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Geographic morphological variation of Gentoo penguin (Pygoscelis papua)

and sex identification: Using morphometric characters and molecular
markers

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DOI: 10.1007/s00300-013-1389-2

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Polar Biol (2013) 36:1723–1734
DOI 10.1007/s00300-013-1389-2
ORIGINAL PAPER
Geographic morphological variation of Gentoo penguin
(Pygoscelis papua) and sex identification: using morphometric
characters and molecular markers
Paulina Valenzuela-Guerra • David Morales-Moraga
Daniel González-Acuña • Juliana A. Vianna
Received: 20 March 2013 / Revised: 27 June 2013 / Accepted: 9 August 2013 / Published online: 29 August 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Gentoo penguins Pygoscelis papua have sexual
dimorphism, which females have smaller morphological
measurements than males. P. papua also shows a geo-
graphic morphological variation, decreasing in size toward
southern latitudes. Therefore, morphological measurements
may vary in space and overlap in the distribution of traits
between females and males, which could lead to sex mis-
identification. A total of 300 blood samples and eight
measurements and weight were obtained for Gentoo pen-
guins from three localities in the South Shetland Islands
and the Antarctic Peninsula. The molecular sex was iden-
tified for all individuals using two primer pairs (P2/P8 and
PL/PR). We obtained higher success amplification for P2/
P8 compared to PL/PR. We used 172 adults to create a
morphological discriminant function using the backward
stepwise method. We established a discriminant function
for sex identification of each locality due to significant
morphological differences found between sexes and
between the three studied localities for adult Gentoo pen-
guins. Using these functions, the percentages of individuals
correctly assigned was 93.87 % in Ardley Island, 88.09 %
in Bernardo O’Higgins, and 83.95 % in Gabriel Gonzalez
Videla. The most southern locality studied was the most
Electronic supplementary material The online version of this
article (doi:10.1007/s00300-013-1389-2) contains supplementary
material, which is available to authorized users.
P. Valenzuela-Guerra  D. Morales-Moraga  J. A. Vianna (&)
Departamento de Ecosistemas y Medio Ambiente, Pontificia
Universidad Católica de Chile, Vicuña Mackenna 4860, Macul,
Santiago, Chile
e-mail: jvianna@uc.cl
D. González-Acuña
Departamento de Ciencias Pecuarias, Facultad de Ciencias
Veterinarias, Universidad de Concepción, Chillán, Chile
•
morphologically divergent from the others and with low
divergence between sexes. Moreover, we also established a
general function for the Gentoo penguin, which can be used
to identify the sex of penguins from a broad number of
localities. Therefore, molecular and biometric are useful
techniques for sexing Gentoo penguins.
Keywords Morphological variation  Molecular
sexing  Discriminant functions  Gentoo penguin
Introduction
Phenotypic changes in response to local environmental
conditions, genetic variation, evolution among populations,
and the biogeographic history of an individual species can
spur geographic morphological variation. It has been
shown that latitudinal clines affects body sizes of animals;
the individuals are either larger at higher latitudes/colder
temperatures (Bergman 1847) or with smaller appendages
at higher latitudes/colder temperatures (Allen 1877).
Morphologically, two subspecies have been described for
Gentoo penguin (Pygoscelis papua), the P. p. papua, J.
R. Forster, 1781 and P. p. ellsworthii, Murphy, 1947
(Stonehouse 1970). P. p. papua subspecies are distributed
in the sub-Antarctic up to 60°S on the Falkland Islands,
Staten, Marion, Heard and Macquarie islands, Kerguelen,
Iles Crozet, and South Georgia; while P. p. ellsworthii
distributed from 60°S up to 65°S, along the South Orkneys,
the South Shetland Islands and the Antarctic Peninsula
(Stonehouse 1970). P. p. ellsworthii has a smaller size and
bill proportion when compared to P. p. papua (Stonehouse
1970). Due to this, at a large scale, geographic morpho-
logical variations in Gentoo penguins do not follow
Bergmann’s rule. On the other hand, morphological
123
1724
differences between populations within subspecies of P.
papua have not been investigated at this scale.
Determining sex of penguins is difficult visually since
they are seemingly sexually monomorphic birds. However,
dimorphism of size between sexes has been observed in
different penguin species. Consequently, these differences
allow us to develop discriminant functions for penguin
species using morphometric data (e.g., Gales 1988; Murie
et al. 1991; Kerry et al. 1992; Amat et al. 1993; Hull 1996;
Zavalaga and Paredes 1997; Renner et al. 1998; Renner
and Davis 1999; Hocken and Russel 2002; Bertellotti et al.
2002; Arnould et al. 2004; Wallace et al. 2008; Hart et al.
2009; Polito et al. 2012). The technique also has been
applied to P. papua (Williams 1990; Renner et al. 1998;
Polito et al. 2012); however, if these discriminant functions
