ORIGINAL ARTICLE Sexual Size Dimorphism And Sex Determination By

original article sexual size dimorphism and sex determination by morphometric measurements in the coscoroba swan cecilia perez calabuig

ORIGINAL ARTICLE
Sexual size dimorphism and sex determination by morphometric measurements
in the Coscoroba Swan
Cecilia Perez Calabuiga∗, Andy J. Greenb , Miguel Ferrera , Roberto
Muriela & Heden Moreirac
a Department of Biodiversity Conservation and Applied Biology,
Estación Biológica de Doñana-CSIC, Sevilla, Spain; b Department of Wetland
Ecology, Estación Biológica de Doñana-CSIC, Sevilla, Spain; c Department
of Genetics and Zoology, Federal University of Pelotas, Pelotas, RS, Brazil
The accuracy of morphological sexing and the occurrence of sexual
dimorphism were analyzed in mature and immature Coscoroba Swans (Coscoroba
coscoroba, Anatidae) near the Estação Ecológica do Taim, southern Brazil.
On the basis of weight and 10 linear measurements of external morphology,
multivariate analysis of vari- ance showed that males were consistently
larger than females (sex confirmed via genetic markers) and mature birds
were consistently larger than immatures. Overall, 38% of immatures and
14% of mature birds were sexed incorrectly by cloacal examination when
compared to genetic data. Therefore, we performed a discriminant
function analysis of different age classes based on morphometric measurements.
Mature birds were sexed with 96% accuracy using head and tarsus
lengths as predictor variables, whereas immatures were sexed with 90%
accuracy based on head and forearm lengths. Method validation
conducted with data for additional mature sampled in a different year
showed that the use of head length alone was as accurate for sexing
(92% correct classification) than discriminant functions based on two
characteristics (91%).
Keywords: Brazil; cloacal examination; Coscoroba Swan; genetic sexing;
morphological sexing; sexual size dimorphism
Introduction
Accurate and easy methods to determine the sex of individuals are valuable
for studies of avian evolu- tionary ecology and genetics, population dynamics,
behavior, migration and conservation management of species and populations
(Clutton-Brock 1986; Newton
1998). In species lacking obvious sexual dimorphism, cloacal sexing is
often used but can be unreliable (Brown et al. 2003; Odwyer et al.
2006, Volodin et al. 2009). Molecular sexing, based on the amplifica-
tion of the chromo-helicase-DNA-binding 1 (CHD1) gene, is the most reliable
method (Ellegren & Sheldon
1997; Griffiths et al. 1998), but is relatively expensive. However,
molecular sexing can complement meth- ods based on discriminant
function analysis (DFA) in species with weak size dimorphism (Dumbell
et al.
1988; Ackerman et al. 2008; Hart et al. 2009). In waterbirds, DFA has
often been used to combine the discriminatory power of single characters
into one formula that best discriminates between sexes (e.g. Weidinger
& van Franeker 1998; Svagelj & Quintana
2007; Ackerman et al. 2008; Quintana et al. 2008; Hart et al. 2009). DFA
can be a reliable, fast and inexpen- sive method for discriminating
the sex of individuals
during non-breeding seasons when sexually dimorphic characters are not
expressed (e.g. Zavalaga & Paredes
1997; Bourgeois et al. 2007).
The Coscoroba (Coscoroba coscoroba) is an unusual member of the Anatidae
found in South America, from the Falkland Islands and Tierra del Fuego,
north through Chile and Argentina, Uruguay and southern Brazil, and as
far north as Paraguay (Kear 2005). In Brazil, C. coscoroba occurs year-round
in Rio Grande do Sul (Belton 2000) with irregular records in other states
(Bornschein et al. 1997). It is traditionally considered to be a swan,
but molec- ular studies suggest it is more closely related to the aberrant
Cape Barren Goose Cereopsis novaehollan- diae (Donne-Goussé et al.
2002). Very little is known about its ecology (see Kear 2005 for a review).
It is a monochromatic species, and previous attempts to separate sexes
based on morphology have been unsuc- cessful (Nascimento et al. 2001).
DFA has previously been applied in some other swan species (e.g. Miller
et al. 1988; Brown et al. 2003). Other studies of swans have compared
different size measures between sexes without applying DFA (e.g. Scott
1972; Mathiasson
1981; 2005; Limpert et al. 1987).

