Using otolith shape and morphometry to identify for Alburnus species

Journal of
Applied Ichthyology
J. Appl. Ichthyol. (2015), 1–10
© 2015 Blackwell Verlag GmbH
ISSN 0175–8659
Received: May 27, 2014
Accepted: April 17, 2015
doi: 10.1111/jai.12860
Using otolith shape and morphometry to identify four Alburnus species
(A. chalcoides, A. escherichii, A. mossulensis and A. tarichi) in Turkish inland
waters
By D. Bostanci1, N. Polat2, G. Kurucu1, S. Yedier1, S. Kontasß1 and M. Darcßin1
1
Department of Biology, Ordu University, Ordu, Turkey; 2Department of Biology, Mayıs University, Samsun, Turkey
Summary
Asteriscus otolith shapes as well as their morphometry and
shape contours were investigated in order to identify four
allopatric Alburnus species: A. chalcoides (G€
uldenst€
adt, 1772)
(Ordu), A. escherichii Steindachner, 1897 (Eskisßehir),
A. mossulensis Heckel, 1843 (Tunceli), and A. tarichi
(G€
uldenst€adt, 1814) (Van) in Turkish inland waters. These
were compared using the shape indices (form factor, roundness, circularity, ellipticity, rectangularity and aspect ratio),
and the morphological characters [otolith weight (OWE),
otolith length (OL), otolith width (OW), otolith perimeter
(OP), and otolith area (OA)]. The overall canonical discriminant analysis (CDA) classification score was 93.8%, with the
lowest score for A. escherichii (82.5%) and the highest for
A. chalcoides (100%). The otolith shapes, morphology and
shape contours of all sampled fish were a clear species differentiator, thereby demonstrating that the otolith shape is species-specific. The current study presents for the first time
comprehensive variation information on interspecific leftright asteriscus otoliths in males and females of each Alburnus species: A. chalcoides from Ordu, A. escherichii from
Eskisßehir, A. mossulensis from Tunceli and A. tarichi from
Van, based on a total of 307 individuals. Scanning electron
microscopy (SEM) images, shape contours and other otolith
characters vary within the same genus; these differences
should be investigated not only in other freshwater fish species or genera but also in the same species living in different
habitats. In addition, further investigation is required not
only with respect to the morphometry, biometry, shape,
geometry, and shape contours of the otoliths, but also
regarding the genetic methods for robust identification of
various sympatric and allopatric fish populations.
Introduction
In predictive linear regressions, the morphometric otolith
parameters such as length, width, thickness, and weight are
used to estimate fish size. Numerous factors such as water
temperature, diet (Torres et al., 2000; Gauldie and Crampton, 2002; Volpedo and Fuchs, 2010), type of substrate and
water depth can affect fish growth, otolith size and shape
(Lombarte et al., 2003; Cardinale et al., 2004; Monteiro
et al., 2005). Consequently, morphometric measurements and
otolith morphology can differ among populations of the
same species in different locations (Reichenbacher et al.,
2009). Individuals of the same species and even of the same
sex as well as their phylogenetic patterns can be reflected in
their morphology (Lombarte and Cruz, 2007).
Studies of otolith morphology have recently increased in
importance with the development of image analysis systems
using a Scanning Electron Microscope (SEM). Thus, otolith
morphology is a most crucial criteria in fisheries, whereby a
number of identification guides, atlases and fisheries studies
on otolith morphology have been published for a variety of
species (Smale et al., 1995; Tuset et al., 2008). Some studies
concern the identification of species, stocks and populations
as well as their relationships, and are dependent on the otolith shape and morphometry analyses (Bird et al., 1986;
Campana and Casselman, 1993; Begg and Brown, 2000; De
Vries et al., 2002; Tuset et al., 2003; Cardinale et al., 2004;
Poulet et al., 2004; Kumar et al., 2012; Radhakrishnan et al.,
2012; Vignon, 2012; Bani et al., 2013; Basusta et al., 2013;
Mohadasi et al., 2014; Yilmaz et al., 2014; Ye et al., 2015).
Otolith shape measurements are considered to be an index
for specific discrimination, stock analysis, and even in testing
the function and ecological significance of shape differences
in the studies of otolith morphology (Aguirre and Lombarte,
1999; Cardinale et al., 2004). Fourier decompositions of the
otolith or sulcus outlines were used in most studies of otolith
morphology on shape variables (Torres et al., 2000; De Vries
et al., 2002; Gauldie and Crampton, 2002; Cardinale et al.,
2004).