are used as a general rule to determine the sex of both
subspecies, this could cause misidentification due to their
geographic morphological variations. Therefore, different
discriminant functions have been developed for each sub-
species and one specific locality. The discriminant func-
tions obtained to sex adult Gentoo penguins from the North
(South Georgia) and the South (King George Island) have
been highly significant (Williams 1990; Polito et al. 2012).
Renner et al. (1998) also generated functions to evaluate
sex identification for 26 Gentoo breeding pairs nesting on
Ardley Island with more than 70 % of efficacy. However,
to develop these functions Renner et al. (1998) utilized the
maximum gap. This morphological measurement was
taken opening the individual’s bill manually until its
maximum aperture, which can be stressful for the animals,
and with measurement high variability. Nevertheless, spa-
tial morphological variation has not been evaluated for P.
papua, which is important to establish the applicability of
the different discriminant function across different
localities.
A complementary method for sex determination of
penguins and to validate discriminant function is using
DNA-based molecular sexing. Universal primers have been
developed to determine avian molecular sex, with P2/P8
being the most widely used pair (Griffiths et al. 1998).
These primers are used to amplify regions of the CHD1
gene found in the sex chromosomes. The P2/P8 amplifi-
cation has been evaluated for a few individuals of four
penguin species: Aptenodytes patagonicus, Eudyptes
chrysocome, P. papua, and Spheniscus magellanicus. Also,
primers such as PL/PR have been developed specifically
for these penguin species (Zhang et al. 2012). Molecular
procedures have the advantage of determining the sex of
individuals at any stage of life; however, they can lead to
an error in sex assignment, such as allelic dropout where
females are misidentified as males (Arnould et al. 2004;
Robertson and Gemmell 2006) or Z-polymorphism.
Z-polymorphism was documented for P2/P8 in a few
123
Polar Biol (2013) 36:1723–1734
species (Arnould et al. 2004; Robertson and Gemmell
2006), which can generate different alleles and interact
together during PCR generating heteroduplex (Casey et al.
2009). For this reason, more than one primer pairs should
be used for avian molecular sex identification.
Thus, to ensure the correct sex assignment for the
Gentoo penguins, we will compare and evaluate two
molecular methods of sex identification on individuals
from different geographic localities along the South Shet-
lands Islands and the Antarctic Peninsula. Moreover, we
will evaluate morphometric variation between sex and
geographic location. Finally, we develop discriminants
function to determine sex for the adult Gentoo penguins
using morphometric measurement supported by the
molecular sex identification.
Methods
To avoid disturbance on the Gentoo penguin breeding
colonies, the adults animals (n = 200) were captured using
hand-held nets when they were walking into the water. To
ensure they were breeding adult, we observed when the
penguins were leaving the breeding pair on the nest. Each
animal-handling procedure was done following Wilson
(1997). Additionally, some juveniles fledglings (n = 85)
and chicks (n = 15) were also captured. Blood samples of
all individuals (n = 300) were collected for molecular sex
identification from the brachial vein using a 23G needle
and stored in 96 % ethanol. All captures were done during
the summer of 2011 on three localities close to the Ant-
arctic Chilean base (Fig. 1): Professor Julio Escudero
(62°120 S–58°550 W) in Ardley Island-South Shetland
Islands (A. Island) (individuals sampled: adults and juve-
niles; n = 50, n = 50, respectively), Bernardo O’Higgins
(B. O’Higgins) (63°190 S–57°510 W) in Cabo Legoupil-
Antarctic Peninsula (individuals sampled: adults, juveniles,
and chicks; n = 50, n = 35, n = 15, respectively), Gabriel
González Videla (G. G. V.) (64°490 S–62°510 W) in Bahı́a
Paraiso-Antarctic Peninsula (individuals sampled: only
adults; n = 100).
Molecular sex determination
For sex identification, we used two different primer pairs to
amplify regions of the CHD1 gene found on the sex chro-
mosomes. We used P8 (50 -CTC CCA AGG ATG AGR AAY
TG-30 ) and P2 (50 -TCT GCA TCG CTA AAT CCT TT-30 )
which have been established for most bird species (Griffiths
et al. 1998). We also used the primer pair PL (50 -CCC AAG
GAT GAT AAA TTG TGC-30 ) and PR (50 -CAC TTC CAT
TAA AGC TGA TCT GG-30 ) developed for a few penguin
species (Zhang et al. 2012). All PCR reactions were
Polar Biol (2013) 36:1723–1734
Fig. 1 Map of Antarctic Peninsula and the three studied area: Ardley Island, Bernardo O’Higgins and Gabriel González Videla
conducted on an applied biosystems machine, according to
Reis et al. (2011). In penguins, for the CHD1-Z and CHD1-
W bands, the primers P2/P8 amplified a fragment of 374 and
392-bp and PL/PR a fragment of 276 and 294-bp, respec-
tively (Zhang et al. 2012).
All amplifications were performed with 30-ll reactions and