∗ Corresponding author. Email: [email protected]
The present study describes sexual dimorphism of immature and
mature Coscoroba in Brazil, and compares the accuracy of alternative sexing
methods. The main objectives were to: (a) determine the accu- racy of
cloacal examination, by comparing the results with those from
molecular sexing; (b) present typical morphometric measures of Coscoroba,
and determine differences between the sexes and two age classes iden-
tifiable by plumage characteristics; (c) develop discrim- inant models
to facilitate the sexing of birds based on morphometry; and (d) compare
the accuracy of DFA with that of sexing by cloacal examination.
Materials and methods
Study area
Rio Grande do Sul State in the southern tip of Brazil has a big lagoon
complex in the southern portion of its coastal plain, formed by Patos,
Mirim, and Mangueira Lagoons and other smaller lakes. All birds were cap-
tured near the “Estação Ecológica do Taim”, an area located in the
southern coastal plain of RS, between the counties of Rio Grande and
Santa Vitória do
Palmar (∼32◦23 W, 52◦32 W). This area was cho-
sen because it is within the most important area for
breeding and molting of Coscoroba in Brazil (Seijas
2001).
Capture of birds and sampling
Birds were captured by hand from a boat during flightless wing molt
between August and January (Nascimento et al. 2001; Seijas 2001) in three
consecu- tive seasons (2005, 2006 and 2007). Birds were divided into two
groups: immatures (fledged but less than two years old) and matures (>2
years old) according to plumage characteristics (Johnsgard 1978). Coscoroba
Swans have red eyes, legs, feet and bill from imma- ture to mature age
classes but there are differences in the plumage pattern. The adult’s
definitive plumage is totally white with the exception of a black tip
on the first six external primaries.
Although plumage is mainly white, immature Coscoroba Swans have brownish
down feathers and brightish brown plumes all over the body and have grayish
brown stains on the tail, back and wings (in primaries, secondaries
and upper wing coverts). Mature swans have gray down feathers only on
the back and under the wings (axillaries). They usually display more
than six primaries with grayish brown fleckings but very rarely on the
secondaries (Calabuig et al. 2010).
The immature group consisted of 41 individuals
(19, 11 and 11 for 2005, 2006 and 2007, respectively).
The mature group consisted of 345 individuals (120,
123 and 102 for 2005, 2006 and 2007, respectively).
Morphometric measurements
Birds were weighed with a balance (to the nearest 20 g) and 10
biometric measures were taken by the same person (CPC). A digital
caliper was used to measure the following (to the nearest 0.1 mm):
head length (length of head to the occipital-tip of the bill), bill
height, bill depth (maximum width of the bill), nos- tril (distal edge
of a nostril to the end of the bill), total culmen and tarsus length
(on the left side of the body). A ruler was used to measure the following
(to the near- est 1 mm): tail (from the preen gland), wing length
without feathers (metacarpophalangeal articulation), forearm (Ferrer &
De le Court 1992) and neck length. Before releasing, all swans were
banded with a num- bered metal CEMAVE-IBAMA ring. Recaptures were readily
identified, and data were only used for each individual on the first capture.
Cloacal and genetic sex determination
Genital identification
Cloacal examination was carried out by CPC, identify- ing “males” by
the presence of a visible erectile groove penis on the ventral wall of
the cloaca (Proctor & Lynch 1993; Brown et al. 2003; Mathiasson 2005).
DNA identification
Blood (3 ml) was taken from each bird from the wing vein and samples were
stored in Vacutainer tubes with EDTA and kept cool in ice, until processing.
Blood samples were analyzed in the Biotechnology Center (CenBiot), Federal
University of Pelotas, Brazil. DNA extraction was performed according
to the procedures of Lahiri and Nurnberger (1991) and DNA samples were
amplified using primers described by He et al. (2005) for the Tundra Swan
(Cygnus atratus). The PCR amplifications were carried out in the reaction
mixture of Ito et al. (2003). After amplification, the PCR products were
separated on a 1% agarose gel (synthesized by Sangon Co., Shangai,
China), stained with ethidium bromide (Sangon Co.) and visualized
under UV light.
Data analyses
We used multivariate analysis of variance (MANOVA) to compare mean differences
between age and sex groups for morphological measurements and body mass.
