicted from the cowbird pre-
dation hypothesis only if cowbird egg-laying ranges
overlap. Otherwise, a female probably remembers
the nests she has parasitized (Sherry et al. 1993), and
predation of these nests would be a form of the ``Ma-
?a'' behavior described in parasitic cuckoos (Zahavi
1979, Soler et al. 1995). If Ma?a-like behavior occurs,
failures due to predation of parasitized Yellow War-
bler nests should occur more often when egg rejec-
tion (i.e. burial) has taken place than when it has not.
This was not the case in our study.
Another prediction of the cowbird predation hy-
pothesis is that unparasitized hosts whose nests are
depredated should have a high probability of their
replacement nest being parasitized. We could not
test this prediction because the identities of Yellow
Warblers that renested were not known. However, in
?ve cases where a ?rst nest was depredated (two
during laying, three during incubation), the same
nests were reused for the new nesting attempts, pre-
sumably by the same adults. None of these replace-
ment clutches was parasitized.
We present two explanations for our failure to sup-
port predictions of the cowbird predation hypothe-
sis. First, host density is high at Delta Marsh, and op-
portunities for parasitism should not be limiting. In
October 2000] 1059Short Communications
FIG. 1. Percent of nests depredated versus per-
cent parasitized in seven populations of Yellow War-
blers, weighted by the number of nests studied. Size
of open circles indicates sample sizes for each pop-
ulation: Ontario (ON, n 5 74; S. Yezerinac unpubl.
data); Manitoba (MB, n 5 331; this study); northern
Colorado (nCO, n 5 73; Howe and Knopf 2000);
southwestern Colorado (swCO, n 5 93; C. Ortega
and J. Ortega unpubl. data); southern Michigan (sMI,
n 5 20; Batts 1958); Montana (MO, n 5 358; J. Tewks-
bury unpubl. data); southeastern Michigan (seMI, n
5 25; DellaSala 1985).
contrast, this may not be the case on Mandarte Is-
land, where few alternate host species occurred.
However, Smith and Taitt (unpubl. data) document-
ed further support for the cowbird predation hy-
pothesis in Song Sparrows on Westham Island, Brit-
ish Columbia, where alternate hosts were present.
Despite this range of opportunity, alternate hosts are
rarely used on Westham Island (J. N. M. Smith pers.
comm.). Second, the timing of breeding of alternate
host species is less synchronous at Delta Marsh than
on Mandarte Island, and the presence of hosts late in
the breeding season means that opportunities for
parasitism are greater there. On Mandarte Island,
cowbirds sometimes continue to lay until the end of
the Song Sparrow laying season (Smith and Arcese
1994), but after this date, they are rarely seen on the
island. At Delta Marsh, however, several other fre-
quently parasitized hosts continue to breed later in
the season, including Song Sparrows that lay second
clutches. Both of the explanations noted above pre-
suppose that nest predation confers a cost for cow-
birds. If a cowbird incurs no cost by depredating a
host nest, nests discovered too late for parasitism
should always be destroyed regardless of the num-
ber of opportunities for parasitism.
Surprisingly, we found that across seven popula-
tions of Yellow Warblers, predation and parastism
were signi?cantly negatively correlated (rs 5 20.85,
P 5 0.008; Fig. 1), in contrast to what has typically
been documented (e.g. Johnson and Temple 1990,
Donovan et al 1997, Arcese and Smith 1999). We sug-
gest that the frequently observed link between pre-
dation and parasitism cannot be explained by the
cowbird predation hypothesis or the cowbird facili-
tation hypothesis, but instead represents a coinci-
dental preference by nest predators and brood par-
asites for particular habitat features (Donovan et al.
1997, Tewksbury et al 1998). The only way to sepa-
rate the independent in?uence of cowbirds as pred-
ators is to compare predation frequencies in popu-
lations where cowbirds are naturally or experimen-
tally variable in abundance (Arcese and Smith 1999).
In Song Sparrows, this test suggests that changes in
the frequency of parasitism are directly linked to
changes in the frequency of nest predation (Arcese
and Smith 1999). Further experiments on other lo-
cally abundant hosts, such as Yellow Warblers, are
needed.
Acknowledgments.?We thank J. P. Goossen for col-
lecting the original data and the staff of the Univer-
sity of Manitoba Field Station at Delta Marsh for lo-
gistical support. The comments of P. Arcese, A. L. A.
Middleton, S. I. Rothstein, J. N. M. Smith, J. J. Tewks-
bury, and an anonymous reviewer greatly improved
the manuscript. We also thank C. P. Ortega, J. N. M.
Smith, J. J. Tewksbury, and S. Yezerinac for kindly
supplying unpublished data. Funds were provided
by a research grant to SGS from the Natural Sciences
and Engineering Research Council of Canada.
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Received 27 September 1999, accepted 28 April 2000.
Associate Editor: S. I. Rothstein