the mixture contained 10–100 ng genomic DNA. For both
primer pairs, the mixture reaction also contained: 1 mM of
each primer, 0.25 U Taq DNA polymerase, 2 mM MgCl2, 1X
PCR buffer, and 0.2 mM dNTPs. The PCR amplification
profile for this was 94 °C for 2 min, followed by 40 cycles of
94 °C for 45 s, 50 °C for 45 s, 72 °C for 45 s, and a final
extension step of 72 °C for 5 min (Reis et al. 2011). All PCR
products were loaded on 3 % agarose gel with SB buffer
(Brody and Kern 2004), but also on non-denaturing 12 %
acrylamide gel (99:1 acrylamide: bis-acrylamide), and run for
0.5 h at 300 V, and 3 h at 300 V, respectively. Afterward, the
agarose gels were visualized with the UV transilluminator,
while the acrylamide gels were dyed with silver nitrate.
Morphometric measurements
All adult individuals were weighed using Pesola Woltkraft
HS-30 of 0.1 g sensibility, and body measurements were
1725
taken using Mitutoyo Digimatic Caliper of 0.01 mm.
A total of eight measurements were taken (Fig. 2): flipper
length (FL), tail length (TL, from the base to the longest
feather), tarsus length (TAL), bill length (BL, to the cul-
men), bill width (BW, taken across the center of the cul-
men), bill depth (BD, taken through the center of the
culmen), total length (TOL, including the bill and tail), and
the body mass (BM). All the measurements and the body
mass were taken by the same person. The animals were
immobilized from the feet and cheek, and all measure-
ments were taken from above the animal. The raw mea-
surement data from adults used for the discriminant
function are in Appendix 1 available on line.
Discriminant function
To obtain the discriminants function, we considered all
adult individuals (n = 172) which sex was identified at
least for one primer pair and excluded the individuals
which the sex was not identified (n = 6), the sex misi-
dentified individuals using the two primer pairs (n = 11)
and the individuals which the BD data was not obtained
(n = 11). The discriminant functions were obtained for
each locality and for all observations (n = 172) after
123
1726
Fig. 2 Papua penguin
measurements taken: flipper
length (FL), tail length (TL),
tarsus length (TAL), bill length
(BL), bill width (BW), bill
depth (BD), total length (TOL)
TL
TAL
verification of statistically significant differences between
the mean of measurements for each variable per sex and
locality using two-way ANOVA with post hoc compari-
sons using Bonferroni correction. We calculated the nor-
mality of each variable individually using Kolmogorov–
Smirnov test with Lilliefors correction, homogeneity of
variances between groups using Bartlett test and multi-
variate normality of the variables including its respective
model using Bootstrap technique with 999 replications for
E-statistic calculation. It is noteworthy that the Bartlett test
aims to test the null hypothesis that the variances between
groups are equal; therefore, the homogeneity of variance
occurs in this case when p value [0.05, also the same
occurs with multivariate normality test because its null
hypothesis is that the combination of variables follow a
normal multivariate distribution.
The order of entry for each of the variables by locality
was identified using the backward stepwise method from
Wilk’s Lambda statistic, a measure of the predictive power
of a variable based on the amount of total variability
explained by the within-group (male vs. female) variabil-
ity, if the value is close to zero, then groups are spread
further apart and therefore greater variability explained.
For each new variable included in a new step, we calcu-
lated the predictive power of the resulting function to
complete the total morphometric measures included in it.
The order of entry of each variable to a new model was
123
Polar Biol (2013) 36:1723–1734
BL
BD
BW
FL
TOL
obtained from the individual contribution of each variable
of each step of the backward stepwise regression (Wilk’s
lambda value). Also, we calculated whether the inclusion
of a new variable resulted in a model with means statisti-
cally different between groups. With the scores obtained in
each function, females were classified as those individuals
whose score was positive and males whose scores were
negative. We used the same classification procedure in the
discriminant functions for each locality, and all observa-
tions combined.
We did a multivariate approximation from principal
component analysis (PCA) to measure the amount of var-
iance contributed by each morphological character in each
study site and for all sites combined. The eigenvalues
(variance explained by each principal component) were
calculated, and the loadings (the weights of the eigenvec-
tors) were used to verify the variability contributed by each
character from the correlation matrix in the first two prin-
cipal components.
Within-group correlation matrices were obtained for each
site to assess possible effects of collinearity (r [ 0.5 between
pairs of variables). Jackniffe procedure was applied to obtain
the rate of correctly classified individuals in selected func-
tions, which leaves out one of the data to calculate a new
