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The Wilson Journal of Ornithology 127(3):498–502, 2015
Genetic Verification of Dizygotic Twin Embryos in the White-throated
Sparrow (Zonotrichia albicollis)
Adam M. Betuel,
1,2
Elaina M. Tuttle,
1
and Rusty A. Gonser
1
ABSTRACT.—The incidence of twinning in avian
species is a phenomenon that has been rarely encoun-
tered. A number of domestic species have been shown
to produce twins but in very low numbers. In wild
populations, only 14 species have been documented
producing twin embryos or nestlings. Despite this, it has
been postulated that birds are just as likely as any other
vertebrate to produce twin offspring. Here we describe
the discovery of dizygotic twins in a long-term study of
breeding ecology in the White-throated Sparrow
(Zonotrichia albicollis). The twin containing egg was
12%heavier than the mean and had a mass greater than
94%of eggs collected. The twin containing egg was
wider and longer than the majority of other eggs
collected during 2010 but still within the expected range
for White-throated Sparrows. Genetic analysis demon-
strated that the twin embryos were full siblings but of
different morph and sex. This is the first documented
case of twinning in our study site out of over 2000
samples over 25 years of study, and likely the first
confirmed case of twinning in this species. Received 30
June 2014. Accepted 27 March 2015.
Key words: blastoderm, dizygotic twins, egg, PCR,
twinning, White-throated Sparrow.
Avian twinning is the result of embryonic
cleavage causing identical monozygotic twins,
two fertilized blastoderms within a single yolk, or
two blastoderms within their own yolk (Hollander
and Levi 1940, Romanoff and Romanoff 1972).
Monozygotic twinning results in identical sib-
lings, while the other developmental route in-
cluding two blastoderms (dizygotic twinning)
leads to fraternal or nonidentical twins. Causation
of twinning is not well known but has been
attributed to low temperatures and hypothermia
effects on cleavage (Sturkie 1946, Batt et al.
1975) as well as the effects of hormonal changes
or enzymes on ovulation (Sarvella 1975).
It has been proposed that the likelihood of
twining in bird species is as common as in other
vertebrates (Romanoff and Romanoff 1972);
however, Byerly and Olsen (1934) reported an
incidence rate of less than one percent in the
domestic chicken (Gallus gallus) as did Riddle
(1923) in the Rock Pigeon (Columba livia). Along
with the chicken and pigeon, domesticated Emus
(Dromaius novaehollandiae; Bassett et al. 1999)
and Ostriches (Struthio camelus; Horban
´czuk
et al. 2003) have successfully produced eggs
containing multiple embryos, with some hatching
success through the aid of artificial incubation.
Occurrences of natural twinning in avian
species have rarely been recorded, particularly in
wild populations. A variety of wild species such
as the American Goldfinch (Spinus tristis; Berger
1953), Peregrine Falcon (Falco peregrinus; Pattee
1
Department of Biology, Indiana State University, 600
Chestnut Street, Terre Haute, IN 47809, USA.
2
Corresponding author; e-mail:
abetuel@sycamores.indstate.edu
498
THE WILSON JOURNAL OF ORNITHOLOGY NVol. 127, No. 3, September 2015
et al. 1984), Eastern Bluebird (Sialia sialis; Bailey
and Clark 2014) and Gadwall (Anas strepera;
Lokemoen and Sharp 1981) have all been
documented producing natural twin embryos. Of
the 14 known cases of wild twinning events (see
Bailey and Clark 2014 for review), only two
studies used molecular analyses to study the
genetic background of the twinning event: the
House Sparrow (Passer domesticus; Griffith and
Stewart 1998) as well as the Hihi (Notiomystis
cincta; Thorogood and Ewen 2006). During the
summer of 2010, a single unhatched White-
throated Sparrow (Zonotrichia albicollis)egg
was harvested and found to contain a pair of
embryos. Through molecular techniques, we were
able to determine that these unhatched embryos
represented both sexes but also both morph types
found within White-throated Sparrows. Parentage
analysis revealed that the embryos were full
siblings and the offspring of the social parents.
We detail the rare event of natural twinning, the
first documented case in the White-throated
Sparrow, and use molecular findings to not only
provide detail to the observation but also to
confirm the dizygotic nature of the embryos.
METHODS
Study System and Field Work.—The White-
throated Sparrow is a socially monogamous
migratory passerine that breeds throughout the
boreal forests of North America (Falls and
Kopachena 2010). A stable genetic polymorphism
caused by a chromosomal inversion produces
individuals of two color morphs: tan and white.
White birds are heterozygous for the inversion
while tan birds are homozygous non-carriers
(Thorneycroft 1966, 1975). In addition to pheno-
typic variations, individuals of each morph
generally demonstrate different life history
strategies and behaviors (Tuttle 2003). White-
throated Sparrows mate in a disassortative man-
ner, where an individual of one color type almost
exclusively mates with the other morph (Lowther
1961, Thorneycroft 1975). This mating system
insures the presence of both morphs following
Mendelian inheritance (Thorneycroft 1975) at
a 50:50 ratio.