Alburnus, a genus known as bleaks in the family of Cyprinidae (Buj et al., 2010), is represented by 42 species worldwide (Froese and Pauly, 2014). The systematic and actual
distribution of the Alburnus species is still not well understood (Buj et al., 2010). The asteriscus otolith shape, morphology and shape contours of four Alburnus species:
Danube bleak, A. chalcoides (G€
uldenst€
adt, 1772); Anatolian
bleak, A. escherichii Steindachner, 1897; Mosul bleak,
A. mossulensis Heckel, 1843; and Tarek, A. tarichi
(G€
uldenst€
adt, 1814) were investigated in the current study.
Despite the increase in otolith morphology studies
throughout the world, the otolith morphology of salt and
freshwater fish species’ and their shapes and relationships
with other variables remains largely unknown or is limited in
2
D. Bostanci et al.
Fig. 1. Map of Turkey with sampling
locations; I- Ordu (A. chalcoides), IIEskisßehir (A. escherichii), III- Tunceli
(A. mossulensis),
and
IV
Van
(A. tarichi).
Table 1
Sampling locations with water parameters, sample size, sampling date, weight and total length ranges of each Alburnus species
Species
A. chalcoides
A. escherichii
A. mossulensis
A. tarichi
Common name
Location
Capture date
Latitude
Longitude
Temp (°C)
pH
DO mg/L
n
Weight range (g)
TL range (cm)
Danube bleak
Curi Stream (Ordu)
May 2013
41° 080 3600
37° 130 4100
17.50
7.62
5.25
26
13.8–52.4
11.8–17.8
Anatolian bleak
Seydi Suyu Stream (Eskisehir)
July 2013
39° 240 0000
31° 120 0000
18.65
8.1
9.45
46
2.4–25.7
7–13.7
Mosul bleak
Munzur River (Tunceli)
Oct-May 2013
39° 060 0500
39° 330 1400
8.32
8.27
10.21
130
10.98–82.72
11.3–21
Tarek
Lake Van (Van)
Jan 2014
38° 380 2800
42° 480 4500
5.77
9.36
9.46
105
44.8–112.2
17.8–23.5
Temp, Temperature; DO, Dissolved oxygen; TL, Total length.
Alburnus species (Buj et al., 2010). The objectives of the present study were therefore to: (i) evaluate the importance of
the asteriscus otolith shape in identifying the four Alburnus
species: A. chalcoides, A. escherichii, A. mossulensis and
A. tarichi; (ii) analyze the right and left pairs of the asteriscus shape and morphology in males and females in the four
Alburnus species. This is one of the first otolith morphology
studies to use right and left asteriscus pairs of males and
females for the four Alburnus species in Turkey.
Materials and methods
All Alburnus chalcoides (from Ordu), A. escherichii (from
Eskisßehir), A. mossulensis (from Tunceli), and A. tarichi
(from Van) were obtained from commercial anglers and
transported to the Hydrobiology Laboratory, Ordu University, Ordu, Turkey for further analyses. Total length (TL)
of each of fish was measured to the nearest 0.1 cm, and
weight (W) was recorded to the nearest the 0.1 g. The
asteriscus otolith pairs were removed, cleaned and the asteriscus weighed to the nearest 0.0001 g, then stored dry for
further examination. Sampling locations of the four Alburnus species (Fig. 1) with the water parameters are shown in
Table 1.
A t-test was applied in order to determine differences
between males and females otoliths. In addition, right and
left asteriscus otoliths were compared; any variables in pairs
of asteriscus were investigated with a paired t-test for all
individuals. A MINITAB 14.0 statistical analysis program
was used for calculations. Based on the TL ranges of the
four Alburnus species, the asteriscus otolith was arbitrarily
divided into four categories: I (7.0–11.0 cm TL), II (11.1–
15.0 cm TL), III (15.1–19.0 cm TL), and IV (19.1–23.5 cm
TL).
Each asteriscus otolith pair was photographed; to assess
shape factors, the otolith images were analyzed with Leica
Application Suit (Version 3.7.0) software. The otolith length
(OL, mm), otolith width (OW, mm), otolith area (A, mm2),
and otolith perimeter (P, mm) were measured using the Leica
S8APO microscope and computer-connected camera system.
These measurements allowed the calculation of six shape
indices: form factor (FF), roundness (RD), aspect ratio
(AR), circularity (C), rectangularity (R), and ellipticity (E)
(Tuset et al., 2003; Ponton, 2006). These shape indices were
calculated for the right and left asteriscus otoliths of both
males and females of each Alburnus species. Moreover, realtionships between shape indices and asteriscus otolith length
of each Alburnus species are shown graphically (Table 2).