function with the other observations, and then the model is
validated with the missing data. The procedure is repeated as
many times as there are observations. This procedure should
Polar Biol (2013) 36:1723–1734
demonstrate the power of classification given by individual
Wilk’s Lambda values using each of the variables and those
obtained by the backward stepwise method.
Despite satisfying the assumptions of normality and
homogeneity of variance–covariance matrices, the variable
body mass (BM) was removed for a final discriminant
function given its high daily and seasonal variability (Za-
valaga and Paredes 1997), so only was used in the two-way
ANOVA, being discarded in the other procedures. Finally,
only models from the backward stepwise method that met the
following criteria were considered: different means (two-
way ANOVA), univariate normal distribution (Kolmogo-
rov–Smirnov test with Lilliefors correction), correlation of
variables within groups less than 0.5, homogeneity of vari-
ances, Wilk’s Lambda value significant (p value \0.05) for
the variables in selected function, and multivariate normal
distribution for the variables included in it. All of them, for
different locations and all together.
The within-class correlation of the group matrices, the
order of the variables in the stepwise regression, and the
jackknife validation were done in SSPS Statistics 21. The
rest of the statistical procedures were performed in the
software R (R Development Core Team 2011) from the
packages: DiscriMiner (Sanchez 2012), MASS (Venables
and Ripley 2002), Nortest (Gross and Ligges 2012), Bio-
stats R (McGarigal 2008), Klar (Weihs et al. 2005), and
Energy (Rizzo and Szekely 2012).
Results
The two primer pairs used to amplify regions of the CHD1
gene successfully amplified fragments from Gentoo Penguins.
Both P2/P8 and PL/PR showed the expected, two bands for
females and only one for males. Since the differences between
the bands CHD1-Z and CHD1-W were so small, long runs on
12 % acrylamide gel was required to be adequately resolved.
However, we were able to obtain the same results using a
faster procedure on 3 % agarose gel with SB buffer.
The success of amplification and efficacy for sex
determination of both primer pairs varied. The P2/P8
primers had approximately an 89 % success rate, while the
Table 1 p values from Two-way ANOVA for morphometric data of Gentoo penguins held in 3 different sites in Antarctica. BM is in Kg, all the
others measures are in cm
Source d.f. FL TL TAL
Sex 1 \0.001* 0.839 0.004*
Locality 2 \0.001* 0.711 0.345
Sex 9 locality 2 0.209 0.516 0.705
Residuals 166
* Significance with p value \0.05
1727
PL/PR primers had approximately an 81 % success rate.
The P2/P8 DNA-based test identified a total of 266 indi-
viduals (n = 300) at all stages of life, 54 females and 42
males from G. G. V. (n = 100), 37 females and 47 males
from B. O’Higgins (n = 100), and 44 females and 42
males from A. Island (n = 100). The PL/PR DNA-based
test identified a total of 244 individuals: 43 females and 41
males from G. G. V., 42 females and 39 males from B.
O’Higgins, and 40 females and 39 males from A. Island.
A comparison between the results obtained for both primer
pairs revealed a degree of sex misidentification (n = 11).
Accordingly, the lack of sufficient dimorphism in juveniles
and chicks, we only used the morphometric data of the
adults (n = 172) to generate discriminant functions.
Except for BW on G. G. V., males were larger than
females for all variables. The means of FL, TAL, BL, BW,
and TOL was found to be statistically significant (two-way
ANOVA; p value \0.05) between sex of penguins
(Table 1), and FL, BL, BW, BD, TOL, and BM were sta-
tistically different between sites (Table 1). Furthermore,
other variables were also found to be statistically different
in some sites, but this was not generalized in all localities
(Table 2). When we combine the three sites together, we
see that males are larger than females for TOL (Mean:
73.94 cm females and 76.13 cm males; p value \0.001);
result also found for FL (Mean: 21.21 cm females and
21.86 cm males; p value \0.001), TAL (Mean: 4.26 cm
females and 4.35 cm males; p value = 0.042) and for
BL (Mean: 4.53 cm females and 4.88 cm males;
p value \0.001). When we compared all variables between
the three sites, we found that B. O’Higgins and A. Island do
not have significant morphological differences except for
BD and BM (Table 2). However, G. G. V. has significant
differences with B. O’Higgins and A. Island for FL, BL,
TOL, and BM, while BW was different only G. G. V. vs B.
O’Higgins (p value \0.043) (Table 2). In addition, the
variances were homogeneous (Bartlett test; p value [0.05)
for the variables FL, TAL, BL, BD, and TOL in the three
sites combined, while other variables had this property in
particular sites (Table 3).
To G. G. V., the first two principal components (PC)
account for 48.3 % of the variability of the data set
BL BW BD TOL BM
\0.001* \0.001* 0.001* \0.001* 0.191
\0.001* 0.015* \0.001* \0.001* \0.001*
0.361 0.301 0.902 0.198 0.557
123
1728 Polar Biol (2013) 36:1723–1734