Also, one-way analysis of variance was used to
determine whether individual measures varied with sex in each age group
(Sokal & Rohlf 1995). For both analyses, we used only the 225 mature
Coscorobas sampled during the 2006 and 2007 seasons (those used for DFA,
see below). Differences were considered sig-
nificant at p ≤ 0.05. All data satisfied Lilliefors and
Levene tests of normality.
For all variables, we calculated a sexual size dimorphism index
as: SSD = {(xm − x¯ f )/x¯ f }× 100 (Weidinger & van Franeker 1998; Svagelj
& Quintana
2007); where x¯ m and x¯ f are the mean values of differ- ent age
males and females, respectively. The coefficient of variation (CV = (SD/x¯
) × 100) was calculated for each sex and averaged between them
(Fletcher &
Hamer 2003) to indicate the degree of variability of each measurement
(Sokal & Rohlf 1995).
DFAs were developed separately for immature and mature birds. We excluded
body mass from these anal- yses since it varies greatly over time (Croxall
1995; Svagelj & Quintana 2007). The performance of each single measurement
as a discriminating variable (uni- variate DFA) was evaluated. Forward
DFAs were applied to obtain combinations of characteristics (dis-
criminant functions) that best distinguished the sexes (see Tabachnick
& Fidell 1996; Phillips & Furness
1997).
For immature birds, the DFA was applied to all individuals. For mature
birds, the DFA was applied to 225 Coscorobas sampled in 2006 and 2007.
For both age classes, the effectiveness of the analyses was assessed,
first in terms of the proportion of birds of known sex that were
classified correctly, and second by jackknife validation. Correct
classification rates tend
immature birds; overall, 38% of immature Coscoroba were sexed incorrectly.
Among mature birds, overall,
14% of mature Coscoroba were sexed incorrectly.
Morphometric differences according to sex and age
Analysis of the whole dataset showed that mature Coscoroba were larger
than immatures and males were bigger than females, with no age × sex
interaction
(MANOVA: age: F11, 242 = 4.6, p < 0.01, Wilks = 0.82;
sex: F = 29, p < 0.01, Wilks = 0.43; and age × sex:
F11, 354 = 1.5, p = 0.1, Wilks = 0.93). Thus, in each
age cohort, males were larger than females in all mea-
surements, except in tail length for immature birds
(Tables 1 and 2).
In immature birds, characteristics that showed the highest sexual size
dimorphism were mass and tail, whereas bill height, wing and tail were
less dimorphic. Mass showed the highest within-sex variation whilst
head length had the lowest (Table 1). In mature birds, mass, tarsus
and neck length showed the highest sexual size dimorphisms whereas
bill depth and tail were less dimorphic. Mass and neck length showed
the highest within-sex variation whilst bill depth and head length had
the lowest (Table 2).
Table 1. Body measurements (mean ± SD and range, in mm), coefficients
of variation (CV) and sexual size dimorphism index (SSD) for immature
male and female Coscoroba Swans (Coscoroba coscoroba) from southern
Rio
Grande do Sul, Brazil. All measured characteristics differed
between the sexes (p < 0.05) except tail length. CV was first calculated
for each sex and then averaged.
One-way
to be overestimated when DFAs are validated with the
same sample used to generate them (Tabachnick &
Male Female
Body
ANOVA (%)
Fidell 1996). The jackknife validation is a process
in which each individual case is classified using a function obtained
from the total sample, excluding the individ- ual case to be
classified (Tabachnick & Fidell 1996). Furthermore, the accuracy of DFAs
for mature birds was confirmed by applying the resulting functions to
a novel dataset, composed of 120 birds captured in the
2005 season.
Results
Cloacal and genetic sex determination
Genetic sexing showed that there were 14 males and
27 females among young birds, and 189 males and 156 females among mature
birds. Some birds were incor- rectly classified by cloacal examination.
