Sipe and Chittenden: A comparison of calcified structures for aging Paralichthys dentatus 
631 
et al. (1992). Briefly, readers enumerated marks (Fig. ID) 
that exhibited “cutting over” in both lateral fields of the 
scale that was accompanied by a clear narrow zone in the 
anterior portion of the scale. Scale radial length (ScRL) 
was measured to 0.001 mm from the center of the focus to 
the anterior edge of the scale by using a compound video 
microscope with the Optimas image analysis system (Me- 
dia Cybernetics, 1999). 
Evaluation of calcified structures 
Each structure was examined for age by two readers — 
twice by reader 1 and once by reader 2. Structures were 
read in a randomly selected order with no knowledge of 
fish size or collection date. Ages were assigned on the 
basis of presumed annual mark counts. Different struc- 
tures from the same fish were read independently, includ- 
ing right and left otoliths, and at least one week separated 
the first and second readings of the same structure. 
Preliminary evaluations of structures included prepara- 
tion times, reading times, confidence in the clarity of pre- 
sumed annual marks, growth of the structures with size 
of the fish, and agreement in repeated age readings of 
the same structure (precision). Structures judged accept- 
able based on those criteria were then evaluated further 
for agreement in age readings between different struc- 
tures from the same fish and to see if the number of pre- 
sumed annual marks increased with structure size and 
fish size. Our preliminary evaluation indicated otoliths 
and scales to be superior to opercular bones; therefore 
opercular bones were not evaluated further. 
Preparation time, a measure of the processing efficien- 
cy of a structure, was evaluated as the time taken to pre- 
pare structures for reading. Clarity of presumed annual 
marks on a structure was evaluated using both reading 
times and confidence scores. Reading time was measured 
as the time taken to read a given structure in an indi- 
vidual fish. Confidence scores, expressed on a scale of 1 
(low) to 5 (high), were assigned by the reader to each read- 
ing based on the clarity of the marks. Differences in confi- 
dence scores between structures were tested at a = 0.05 
by using the normal approximation to the Mann-Whitney 
test for ordinal data (Zar, 1996). 
The assumption that structure growth is directly relat- 
ed to fish growth was evaluated using regression analysis 
(Zar, 1996). Structure sizes (ScRL, OpRL, WOTL, WORL, 
SORL) were regressed on fish TL to determine if the re- 
lationships were significant and increasing. Sample sizes 
varied in these regressions, and in regressions of the num- 
ber of presumed annual marks on structure size described 
below because some structures were broken in prepara- 
tion and could not be measured. 
Precision in age determinations for a given structure 
was evaluated using simple percent agreement in repeat- 
ed readings within and between readers. Within-reader 
agreement compared the first and second readings by 
reader one, and between-reader agreement compared the 
first readings of each of the two readers. Reader comments 
on structure features were evaluated to determine the 
proximal causes of disagreements. 
Scales that disagreed in the initial two readings by reader 
1 were reread independently a third time by reader 1 
to reach a consensus for use in between-structure compar- 
isons. Likewise, right and left otoliths that disagreed in 
the initial two readings by reader 1 were read a third time 
to reach a consensus. Structures that showed no agree- 
ment in three readings (1 of 81 for scales, 1 of 81 for sec- 
tioned otoliths) were not included in between-structure 
comparisons. 
Agreement in presumed annual mark counts between 
different structures of an individual fish was evaluated by 
using simple percent agreement between structures and 
simple linear regression procedures. For the regressions, 
ages determined by one structure were regressed on ages 
determined by another structure, and the slope of the re- 
gression line was tested to see if it differed significantly 
from one. A slope of one implies that y = x and that the two 
structures give the same age. For each regression, we used 
as the x-variable the structure judged to be superior in the 
preliminary evaluations. 
The assumption that the number of presumed annual 
marks on a structure is directly related to structure size 
and to fish size was evaluated using regression analysis 
(Zar, 1996). The number of presumed annual marks on a 
structure was regressed on structure size (ScRL, WOTL, 
WORL, SORL) and on fish TL to determine if the relation- 
ships were significant and increasing. 
Results 
Comparative appearance of calcified structures 
All four calcified structures showed concentric marks that 
were interpreted as annual (Fig. 1). However, these struc- 
tures differed greatly in the clarity of presumed annual 
marks. 
Presumed annual marks on both whole and sectioned 
otoliths (Fig. 1, A and B) were typically clear, consistent, 
and easy to interpret, especially for sectioned otoliths. The 
right-left difference in the location of the focus had moder- 
ate effects on mark clarity for both whole and sectioned 
otoliths, as described below. Whole otolith marks were 
most easily read at younger ages, but age had little effect 
on sectioned otolith mark clarity. The few disagreements 
in otolith ages were primarily caused by an early, presum- 
ably false, mark that often occurred prior to the first pre- 
sumed annual mark (Fig. 2). This early mark appeared as 
a thin opaque band close to, but distinct from, the focus 
and was found on both young (Fig. 2A) and older (Fig. 2B) 
fish. We tried not to count this early mark in our age read- 
ings, because it did not occur consistently in all fish. Fi- 
nally, only one otolith of 81 pairs was poorly calcified and 
unable to be read whole, although its age was easily deter- 
mined upon sectioning. 
Presumed annual marks on opercular bones (Fig. 1C) 
were fairly clear in some fish, but they were more often 
poorly defined, inconsistent, and difficult to follow across 
the structure, making age interpretation difficult and high- 
ly subjective. Opercular bones commonly exhibited un- 
