Single-copy DNA-DNA hybridization was used to establish phylogenetic relationships among 13 species of waterfowl (Anatidae) chosen from 10 tribes. On the basis of UPGMA clustering of [delta]T m to distances, we suggest that the tribes Anatini, Aythyini, Tadornini, Mergini, and Cairinini diverged more recently than the Anserini, Stictonettini, Oxyurini, Dendrocygnini, and Anseranatini. The Freckled Duck (Stictonetta naevosa, tribe Stictonettini)
is only distantly related to the other Anatidae. Presumably the lineage diverged very early. The sheldgeese (tribe Tadornini) and the true geese (Anserini) are only remotely related. The Oxyurini, considered to be in the subfamily Anatinae, is remotely related to the other Anatidae. The Dendrocygnini form an isolated tribe with no close relationship to the swans and geese (subfamily Anserinae). We found that the screamers (Anhimidae) are distantly related to the Anatidae.
A method to estimate missing cells in a data matrix of pairwise distances is presented. Received 1 May 1987, accepted 16 February 1988.
Institute of Molecular Biophysics, The Florida State University, Tallahassee, Florida 32306 USA
RECENTLY, nucleic acid sequence analysis has
been used to estimate relationships of many or-
ganisms. The analysis can include direct se-
quencing of individual cloned genes or gene
products, restriction enzyme analysis of ho-
mologous DNA fragments, and general analysis
of unique DNA by single-copy DNA-DNA hy-
bridization. In higher eukaryotes, perhaps only
5-10% of the DNA (including gene-coding re-
gions and a small fraction of the single-copy
DNA) is under selection (Britten 1986). The ma-
jor proportion of DNA, unique as well as re-
petitive, consists of nucleotide sequences that
are never expressed as proteins, have no known
function, and are therefore presumably free to
evolve (Britten 1986).
Although both single-copy and repeated DNA
comparisons have been used in studies of phy-
logenetic relationships in birds (Shields and
Strauss 1975; Eden et al. 1978; Sibley and Ahl-
quist 1982, 1983), the total DNA of higher eu-
karyotes is composed of many subsets of DNA
that show varying degrees of reiteration (Brit-
ten and Kohne 1968). These include the ex-
tremes of specific sequences, present as only
one copy per genome through sequences re-
peated millions of times. The repeated families
show sequence as well as copy-number evolu-
tion. Sequences that occur only a few times in
one organism may occur many times even in a
closely related species (Zwiebel et al. 1982). De-
pending on reassociation conditions, the re-
petitive DNA fraction in bird genomes makes
up about 10-40% of the total DNA (Eden et al.
1978) but contains only a small percentage of
the total sequence diversity. The remaining 60-
90% are unique sequences and represent about
98% of the total sequence complexity (Sibley
and Ahlquist 1983).
The most convenient procedure for sequence
comparison of total DNA of different species
involves measurement of the thermal stability
of reannealed heteroduplex single-stranded
DNA. Because repetitive DNA sequences rean-
neal first, such sequences might contribute in
a disproportionate way to the thermal stability
of resulting duplexes (Sibley and Ahlquist 1983).
For this reason the repetitive DNA fraction is
usually removed. Nevertheless, repetitive se-
quences, because of rapid evolution, can also
provide valuable data on the relationship of
recently evolved species (Gillespie 1977, Ar-
nason and Widegren 1986).
Although the fossil record of birds is incom-
plete, that of waterfowl appears better than in
many other families (Olson and Feduccia 1980).
Paleontological investigations show that the
major waterfowl radiation probably took place
within the last 60 million years (Delacour and
Mayr 1945, Olson and Feduccia 1980). These
events fall within the sensitivity range of DNA-
DNA hybridization proposed by Sibley and
Ahlquist (1983).
We propose a phylogeny of the Anatidae
based on single-copy DNA relationships among
samples from 13 of the 148 species of waterfowl
belonging to 10 traditionally defined tribes.
Single-copy DNA sequence relationships among
these waterfowl and three other avian species
(Northern Screamer, Chauna chavaria; Spur-
winged Plover, Vanellus spinosus; and domestic
chicken, Gallus gallus) were investigated.