Using otolith shape and morphometry to identify four Alburnus species
The SHAPE (Version 1.3) software program package was
used to extract the contour shape of the otoliths based on
the elliptic Fourier descriptors (Iwata and Ukai, 2002). The
SPSS 21.0 statistical analysis program was used for calculations; canonical discriminant analysis (CDA) was then performed to detect morphometric differences in the asteriscus
otoliths of A. chalcoides (Ordu), A. escherichii (Eskisßehir),
A. mossulensis (Tunceli), and A. tarichi (Van). In addition,
the Wilks k assessed the performance of the discriminant
analyses. Interspecific variance, total variance and their percentage of agreement between real and predicted groups
membership were calculated for each Alburnus species. SEM
Table 2
Formulae for shape indices
Shape indices
Formula
Form factor
Roundness
Aspect ratio
Ellipticity
Circularity
Rectangularity
4 p A P2
4 A (p OL2)1
OL OW1
(OL OW) (OL + OW)1
P2 A1
A (OL OW)1
Otolith: A, Area; P, perimeter; OL, length; OW, width.
3
was used to record the morphological characteristics of the
asteriscus otolith of each Alburnus species. For scanning, the
asteriscus otoliths were attached to a stub using double-sided
carbon tape. The immobilized otoliths on the stub were
coated with 13.5 nm gold. The right and left asteriscus were
analyzed using SEM (JMS-6060LV microscope) at 5.0 KV.
Results
The asteriscus of A. chalcoides (n = 26; 76.9% females,
19.2% males), A. escherichii (n = 46; 41.3% females, 34.7%
males), A. mossulensis (n = 130; 61.5% females, 38.5%
males), and A. tarichi (n = 105; 22.9% females, 77.1% males)
were evaluated. Table 3 shows a summary of descriptive
statistics (mean, standard deviation, standard error, minimum and maximum) and paired-t test results. Interpretations
of the left and right asteriscus otolith pairs were not statically different (P > 0.05) in terms of otolith weight and
perimeter for each Alburnus species (Table 3). Left and right
asteriscus otolith pairs were not statically different (P > 0.05)
in terms of otolith width and area for A. chalcoides,
A. mossulensis and A. escherichii, however, they were statically different for A. tarichi (P < 0.01, P < 0.001, respectively) (Table 3). For the otolith length, the left and right
otolith pairs were statically different for A. escherichii
Table 3
Summary of descriptive statistics and paired t-test results for left and right asteriscus otoliths of A. chalcoides (Ordu), A. escherichii
(Eskisßehir), A. mossulensis (Tunceli), and A. tarichi (Van)
Mean
Otolith variables
A. chalcoides
Weight P > 0.05
Length P > 0.05
Width P > 0.05
Area P > 0.05
Perimeter P > 0.05
A. escherichii
Weight P > 0.05
Length P < 0.05
0.028*
Width P > 0.05
Area P > 0.05
Perimeter P > 0.05
A. mossulensis
Weight P > 0.05
Length P > 0.05
Width P > 0.05
Area P > 0.05
Perimeter P > 0.05
A. tarichi
Weight P > 0.05
Length P > 0.05
Width P < 0.01
0.008**
Area P < 0.001
0.001***
Perimeter P > 0.05
SE
SD
Min.
Max.
L
R
L
R
L
R
L
R
0.0017
2.33
2.08
3.18
7.39
0.0018
2.32
2.06
3.17
7.48
0.00009
0.0521
0.0389
0.105
0.185
0.00010
0.0573
0.0396
0.117
0.204
0.00047
0.2603
0.1943
0.525
0.927
0.00051
0.2866
0.1981
0.584
1.018
0.0006
1.826
1.684
2.239
5.691
0.0009
1.841
1.684
2.181
5.677
0.0027
2.882
2.397
4.355
9.033
0.003
3.010
2.368
4.507
9.657
0.0011
1.65
0.0011
1.62
0.00005
0.0327
0.00005
0.0310
0.00037
0.2146
0.00037
0.2083
0.0005
1.119
0.0004
1.096
0.0019
2.089
0.002
2.123
1.79
2.17
5.94
1.80
2.16
5.91
0.0371
0.0763
0.122
0.0345
0.0739
0.114
0.2434
0.5001
0.800
0.2312
0.4955
0.767
1.130
0.949
3.753
1.141
0.918
3.788
2.238
2.953
7.370
2.226
3.016
7.118
0.0018
2.23
2.03
3.17
7.08
0.0018
2.24
2.04
3.18
7.09
0.00005
0.0227
0.0193
0.0593
0.0807
0.00005
0.0231
0.0192
0.0591
0.0791
0.00058
0.2462
0.2093
0.6446
0.8769
0.00056
0.2502
0.2072
0.6392
0.8560
0.0005
1.693
1.623
2.019
5.395
0.0007
1.712
1.578
2.054
5.440
0.0043
3.228
2.711
5.492
10.360
0.0041
3.112
2.711
5.538
10.791
0.0029
2.60
2.45
0.0029
2.61
2.38
0.00008
0.0219
0.0216
0.00008
0.0201
0.0208
0.00085
0.2222
0.2188
0.00083
0.2028
0.2104
0.0015
1.597
1.926
0.0018
2.169
1.926
0.0057
3.196
3.068
0.0057
3.239
2.939
4.49
4.38
0.0693
0.0720
0.7037
0.7268
3.240
2.543
6.663
6.655
9.01
8.97
0.0816
0.0828
0.8280
0.8364
7.347
7.431
11.777
11.533
R, Right asteriscus; L, Left asteriscus, SE, Standard error; SD, Standard deviation, Min., Minimum; Max., Maximum.
