Determining Sex of Magellanic Penguins Using Molecular Procedures and Discriminant

Functions
Author(s): Marcelo Bertellotti, José L. Tella, José A. Godoy, Guillermo Blanco, Manuela G.
Forero, José A. Donázar and Olga Ceballos
Source: Waterbirds: The International Journal of Waterbird Biology, Vol. 25, No. 4 (Dec.,
2002), pp. 479-484
Published by: Waterbird Society
Stable URL: http://www.jstor.org/stable/1522534
Accessed: 17-09-2016 06:25 UTC

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 Determining Sex of Magellanic Penguins Using Molecular
Procedures and Discriminant Functions

MARCELO BERTELLOTTI1, JOSs L. TELLA2, JOSE A. GODOY3, GUILLERMO BLANCO4,

MANUELA G. FORERO5, JOS A. DONAZAR2 AND OLGA CEBALLOS6

'Centro Nacional Patag6nico (CONICET), Brown 3500, U9120ACV Puerto Madryn Chubut, Argentina
Internet: bertello@cenpat.edu.ar

2Department of Applied Biology, Estaci6n Biol6gica de Dofiana (CSIC), Av. de M Luisa s/n
Pabell6n del Perui, 41013 Sevilla, Spain

3Laboratory of Molecular Ecology, Estaci6n Biol6gica de Dofiana (CSIC), Av. de M- Luisa s/n
Pabell6n del Perui, 41013 Sevilla, Spain

4Instituto de Investigaci6n en Recursos Cinegeticos (CSIC-UCLM), Ronda de Toledo s/n 13005 Ciudad Real, Spa

5Instituto Mediterrineo de Estudios Avanzados (CSIC-UIB), Department of Natural Resources

07190 Esporles, Mallorca, Spain

6Grupo de Estudios Biol6gicos Ugarra, Carlos III 19 4?-I, 31002 Pamplona, Spain

Abstract.-Magellanic Penguins (Spheniscus magellanicus) show little sexual dimorphism, and although males a
usually larger than females, sexing by direct observation may be difficult, especially in the case of chicks. In this p
per we evaluate the utility of four different PCR-based sex determination techniques using genomic DNA for sex
Magellanic Penguins. We found that the primer set designed for sex determination in Collared Flycatcher (Ficedul
albicollis) also provided a reliable, simple and convenient sexing procedure for Magellanic Penguins. Additionall
we obtained discriminant functions for sexing adults and chicks, sampled at six colonies differing in size and othe
ecological characteristics. Discriminant function for adults used two variables, bill length and bill depth that c
rectly classified 97% of the birds. Discriminant function for chicks included bill length and flipper length and co
rectly classified 78% of the individuals. Although molecular and biometric approaches could be useful for sex
adult Magellanic Penguins, only molecular procedures proved appropriate for accurately sexing chicks. Received 24
March 2002, accepted 5 August 2002.
Key words.-Magellanic penguins, Spheniscus magellanicus, DNA, sexing birds, discriminant functions.
Waterbirds 25(4): 479-484, 2002

determination derived from biometric data

Knowledge of the sex of individuals is an
important requirement in many field stud- (Amat et al. 1993; Renner et al. 1998).
ies, but it is difficult for monomorphic spe- The Magellanic Penguin (Spheniscus ma-
cies such as most seabirds. Penguins show gellanicus) is a slightly dimorphic seabird
little sex-linked size and plumage dimor-
widely distributed along the Patagonian
phism, and although males are usually big- coast of Argentina (Yorio et al. 1998). Dis-
ger than females (Martinez 1992; Agnew and criminant functions obtained from biometry
Kerry 1995), changes with age and overlap of in individuals of known sex have been the
size between members of a pair make sexing commonest method of sexing adult Magel-
by direct observation difficult (Scolaro et al.
lanic Penguins, since two discriminant func-
1983; Amat et al. 1993). For several penguintions, obtained in two colonies, are available
species, the sex has been estimated by ob-for sexing adults (Scolaro et al. 1983; Gan-
serving copulation (Scolaro et al. 1990), clo-
dini et al. 1992). However, numerous studies
acal examination (Boersma and Davies 1987; have shown geographic body size variation
Gales 1988; Renner et al. 1998; Renner and for other penguin species inhabiting a wide
Davies 1999), or dissection (Scolaro et al.geographical area (Renner et al. 1998; Ren-
1983; Scolaro 1987; Zavalaga and Paredes ner and Davis 1999), which may limit the ap-
1997). In addition, in some studies discrimi-
plicability of discriminant models to colonies
nant functions have been developed for sex or localities where the functions were ob-