MATERIALS AND METHODS
Material came from Mergus Waterfowl Farms (Tal-
lahassee, Florida). Species were the White-faced
Whistling-Duck (WW) (Dendrocygna viduata), Plumed
Whistling-Duck (D. eytoni), Bar-headed Goose (BG)
(Anser indicus), Red-breasted Goose (Branta ruficollis),
Ruddy Duck (RD) (Oxyura jamaicensis), Orinoco Goose
(OG) (Neochen jubata), Hooded Merganser (HM) (Lo-
phodytes cucullatus), Wood Duck (WD) (Aix sponsa),
White-eyed Pochard (WP) (Aythya nyroca), Mallard
(ML) (Anas platyrhynchos), White-cheeked Pintall (WC)
(Anas bahamensis), and the nonanatid Spur-winged
Plover and domestic chicken. Northern Screamer (SC)
material came from Mary Dam (Haines City, Florida),
the Magpie Goose (MG) (Anseranas semipalmata) and
Trumpeter Swan (TS) (Cygnus buccinator) from Mi-
chael Lubbock (Sylva, North Carolina), and the Freck-
led Duck (FD) (Stictonetta naevosa) from the Wildfowl
Trust (Slimbridge, United Kingdom). Gamma-labeled
ATP 32 was obtained from ICN (Irvine, California).
Isolation of DNA.--DNA was isolated from blood
cells by a procedure modified from Blin and Stafford
(1976). Blood was obtained from a wing vein and
collected in a heparinized syringe. After one-fold di-
lution with 0.15 M NaC1 containing 0.05 M EDTA at
pH 7.6 (buffer A), cells were collected by centrifu-
gation, resuspended in 25 volumes of buffer A, and
lysed by the addition of 0.3% sodium dodecyl sulfate.
An equal volume (with respect to the total lysate) of
redistilled phenol, equilibrated with 0.1 M Tris-HCL
buffer pH 9.0, was added, and the mixture was shaken
in a gyrotory waterbath overnight at room tempera-
ture (25øC). After the addition of one-half volume of
chloroform, the phases were separated by centrifu-
gation, the aqueous phase collected, and the crude
DNA precipitated by the addition of an equal volume
of absolute ethanol. The DNA was washed with 70%
ethanol and dissolved in TE (0.01 M Tris-HCL buffer
pH 7.6, 0.001 M sodium EDTA) (5 ml/ml original
blood). After complete solution was obtained, 0.5 ml
of 1.0 M Tris-HCL buffer pH 8.0 was added followed
by 100 g of pancreatic ribonuclease. The mixture was
incubated for 30 min at 37øC, 5 mg of pronase was
added, followed immediately by 0.2 ml of 10% sodium
dodecyl sulfate, and the solution was incubated for 2
h at 60øC. The mixture was again deproteinized by
shaking overnight with an equal volume of phenol
saturated with 0.1 M Tris-HCL buffer pH 9.0, the
phases were separated by centrifugation, and the DNA
was precipitated with two volumes of cold ethanol
after the addition of ammonium acetate to a final
concentration of 2.5 M. After briefly washing with
70% ethanol, the DNA was dissolved in TE buffer and
the concentration adjusted to 1.0 mg/ml.
Isolation of single-copy DNA.--A DNA fraction en-
riched for single-copy material was isolated as de-
scribed by Sibley and Ahlquist (1983). DNA was
sheared by sonication into fragments with an average
length of 500 nucleotides. Fragment size was deter-
mined by electrophoretic comparison with calibrated
markers. The DNA was denatured by immersion in
boiling water for 10 min and reannealed at 50øC in
0.48 M sodium phosphate buffer (pFI 7.0) to a Cot of
1,000. A single stranded fraction was isolated by hy-
droxyapatite chromatography at 50øC. The fraction
was dialyzed against TE buffer, concentrated with iso-
butanol (Maniatis et al. 1982), and precipitated with
ethanol. After washing with 70% ethanol the DNA
was dissolved in TE buffer and the concentration ad-
justed to 1.0 mg/ml.
Radio-labeling of single-copy DNA.--Single-copy DNA
was labeled with 32P at the 5' terminus by incubation
with polynucleotide kinase and gamma-labeled ATP 32.
A 5' terminus labeling kit was obtained from Bethesda
Research Laboratories and used according to the in-
structions provided by the manufacturer. The specific
activity of the radio-labeled DNA prepared by this
procedure averaged 5 x 106 counts/g of DNA. The
labeled DNA was freed of unincorporated labeled
ATP by the spin-column technique (Maniatis et al.
