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Geographic morphological variation of Gentoo penguin (Pygoscelis papua)

and sex identification: Using morphometric characters and molecular

Article  in  Polar Biology · December 2013

DOI: 10.1007/s00300-013-1389-2


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David Morales Daniel González-Acuña

Pontificia Universidad Católica de Chile University of Concepción


Juliana A Vianna
Pontificia Universidad Católica de Chile


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Polar Biol (2013) 36:1723–1734
DOI 10.1007/s00300-013-1389-2


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 morphologically divergent from the others and with low
dimorphism, which females have smaller morphological divergence between sexes. Moreover, we also established a
measurements than males. P. papua also shows a geo- general function for the Gentoo penguin, which can be used
graphic morphological variation, decreasing in size toward to identify the sex of penguins from a broad number of
southern latitudes. Therefore, morphological measurements localities. Therefore, molecular and biometric are useful
may vary in space and overlap in the distribution of traits techniques for sexing Gentoo penguins.
between females and males, which could lead to sex mis-
identification. A total of 300 blood samples and eight Keywords Morphological variation  Molecular
measurements and weight were obtained for Gentoo pen- sexing  Discriminant functions  Gentoo penguin
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 Introduction
PL/PR). We obtained higher success amplification for P2/
P8 compared to PL/PR. We used 172 adults to create a Phenotypic changes in response to local environmental
morphological discriminant function using the backward conditions, genetic variation, evolution among populations,
stepwise method. We established a discriminant function and the biogeographic history of an individual species can
for sex identification of each locality due to significant spur geographic morphological variation. It has been
morphological differences found between sexes and shown that latitudinal clines affects body sizes of animals;
between the three studied localities for adult Gentoo pen- the individuals are either larger at higher latitudes/colder
guins. Using these functions, the percentages of individuals temperatures (Bergman 1847) or with smaller appendages
correctly assigned was 93.87 % in Ardley Island, 88.09 % at higher latitudes/colder temperatures (Allen 1877).
in Bernardo O’Higgins, and 83.95 % in Gabriel Gonzalez Morphologically, two subspecies have been described for
Videla. The most southern locality studied was the most 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
Electronic supplementary material The online version of this
article (doi:10.1007/s00300-013-1389-2) contains supplementary in the sub-Antarctic up to 60°S on the Falkland Islands,
material, which is available to authorized users. Staten, Marion, Heard and Macquarie islands, Kerguelen,
Iles Crozet, and South Georgia; while P. p. ellsworthii
P. Valenzuela-Guerra  D. Morales-Moraga  J. A. Vianna (&)
distributed from 60°S up to 65°S, along the South Orkneys,
Departamento de Ecosistemas y Medio Ambiente, Pontificia
Universidad Católica de Chile, Vicuña Mackenna 4860, Macul, the South Shetland Islands and the Antarctic Peninsula
Santiago, Chile (Stonehouse 1970). P. p. ellsworthii has a smaller size and
e-mail: bill proportion when compared to P. p. papua (Stonehouse
1970). Due to this, at a large scale, geographic morpho-
D. González-Acuña
Departamento de Ciencias Pecuarias, Facultad de Ciencias logical variations in Gentoo penguins do not follow
Veterinarias, Universidad de Concepción, Chillán, Chile Bergmann’s rule. On the other hand, morphological