Table 2 p values adjusted by

Variable Female versus G. G. V. versus B. G. G. V. versus B. O0 Higgins versus
Bonferroni correction for post
male O0 Higgings A. Island A. Island
hoc comparisons of each
p value p value p value p value
morphometric character after
Two-way ANOVA Flipper length \0.001* 0.007* \0.001* 0.886
Tail length 0.840 0.997 0.998 0.740
Tarsus length 0.003 0.450 0.610 0.930
Bill length \0.001* 0.007* 0.003* 0.997
Bill width \0.001* 0.043* 0.343 0.997
Bill depth 0.004* \0.001* 0.997 \0.001*
Total length \0.001* \0.001* \0.001* 0.998
* Significance level: Body mass 0.390 \0.001* \0.001* \0.001*
p value \0.05

Table 3 K-squared from Bartlett test for homogeneity of variances between the sexes for all variables en each study site and in all sites
combined
Variable G. G. V. B. O’Higgins A. Island All sites
K square p value K square p value K square p value K square p value

Flipper length 4.357 0.317 0.036 0.850 0.389 0.533 2.377 0.123
Tail length 5.310 0.021* 2.660 0.103 0.127 0.722 7.117 0.008*
Tarsus length 1.485 0.223 1.872 0.171 0.682 0.409 1.781 0.182
Bill length 2.423 0.120 0.001 0.995 2.339 0.126 1.168 0.280
Bill width 9.103 0.003* 0.177 0.674 0.215 0.643 5.169 0.023*
Bill depth 1.238 0.266 0.651 0.420 3.727 0.054 1.319 0.251
Total length 0.119 0.730 0.284 0.594 0.383 0.536 0.629 0.428
* Significance level: p value \0.05
Non-significant p values indicate that the variances are statistically homogeneous between sexes

(Fig. 3), while only in the sixth PC is reached to explain
more than 90 % of the variability (94.2 %). In B. O’Hig-
gins and A. Island, the first two PC explain 51.4 and
57.3 % of the variance, respectively. Only A. Island
reached more than 90 % of the variation explained in the
fifth PC; therefore, almost all the variables have a similar
degree of importance in terms of variability explained.
Considering all sites together, the first PC explains 29.1 %
of the proportion of the variance while the second 19.7 %.
There is no strong correlation with any PC with a variable
in the first component, in fact the highest TL occurs for the
second PC in A. Island (r-Pearson = 0.733). TOL variable
seems to be the most important to have relatively high
correlations in the first two principal components in each
location and all together (Fig. 3).
The backward stepwise analysis gives a Wilk’s Lambda
value for each locality and all observations by adding each
variable sequentially. In B. O’Higgins a greater differences
between groups is explained when considering the seven
variables (Wilk’s Lambda = 0.311), but only the first three
variables are significant (in order of entry: BL, TOL and TL)
(Table 4). Using all seven variables, the least explanatory
power was found in G. G. V. (Wilk’s Lambda = 0.575)
because only two of them had univariate normal distribution:
FL (KS-test, D-value = 0.087, p value = 0.128) and TOL
(KS-test, D-value = 0.088, p value = 0.118). On A. Island,
all variables had homogeneity of variances (Table 3), but
only three of them: FL, BL, and TOL were significant by
including one by one to the function (Table 4) This mea-
surements also presented a median statistically different
between males and females (Table 1). Finally, if all localities
are combined using the stepwise Wilk’s Lambda, the statistic
is better than the value found for G. G. V. using seven vari-
ables (Wilk’s Lambda = 0.523), but only FL, BL, and TOL
had significant univariate normality (D-statistic = 0.052,
p value = 0.332).
Given the values of the Wilk’s Lambda method obtained
from the stepwise method, three discriminant functions
were calculated per site and considering all sites combined
allowing for the order of entry of variables for each of the
steps, including the individual value of Wilk’s Lambda per
variable as criteria of selection (Table 4). Finally, for each
locality and all observations, we obtained the following
discriminant functions:

123
Polar Biol (2013) 36:1723–1734 1729

a b
TOL
TL

0.5

0.5
TOL BW
Comp 2(18.2%)

Comp 2(17.9%)
FL
BL
0.0

0.0
BW
BD
FL
BD TL
-0.5

-0.5
TAL
BL TAL

-1.0 -0.5 0.0 0.5 -1.0 -0.5 0.0 0.5

Comp 1(30.1%) Comp 1(35.1%)

c TL d
TOL
0.5

0.5
TOL TL
TAL
Comp 2(19.8%)

Comp 2(19.7%)
FL
BD
0.0

0.0
FL
BL
BW BL
BW
-0.5

-0.5 BD
TAL

-1.0 -0.5 0.0 0.5 -1.0 -0.5 0.0 0.5

Comp 1(37.6%) Comp 1(29.1%)

Fig. 3 Biplot of loadings of first and second principal component for each morphometric measurement in Gabriel Gonzalez Videla (a),
O’Higgins Base (b), Ardley Island (c) and the combination of all study sites (d). Loadings were calculated from the correlation matrix

Gabriel González Videla (G.G.V.): All observations:

D1.1= 54.06 – 1.42 *FL – 4.86 *BL (1) D4.1= 62.22 – 1.80 *FL – 4.95 *BL (10)

D1.2= 64.69 – 1.41*FL – 4.88* BL – 2.49*TAL (2) D4.2= 72.47 – 1.42*FL – 5.28*BL – 0.22*TOL (11)

D1.3=58.99 – 1.33*FL – 4.82*BL – 0.09*TOL (3) D4.3= 73.53 – 1.37*FL + 0.47*TL – 5.40*BL – 0.33*TOL (12)

Bernardo O’Higgins (B. O’Higgins): Where D \ 0 identifies male individuals, and D [ 0