1982). The maximum amount of unincorporated la-
beled ATP contamination was less than 3%. Terminal
labeling was substituted for iodination (Sibley and
Ahlquist 1983) for safety reasons.
Hybridization of single-copy tracer DNA with driver
DNA.--Hybrids were formed from a mixture com-
posed of 1 part (=200 ng) radio-labeled single-copy
DNA and 1,000 parts (=200 g) sheared, whole DNA
(Sibley and Ahlquist 1983). Hybrids were denatured
by immersion in boiling water for 10 min, precipi-
tated with ethanol, and redissolved in 0.5 M sodium
phosphate (pH 7.0). Reannealing was carried out at
60øC until a Cot of 8,000 was reached. The formation
of double-stranded hybrid DNA was analyzed by
thermal elution from hydroxyapatite. Thermal elu-
tion columns were submerged within an insulated
column box connected to a Haacke temperature-con-
trolled (ñ0.1øC) circulating waterbath. After appli-
cation of the samples to the column (200 g of DNA/
ml of hydroxyapatite) at 55øC in 0.03 M sodium phos-
phate, the column was washed with 40 ml of 0.12 M
sodium phosphate (pH 7.0) to remove the unbound
tracer. The last fraction of the wash (5 ml) was counted
in a liquid-scintillation counter, and radioactivity was
determined to be no higher than the normal back-
ground, indicating that the unbound tracer was elut-
ed from the column. At each of the eight temperatures
(60-95øC in 5øC increments) the single-stranded DNA
was eluted with 10 ml of 0.12 M sodium phosphate
8C
r
LU
r
Z
80
z c
2c
IOO
40
z
20
2
4
õ \
70 80 90
T,C
(pH 7.0) and counted in a liquid-scintillation counter.
The average number of samples chromatographed si-
multaneously was 6, with a maximum number of 10.
We used Tm to contrast homoduplex thermal disso-
ciation distribution with those of heteroduplexes
(Sibley and Ahlquist 1983, Sheldon 1987). Figure 1
gives examples of thermal elution curves from which
ATto values were extrapolated. Each 1 ø difference for
AT values indicates a 1% divergence in nucleotide
sequences (Bonner et al. 1973). All other statistics
(percentage hybridization [%H], normalized percent-
age hybridization [NPH], and T5oH) were calculated
as described by Sheldon (1987).
To calibrate AT and ToH values with time, one
must assume that DNA evolves at a constant rate (Sib-
ley and Ahlquist 1983). In view of recent evidence
suggesting a nonconstant rate (Britten 1986, Sheldon
1987), we use ATto values only to estimate the branch-
ing order.
Construction of the data matrix and phylogenetic tree.--
A 13 x 13 matrix was constructed (Table 1) from the
average ATto values for each pairwise comparison. The
23 missing AT values required for constructing a
complete matrix were estimated using the following
rationale. First, a distance function must satisfy the
triangle inequality:
d, < d,k + di.
Second, some distance functions satisfy a stronger
rule called the "ultrametric inequality" (Hartigan
1975):
d. < max(d,k, djk).
If a distance function satisfies the ultrametric in-
equality, then a matrix of such pairwise distances has
an exact representation as a tree.
Conversely, any tree has an exact representation in
terms of a matrix of distances that satisfy the ultra-
metric inequality (Hartigan 1975). We estimated miss-
ing values from the ultrametric inequality. For ex-
ample, if d23 was missing, then d2, < max(d2k, dsk) for
i I
60 70 80 90
T, C
Fig. l. Top: Thermal dissociation curves in which
the tracer DNA of the Hooded Merganser (Lophodytes
cucullatus) was hybridized with the driver DNAs of
Hooded Merganser (1), Orinoco Goose (Neochen ju-
bata; 2), Mallard (Anas platyrhynchos; 3), Wood Duck
(Aix sponsa; 4), Ruddy Duck (Oxyura jamaicensis; 5),
and White-faced Whistling-Duck (Dendrocygna vidua-
ta; 6). Bottom: Thermal dissociation curves in which
the tracer DNA of the White-faced Whistling-Duck
was hybridized with the driver DNAs of White-faced
Whistling-Duck (1), Mallard (2), Hooded Merganser
(3), Orinoco Goose (4), White-eyed Pochard (Aythya
nyroca; 5), Wood Duck (6), Ruddy Duck (7), and Spur-
winged Plover (Vanellus spinosus; 8).