479

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 480 WATERBIRDS

tained Molecular
(Coulson et Sexingal. 1983; Eva
Mawhinney and Diamond
A drop of 1999
blood was taken by venipuncture of the
brachial or foot veins, and stored in 1-ml ethanol. Crude
A potential alternative for accu
DNA extract was prepared by boiling 5 gl of the blood/
ing penguins is
ethanolthe recently
mixture in 100 gl of a 100mM NaOH solutiond
based methods (reviewed
for 10 min. in
After centrifugation, 0.5 gl of the superna-
Sheldon 1997). Among
tant was used directly as template in PCR. thes
PCR was performed in a final volume of 25 gl con-
methods targeting CHD1-Z
taining 67 mM Tris-HC1 pH 8.8, 16 mM (NH4)2SO4, 3.5
genes are purported
mM MgCl2, 0.01% Tween-20,to0.01% be gelatin,of
0.2 mM u
each dNTP,
cation to birds, with0.2 gM each primer,
the and 0.5 Uexcep
of Taq DNA
polimerase. The thermal profile comprised an initial
species. Different primer sets
denaturation step of 94?C for 2 min, followed by a single
signed for amplifying differe
cycle of 2 min at 940C, 30 s at 55?C and 1 min at 72?C,
CHD1 genes. Amplification
and 34 cycles of 30 s at 92?C, 30 s at 50'C, 45 s at 72?C. p
A final extension step of 72oC for 5 min was added after
ing from CHD1-Z and
the last cycle. The same cyclingCHD1-W
parameters were used
tinguished through differe
with all primer sets. Twenty tl of the PCR reaction was
analyzed by
procedures. The electrophoresis in a 2% or 3% agarose
procedure usgel
containing 0.5 gg/ml ethidium bromide. PCR products
level of technical expertise de
were examined and photographed under UV light.
tive cost, and applicability to lar
samples. Although
Statistical Analyses some gene
have been applied to some
After knowing the sex of all the birds from molecu- sp
guins (Dubachlar analyses,
1996), we performed these
two-way ANOVAs to tec study
colony (random
not been tested for effect) and sex (fixed effect) differenc-
Magellanic
es in body size of adults and chicks separately. We de-
The main objective
rived discriminant functionsof this
(separately for adults and p
evaluate the utility ofprocedure
chicks) using DISCRIM four diff
of the SAS System
lar programfor
procedures (version 6.12).sexing
In some previous cases, dis-
M
criminant functions were tested against the same sam-
guins. Additionally, we
ple from which it obtained
was derived and not from an
functions derived from both adults andindependent sample, resulting in an exaggerated effec-
tiveness.
chicks previously sexed by DNA. These birdsTo avoid this, we applied ajackknife procedure
(Amat et al. 1993), in which each individual in the sam-
were sampled at six colonies of different size
ple was classified using a discriminant function derived
and ecological characteristics, and thus
from thethe total sample, excluding the individual being
classified (Chardine and Morris 1989; Amat et al. 1993).
discriminant functions could be applied to a
This algorithm chooses the function that had the lowest
wide range of the species' distributions.
percentage of misclassification. Values reported are
means ?SD.