1724 Polar Biol (2013) 36:1723–1734

differences between populations within subspecies of P. species (Arnould et al. 2004; Robertson and Gemmell
papua have not been investigated at this scale. 2006), which can generate different alleles and interact
Determining sex of penguins is difficult visually since together during PCR generating heteroduplex (Casey et al.
they are seemingly sexually monomorphic birds. However, 2009). For this reason, more than one primer pairs should
dimorphism of size between sexes has been observed in be used for avian molecular sex identification.
different penguin species. Consequently, these differences Thus, to ensure the correct sex assignment for the
allow us to develop discriminant functions for penguin Gentoo penguins, we will compare and evaluate two
species using morphometric data (e.g., Gales 1988; Murie molecular methods of sex identification on individuals
et al. 1991; Kerry et al. 1992; Amat et al. 1993; Hull 1996; from different geographic localities along the South Shet-
Zavalaga and Paredes 1997; Renner et al. 1998; Renner lands Islands and the Antarctic Peninsula. Moreover, we
and Davis 1999; Hocken and Russel 2002; Bertellotti et al. will evaluate morphometric variation between sex and
2002; Arnould et al. 2004; Wallace et al. 2008; Hart et al. geographic location. Finally, we develop discriminants
2009; Polito et al. 2012). The technique also has been function to determine sex for the adult Gentoo penguins
applied to P. papua (Williams 1990; Renner et al. 1998; using morphometric measurement supported by the
Polito et al. 2012); however, if these discriminant functions molecular sex identification.
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 Methods
discriminant functions have been developed for each sub-
species and one specific locality. The discriminant func- To avoid disturbance on the Gentoo penguin breeding
tions obtained to sex adult Gentoo penguins from the North colonies, the adults animals (n = 200) were captured using
(South Georgia) and the South (King George Island) have hand-held nets when they were walking into the water. To
been highly significant (Williams 1990; Polito et al. 2012). ensure they were breeding adult, we observed when the
Renner et al. (1998) also generated functions to evaluate penguins were leaving the breeding pair on the nest. Each
sex identification for 26 Gentoo breeding pairs nesting on animal-handling procedure was done following Wilson
Ardley Island with more than 70 % of efficacy. However, (1997). Additionally, some juveniles fledglings (n = 85)
to develop these functions Renner et al. (1998) utilized the and chicks (n = 15) were also captured. Blood samples of
maximum gap. This morphological measurement was all individuals (n = 300) were collected for molecular sex
taken opening the individual’s bill manually until its identification from the brachial vein using a 23G needle
maximum aperture, which can be stressful for the animals, and stored in 96 % ethanol. All captures were done during
and with measurement high variability. Nevertheless, spa- the summer of 2011 on three localities close to the Ant-
tial morphological variation has not been evaluated for P. arctic Chilean base (Fig. 1): Professor Julio Escudero
papua, which is important to establish the applicability of (62°120 S–58°550 W) in Ardley Island-South Shetland
the different discriminant function across different Islands (A. Island) (individuals sampled: adults and juve-
localities. niles; n = 50, n = 50, respectively), Bernardo O’Higgins
A complementary method for sex determination of (B. O’Higgins) (63°190 S–57°510 W) in Cabo Legoupil-
penguins and to validate discriminant function is using Antarctic Peninsula (individuals sampled: adults, juveniles,
DNA-based molecular sexing. Universal primers have been and chicks; n = 50, n = 35, n = 15, respectively), Gabriel
developed to determine avian molecular sex, with P2/P8 González Videla (G. G. V.) (64°490 S–62°510 W) in Bahı́a
being the most widely used pair (Griffiths et al. 1998). Paraiso-Antarctic Peninsula (individuals sampled: only
These primers are used to amplify regions of the CHD1 adults; n = 100).
gene found in the sex chromosomes. The P2/P8 amplifi-
cation has been evaluated for a few individuals of four Molecular sex determination
penguin species: Aptenodytes patagonicus, Eudyptes
chrysocome, P. papua, and Spheniscus magellanicus. Also, For sex identification, we used two different primer pairs to
primers such as PL/PR have been developed specifically amplify regions of the CHD1 gene found on the sex chro-
for these penguin species (Zhang et al. 2012). Molecular mosomes. We used P8 (50 -CTC CCA AGG ATG AGR AAY
procedures have the advantage of determining the sex of TG-30 ) and P2 (50 -TCT GCA TCG CTA AAT CCT TT-30 )
individuals at any stage of life; however, they can lead to which have been established for most bird species (Griffiths
an error in sex assignment, such as allelic dropout where et al. 1998). We also used the primer pair PL (50 -CCC AAG
females are misidentified as males (Arnould et al. 2004; GAT GAT AAA TTG TGC-30 ) and PR (50 -CAC TTC CAT
Robertson and Gemmell 2006) or Z-polymorphism. TAA AGC TGA TCT GG-30 ) developed for a few penguin
Z-polymorphism was documented for P2/P8 in a few species (Zhang et al. 2012). All PCR reactions were