July 1988] Waterfowl Phylogeny 455
TABLE 2. Reciprocal ATto values.
Mean a n b Range
Anas platyrhynchos 0.7 12 1.1
Anas bahamensis 0.1 4 0.3
Aythya nyroca 0.5 7 0.8
Aix sponsa 0.3 9 0.7
Neochen jubata 0.4 8 1.7
Lophodytes cucullatus 0.3 8 0.8
Anser indicus 1.3 7 2.7
Cygnus buccinator 1.0 6 1.6
Stictonetta naevosa 1.0 5 0.6
Oxyura jamaicensis 0.5 9 0.9
Dendrocygna viduata 0.7 11 1.7
Anseranas semipalmata 1.0 6 2.4
Chauna chavaria 1.1 6 2.8
Average distance between average of a labeled Anas platyrhynchos
distance from another species and average of the reciprocal test.
b Number of species for which reciprocal tests were made with Anas
platyrhynchos.
Anas platvrhvnch (ML)
[-- Arias bahomen$i$ (WC)
I Ap? .yoc= (wP)
Aix sponsa (WD)
-- Neoche_n ]ubato (OG)
I Lophodytes cucullatus (HM)
Anse.__._[r indlcus (BG)
Cygnus buccinotor (T$)
$tictonetta hoerosa (FD)
Cyura jamolcensis (RD)
Dendrocyna viduata (WW)
Anseran0$ $emiplmata (MG)
Cheuna Choyaria ($C)
12.0 I0.0 8.0 6.0 4.0 2.0 0.0
% DIVERGENCE
Fig. 2. Dendrogram generated by UPGMA based
on DNA-DNA hybridization (ATto) of 13 species of
waterfowl representing 10 tribes in 3 subfamilies. See
Table 1 for the distance matrix and abbreviations.
all k and hence is bounded above by the minimum
of the maxima (Meeter pets. comm.). This estimate,
the largest distance consistent with the ultrametric
inequality, was used for the missing distances. In the
estimate of the ATto for WC and WP (Table 1), 1.7 was
the smallest maximum value of all distances between
other taxa compared individually with WC and WP.
To verify the accuracy of this method, a data matrix
was constructed using only species for which com-
plete data were available (Anas platyrhynchos, Aythya
nyroca, A ix sponsa, Neochen jubata, Lophodytes cucullatus,
Oxyura jamaicensis, and Dendrocygna viduata ). Five cells
within this 7 x 7 complete matrix were chosen ran-
domly, and estimated values were compared with ac-
tual values. The mean difference between measured
and estimated values was 0.26 (SD = 0.11). The dif-
ference between measured and estimated values
ranged from 0.1 to 0.4.
The distance matrix was clustered by the unweight-
ed pair-group method using arithmetic averages
(UPGMA; Sneath and Sokal 1973).
RESULTS
Delta Tm values for a pair of taxa varied from
10% to 30% of the average value (Table 1). In
almost all individual experiments, however, the
order of the ATm was the same. Discrepancies
occurred in some reciprocal values (Table 2).
Many of the higher reciprocal ATto values were
influenced by the one or two aberrant values
relating to a particular species or an abnormally
low homoduplex T value (see below and Dis-
cussion). The NPH values ranged from 90% for
species with a AT of less than 3.0, to 75-90%
for those with a AT of less than 8.0 and more
than 3.0, to about 70% for the hybrids of single-
copy DNA between the Mallard and the Magpie
Goose (Anseranas) or the screamer (Chauna). Hy-
brids between single-copy DNA of the Mallard
and the total DNA of the chicken or the Spur-
winged Plover (Vaneflus) had NPH values of
40-50%. For unknown reasons NPH values
among closely related species often exhibit a
high degree of variance (Sheldon 1987). We
found a high degree of NPH variance (10-20%)
for both distantly related and closely related
species. For this reason we used the T statistic
in lieu of ToH. The T statistic does not incor-
porate NPH values when calculated; therefore,
any variance in NPH will not obscure the
branching pattern.
Species in the subfamily Anatinae (Mallard
[Anatini], White-eyed Pochard [Aythyini],
Hooded Merganser [Mergini], Wood Duck
[Cairini], and Orinoco Goose [Tadornini]) ap-
parently diverged more recently than the other
taxa (Fig. 2). These birds exhibited AT values
smaller than 3.2. In addition, the White-eyed
Pochard was more closely related to the Mallard
(ATto = 1.7) than to the Wood Duck, Hooded
Merganser, or Orinoco Goose. The latter two
species were consistently more closely related
(AT = 2.0) to each other than to other species.