*P < 0.05; **P < 0.01, ***P < 0.001.
L
R
4
D. Bostanci et al.
P values
Species
Sex
Alburnus
chalcoides
♀
♂
♀
♂
♀
♂
♀
♂
♀
♂
♀
♂
♀
♂
♀
♂
R
L
Alburnus
escherichii
R
L
Alburnus
mossulensis
R
L
Alburnus
tarichi
R
L
Mean weight
asteriscus
0.0018
0.0015
0.0018
0.0013
0.0012
0.0010
0.0012
0.0011
0.0019
0.0017
0.0019
0.0016
0.0036
0.0027
0.0035
0.0027
Weight
Length
Width
Area
Perimeter
*
0.412
0.572
0.041
0.173
0.182
0.218
0.334
0.036*
0.063
0.087
0.069
0.425
0.328
0.271
0.364
0.442
0.480
0.701
0.410
0.561
0.043
0.134
0.100
0.115
0.138
0.010**
0.260
0.016*
0.031*
0.123
*
***
***
*
***
0.001
0.000
0.032
0.000
0.020*
0.001***
0.000***
0.010**
0.000***
0.001***
Table 4
Statistical comparisons for left and
right asteriscus otoliths of male and
female
A. chalcoides
(Ordu),
A. escherichii (Eskisßehir), A. mossulensis (Tunceli), and A. tarichi (Van)
R, Right asteriscus; L, left asteriscus.
*P < 0.05; **P < 0.01; ***P < 0.001.
Shape indices
Species and TL
ranges
TL
Categories
FF
RD
AR
E
C
R
A. chalcoides (11.8–
17.8 cm)
A. escherichii (7–
13.7 cm)
A. mossulensis (11.3
–21 cm)
II
III
I
II
II
III
IV
III
IV
0.77390
0.74710
0.77801
0.75506
0.80946
0.77462
0.71180
0.69800
0.69579
0.77340
0.74350
0.98330
1.01040
0.81748
0.79727
0.74590
0.81960
0.8604
1.0940
1.1414
0.9467
0.9130
1.0914
1.1017
1.1427
1.0870
1.0739
0.04410
0.06400
0.24890
0.04688
0.04335
0.04765
0.06610
0.04056
0.03369
16.302
18.356
16.177
16.686
15.568
16.253
17.782
18.074
18.246
0.66154
0.66041
0.72763
0.72010
0.69958
0.68725
0.6668
0.69601
0.71584
A. tarichi (17.8–
23.5 cm)
Table 5
Shape indices values of asteriscus
otoliths of total size classes for Alburnus species; A chalcoides (Ordu),
A escherichii (Eskisßehir), A. mossulensis (Tunceli), and A. tarichi (Van)
FF, Form factor; RD, roundness; AR, aspect ratio; E, ellipticity; C, circularity; R, rectangularity, and TL, total length.
(P < 0.05) but not (P > 0.05) for A. chalcoides A. mossulensis
or A. tarichi (Table 3). Differences between right and left
otoliths were tested using a paired t-test; right asteriscus otoliths for A. chalcoides and A. mossulensis, and left asteriscus
otoliths for A. escherichii and A. tarichi were preferred and
used for further calculations.
Statistical comparisons of otolith variables for left and
right asteriscus otoliths of male and female A. chalcoides
(Ordu), A. escherichii (Eskisßehir), A. mossulensis (Tunceli),
and A. tarichi (Van) are summarized in Table 4. Interpretations of these results are statistically different for male and
female A. tarichi while not statically different for male and
female A. escherichii (Table 4). In addition, the differences in
right and left otolith variables and the differences for male
and female A. chalcoides and A. mossulensis (Table 4). Asteriscus otolith shape indices per size category and total length
(TL) ranges of each Alburnus species are shown in Table 5.