METHODS We took precautions to avoid handling and labelin

errors, and we repeated every individual assay for whi
diagnostic bands were not clearly seen. In addition,
During January, 1999 we randomly sampled Magel-
lanic Penguins adults and chicks at six breeding repeated
colo- assays for 48 chicks and 60 adults chosen
random and obtained the same result in all cases.
nies on the Patagonian coast of Argentina: San Lorenzo
(42005'S, 63051'W), Asentamiento Oeste (42006'S,
63056'W), Isla Primera (Caleta Valdes) (42021'S,
63037'W), Caleta Interna (42027'S, 63036'W), Punta RESULTS
Tombo (44002'S, 65011'W) and Cabo Dos Bahias
(44054'S, 65032'W) (Yorio et al. 1998). These colonies
Molecular
were located along 500 km of coastline and colony size
Sexing
varied from 483 to 175,000 pairs, thus satisfactorily cov-
ering the range of colony size for the species (Tella et al. Primers 2917F/3088R (Ellegren 1996),
2001; for a detailed description of these colonies see Yo-
P2/P8 (Griffiths et al. 1998) and 2550F/
rio et al. 1998). Chicks were sampled when they were
2718R (Fridolfsson and Ellegren 1999), de-
about 70 days old (68.1 + 7.0),just few days before leav-
signed to detect intronic size differences,
ing the nests (for sampling details see Tella et al. 2001;
Forero et al. 2001; Forero et al. 2002; Forero et al. in
were all tested initially on four breeding
press). Both adults and chicks were weighed on a spring
balance to the nearest 25 g. Bill length and bill depthpairs whose members showed large size dif-
ferences, and whose sex could therefore be
were measured as described by Scolaro et al. (1983),
with a digital calliper to the nearest 0.01 mm. Flipper
confidently determined. The three primer
length was measured to the nearest mm from the hume-
ro-radial joint to the tip of the flipper (as by pairs
Zavalaga designed to amplify intron-spanning
and Paredes 1997) with a ruler. sequences successfully amplified fragments