D2.1= 51.22 – 9.38*BL – 0.10*TOL
D2.2= 70.44 + 1.05*TL – 9.88*BL – 0.51*TOL
D2.3= 56.72 – 9.07*BL – 5.22*BW– 0.06*TOL
Ardley Island (A. Island):
D3.1= 125.47 – 3.55 *FL – 0.63 *TOL
D3.2= 132.94 –3.37*FL + 1.35*TL – 1.04*TOL
D3.3= 125.99 – 2.96*FL – 3.87*BL – 0.57*TOL
females. Functions that met all assumptions made were
(4)
Eq. 3 (G. G. V.), Eq 4 in B. O0 Higgins and Eq. 9 (A.
(5) Island), while we consider all observations Eq. 11
responded more robustly to the assumptions (Tables 1, 3
(6) and 4). Although TL in A. Island function had a significant
Wilk’s Lambda, this variable had no significant differences
between group means (p value = 0.849). Thus, these three
functions are best discriminate for their respective locali-
(7)
ties. The best function obtained by combining the localities
(8) was Eq. 11, which had multivariate normality (E-statis-
tic = 0.591, p value = 0.656) and different means and
(9) equal variances for its variables. The others functions also

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1730 Polar Biol (2013) 36:1723–1734

Table 4 Order of entry of variables to the discriminant function by backward stepwise method of regression
Variable G. G. V. B. O’Higgins A. Island All sites
W‘s L Order W‘s L Order W‘s L Order W‘s L Order

Flipper length 0.611* 2 0.315 6 0.565* 1 0.578* 2

Tail length 0.575 7 0.376* 3 0.411* 3 0.535* 4
Tarsus length 0.586* 3 0.326 5 0.369 7 0.530 5
Bill length 0.699* 1 0.464* 1 0.389 4 0.692* 1
Bill width 0.576 6 0.344 4 0.378 5 0.524 6
Bill depth 0.577 5 0.311 7 0.373 6 0.523 7
Total length 0.581* 4 0.419* 2 0.464* 2 0.549* 3
Wilk’s lambda (W‘sL) values to add an additional variable are shown. With each new variable added the value decreased because the function
explain more variability between groups (male vs female). Values with * indicate that the means of the variables included in the function are
statistically different between groups
Significance level: p value \0.05

had multivariate normality for the variables used in it, for
G. G. V. the E-statistic was 0.961 (p value = 0.174) a little
less significant that the others sites (E-statistic was 0.478
(p value = 0.901) and 0.874 (p value = 0.339) for B.
O’Higgins and A. Island, respectively.
For the 12 functions calculated, an overlap in discrimi-
nant scores was observed. (Fig. 4) For the four functions
chosen the misclassification was 16.05 % (F = 11.36 %,
M = 21.62 %) in G. G. V., 11.91 % (F = 14.28 %,
M = 9.52 %) in B. O’Higgins, 6.13 % (F = 5.00 %,
M = 6.89 %) for A. Island and 18.6 % (F = 16.47 %,
M = 20.69 %) for all sites combined. The Fig. 4 shows the
precision of the Jackknife method and the degree of
overlapping of each function selected in each study site and
when we combined all the locations.
The centroids of the groups (mean scores) were 1.454
for females and -1.108 for males in Eq. 3, 2.229 for
females and -2.231 for males in Eq. 4, 2.312 for females,
and -3.055 for males in Eq. 9, and 1.596 (females) and
-1.643 (males) in Eq. 11. Misclassification ranges for each
function were: (-1.934–2.685) in Eq. 3, (-1.015–2.098)
in Eq. 4, (-2.516–0.213) in Eq. 9 and (-2.489–2.766) in
Eq. 11. Any penguin whose calculated score lies in the
misclassification range of its respective function should be
measured again, or use another more robust function or
simply not classify it to increase the precision of the
function.
Discussion
Geographic morphologic variation is very important to
understand the species biology, ecology, and biogeogra-
phy. Gentoo penguins have been divided into two
subspecies based on morphological characteristics, where
P. p. ellsworthii (Southern Gentoo penguins) is character-
ized by its smaller size compared to P. p. papua (Northern
Gentoo penguins; Stonehouse 1970). Accordingly, geo-
graphic morphological variations of Gentoo penguins at a
larger scale (subspecies) and at a smaller scale (populations
within P. p. ellsworthii) follow a converse Bergmann’s
rule. When we compared the morphometric among popu-
lations of Southern Gentoo penguins, we observed a similar
trend. The P. papua from the G. G. V. (the most southern
locality) are morphologically smaller than the other local-
ities (especially for FL and TOL) (Table 2). Variation in
body size among populations within species may reflect
phenotypic or genetic responses to environmental gradients
or geographic distances. Natural selection can promote
population divergence in a variety of traits promoting local
adaptation to different environmental conditions. The
extreme environmental conditions, such very low temper-
atures and intense seasonality combined with a period of
the year with complete or near darkness, requires adapta-
tion mechanisms which can be crucial for the persistence of
a species such as birds (Peck et al. 2005). On the other
hand, phenotypic differentiation can arise solely due to the
action of random microevolutionary processes of genetic
drift and mutation (Wright 1931). However, the degree of
ongoing gene flow between geographically adjacent pop-
ulations can homogenize phenotypic differences and delay
the degree of local adaptation. Signature of natural selec-
tion has been evidenced in morphological traits of bird
species in Antarctic. The selection pattern was investigated
on several body size traits in a population of Antarctic bird
species, the snow petrels (Pagodroma nivea), over an
11-year period (Barbraud 2000). The author found evi-
dence of directional selection on bill length and stabilizing
selection on tarsus length, associated with reproductive