Although genetic females were rarely misclassified as “males”, males were
often wrongly identified as “female”. Cloacal inspection was particularly
unreliable for
measurement n = 14 n = 27 F1.39 CV SSD
Mass (g) 3760 ± 460 3230 ± 400 14.4 14.4 16.1
2650 – 4400 2250 – 4100
Total culmen 70.6 ± 4.6 65.4 ± 2.4 22.7 6.1 8.0
62.5 – 79.2 62 – 73
Nostril 52.7 ± 2.8 48.7 ± 2.6 21.4 6.6 8.2
48.6 – 56.6 41.3 – 56.3
Bill height 25.7 ± 1.0 24.5 ± 1.5 6.7 6 4.9
23.6 – 27 22.2 – 28.8
Bill depth 30.2 ± 1.4 28.5 ± 1.3 13.7 5.2 6.0
27.2 – 32.7 24 – 30.1
Head length 138 ± 4.9 129.6 ± 3.1 46.6 4.2 6.5
129 – 146.3 123.1 – 135
Tarsus 101.6 ± 5.6 95.4 ± 4.4 14.6 5.7 6.5
87.8 – 108.2 85 – 104
Wing 173.8 ± 7.1 164.1 ± 9.7 10.9 6 5.9
162 – 185 145 – 185
Forearm 213.1 ± 8.3 195.9 ± 8.8 37.7 5.9 8.8
200 – 225 175 – 215
Tail 183.1 ± 14.7 173.2 ± 21.9 2.5 11.4 5.7
161 – 205 107 – 220
Neck length 345.4 ± 24.7 315 ± 21.8 16.1 8.3 9.6
305 – 390 260 – 370
Table 2. Body measurements (mean ± SD and range, in mm), coefficients
of variation (CV) and sexual size dimor- phism index (SSD) for mature
male and female Coscoroba Swans (Coscoroba coscoroba) sampled in 2006
and 2007 in
southern Rio Grande do Sul, Brazil. All measured charac-
teristics differed between the sexes (p < 0.05). CV was first calculated
for each sex and then averaged.
Table 3. Accuracy of sexing of immature Coscoroba Swans using single
measurements or combinations in a discrimi- nant function (DF1 = head
length and forearm). Identical results were obtained by the jackknife
method (see text).
Original sample (n = 41) (% correct classification)
Body
measurement Males Females
One-way
ANOVA (%)
Variables
Wilks’
lambda Males Females Overall
Cut-off point (mm)
2006–2007 n = 125 n = 100 F 1.223 CV SSD
Mass (g) 4000 ± 335 3340 ± 360 197.2 12.9 19.8
3050 – 5140 2400 – 4500
Total culmen 70.6 ± 2.6 65.5 ± 2.3 241.5 5.1 7.8
62.8 – 78.8 59.8 – 71.8
Nostril 52.8 ± 1.6 49 ± 2.2 221.7 5.3 7.8
48.4 – 58.8 40 – 59.2
Bill height 26.3 ± 1.1 24.6 ± 1.1 136.3 5.5 7.1
23.7 – 29 22.2 – 27.5
Bill depth 30.7 ± 0.8 29.2 ± 0.9 152.2 3.7 4.9
29 – 33.3 26.4 – 31.3
Head length 140.1 ± 2.8 131.2 ± 2.8 548.9 3.9 6.8
132.2 – 147 123.8 – 138.8
Tarsus 105.8 ± 3.8 96.1 ± 3.4 397.3 6 10.1
93.3 – 117.2 84.6 – 104.5
Wing 178.7 ± 7.5 165 ± 8.6 162.8 6.1 8.3
152 – 200 130 – 186
Forearm 212.6 ± 8 196.4 ± 7.7 235.3 5.5 8.2
177.0 – 230 168 – 217
Total culmen 0.63 64.3 92.6 83 69.3
Nostril 0.65 71.4 89 83 51.9
Bill height 0.85 64.3 74.1 70.7 26.2
Bill depth 0.74 78.6 70.4 73.2 30.1
Head length 0.45 86 89 88 135.0
Tarsus 0.73 78.6 66.7 70.7 101.0
Wing 0.78 78.6 70.4 73.2 173.8
Forearm 0.51 78.6 81.5 80.5 207.1
Tail 0.94 64.3 66.7 66 202.4
Neck length 0.74 78.6 70.4 73.2 339.0
DF1 0.42 78.6 96.3 90.2
only two Coscorobas differed between classifications (changing the correct
classification to 86% for males and 85% overall). Validation with a novel
sample of birds provided slightly different classifications when compared
with DFA, decreasing the accuracy for
Tail 175.4 ± 6.9 166.4 ± 8.5 76.2 5.1 5.4
145 – 190 141 – 187
all measures except for bill height and
bill
depth
Neck length 355.9 ± 18.6 317.3 ± 20.2 221.1 8 12.2
275 – 400 265 – 365
Discriminant analysis
Immature swans
Head length was the most accurate single indicator of sex, correctly
classifying 88% of birds (Table 3). Coscorobas with head lengths
longer than 135 mm were classified as males. Jackknife validation provided
exactly the same classifications as those produced by DFAs for all
single measurements. DFA retained head length and forearm as the best
predictor variables and excluded others. This model correctly
classified 79% of females and 96% of males (Table 3). Only three
females and one male were misclassified. The discrimi- nant function
obtained for immature Coscorobas was:
DF1 = 0.43 (head length) + 0.13 (forearm) – 84.6.
