Vasconcelos et al.: Population identification of Trachurus picturatus in the Northeast Atlantic 
85 
Figure 4 
Illustration showing the 12 anatomical landmarks of the blue jack mackerel (Tra¬ 
churus picturatus ): 1) anterior tip of the mouth; 2) origin of the 1 st dorsal fin; 3) 
posterior end of the 1 st dorsal fin; 4) origin of the 2 nd dorsal fin; 5) posterior end of 
the 2 nd dorsal fin; 6) caudal peduncle; 7) middle point of the forked caudal fin; 8) 
inflection point of the lateral line; 9) origin of the anal fin; 10) origin of the pelvic 
fin; 11) posterior insertion of the operculum; and 12) insertion of the pectoral fin. 
Otolith shape analysis 
A total of 277 otoliths were analyzed (Fig. 3): 100 from 
waters off Peniche (22-37 cm TL, 40 females and 60 
males), 100 off Madeira (15-27 cm TL, 50 females and 
50 males), and 77 off the Canary Islands (14-23 cm 
TL, 34 females and 43 males). 
The otoliths were positioned on a microscope slide 
and photographed with the sulcus acusticus facing 
down and the rostrum to the left on the horizontal 
plane to reduce distortion errors in the normalization 
process. High-contrast digital images were captured by 
a Leica EC3 digital camera (Leica Microsystems, Wetz- 
lar, Germany) linked to a Leica MZ9.5 stereomicroscope 
that uses transmitted light and with the software Leica 
Application Suite X Core, vers. 4.5, that delivers dark 
2-dimensional objects against a white background. The 
microscope magnification was adjusted to the largest 
otolith size to ensure the same magnification (8x) for 
all otoliths. 
The image processing program SHAPE, vers. 1.3 
(Iwata and Ukai, 2002), was used to enhance contrast 
of the images, transforming color or grayscale images 
to black-and-white silhouettes of the structure (Cadrin 
and Friedland, 2005). Otoliths contours were extracted 
as chain-codes from the digital image by means of the 
ChainCoder package that is part of SHAPE. The el¬ 
liptic Fourier descriptors, or harmonics, were obtained 
from the SHAPE package Chc2Nef by using a discrete 
Fourier transformation of the chain-coded contour 
(Kuhl and Giardina, 1982; Lestrel, 1989, 1997). The 
four coefficients of each harmonic were normalized 
in relation to the first harmonic to become invariant 
to otolith size, rotation, and starting point (Kuhl and 
Giardina, 1982; Iwata and Ukai, 2002). The first three 
coefficients of the first harmonic were degenerated to 
fixed values: aj=l, b 1 =c 1 =0 (Tracey et ah, 2006). 
A random subsample of 10 otoliths for each sex 
and area was used to define the minimum number of 
harmonics needed (99% accumulated variance) for the 
multivariate analysis. For each otolith, 100 harmon¬ 
ics were generated. Possible effect of area and sex on 
otolith normalized elliptic Fourier descriptors (NEFDs) 
was tested by using MANOVA. To detect possible mor¬ 
phometric differences in the contour shape of otoliths 
from the three studied areas, a canonical discriminant 
analysis was performed and a jackknife cross-valida¬ 
tion procedure was carried out with IBM SPSS soft¬ 
ware to validate similarities between groups by listing 
the misclassification of individuals within other areas 
(Neves et al., 2011; Vieira et al., 2014). 
A significance level of 0.05 was set for all statistical 
tests used. 
Results 
Body shape analysis 
No shape differences were found between males and 
females within the three areas under study (Madeira: 
F=0.15, P=0.72; Peniche: F=0.68, P=0.83; Canary Is¬ 
lands: F=0.40, P=0.59), and therefore sexes were pooled 
for each region. 
A significant difference in the mean body shape of 
blue jack mackerel was found for each of the three 
study areas (Table 1). The overall assignment of blue 
jack mackerel individuals in their original sample was 
correctly classified for 78.0% of the total number of 
specimens (Table 2). In the jackknifed classification 
matrix of the discriminant analysis, a proportion of 
33.0% of the specimens from Madeira were incorrectly 
assigned to the Canary Islands (23.0%) and Peniche 
(10.0%). Misclassifications also occurred between the 