Polar Biol (2013) 36:1723–1734 1725

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 taken using Mitutoyo Digimatic Caliper of 0.01 mm.
Reis et al. (2011). In penguins, for the CHD1-Z and CHD1- A total of eight measurements were taken (Fig. 2): flipper
W bands, the primers P2/P8 amplified a fragment of 374 and length (FL), tail length (TL, from the base to the longest
392-bp and PL/PR a fragment of 276 and 294-bp, respec- feather), tarsus length (TAL), bill length (BL, to the cul-
tively (Zhang et al. 2012). men), bill width (BW, taken across the center of the cul-
All amplifications were performed with 30-ll reactions and men), bill depth (BD, taken through the center of the
the mixture contained 10–100 ng genomic DNA. For both culmen), total length (TOL, including the bill and tail), and
primer pairs, the mixture reaction also contained: 1 mM of the body mass (BM). All the measurements and the body
each primer, 0.25 U Taq DNA polymerase, 2 mM MgCl2, 1X mass were taken by the same person. The animals were
PCR buffer, and 0.2 mM dNTPs. The PCR amplification immobilized from the feet and cheek, and all measure-
profile for this was 94 °C for 2 min, followed by 40 cycles of ments were taken from above the animal. The raw mea-
94 °C for 45 s, 50 °C for 45 s, 72 °C for 45 s, and a final surement data from adults used for the discriminant
extension step of 72 °C for 5 min (Reis et al. 2011). All PCR function are in Appendix 1 available on line.
products were loaded on 3 % agarose gel with SB buffer
(Brody and Kern 2004), but also on non-denaturing 12 % Discriminant function
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 To obtain the discriminants function, we considered all
agarose gels were visualized with the UV transilluminator, adult individuals (n = 172) which sex was identified at
while the acrylamide gels were dyed with silver nitrate. least for one primer pair and excluded the individuals
which the sex was not identified (n = 6), the sex misi-
Morphometric measurements dentified individuals using the two primer pairs (n = 11)
and the individuals which the BD data was not obtained
All adult individuals were weighed using Pesola Woltkraft (n = 11). The discriminant functions were obtained for
HS-30 of 0.1 g sensibility, and body measurements were each locality and for all observations (n = 172) after

1726 Polar Biol (2013) 36:1723–1734

Fig. 2 Papua penguin BL

measurements taken: flipper
length (FL), tail length (TL),
tarsus length (TAL), bill length
(BL), bill width (BW), bill
depth (BD), total length (TOL)






verification of statistically significant differences between obtained from the individual contribution of each variable
the mean of measurements for each variable per sex and of each step of the backward stepwise regression (Wilk’s
locality using two-way ANOVA with post hoc compari- lambda value). Also, we calculated whether the inclusion
sons using Bonferroni correction. We calculated the nor- of a new variable resulted in a model with means statisti-
mality of each variable individually using Kolmogorov– cally different between groups. With the scores obtained in
Smirnov test with Lilliefors correction, homogeneity of each function, females were classified as those individuals
variances between groups using Bartlett test and multi- whose score was positive and males whose scores were
variate normality of the variables including its respective negative. We used the same classification procedure in the
model using Bootstrap technique with 999 replications for discriminant functions for each locality, and all observa-
E-statistic calculation. It is noteworthy that the Bartlett test tions combined.
aims to test the null hypothesis that the variances between We did a multivariate approximation from principal
groups are equal; therefore, the homogeneity of variance component analysis (PCA) to measure the amount of var-
occurs in this case when p value [0.05, also the same iance contributed by each morphological character in each
occurs with multivariate normality test because its null study site and for all sites combined. The eigenvalues
hypothesis is that the combination of variables follow a (variance explained by each principal component) were
normal multivariate distribution. calculated, and the loadings (the weights of the eigenvec-
The order of entry for each of the variables by locality tors) were used to verify the variability contributed by each
was identified using the backward stepwise method from character from the correlation matrix in the first two prin-
Wilk’s Lambda statistic, a measure of the predictive power cipal components.
of a variable based on the amount of total variability Within-group correlation matrices were obtained for each
explained by the within-group (male vs. female) variabil- site to assess possible effects of collinearity (r [ 0.5 between
ity, if the value is close to zero, then groups are spread pairs of variables). Jackniffe procedure was applied to obtain
further apart and therefore greater variability explained. the rate of correctly classified individuals in selected func-
For each new variable included in a new step, we calcu- tions, which leaves out one of the data to calculate a new
lated the predictive power of the resulting function to function with the other observations, and then the model is
complete the total morphometric measures included in it. validated with the missing data. The procedure is repeated as
The order of entry of each variable to a new model was many times as there are observations. This procedure should