Values of DF1 > 0 identified males and values of
DF1 < 0 identified females.
Mature swans
Head length was the most accurate single indica- tor of sex, correctly
classifying 93% of Coscorobas (Table 4). Jackknife validation provided
exactly the same classifications as those produced by DFAs for all
single measurements except for nostril length, where
(Table 4). DFA retained head length and tarsus as the
best predictors. This model correctly classified 96% of females and
95% of males (overall success, 96%, three females and four males
misclassified) with a low value for Wilks’ lambda (Table 4). The DF1
obtained for
mature Coscorobas was: DF1 = 0.84 (head length)
+ 0.38 (tarsus) – 152.6. This model was represented
in Figure 1 where mature Coscorobas were classified
according to head length and tarsus measurements. Misclassifications were
unusually large females mis- classified as males.
A DFA with only head length as predictor vari- able classified 93% of
cases correctly, with a value of Wilks’ lambda close to the best model
with head length and tarsus together. This alternative discrim-
inant function (DF2) was: DF2 = 1.1 (head length)
– 150. DF2 performed slightly better than DF1 when
validated against a new sample of birds (Table 4), with
92% of individuals sexed correctly.
Discussion
Sex determination by cloacal inspection
Although cloacal examination is a widely used method for sexing waterfowl
(e.g. Green 2000; Gray & Hamer
2001; Nascimento et al. 2001), it has several disadvan- tages. This
method requires observers with consider- able experience, is only
possible during the breeding

Table 4. Accuracy of sexing of adult Coscoroba Swans using
single measurements and a discriminant function (DF1 = head length and
tarsus) for 2006 and 2007 (original sample included in discriminant
analyses) and 2005 (used for validation, see text). A jackknife method
produced identical results to those shown here for the original sample,
with the exception of nostril (see text).
Original sample (n = 225) (% correct classification) New sample cross-validation
(n = 120)
Variables
Wilks’ lambda
Males
Females
Overall
Males
Females
Cut-off point (mm)
Total culmen
0.48
88.8
87.9
88.4
92.2
75
67.8
Nostril
0.5
88
83.8
86.2
98.4
54
48.8
Bill height
0.62
75
81
77.7
67.2
90.2
25.3
Bill depth
0.59
80
77
78.7
100
78.4
19.4
Head length
0.29
94.4
92
93.3
91
93
136.3
Tarsus
0.36
90.4
89.9
90.1
86
87.5
100.4
Wing
0.58
83.2
78
80.9
87.5
60.3
173.3
Forearm
0.49
87.9
84.8
86.5
97
66.1
203.4
Tail
0.74
76.6
69.8
73.6
98.4
48
169.2
Neck length
0.5
87.2
82
84.9
73.5
80.4
330.8
DF1
0.26
95.2
95.9
95.5
91
91.1

Figure 1. Plot of 329 mature Coscoroba Swans from southern Rio Grande
do Sul, Brazil, according to head length and forearm length. Males and
females were identified by genetic sexing. The straight line represents
50% probability of sex classification according to the discriminant
function.
period for some species, and can cause internal dam- age to
the bird due to the pressure applied during penis visualization (e.g.
Sax & Hoi 1998; Lombardo 2001; Oliveira et al. 2004). We found it to
be an unreliable method for the Coscoroba, owing to the difficulties
of observing the penis, especially in young birds where penis development
is incomplete (Odwyer et al. 2006). Similar levels of inaccuracy have
been recorded in petrels (Odwyer et al. 2006) and in whistling ducks
(genus Dendrocygna, Volodin et al. 2009). We do not believe that the
error in sexing can be attributed to an observer effect in our study
because the person who sexed the swans was an experienced specialist who
has been ringing waterbirds for 20 years. Previous studies relying exclusively
on cloacal inspection to sex Anatidae (e.g. Green 2000) may also
contain errors.