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 SEX DETERMINATION IN MAGELLANIC PENGUINS 481

and 266 chicks

from Magellanic Penguins, although from six different colonies,
their
which
utility for gender determination provided
varied. the sample set for discrimi-
Both
P2/P8 and 2917F/3088R yielded nant the
functions.
expect-
ed patterns consisting of two bands from fe-
males and only one from males. The Discriminant Functions
estimated sizes of the bands were 490 and
470 bp for 2917F/3088R, and 360 and 380We found significant effects of both colo-
ny and sex on adult body mass and bill
bp for P2/P8, for Z and W genes respectively.
Such small differences required long runs depth, while bill length and flipper length
on 3% agarose gels to be adequately re- only differed between sexes (Table 1, see al-
so Forero et al. 2001). Male chicks were also
solved, but proved sufficient for the applica-
tion of these methods to Magellanic significantly larger in all morphometric mea-
Penguins in a relatively simple procedure. Asurements than females (Table 1). We found
single band was obtained with primerdifferences in chick bill length, bill depth
2550F/2718R from both males and females, and flipper length between sexes but not
indicating that sizes of the products from among colonies, while we found differences
CHD1-Z and CHD1-W genes were identical, in body mass among colonies but not be-
or too close to be adequately resolved bytween sexes (Table 1).
these gels. The use of these primers for sex The parameter that gave the best single
determination in this species would requirefactor correlation with sex was bill depth
different electrophoretic conditions or the(canonical correlation 0.838), and the dis-
differentiation of both products based on se- criminant function with only this variable
quence differences. We also tested an alter-classified correctly 94.9% of the cases. On
native approach originally used by Ellegrenthe other hand, bill length considered as the
(1996) to sex Collared Flycatcher (Ficedula only variable correctly classified 85.5% of the
albicollis). He designed a primer (cfR) tocases (canonical correlation 0.721). The bet-
match an intronic sequence specific of the ter discriminant function using jack-knife
CHD-W gene, which when used in combina-procedure for adults included three vari-
tion with primer 2945F yields a 210 bp W-spe-ables: bill length, bill depth and body mass
cific band. On the other hand, a third (all P-values <0.001). However, the model
primer 3224R, together with 2945F yields excluding
a body mass classified correctly a
higher number of individuals (canonical
band of 630 bp from both genes (sizes corre-
correlation 0.857) and thus we selected the
spond to Collared Flycatcher, Ellegren 1996).
This primer set amplified products of similarfollowing function:
sizes in Magellanic Penguin. A 210 bp W-spe-
cific fragment was obtained only from fe- D1 = -85.7425 + 2.4267 * bill depth +
0.5653 * bill length
males, while a 680 bp fragment was obtained
from both males and females. Both bands
Where an adult bird would be male if D1 > 0
were readily resolved by simple agarose gel and female if D1 < 0. This discriminant func-
electrophoresis, resulting in a simple, conve-
tion correctly classified 97.0% of the adult in-
nient and low-cost method for sexing Magel- dividuals (N = 331) sexed using molecular
lanic Penguins. techniques (95% of 192 males and 99% of
We tested the reliability of the assay based
139 females), improving functions previous-
on primers 2945F, cfR and 3224R by apply- ly obtained by Scolaro et al. (1983) and Gan-
ing it to a subset of 32 breeding pairs for dini et al. (1992) in five out of our six study
which sex could be confidently assigned colonies (Table 2).
based on morphology. In all pairs, one indi- The parameter that gave the best single
vidual was identified as male and the other as
factor correlation with sex of chicks was flip-
female, with the larger bird always sexed per
as length (canonical correlation 0.544),
male. We therefore applied these molecularand the discriminant function obtained with
methods for sex determination of 331 adults
only this variable classified correctly 73.7%

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 482 WATERBIRDS

Table 1. Mean values (?SD, sample size

Patagonia, Argentina, sexed by molec
ANOVAs, with sex as a fixed effect an
sented in grams.

Two-way AN

Variable Males Females Colonies Sex

Adults

Bill length 58.7 ? 2.4 53.8 ? 2.3 F5,319 = 1.5 F1,319 = 294.6
(192) (139) ns P < 0.001
Bill depth 24.0 ? 1.2 20.5 ? 1.0 F5,319 = 10.3 F1,31, = 1293.4
(192) (139) P < 0.02 P < 0.001
Flipper length 166.9 ? 6.6 158.6 ? 6.3 F5,310 = 3.8 FI, 30= 69.8
(189) (133) ns P < 0.001

Body mass 4501 + (183)

402 3708 + 355 F, 300 = 5.79 F1,300 =536.9
(129) P < 0.05 P < 0.001

Chicks

Bill length 47.0 ? 2.6 45.4 ? 2.0 F5, 254 = 0.7 F,254= 6.8
(143) (123) ns P < 0.05
Bill depth 15.8 + 1.1 14.7 ? 1.0 F5,254 = 4.0 F,254 = 47.0
(143) (123) ns P < 0.001
Flipper length 165.6 ? 5.6 158.3 ? 5.7 F5, 254 = 3.3 F1,254=81.1
(143) (123) ns P < 0.001
Body mass 3011 + 566 2709 ? 519 F5, 251 = 9.1 F1,251 = 4.6
(141) (122) P < 0.02 ns