Ellipticity increases with the increase in TL of A. mossulensis,
and becomes more elliptic (Table 5). However, ellipticity
decreases with an increase in TL of A. tarichi, and becomes
less elliptic (Table 5). Circularity increases with an increase
in TL of each Alburnus species and becomes more circular
(Table 5). Form factor decreases with the TL increase in
each Alburnus species (Table 5). A third category includes
three Alburnus species: A. chalcoides, A. tarichi and
A. mossulensis; the first category contains only A. escherichii
because of their small size. The second category contains the
three other Alburnus species; a fourth category is A. tarichi
and A. mossulensis. Table 6 gives summaries of descriptive
statistics of shape indices for left asteriscus otoliths of
A. escherichii (Eskisßehir) and A. tarichi (Van), and for right
asteriscus otoliths of A. chalcoides (Ordu) and A. mossulensis
(Tunceli). Indices such as FF, AR, RD, R, C and E values
are associated with asteriscus length (Fig. 2). According to
these data, the relationship between otolith length and AR,
C, E was determined as a linear relationship, despite the relationship between otolith length and FF, RD, R being determined as a nonlinear relationship (Fig. 2).
The discriminant function included all basic otolith morphometric characteristics: otolith weight (OWE), otolith
length (OL), otolith width (OW), otolith perimeter (OP), and
otolith area (OA) to explain the interspecific variability.
Three and two canonical varieties were derived for differentiation of these Alburnus species; discrimination among them
Using otolith shape and morphometry to identify four Alburnus species
Table 6
Summary of descriptive statistics of
shape indices for left asteriscus otoliths of A. escherichii (Eskisßehir) and
A. tarichi (Van), and for right asteriscus otoliths of A. chalcoides (Ordu)
and A. mossulensis (Tunceli)
5
Species
Shape indices
Mean
SE
SD
Min.
Max.
n
Alburnus
chalcoides
right
asteriscus
Form factor
Roundness
Aspect ratio
Ellipticity
Circularity
Rectangularity
Form factor
Roundness
Aspect ratio
Ellipticity
Circularity
Rectangularity
Form factor
Roundness
Aspect ratio
Ellipticity
Circularity
Rectangularity
Form factor
Roundness
Aspect ratio
Ellipticity
Circularity
Rectangularity
0.7168
0.7530
1.1262
0.05760
17.699
0.66077
0.76602
0.9990
0.9283
0.03864
16.448
0.72344
0.79218
0.80682
1.0975
0.04594
15.920
0.69354
0.69629
0.8513
1.0768
0.03522
18.208
0.71142
0.0147
0.0145
0.0192
0.00811
0.360
0.00468
0.00653
0.0142
0.0113
0.00592
0.144
0.00500
0.00461
0.00434
0.00458
0.00207
0.0972
0.00206
0.00636
0.0163
0.00848
0.00425
0.185
0.00595
0.0735
0.0725
0.0961
0.04053
1.799
0.02341
0.04281
0.0929
0.0739
0.03879
0.941
0.03278
0.04992
0.04697
0.0496
0.02238
1.051
0.02230
0.06455
0.1652
0.0860
0.04316
1.876
0.06042
0.5936
0.5564
1.0062
0.00308
14.488
0.60201
0.65595
0.7937
0.7836
0.12132
14.592
0.65253
0.59734
0.65850
1.0031
0.00154
13.688
0.62714
0.44617
0.6691
0.6508
0.21155
15.199
0.63682
0.8669
0.8501
1.4354
0.17877
21.159
0.71582
0.86074
1.1878
1.1366
0.06392
19.148
0.78504
0.91762
0.93161
1.2360
0.10556
21.027
0.76031
0.82637
2.3496
1.3840
0.16107
28.151
1.20029
26
26
26
26
26
26
46
46
46
46
46
46
130
130
130
130
130
130
105
105
105
105
105
105
Alburnus
escherichii left
asteriscus
Alburnus
mossulensis
right
asteriscus
Alburnus tarichi
left asteriscus
SE, Standard error; SD, standard deviation.
was high. The canonical discriminant analysis revealed that
93.8% of the fish were correctly classified. The two discriminant functions were significant (P < 0.05), with Wilks k being
0.022 for function 1 and 0.146 for function 2. The first discriminant function of the CDA explains 56.7% of the variability and the second function of the CDA explains 39.1%
of the variability for the four Alburnus species, which are
correctly differentiated in this analysis (Fig. 3).
Jack-knifed cross-validation classification scores for predicted group membership of each Alburnus species are listed
in Table 7. The overall classification score of the discriminant analysis was 93.8%, with the lowest score obtained for
A. escherichii (82.5%) and the highest for A. chalcoides
(100%). Classification scores were 97.8 and 93.1% for
A. tarichi and A. mossulensis, respectively (Table 7). A. chalcoides is well separated from A. escherichii, A. tarichi and
A. mossulensis on the first discriminant function, while
A. escherichii, A. tarichi and A. mossulensis are mainly separated along the second discriminant function.
SEM images of proximal-left and distal-right asteriscus
otolith surfaces of each Alburnus species were prepared for
comparative investigation of the morphology (Fig. 4). This is
the first time that SEM images were used to determine asteriscus shape diversity among the four Alburnus species
(Table 8). The shape contours of proximal-left and distalright asteriscus otoliths of each Alburnus species are clearly
shown in Fig. 5.