Sexual size dimorphism and age effects
Few previous data were available on the morphomet- rics of Coscoroba (Kear
2005). Our results indicate significant size dimorphism, with male
Coscoroba generally being larger than females, both for immature and mature
birds. In general, large size may be advan- tageous in male swans and
geese because they are responsible for nest protection (Scott 1972; Veselovsky
1973; Hawkins 1986; Whitehead & Tschirner 1990) or may need to defend
females against males seek- ing extra-pair copulations (Mineau & Cooke
1979; McKinney et al. 1983; Welsh & Sedinger 1990; Gauthier & Tardif
1991; Choinière & Gauthier 1995). As in the Cape Barren Goose, both
Coscoroba sexes care for their cygnets and maintain long-term pair
bonds, but the male is primarily a guardian and helps with nest building
but not incubation.
Mass and neck length showed the highest degree of sexual size
dimorphism in Coscoroba, but head length was the most useful single variable
to distinguish between males and females, because it had an interme-
diate level of dimorphism combined with particularly low CV. In most
bird species, intraspecific variation is markedly lower in the bill
and other body parts asso- ciated with food intake than in the rest of
the body, perhaps as a result of adaptation to particular forag- ing
behavior and diet (Miller et al. 1988). However, different CVs between
measures can also reflect dif- ferences in measurement error (Yezerinac
et al. 1992), and neck length is likely to have had a relatively high
measurement error in our study. Our findings were consistent with previous
studies that showed that head (e.g. Veselovsky 1973; Miller et al.
1988; Brown et al.
2003), tarsus (e.g. Veselovsky 1973; Brown et al. 2003; Mathiasson
2005) and forearm (e.g. Mathiasson 2005) lengths are key characteristics
to differentiate sexes in Anseriformes.
Coscoroba Swans reach maturity when they are two years old and start breeding
at three to four years old (Wilmore 1979; Silva-García & Brewer
2007). Although birds are generally assumed to have determinate growth,
our results showed that mature Coscoroba differed significantly in size
from swans less than two years of age, implying that growth continues
in this species for an extended period prior to reach- ing sexual maturity
(Kirkwood et al. 1989; Carrier & Auriemma 1992; Tumarkin-Deratzian et
al. 2006). ANOVAs of individual measures showed that mass, bill depth,
head length, tarsus, tail and neck length were all significant greater
in mature birds (results not shown). Growth rates of large, precocious
birds such as swans are particularly slow (Ricklefs 1973; Carrier & Auriemma
1992).
Sex determination by discriminant function analysis
Discriminant functions were developed using only two morphometric variables:
head and forearm lengths for immature birds, and head and tarsus
lengths for mature birds, resulting in 90% and 96% of correct sexual
classification, respectively. These classification rates are much
higher than those based on cloacal inspection. However, cross-validation
with a new sam- ple of mature swans suggested that a single measure
(head length) was the most reliable sexing method, with 92% correct
classification. While jackknife val- idation reveals influential observations
that can bias DFA, cross-validation is a more rigorous validation process
and should ideally involve a new sample of individuals measured at different
times, locations and by different observers (Tabachnick & Fidell
1996). We suspect that discriminant functions would provide a better
alternative to cloacal sexing in many other bird species.
More research is required to calibrate the use of our discriminant
functions in other Coscoroba popu- lations with other observers. Given
the results of the validation with novel data, sex determination based
on head length is likely to be the most robust dis- criminant function
method. When cloacal inspection is carried out without genetic sexing,
these data can be plotted together with head length data to establish
the best cut-off point for sex determination based on morphometrics. For
example, in a population in which Coscoroba were physically smaller,
the cut-off points of Tables 3 and 4 for head length might be too
high.
Acknowledgments
We would like to thank S. Scherer for his valuable assistance in the
field. We thank Dr. B. Vaz, R. Tavares, J. Camacho and L. Bassini from
the Department of
Genetics and Zoology, Federal University of Pelotas, RS, Brazil
for technical help in carrying out DNA extraction.
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