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Fishery Bulletin 1 12(4) 
Figure 2 
Map with sample sizes of age-1 female southern flounder ( Paralich - 
thys lethostigma) collected within the state waters of North Caro- 
lina in 2009 and 2010 as part of a sampling program of the North 
Carolina Division of Marine Fisheries (NCDMF) and from seafood 
dealers. The 3 regions are based on NCDMF sampling areas; howev- 
er, they are characterized also by different estuaries. The northern 
region includes Albemarle Sound, the central region consists of the 
Neuse-Pamlico Estuary, and the southern region is defined by the 
Cape Fear and New River estuaries. 
dots per inch), yielding images comparable in quality 
to those from high-resolution photographs. Use of a 
flatbed scanner permitted us to scan relatively large 
batches of samples simultaneously (without the bend- 
ing error that is a concern with optical microscopy). 
Southern flounder otoliths are relatively flat, and all 
otoliths were scanned sulcus-side down in a uniform 
orientation. 
Otolith shapes were described by using 1) ellipti- 
cal Fourier analysis (EFA) and 2) calculation of uni- 
variate shape indices. Compared with other types of 
Fourier transforms (e.g., fast Fourier transform), EFA 
is thought to provide the most consistent results for 
this type of application (Merigot et al., 2007). The EFA 
method decomposes the closed-form contour into sepa- 
rate x and y coordinates (Kuhl and Giardina, 1982). 
Generally, a predetermined number of harmonics is de- 
fined for analysis, and each harmonic involves 2 para- 
metric functions that describe 4 coefficients (i.e., ampli- 
tudes), a n , b n , c n , and d n for the n th harmonic. 
In this study, elliptical Fourier coefficients (EFCs) 
were calculated with the program SHAPE, vers. 1.3 
(Iwata and Ukai, 2002). SHAPE software extracts the 
chain-coded contour of each otolith (Freeman, 
1974), and then the software normalizes (i.e., 
removes any size effects) the chain-code data on 
the basis of the first 3 coefficients of the first 
harmonics. As a result of this normalizing, the 
first 3 coefficients of the first harmonic are de- 
graded ultimately and are unfit for analysis; the 
total number of coefficients = (4xi7 n ) - 3, where 
H n represents the number of harmonics investi- 
gated. This study extracted 10 harmonics, or 37 
EFCs, for analysis. 
Several size parameters and shape indices 
(Table 1) were also calculated for each otolith. 
Otolith area (in square millimeters) was provid- 
ed as part of the SHAPE output analysis, and 
perimeter data (in millimeters) were calculated 
from the chain-code output file. Both otolith 
length and otolith width were measured (in mil- 
limeters) with ImageJ software, vers. 1.45 (Na- 
tional Institutes of Health, Bethesda, MD) ac- 
cording to otolith dimension definitions provided 
in Stevenson and Campana (1992). These size 
variables were then used to calculate several 
shape indices, including circularity, rectangular- 
ity, ellipticity, aspect ratio, and form function. 
Each of these variables is a common shape index 
used routinely in otolith morphometric investi- 
gations (Table 1; Russ, 1990). 
Statistical procedures 
Univariate shape indices were examined for 
normality through the use of normal quantile- 
quantile plots, and any non-normal distributions 
underwent log transformation in an attempt to 
satisfy the assumption of normality (Cadrin et 
al. 2005). To identify and control for the effect of oto- 
lith size on the shape indices, an analysis of covari- 
ance (ANCOVA) was used with geographic region as 
Table 1 
Size parameters, shape indices, and shape formulae 
used to describe otoliths from female southern flounder 
(Paralichthys lethostigma ) collected in the U.S. South 
Atlantic and Gulf of Mexico in 1996 and in 2009-12 as 
part of state or federal agency sampling programs and 
from seafood dealers for this study of the population 
structure of southern flounder in these basins. 
Size parameter 
Shape index and formula 
Area (A) 
Perimeter (P) 
Otolith length (OL) 
Otolith width (OW) 
Circularity = P 2 /A 
Rectangularity = AKOLxOW) 
Form function = AnAJP 2 
Aspect ratio = OL / OW 
Ellipticity = ( OL-OW)/(OL+OW) 