of the cases. Theet second

al. 1990), is restricted to a small window
single factoof
depth (canonical correlation
the breeding 0.44
season. The use of vent mea-
discriminant function obtained with this surements, which in Magellanic Penguins
variable classified correctly 70.3% of correctly
the cas- predicted the sex of 92% of the in-
es. The best discriminant function obtained dividuals, is also restricted to a short period
for chicks (N = 266) included flipper length(few days after egg-laying) and only applica-
and bill depth (all P-values < 0.001). The blere-
to breeding adults (Boersma and Davies
sulting function was: 1987). Gonadal inspection through laparos-
copy requires surgery and usually works only
D2 = -42.47 +0.6869 * bill depth +
0.1976 * flipper length
Table 2. Percentage of adult Magellanic Penguins sexed
A chick would be a male if D2 > 0, and female
by DNA (192 males and 139 females) and later correctly
sexed by A) our morphometric discriminant function,
if D2 < 0. This function correctly classified
and by those previously published by B) Scolaro et al.
78% of the sexed chicks (78% of 143 males
1983 (22.17 * bill depth + 7.73 * bill length - 95.4), and
and 78% of 123 females). C) Gandini et al. 1992 (if bill depth > 22.3, male, if not,
female). The functions best classifying the sex of pen-
guins for each colony and for the whole sample are
DISCUSSION shown in bold.

Molecular procedures show clear advan-Colony A B C

tages than more conventional methods such Asentamiento Oeste 100 92.6 92.6
as behavioral observations, cloacal examina-
San Lorenzo 97.2 88.9 91.7
tion, and internal observation of gonads.
Isla Primera 94.2 88.5 90.4
Caleta
Dissection obviously involves killing the indi- Interna 98.4 88.5 100
Punta Tombo 94.4 90.0 93.3
viduals, thus being ethically questionable.
Cabo Dos Bahfas 100 92.3 96.9
Sexing penguins through behavioral obser-
Total 97.0 90.0 94.3
vations, such as copulation position (Scolaro

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 SEX DETERMINATION IN MAGELLANIC PENGUINS 483

for sexually mature individuals 2001),

(M. A.weQue-
obtained a satisfactory
vedo, pers. comm.). All of the nant
abovefunction
meth- for sexing adult in
ods are only useful for breeding Our discriminant
birds, and function techni
plicable
during particular periods of the year. to a wider geographic ra
Karyo-
the of
type analysis extends the possibility two previously published te
sexing
(Scolaro
any individual independent of age et al. 1983; Gandini et al. 1
and sam-
pling date, but with the constraint of need-
slightly improves sex determinatio
ing a blood cell culture (Dubach 1996) that
correctly classifying 97% of the ad
is more time-consuming that theall. molecular
Therefore, this technique coul
procedures we used here. for studies with large sample sizes, w
We tested four molecular assays
needfor
for sex-
total accuracy is not impera
ing Magellanic Penguins (Ellegren
Our 1996;
discriminant function ca
Griffiths et al. 1998; Fridolfsson plied satisfactorily to birds in colon
and Ellegren
ed between
1999), finding that three of them work well 42'-45'S, on the Atlanti
for this species. Thus, molecular sexing is
However, the species' range exten
confirmed for the Magellanic further:
Penguin.to We550S on the Atlantic coas
found that the primer set originally designed
Falkland Islands and up to about 45
for sex determination in the Collared Fly- (Martinez 1992). If M
Pacific coast
catcher (Ellegren 1996) was also useful for
Penguins show extra geographic v
the Magellanic Penguin. This isoutside
surprising,
of our study area that wou
since this assay was based onfere
intronic se-
with sexing by morphometrics,
quences that tend to be highly variable
criminant function could be not a
among species. Low substitution
otherrates in
colonies with the same acc
CDH1-W introns (Ellegren andshowed here.
Fridolfsson
1997), and the high magnesium concentra-
On the other hand, although Scolaro
tions, might contribute to the (1987)observed
found satisfactory functions for fledg-
cross-amplification between highly divergent
lings and yearlings (93% and 97% of the cas-
species. We have obtained similar results
es correctly classified, respectively) for a
with other avian species (Balbontin et of
single colony al.Magellanic Penguins, there
are no discriminant
2001;J. A. G., unpubl. results) indicating the functions available for
method may have a wider application than
growing chicks in this species. Moreover, the
previously suspected. This method was
reliability the chicks through biometry
of sexing
simplest and most convenient for mightsexing Ma-
be questioned, since our discriminant
gellanic Penguins, requiring only a simple
function only classified correctly 78% of the
PCR and agarose gel electrophoresis.
birds. The lowFur-percentage of our classifica-
thermore, by combining quicktion DNA prepa-
invalidates its use in many cases (e.g.,
rations with short runs in 1.5-2% agarosesex allocation in birds), given
when studying
gels, a high output can be achieved with
that the actuallim-
brood sex ratio needs to be
ited investment of labor or reagents. Al-
known (e.g., Ellegren and Sheldon 1997).
though molecular techniques are often
Although our discriminant functions
regarded as complicated, requiring
based ontechni-
morphometry might still prove use-
cal expertise and expensive infrastructure,
ful with some limitation, we found molecular
the particular protocol proposed here
analysis for
to be the most reliable and effective
sexing Magellanic Penguins involves simple
method for sexing Magellanic Penguins.
techniques, requires limited infrastructure
The usefulness of this technique is of impor-
that is common and basic in any DNA
tance analy-
in the development of ecological stud-
ies that imply the inclusion of sex as a
sis laboratory, and is relatively inexpensive.
Although we have found significant differ-
potential factor causing variation in the phe-
ences among colonies for several morpho-
nomenum studied as have been showed in
logical measurements of Magellanic
severalPenguin
studies carried out with this species
adults and chicks (see also Forero
(Forero et al.
et al. 2001; Tella et al. 2001; Forero