The asteriscus shapes of A. chalcoides and A. mossulensis
are discoidal, whereas A. escherichii and A. tarichi are ovoid.
The dorsal margin, otolith width and depth are also considerably different for the four Alburnus species. For instance,
the asteriscus of A. mossulensis have a crenated dorsal mar-
gin, whereas the dorsal margin of A. chalcoides is serrated
with a more or less regular margin. However, the mesial and
lateral surfaces of the asteriscus are not different.
Discussion
The current study results suggest that differences in asteriscus
otolith measurements and shape indices are detectable in
A. chalcoides (Ordu), A. escherichii (Eskisßehir), A. mossulensis (Tunceli), and A. tarichi (Van). Otolith properties are useful tools to identify their interspecific relationships. The uses
of their morphological and morphometric characterization
have been shown to be important to discriminate fish species
(Lombarte et al., 1991; Smale et al., 1995), and the otoliths
have a high morphological specificity for species (Aurioles,
1991; Martınez et al., 2007). The current study must be verified to support this information. The otolith weight is most
sensitive to variations in the growth rate and is a powerful
discriminator (Reznick et al., 1989; Pawson, 1990; Foresberg
_
and Neal, 1993; Tuset et al., 2006; Ilkyaz
et al., 2010; Zorica
et al., 2010; Amouei et al., 2014); however, it is not a powerful discriminator in identifying the four Alburnus species.
Moreover, asteriscus measurements and shape indices are
compromised by the high variability in otolith shape and
morphometry. Considering the findings of this study, it is
evident that the asteriscus shape and morphometry are useful
for the encouragement of further research on verifying the
role of the otolith in identification, discriminating and taxonomy of fish. The results of this study show that the shape
indices significantly differed in analysis from species to species, although they indicate a similar pattern of otoliths
(Table 4). These results correlate with Tuset et al. (2008) that
6
D. Bostanci et al.
(a)
(d)
(b)
(e)
(c)
(f)
Fig. 2. Shape indices: (a) Circularity,
(b) Roundness, (c) Form factor, (e)
Aspect ratio, (d) Ellipticity, and (f)
Rectangularity vs Asteriscus otolith
length of four Alburnus species:
A. chalcoides, A. escherichii, A. mossulensis and A. tarichi (2013–2014).
Fig. 3. Scatter plot showing scores for canonical discriminant analysis from four Alburnus species: A. chalcoides (Ordu), A. escherichii
(Eskisßehir), A. mossulensis (Tunceli), and A. tarichi (Van) for pooled
year classes (2013–2014).
the otoliths are the most widely used for discrimination of
fish species because of their form, weight, growth, consistency and chemical composition.
The overall classification of the discriminant analysis was
93.8%, with the lowest score obtained for A. escherichii
(82.5%) and the highest for A. chalcoides (100%), and 97.8
and 93.1% for A. tarichi and A. mossulensis, respectively.
Interpretation of the overall classification was 93.8% for the
four Alburnus species; this is a high rate within the same
genus using the morphometric character of asteriscus.
A. chalcoides is well separated (100%) from A. escherichii,
A. tarichi and A. mossulensis (Table 7). Canonical discriminant analysis (CDA) was very useful to determine differentiation for each Alburnus species: A. chalcoides (100%),
A. tarichi (97.8%), A. mossulensis (93.1%), and A. escherichii
(82.5%). CDA analysis was used in most studies such as in
analysis of species and population differentiations (Lord
et al., 2012; Skeljo and Ferri, 2012), and is one of the multivariate statistical analyses in evolutionary biology and systematics (Campbell and Atchley, 1981). According to the
Parmentier et al. (2001) study, the otolith shape may be similar in fish inhabiting the same ecological pattern, but may
change in a different habitat. Thus, the ecological patterns
and water parameters may affect the rates of these four Alburnus species directly or indirectly. For instance, 97.8% separated A. tarichi as an endemic fish species living in Lake
Van with saline soda lake water, while A. escherichii
(82.5%), A. mossulensis (93.1%) and A. chalcoides (100%)
live in freshwater.