Field work was conducted at Cranberry Lake
Biological Station (CLBS) in the Adirondack
Mountains of upstate New York (44u159N, 74u
789W). Data at this location have been gathered
annually in a consistent manner since 1988.
Sparrows arrive at the study site in late April to
early May and disperse onto territories. Research-
ers observe paired birds to identify territory
boundaries, note various behaviors, and search
for nests. All located nests are monitored until
hatching, at which time nestlings are measured and
weighed daily until 6 days of age. Eggs that have
not hatched by that day are collected for processing
in the lab (Federal collection permit SCCL128606,
NY State permit # 936). Eggs from abandoned
nests are also collected.
Laboratory Techniques.—All harvested eggs
are photographed, weighed, dissected, and sepa-
rated by component (i.e., yolk, shell, etc.). Length
and width of a select number of eggs (from years
2010 to 2012) were collected using ImageJ
(Rasband 2013). Embryo body length and an
age estimate are recorded for all fertile eggs.
Approximately 1mg of tissue is extracted from the
embryo, focusing on the abdominal region in
larger embryos or the entire body in small
embryos, and then placed in 100ml incubation
buffer (Promega, Madison, WI, USA). After the
tissue is broken-down manually with a pestle,
samples are kept frozen until return to the
molecular ecology lab at Indiana State University.
DNA is then extracted from the tissue sample
following the magnetic extraction DNA IQ
TM
System protocol (Promega, Madison, WI).
Genetic data from each embryo are used to
determine both morph and sex. Morph analysis is
conducted using a modification of the protocol
described in Michopoulos et al. (2007). Sex
determination is first attempted by using primers
P2 and P8 (Griffiths et al. 1998). Embryonic
samples from this project are often in poor quality
and replicating the relatively large fragment size
found in the P2/P8 primer set can be difficult. For
troublesome samples (i.e., those with degraded
DNA), the 1237L/1272H primer set designed by
Kahn et al. (1998) is used because of the shorter
fragment size. Additionally, using the White-
throated Sparrow BAC CH264-158I02 sequence
(GenBank accession number AC237007) we
modified the Kahn reverse primer to match the
White-throated Sparrow target sequence exactly,
improving the sex assay.
In addition to morph and sex assignment,
parentage is assigned to all individuals by using
a set of 8 microsatellites; Gf01 and Gf12 (Petren
1998), MME1 (Jeffery et al. 2001), Dpm01 and
Dpm03 (Dawson et al. 1997), H02, C02, and C07
(Poesel et al. 2009). After PCR amplification all
morph, sex, and parentage samples are run on an
SHORT COMMUNICATIONS
499
ABI PRISM 310H(Life Technologies, Carlsbad,
CA, USA) automated sequencer and analyzed with
the GeneScanH(Life Technologies, Carlsbad, CA,
USA) program. Parentage data from an embryo are
then compared to the social parents and confirmed
with CERVUS (version 3.0, Field Genetics Inc.,
London, UK; Kalinowski et al. 2007) using the
calculated likelihood scores (LOD).
RESULTS
On 8 July 2010, a White-throated Sparrow nest
(tan male x white female) was found containing
a pair of large chicks (5 and 6 days post hatching)
as well as an unhatched egg. Nestling age was
determined by comparing tarsus length to known
age standards, and so we calculated that the egg
was a minimum of 18 days old, laid between 17
and 20 June. Egg age was determined by adding
nestling age to the expected White-throated
Sparrow incubation length (average 512 days)
and accounting for uncertainty in the order of
laying. The egg was collected and kept in an
empty film canister inside a refrigerator until
dissection on 8 August 2010. The mass of the egg
was 2.84 g which was larger than 94%of the other
collected eggs and 12%heavier than the mean of
the other eggs collected during the 2010 breeding
season (n536, mean 52.54 g, SE 50.20). The
twin egg was 2.22 cm long and 1.65 cm wide.
These measurements fall within those expected for
White-throated Sparrows (Falls and Kopachena
2010) but are 5%longer and 8%wider than
the observed mean measurements of other un-
hatched eggs of white morph females at CLBS in
subsequent years (n59, length mean 52.10 cm,
SE 50.12; width mean 51.53 cm, SE 50.10).
Shape and coloration of the egg appeared to be
typical with those found at the CLBS study site
(Fig. 1).
Upon dissection, a pair of embryos were found
inside the egg (Fig. 2). Embryo #1 registered
a body length of 12.06 mm, while embryo #2 was
much smaller, measuring 6.47 mm in length. Age
estimates of both embryos based on body length
correspond to 5 and 3 days. Molecular analyses
revealed that embryo #1 was a female white
morph, while embryo #2 was a male tan morph.
The nest mates of the embryos were both white
individuals, one male and the other female,
comprising a 3:1 morph ratio favoring white
individuals and a 1:1 sex ratio. Parentage analysis
confirmed that the observed tan male and white
female pair were the biological and social parents.