The White-cheeked Pintail (Anatini) was also
more closely related to the Mallard (AT = 1.1)
than to the other species tested.
The remaining species showed large AT val-
ues with respect to each other and to the five
Anatinae species. The Magpie Goose (Anser-
anatinae) was the most distant species, with an
average ATm value of 9.5 with respect to all other
waterfowl. With respect to the Mallard, the
White-faced Whistling-Duck (Dendrocygnini)
showed a very early divergence (ZTm = 7.8),
followed by the Ruddy Duck (Oxyurini; ZTm =
7.5), Freckled Duck (Stictonettini; zTm = 6.5),
and Bar-headed Goose (Anserini; ZTm = 6.0).
Discrepancies among reciprocal values were
higher when the number of hybridizations for
each comparison was limited. Yet, when the
Bar-headed Goose and White-faced Whistling-
Duck were compared (n = 6), a large discrep-
ancy in reciprocal values remained. When the
Bar-headed Goose tracer DNA was hybridized
to the White-faced Whistling-Duck driver DNA,
the average ZTm was 5.9 (n = 2, SD = 0.14). The
reciprocal experiment was repeated twice and
had an average ZTm of 7.7 (SD = 0.49). The
experiment was repeated with a new tracer DNA
for each species. The ZTm values were within
0.3 ø of the previous values. The overall ZTm av-
erages for the tracer DNAs of the Bar-headed
Goose and White-faced Whistling-Duck were
5.9 (n = 3, SD = 0.10) and 7.7 (n = 3, SD = 0.38).
A similar condition existed in the Red-breasted
Goose and the Plumed Whistling-Duck. The
Trumpeter Swan was more closely related to
the Bar-headed Goose and the Red-breasted
Goose (ZTm = 4.0) than any of the waterfowl
tested (ATmS > 5.8). The AT m value (4.0) indi-
cates that this swan and goose lineage diverged
relatively long ago.
Species from the family Anatidae were com-
pared with the screamers (Anhimidae), Spur-
winged Plover (Charadriidae), and domestic
chicken (Phasianidae) (data not shown). These
data (including reciprocal values) indicated that
ducks, geese, and Anserarias semipalmata are more
closely related to screamers (average ZXTm = 11.3)
than to Spur-winged Plovers (ZXTm = 14.4) or
chickens (average ZXTm = 14.1).
DISCUSSION
We found that a single-copy DNA similarity
based on relationships among different taxa of
waterfowl was in good agreement with results
based on other criteria. The tribes in the
subfamily Anatinae apparently radiated more
recently than the Anserinae, Oxyurini, Den-
drocygnini, Stictonettini, and Anseranatinae,
which diverged much earlier. Our analysis gen-
erally agreed with the schemes advanced by
Delacour and Mayr (1945), Johnsgard (1961),
Brush (1976), and others (Frith 1955, 1964; Kear
1967; Sibley and Ahlquist 1972; Jacob and Gla-
ser 1975; Bottjer 1983; Kessler and Avise 1984;
Livezey 1986) who used anatomical, behavioral,
and other chemical or immunological data. The
branching pattern (Fig. 2) among certain species
(e.g. Lophodytes cucullatus, Neochen jubata, and
Aix sponsa) may not represent the true phylo-
genetic pattern because of the high degree of
variance and the closeness of ZTm values. Yet,
for the species tested two main periods of ra-
diation have occurred. We believe that the Mag-
pie Goose (Anseranas) lineage diverged very
early (von Boetticher 1940) and the whistling-
ducks (Dendrocygnini) and the stifftails (Oxy-
urini) diverged somewhat more recently from
the main lineage. Specific results with respect
to the early divergence of the Ruddy Duck (Oxy-
ura) are interesting because in virtually all
schemes for the classification of waterfowl this
tribe has been included in the subfamily Ana-
tinae. Delacour and Mayr (1945) considered the
Oxyurini a predabbler; Johnsgard (1978) con-
sidered the Oxyurini also to be in the Anatinae
and to have diverged after the shelducks. Feath-
er-protein data (Brush 1976) and morphological
characteristics (Livezey 1986) have supported a
possible link between the Oxyurini and the
seaducks (Mergini). The large ZTm value be-
tween members of these taxa is inconsistent with
this interpretation. Feather protein and mor-
phological similarities may be the result of con-
vergent evolution due to diving in both tribes.