Moreover, the three Alburnus species (A. chalcoides,
A. escherichii and A. mossulensis) have a lotic water habitat,
Using otolith shape and morphometry to identify four Alburnus species
Table 7
Jack-knifed cross-validation classification matrices for predicted group
membership of each Alburnus species:
A. chalcoides (Ordu), A. escherichii
(Eskisßehir), A. mossulensis (Tunceli),
and A. tarichi (Van)
7
Predicted Group Membership (%)
Species
A. chalcoides
A. chalcoides
A. escherichii
A. mossulensis
A. tarichi
Total
100.0
0.0
0.0
0.0
82.5
17.5
0.0
2.6
93.1
0.0
0.0
2.2
93.8% of originally grouped cases correctly classified
A. escherichii
A. mossulensis
A. tarichi
0.0
0.0
4.3
97.8
(a)
(c)
(b)
(d)
Fig. 4. SEM images of proximal-left
and distal-right asteriscus otolith
surfaces from each Alburnus species: (a)
A. chalcoides (Ordu), (b) A. escherichii
(Eskisßehir), (c) A. mossulensis (Tunceli), and (d) A. tarichi (Van).
Table 8
Asteriscus otolith characteristics of
A. chalcoides (Ordu), A. escherichii
(Eskisßehir), A. mossulensis (Tunceli),
and A. tarichi (Van)
Otolith
characteristics
Otolith shape
Otolith width
Mesial surface
Lateral surface
Dorsal margin
Depth
while A. tarichi has lentic water habitat. Another interesting
result regarding the location of the three Alburnus species
[A. escherichii from Eskisßehir (82.5%), A. mossulensis from
Tunceli (93.1%) and A. chalcoides from Ordu (100%)] is that
they have a lotic habitat with freshwater, but their locations
are in different regions (Fig. 1). The 100% separated A. chalcoides live in the Curi Stream (Ordu), close to the Black Sea.
A. escherichii (82.5%) from the Seydi Suyu Stream
(Eskisßehir), and A. mossulensis (93.1%) from the Munzur
River (Tunceli) are not close to the sea (Fig. 1), and therefore their discriminations are different (Tables 7 and 8).
The current study was also planned to compare otolith
shape using the SEM images among the four Alburnus spe-
A. chalcoides
A. escherichii
A. mossulensis
A. tarichi
Discoidal
Moderately
thin
Convex
Convex
Serrate
Shallow
Oval
Moderate
Discoidal
Moderately
thin
Convex
Convex
Crenate
Shallow
Oval
Moderately
thin
Convex
Convex
Serrate
Shallow
Convex
Convex
Rounded
Moderately
shallow
cies in order to observe a variation in otolith shape for these
different species of same genus. Asteriscus otolith characteristics of the four species are well detailed using their SEM
images (Table 8); proximal-left and distal-right surface asteriscus differences are shown in Fig. 4.
The shape contours of proximal-left and distal-right asteriscus otoliths are also powerful for identification of these
four Alburnus species. Use of the otolith shape, morphometry, and shape contours shows their importance. CDA using
otolith shape, morphometry and biometry can be a useful
method for differentiating stocks, populations, and interspecific and intraspecific variations (Stransky et al., 2008;
Neves et al., 2011; Yu et al., 2014; 2015), rather than using
8
D. Bostanci et al.
(a)
(b)
In conclusion, results of the SEM images show that otolith
characters and shape contours vary within the same genus
and can change from species to species; this difference should
be investigated not only in other freshwater fish species or
genera but also in the same species living in different habitats. Additional studies are needed, such as precise and
essential measurements of environmental factors and their
combination on the population connectivity using genetics in
the different streams, rivers, lakes, etc. Further investigation
is needed, including a comparative study not only of morphometry, biometry, shape, geometry, and shape contours of
the otoliths, but also of genetic methods for powerful identification of various sympatric and allopatric fish populations.
Acknowledgements
(c)
For providing the Alburnus species we greatly appreciate Dr.
€ ur Emiro
Ozg€
glu (A. escherichii), Resul Iskender (A. chalcoides), Elif Bekdemir G€
ultepe (A. tarichi) and H€
ulya Alan
(A. mossulensis).
References
(d)
Fig. 5. Contour shapes of proximal-left and distal-right asteriscus
otoliths of four Alburnus species: (a) A. chalcoides (Ordu), (b)
A. escherichii (Eskisßehir), (c) A. mossulensis (Tunceli), and (d)
A. tarichi (Van).
genetic methods (Cardinale et al., 2004; Pothin et al., 2006;
Merigot et al., 2007; Shafee et al., 2013). The shape variability of otoliths is related to genetic (Tuset et al., 2003), ontogenetic, and environmental factors such as temperature,
habitat, seasonal variations and diet (Campana, 2001), and
ecological and biological behavior of the species (Tuset et al.,
2003). Actually, otolith microchemistry, tagging, parasites,
and especially genetics discrimination techniques have proved
to be very accurate in stock separation and structure (Finamore et al., 2004; Tracey et al., 2006; Cheng et al., 2008;
Radhakrishnan et al., 2012; Mohadasi et al., 2014). These
previous studies indicate that genetic techniques are important to evaluate the ontogeny, to identify species and stocks,
even the to evolutionary response of fish populations (Perrier
et al., 2011; Russello et al., 2011; Shafee et al., 2013; Dorafshan et al., 2014). Molecular and genetic techniques are
costly, time consuming and need special laboratory facilities
(Bektas and Belduz, 2009; Bani et al., 2013), whereas the
otolith shape and morphometry are cheaper and more timeefficient techniques (Tracey et al., 2006).