Genetic analysis thus demonstrated that the
embryos were dizygotic twins, although the
origins (i.e., a single yolk with two blastoderms
or two yolks enclosed in a single shell) could not
be determined with absolute certainty.
DISCUSSION
Here, we document the first known case of
twinning in the White-throated Sparrow. Since the
White-throated Sparrow project at CLBS began in
1988, more than 200 eggs have been collected and
over 1800 chicks have been documented hatching
with no confirmed occurrence of twinning. Nests
that were found during incubation and followed
through hatching have never yielded more chicks
than eggs, including those where the hatching
event may have been missed. We are unsure of the
FIG. 1. The White-throated Sparrow egg containing the
dizygotic twins prior to dissection.
FIG. 2. The larger white morph female embryo below
the smaller tan morph male as well as egg contents
after dissection.
500
THE WILSON JOURNAL OF ORNITHOLOGY NVol. 127, No. 3, September 2015
incidence of twinning in nests found post hatching
as the clutch size is unknown in these cases. In
addition, we do not know if twinning occurred in
any eggs depredated prior to hatching. Though
these areas of uncertainty exist, no indication of
twinning at our study site over the course of the
project has been apparent.
Thermal stress placed upon laying females has
been shown to increase the rate of twinning.
Inducing hypothermia in domestic chickens
(Sturkie 1946) as well as chilling the eggs of
Mallards (Anas platyrhynchos; Batt et al. 1975)
both resulted in an elevated rate of twin pro-
duction. Using the age of the hatched nestlings to
estimate the range of dates that the eggs were laid,
we discovered that the weather during that period
was cool (,40uF) and rainy. These conditions are
not outside those normally experienced at CLBS
and they are much warmer than the temperatures
that were shown to affect twinning rates in those
other species. Sarvella (1975) suggested that
twinning may also be stimulated by hormonal or
enzyme effects. In our twinning case, the female
that laid the egg was a white morph mated to a tan
male. Previous studies have shown some variation
in gonadal steroid levels between females of both
morphs (Maney et al. 2009, Horton et al. 2014),
though others have only shown differences
between males (Spinney et al. 2006, Swett and
Breuner 2009), and none have demonstrated
a connection to egg production and embryo
development. Tan males tend to provide more
assistance with nesting attempts as well as guard
their mate during the fertile period (Tuttle 2003).
It can be assumed that white females on average
are less stressed, in a social and energetic context,
by the aid their mates provide. The cause of
twinning in this individual does not seem linked to
temperature or the known aspects of its social
environment, but a lack of a hormone sample
from the mother as well as other condition
measures leaves much unknown regarding the
stress level of this female.
An interesting finding was the similarity in size
between the egg containing the twin embryos and
those of single yolks/embryos. The eggs collected
during the 2010 breeding season range in date
collected, length in the refrigerator prior to
dissection, whether they were fertilized or not, as
well as how long they were incubated. These
variables along with the fact that eggs can lose
weight during incubation may have had an effect
on each individual egg’s mass. It is possible that
the egg containing the twins was significantly
heavier at laying than the others collected, but we
were unable to account for all these variables.
However, the fact that the egg was within the
normal range for mass, length, and width expected
for the species may indicate that the twins
originated from a single yolk with two blastoderms
rather than two separate yolks. A case of dizygotic
embryos arising from a single yolk has been shown
to be a rare occurrence (Levi 1957, Astheimer and
Grau 1985). The fact that the egg was not
noticeably larger upon collection is unlike most
other documented cases of twinning. Unfortunate-
ly the state of the eggs’ contents, specifically the
degradation of the liquid contents, prevented the
determination of the number of yolks present.
In our experience collecting eggs, all those still
unhatched 3 days after the start of hatching in
a nest have been inviable. The cause of death of
these eggs has not yet been studied at CLBS. Why
these twin embryos perished is unknown, al-
though the difference in size suggests that one
embryo died, contaminating the egg environment
which led to the death of the second embryo.
Since both embryos would have to be the same
age and recipients of the same levels of in-
cubation, likely causes of death may include lack
of nutrients due to sharing resources, or the
smaller embryo was inviable. Romanoff and
Romanoff (1972) discovered two peaks of mor-
tality in twins, one a third of the way through
development, and the other just before hatching.
The age estimates of the twin embryos matches
roughly with this first peak of mortality, corre-
sponding to a third of development. Through
molecular analysis including parentage and
morph/sex determination, this description of
twinning in the White-throated Sparrow provides
insight into the rarely observed phenomenon of
dizygotic twinning in wild birds.
ACKNOWLEDGMENTS
We want to acknowledge the many field assistants who
have helped on the White-throated Sparrow project
throughout the years. Our thanks also goes to the Cranberry
Lake Biological Station (CLBS), The Center for Genomic
Advocacy (TCGA) at Indiana State University (ISU), C. A.
T. Gonser, M. A. Betuel, and Zonotrichia Zeke. Funding
sources included the School of Graduate and Professional
Studies at ISU, the Northern New York Audubon Society,
as well as NSF DUE-0934648 and NIH 1R01GM084229
grants (to E. M. Tuttle and R. A. Gonser).
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