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 484 WATERBIRDS

et al. 2002; Forero, M. G., K. A. Hobson,

Forero etG. R.al.
Bortolotti,
inJ. A. p
Donizar, M. Bertellotti and G. Blanco. 2002. Food
ratory studies resource
aimed at testin
utilization by Magellanic penguins evaluat-
of molecular procedures
ed through stable isotope analysis: segregation byfo
with low sex and age
degree of and influence of offspring quality.dim
sexual Ma-
rine Ecology Progress Series. 234: 289-299.
quired as a tool in
Forero, M. G., J. ecologica
L. Tella, K. A. Hobson, M. Bertellotti
and G. Blanco. Conspecific food competition ex-
ACKNOWLEDGMENTS plains variability in colony size: a test using stable iso-
topes in Magellanic penguins. Ecology. In press.
We thank M. Ferrer and S. C. Griffith for comments Fridolfsson, A. K. and H. Ellegren. 1999. A simple and
universal method for molecular sexing of non-ratite
on the manuscript, and to Fundaci6n Patagonia Natural
birds. Journal of Avian Biology 30: 116-121.
for the logistic support. We also thank Secretaria de Tur-
Gales, R. P. 1988. Sexing adult Blue Penguins by exter-
ismo y Areas Protegidas of Chubut Province and Direc-
nal measurements. Notornis 35: 71-75.
ci6n Nacional de Fauna y Flora Silvestre for the permits
Gandini, P., E. Frere and T. Holik. 1992. Implicancias de
to conduct fieldwork. M. B. was supported by a grant
las diferencias en el tamafio corporal entre colonias
from the Consejo Nacional de Investigaciones Cientiff-
cas y Tecnicas (CONICET) of Argentina. During field- para el uso de medidas morfometricas como
work G. B. and M. G. F. joined a grant from the metodo de sexado en Spheniscus magellanicus. El Hor-
nero 13: 211-213.
Ministerio de Educaci6n y Ciencia of Spain in the De-
Griffiths, R., M. C. Double, K. Orr and R. J. G. Dawson.
partment of Biology of the University of Saskatchewan,
1998. A DNA test to sex most birds. Molecular Ecol-
Canada. This study was financially supported by funds
from Caja General de Ahorros de Granada. ogy 7: 1071-1075.
Lachenbruch, P. A. and M. R. Mickey. 1968. Estimation
of error rates in discriminant analysis. Technomet-
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