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Polar Biol (2013) 36:1723–1734 1731

Fig. 4 Histogram of

10
discriminant scores for Gabriel n=81 n=42
a b
Gonzalez Videla (a), Bernardo

10

8
O’Higgins (b), Ardley Island

8
(c) and the combination of all

6
study sites (d) on their

6
respective discriminant

4
functions Selected for sex

4
classification of Gentoo

2
2
penguins from Antarctica. Dark

Number of individuals
Gray represents females, white

0
0
males, gray misidentified
-10 -5 0 5 10 -10 -5 0 5 10

20
n=49 c n=172 d
10

15
8
6

10
4

5
2
0

0
-10 -5 0 5 10 -10 -5 0 5 10
Discriminant score

success among males. Likewise, strong directional pheno-
typic selection on skeletal size of Chinstrap penguins
(Pygoscelis antarctica) comparing carcasses with live
chicks (Moreno et al. 1999). Apparently the target of
selection is weight at emancipation which is correlated
with flipper length of P. antarctica. The geographic vari-
ation in body size was evaluated for an Arctic bird, dovekie
(Alle alle), across the species breeding range associated
with environmental parameters (Wojczulanis-Jakubas et al.
2011). Morphological variation was described for the A.
alle with a longitudinal increase in body size from west to
east; however, the traits association with environmental
factors such air temperature, wind speed and sea surface
temperature is not clear. Nevertheless, there is an absence
of studies evaluating spatial geographic variation in Ant-
arctic bird species at intraspecific level. Our analysis
identified geographic morphological variation among the
P. papua populations studied with higher morphological
divergence between G. G. V. and B. O’Higgins, followed
by G. G. V. and A. Island. Although individuals from A.
Island and B. O’Higgins which are 136 km in a straight
line approximately apart are not morphologically distinct
for most of the traits, the higher distance of G. G. V. from
those two localities (352 km from A. Island and 299 km
from B. O’Higgins in a Euclidean distance approximately)
may limit migration and morphological homogenization.
The great distances between archipelagos that provide
terrain for breeding colonies can be a geographic barrier,
because the Gentoo penguin is a non-migratory species,
adapted to areas free of ice that winters close to their
breeding sites (Trivelpiece et al. 1987). Therefore, the
evaluation of morphological traits along the species dis-
tribution with a larger number of populations would clarify
the existence of a pattern of isolation by distance or the
association with local environmental conditions. Recent
demographic studies have shown the effects of climatic
change on Gentoo penguins, expanding their range toward
higher latitudes (Carlini et al. 2009; Forcada and Trathan
2009). An increase of 2–3 °C on air temperature in the
Antarctic Peninsula Region has been recorded during the
last 50 years (King 1994; Smith et al. 1996); the reduction
in extensive sea ice is associated with the less cold tem-
peratures (Fraser et al. 1992) which may influence this
expansion. There are evidence of the importance of local
environmental conditions to the distribution of the popu-
lations of P. papua which may also spur morphological
trait variation. Whether the pattern of morphological vari-
ation observed for P. papua can be explained by limited
gene flow expected in a pattern of isolation by distance, or
rather reflects local adaptation in response to natural
selection or phenotypic plasticity in response to environ-
ment variation remains to be explored.
The geographic body size variation has limited the
applicability of general discriminant functions for sex
identification of both P. papua subspecies (Renner et al.
1998) resulting in the creation of different models for the
two subspecies. Only one discriminant function has been
obtained to sex P. p. papua (Williams 1990) while different