The Freckled Duck gave large ZTm values
(>6.0) compared with all other waterfowl. This
species was thought to be an aberrant member
of the tribe Anatini (Delacour and Mayr 1945).
Our data support the hypothesis advanced by
Frith (1964) and Brush (1976) that Stictonetta is
only distantly related to the other Anatidae and
that its lineage must have diverged early from
the main lineage leading to other waterfowl.
We found that different values for the ZTmS
between a pair of species were generally stable,
although never as stable as reported by other
investigators (Sibley and Ahlquist 1983, Shel-
don 1987). A number of factors could produce
variance in ZTm values. One is the effect of ho-
moduplex T variance on calibrations of hetero-
duplex ZTm values. The majority of homoduplex
Tm values fell close to the ideal 85øC proposed
by Sheldon (1987), but a few were 1-2 ø below;
none were higher. A correction factor was not
employed (as done by Sheldon 1987) because
the causes of variance (e.g. tracer DNA impurity
or improper labeling) in the homoduplex Tm
might equally affect the heteroduplex Tin. De-
cisions about which values, if any, should be
corrected were difficult because the number of
hybridizations was low. The small number of
experiments for each comparison and mechan-
ical error may contribute to the increased vari-
ance. Producing exact elution rates, column
temperatures, and fraction volumes is difficult
when performing DNA-DNA hybridization ex-
periments. This was evident from the observed
higher degree of interexperimental vs. intraex-
perimental variance.
Variance in reciprocal values was usually no
greater than the degree of variance exhibited
for interexperimental ATto values. The most se-
rious discrepancies were between the geese and
the whistling-ducks. We have no explanation
for this anomalous result. Nonreciprocity in ATto
values has been considered by other investi-
gators as an indication of "experimental qual-
ity" (Sheldon 1987). The consistent difference
in reciprocal values (1.8) for these two species,
however, indicates that some factor other than
experimental error was involved. The rather
large variance precludes the resolution of in-
equalities of DNA evolution rates.
The DNA of several species of waterfowl, in-
cluding the Bar-headed Goose, contains many
"sequence families" that consist of considerable
amounts of repetitive DNA (McHugh, Madsen,
and de Kloet in prep.). The reiteration frequen-
cies of the sequences in these families can differ
considerably even among closely related species.
Certain sequences that are barely detectable in
one species are found in multiple copies in oth-
ers (sometimes accounting for 5-10% of the total
DNA). This phenomenon of sequence differ-
ences among closely related species also occurs
in Drosophila (Zweibel et al. 1982). We have not
determined the actual degree of sequence di-
vergence among the members of such families
of reiterated DNA in any species, but in some
cases such sequences may have diverged farther
than in others. This would create a disparity in
the actual sequence complexity for species' tracer
DNA. The conditions (0.5 M sodium phosphate
at 60øC) for hybridization in general usage for
taxonomic studies are considered to be of low
stringency. Especially for heterologous hybrids,
reciprocal tracer and driver combinations may
give different results. The reiterated sequences
in the driver may be partly responsible for the
discrepancies. In experiments where single-copy
instead of total DNA is used as the driver, the
difference between the reciprocal values is re-
duced by approximately 50% (Madsen and de
Kloet in prep.).
Despite the inconsistencies, the Anserini lin-
eage apparently diverged relatively early in wa-
terfowl evolution, although probably later than
the Dendrocygnini. Although these tribes are
classified in the same subfamily (Anserinae),
they are not as closely related to one another
as geese are to swans. We believe that the Orino-
co Goose (Tadornini) and the Bar-headed Goose
are only remotely related, and that their mor-
phological and behavioral similarity is the re-
sult of convergent evolution (Delacour and Mayr
1945, Johnsgard 1961).
ACKNOWLEDGMENTS
We thank Frances C. James for technical and edi-
torial assistance and Duane A. Meeter for help with
statistical analysis. We are indebted also to Lawrence
G. Abele for technical help. Alan H. Brush, Anthony
Bledsoe, and Jon Ahlquist provided useful criticisms
of the manuscript. We thank Mike Lubbock (Sylva,
North Carolina), Mary Dam (Haines City, Florida),
and the Wildfowl Trust (Slimbridge, Gloucestershire,
U.K.) for cooperation in obtaining blood samples.
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