Polar Biol (2013) 36:1723–1734 1727

demonstrate the power of classification given by individual PL/PR primers had approximately an 81 % success rate.
Wilk’s Lambda values using each of the variables and those The P2/P8 DNA-based test identified a total of 266 indi-
obtained by the backward stepwise method. viduals (n = 300) at all stages of life, 54 females and 42
Despite satisfying the assumptions of normality and males from G. G. V. (n = 100), 37 females and 47 males
homogeneity of variance–covariance matrices, the variable from B. O’Higgins (n = 100), and 44 females and 42
body mass (BM) was removed for a final discriminant males from A. Island (n = 100). The PL/PR DNA-based
function given its high daily and seasonal variability (Za- test identified a total of 244 individuals: 43 females and 41
valaga and Paredes 1997), so only was used in the two-way males from G. G. V., 42 females and 39 males from B.
ANOVA, being discarded in the other procedures. Finally, O’Higgins, and 40 females and 39 males from A. Island.
only models from the backward stepwise method that met the A comparison between the results obtained for both primer
following criteria were considered: different means (two- pairs revealed a degree of sex misidentification (n = 11).
way ANOVA), univariate normal distribution (Kolmogo- Accordingly, the lack of sufficient dimorphism in juveniles
rov–Smirnov test with Lilliefors correction), correlation of and chicks, we only used the morphometric data of the
variables within groups less than 0.5, homogeneity of vari- adults (n = 172) to generate discriminant functions.
ances, Wilk’s Lambda value significant (p value \0.05) for Except for BW on G. G. V., males were larger than
the variables in selected function, and multivariate normal females for all variables. The means of FL, TAL, BL, BW,
distribution for the variables included in it. All of them, for and TOL was found to be statistically significant (two-way
different locations and all together. ANOVA; p value \0.05) between sex of penguins
The within-class correlation of the group matrices, the (Table 1), and FL, BL, BW, BD, TOL, and BM were sta-
order of the variables in the stepwise regression, and the tistically different between sites (Table 1). Furthermore,
jackknife validation were done in SSPS Statistics 21. The other variables were also found to be statistically different
rest of the statistical procedures were performed in the in some sites, but this was not generalized in all localities
software R (R Development Core Team 2011) from the (Table 2). When we combine the three sites together, we
packages: DiscriMiner (Sanchez 2012), MASS (Venables see that males are larger than females for TOL (Mean:
and Ripley 2002), Nortest (Gross and Ligges 2012), Bio- 73.94 cm females and 76.13 cm males; p value \0.001);
stats R (McGarigal 2008), Klar (Weihs et al. 2005), and result also found for FL (Mean: 21.21 cm females and
Energy (Rizzo and Szekely 2012). 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;
Results p value \0.001). When we compared all variables between
the three sites, we found that B. O’Higgins and A. Island do
The two primer pairs used to amplify regions of the CHD1 not have significant morphological differences except for
gene successfully amplified fragments from Gentoo Penguins. BD and BM (Table 2). However, G. G. V. has significant
Both P2/P8 and PL/PR showed the expected, two bands for differences with B. O’Higgins and A. Island for FL, BL,
females and only one for males. Since the differences between TOL, and BM, while BW was different only G. G. V. vs B.
the bands CHD1-Z and CHD1-W were so small, long runs on O’Higgins (p value \0.043) (Table 2). In addition, the
12 % acrylamide gel was required to be adequately resolved. variances were homogeneous (Bartlett test; p value [0.05)
However, we were able to obtain the same results using a for the variables FL, TAL, BL, BD, and TOL in the three
faster procedure on 3 % agarose gel with SB buffer. sites combined, while other variables had this property in
The success of amplification and efficacy for sex particular sites (Table 3).
determination of both primer pairs varied. The P2/P8 To G. G. V., the first two principal components (PC)
primers had approximately an 89 % success rate, while the account for 48.3 % of the variability of the data set