123
1732
studies have used morphometric measurements for sex
identification of P. p. ellsworthii (Renner et al. 1998; Polito
et al. 2012). However, none these studies have evaluated
geographic morphologic variation between Southern Gen-
too penguin populations. Regarding morphological differ-
ences we have found among localities, the discriminant
functions to determine the sex of P. p. ellsworthii adults
developed in this study should be used for each locality.
Nevertheless this function should not be used for sub-
Antarctic populations (up to 60°S), which we have not
studied and have been described as a morphologically
divergent subspecies (P. p. papua; Stonehouse 1970). Even
though this systematic classification in two subspecies have
not been supported by molecular data (de Dinechin et al.
2012), the animals are morphological distinct. The
obtained results with the different functions in this research
could have been compared with previous studies; however,
the type of morphological characters taken by Renner et al.
(1998) and Polito et al. (2012) in A. Island were different to
our measurements and this difficult the comparison. BL
and BD were the most significantly different dimorphic
character described in Polito et al. (2012). In our study BD
did not show significant differences between sexes for
Southern Gentoo penguins at any locality. However, the
BD from our study is measured in the culmen, while Polito
et al. (2012) the BD was obtained in the nostrils. Other-
wise, discriminant function-based morphological sexing
apparently has never been applied to Gentoo penguin
populations from the Antarctic Peninsula (B. O0 Higgins
and G. G. V.). Despite the morphological differences that
we have found among localities, we also developed a
general discriminant function for sex determination of
Southern Gentoo penguins, which should be used in dif-
ferent localities in the Antarctic Peninsula and the South
Shetland Islands with a lower degree of confidence (81 %
approximately) when compared to more specific functions
developed for each locality (93.87 % in A. Island, 88.09 %
in B. O’Higgins and 83.95 % in G. G. V.). However, the
large sample size (n = 172) used to generated this general
discriminant function, and the 81 % success rates are
characteristics which are found as acceptable to use this
function in P. p. ellsworthii (Kerry et al. 1992).
The TOL turns out to be the characteristic that dis-
criminates between sexes to have the lowest degree of
overlap between distributions and therefore must be pres-
ent in all equations chosen; in fact, FL and BL variables are
included in almost all the functions chosen (except FL in B.
O’Higgins), which speaks of a high discriminant power of
these variables (Tables 1, 3; Fig 3). The degree of sorting
efficiency was only slightly lower than that obtained by
Renner et al. (1998) in A. Island (8.6 vs. 9.8 % mis-
classification for top models) but not very different from
those applied to other penguin functions (Gales 1988;
123
Polar Biol (2013) 36:1723–1734
Kerry et al. 1992; Zavalaga and Paredes 1997). Although a
particular model may suggest high accuracy in the classi-
fication of individuals, it is not enough just to evaluate the
statistical differences between the means and variances of
the groups, we must also investigate the level of individual
contribution each variable makes to the model, and the
order in which they should be evaluated to maximize the
differences between groups. It is also important to verify
the assumption of multivariate normality in the variables
included in the model, verification rarely reported in
studies using this class of functions. Similarly, it is nec-
essary to test the homogeneity of variance–covariance
matrices of populations in each of the variables included in
the model. The application of filtering variables allowed us
to generate models that can be applied with a real degree of
confidence in sexing new individuals. Recently, a dis-
criminant function was developed for each of the Pygosc-
elis species using bill measurements in adults from one
locality at South Shetland Islands (Polito et al. 2012).
Therefore, the geographic variation here described for P.
papua should be further evaluated for P. adelia and P.
antarctica which might lead to a sex misidentification
when using a single function without previously evaluated
spatial morphological trait diversity.
Many studies have used molecular procedures as an
alternative for sex identification of penguins (Bertellotti
et al. 2002; Costantini et al. 2008; Hart et al. 2009; Reis
et al. 2011; Zhang et al. 2012; Polito et al. 2012). Also,
these procedures support discriminant function analysis to
sex penguins as shown in this study and have also been
reported for Macaroni Penguins (Hart et al. 2009) and
Pygoscelis species (Polito et al. 2012). We tested two
molecular procedures finding that both were successful for
Gentoo penguins. However, PR/PL primers have the
smallest success rate, even if this tool was developed
specifically for penguin species (Zhang et al. 2012). A little
disagreement found between both primer pairs (P2/P8 and
PL/PR) may occur because of error in sex assignment, due
to allelic dropout, Z-polymorphism or heteroduplex (Ar-
nould et al. 2004; Robertson and Gemmell 2006; Casey
et al. 2009). For this reason, we recommend the use of
more than one primer pair. Another potential method to
improve the molecular sex identification is to enhance the
gel resolution, changing the agarose gel by acrylamide gel
(Wang et al. 2007). However, we used both gel procedures
with the same results. Although 3 % agarose gel with SB
buffer is a simple, faster, cheaper method frequently used
in DNA analysis laboratory.
Finally, the molecular procedures used in this study to
sex Gentoo penguins are a successful tool at any state life
and localities along its distribution. Moreover, these tech-
niques can support discriminant function analysis with a
higher success rate. The discriminant functions developed
Polar Biol (2013) 36:1723–1734
in this study can give instant results in the field when
sexing adults and can be used for diverse studies which
require sex determination along the Antarctic Peninsula
and to increase reproductive success of birds from captivity
breeding programs and to develop conservation plans
(Cheney 1990; Fridolfsson and Ellegren 1999). According to
our result and other studies in different penguins’ species
have shown geographic body size variations (Murie et al.
1991; Gandini et al. 1992; Renner and Davis 1999), which
limit the applicability of discriminant functions across dif-
ferent localities. We recommend the use of the different
discriminant functions considering the geographic morpho-
logical variation. Thus, we also standardized a general
function for South Shetland Islands and the Antarctic Pen-
insula, but we recommend the use of this when a high level of
accuracy is not required for sex determination. Otherwise,
the results of geographic morphological variation in South-
ern Gentoo penguins found in our study are important start
for further investigations to understand the role of micro-
evolutionary process over the morphological traits and
associated with studies on population genetics structure.
Acknowledgments This work was funded by Instituto Antártico
Chileno (INACH-G_06-11; INACH-T-27-10 and INACH MG_02-
12). D. Noll and B. Ramos for help in molecular procedures.
L. Moreno and J. Hernandez for help in the field. F. Rengifo and
N. Sallaberry for comments on the manuscript.
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