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

Sex 1 \0.001* 0.839 0.004* \0.001* \0.001* 0.001* \0.001* 0.191

Locality 2 \0.001* 0.711 0.345 \0.001* 0.015* \0.001* \0.001* \0.001*
Sex 9 locality 2 0.209 0.516 0.705 0.361 0.301 0.902 0.198 0.557
Residuals 166
* Significance with p value \0.05

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

Polar Biol (2013) 36:1723–1734 1729

a b


Comp 2(18.2%)

Comp 2(17.9%)



-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

Comp 2(19.8%)

Comp 2(19.7%)


-0.5 BD

-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

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

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

Polar Biol (2013) 36:1723–1734 1731

Fig. 4 Histogram of

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


O’Higgins (b), Ardley Island

(c) and the combination of all

study sites (d) on their

respective discriminant

functions Selected for sex

classification of Gentoo

penguins from Antarctica. Dark

Number of individuals
Gray represents females, white

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

n=49 c n=172 d




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

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

1732 Polar Biol (2013) 36:1723–1734

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

Polar Biol (2013) 36:1723–1734 1733

in this study can give instant results in the field when Cheney CA (1990) Spheniscus penguins: an overview of the world
sexing adults and can be used for diverse studies which captive population. Spheniscus Penguin Newsl 3:12–17
Costantini V, Guaricci AC, Laricchiuta P, Rausa P, Lacalandra GM
require sex determination along the Antarctic Peninsula (2008) DNA sexing in Humboldt Penguins (Spheniscus hum-
and to increase reproductive success of birds from captivity boldti) from feather samples. Anim Reprod Sci 106:162–167
breeding programs and to develop conservation plans de Dinechin M, Dobson FS, Zehtindjiev P, Metcheva R, Couchoux C,
(Cheney 1990; Fridolfsson and Ellegren 1999). According to Martin A, Quillfeldt P, Jouventin P (2012) The biogeography of
Gentoo Penguins (Pygoscelis papua). Can J Zool 90:352–360
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have shown geographic body size variations (Murie et al. the Southern Ocean. Glob Chang Biol 15:1618–1630
1991; Gandini et al. 1992; Renner and Davis 1999), which Fraser WR, Trivelpiece WZ, Ainley D, Trivelpiece SG (1992)
limit the applicability of discriminant functions across dif- Increases in Antarctic penguin populations: reduced competition
with whales or a loss of ice due to environmental warming?
ferent localities. We recommend the use of the different Polar Biol 11:525–531. doi:10.1007/BF00237945
discriminant functions considering the geographic morpho- Fridolfsson AK, Ellegren H (1999) A simple and universal method for
logical variation. Thus, we also standardized a general molecular sexing of non-ratite birds. J Avian Biol 30:116–121
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accuracy is not required for sex determination. Otherwise, diferencias en el tamaño corporal entre colonias para el uso de
the results of geographic morphological variation in South- medidas morfométricas como método de sexado en Spheniscus
ern Gentoo penguins found in our study are important start magellanicus. Hornero 13:211–213
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Acknowledgments This work was funded by Instituto Antártico Testing and improving the accuracy of discriminant function
Chileno (INACH-G_06-11; INACH-T-27-10 and INACH MG_02- tests: a comparison between morphometric and molecular sexing
12). D. Noll and B. Ramos for help in molecular procedures. in Macaroni Penguins. Waterbirds 32:437–443
L. Moreno and J. Hernandez for help in the field. F. Rengifo and Hocken AG, Russel JJ (2002) A method for determination of gender
N. Sallaberry for comments on the manuscript. from bill measurements in Otago blue penguins (Eudyptula
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