Aguirre, H.; Lombarte, A., 1999: Ecomorphologic comparisons of
sagittae in Mullus barbatus and M. surmuletus. J. Fish Biol. 55,
105–114.
Amouei, F.; Valinassab, T.; Haitov, A., 2014: Aging and morphology of otolith in Alburnus chalcoides (Guldenstaedt, 1772) in
the southern Caspian Sea. Caspian J. Environ. Sci. 12, 205–
214.
Aurioles, G. D., 1991: Otolith size versus weight and body length
relationships for some fishes of the Pacific coast of Baja California Sur. Mexico. Fish. Bull. 89, 701–706.
Bani, A.; Poursaeid, S.; Tuset, V. M., 2013: Comparative morphology of the sagittal otolith in three species of south Caspian gobies. J. Fish Biol. 82, 1321–1332.
Basusta, A.; Bal, H.; Aslan, E., 2013: Otolith biometry-total length
relationships in the population of Hazar Bleak, Alburnus heckeli
(Battalgil, 1943) inhabiting Lake Hazar, Elazig. Turkey. Pak. J.
Zool 45, 1180–1182.
Begg, G. A.; Brown, R. W., 2000: Stock identification of haddock
(Melanogrammus aeglefinus) on Georges Bank based on otolith
shape analysis. Trans. Am. Fish. Soc. 129, 935–945.
Bektas, Y.; Belduz, A. O., 2009: Morphological variation among
Atlantic horse mackerel, Trachurus trachurus populations from
Turkish coastal waters. J. Anim. Vet. Adv. 8, 511–517.
Bird, J. L.; Eppler, D. T.; Checkley, D. M., 1986: Comparisons of
herring otoliths using Fourier-series shape-analysis. Can. J. Fish
Aquat. Sci. 43, 1228–1234.
Buj, I.; Vukic, J.; Sanda, R.; Perea, S.; Caleta, M.; Marcic, Z.;
Bogut, I.; Povz, M.; Mrakovcic, M., 2010: Morphological comparison of bleaks (Alburnus. Cyprinidae) from the Adriatic
Basin with the description of a new species. Folia Zool. 59, 129–
141.
Campana, S. E., 2001: Accuracy, precision and quality control in
age determination, including a review of the use and abuse of
age validation methods. J. Fish Biol. 59, 197–242.
Campana, S. E.; Casselman, J. M., 1993: Stock discrimination using
otolith shape analysis. Can. J. Fish Aquat. Sci. 50, 1062–1083.
Campbell, N. A.; Atchley, W. R., 1981: The geometry of canonical
variate analysis. Syst. Zool. 30, 268–280.
Cardinale, M.; Doering-Arjes, P.; Kastowsky, M.; Mosegaard, H.,
2004: Effects of sex stock and environment on the shape of
known-age Atlantic cod (Gadus morhua) otoliths. Can. J. Fish
Aquat. Sci. 61, 158–167.
Cheng, Q.; Cheng, H.; Wang, L.; Zhong, Y.; Lu, D., 2008: A preliminary genetic distinctness of four Coilia fishes (Clupeiformes
Using otolith shape and morphometry to identify four Alburnus species
Engraulidae) inferred from mitochondrial DNA sequences.
Russ. J. Genet. 44, 339–343.
De Vries, D. A.; Grimes, C. B.; Prager, M. H., 2002: Using otolith
shape analysis to distinguish eastern Gulf of Mexico and Atlantic Ocean stocks of king mackerel. Fish. Res. 57, 51–61.
Dorafshan, S.; Shafee, Z.; Keivany, Y., 2014: A study on genetic differentiation in two species of Iranian bleaks, Alburnus mossulensis and Alburnus caeruleus (Teleostei, Cyprinidae) using simple
sequence repeats. Caspian J. Env. Sci. 12, 197–204.
Finamore, F.; Bianco, P.G.; Milone, M., 2004: Genetic diversity
among Italian bleak, Alburnus albidus populations from Southern Italy: preliminary analysis of cytochrome b sequence variation. In: Ecologia. Atti del XIII Congresso Nazionale della
Societa Italiana di Ecologia. R. Cassagrandi and P. Meli (Eds).
Arcane, Rome, pp. 1–4.
Foresberg, J. E.; Neal, R., 1993: Estimating sex of Pacific halibut
(Hippoglossus stenolepis) using Fourier shape analysis of otoliths
Technical Report. Internat. Pacific Halibut Comm 29, 5–23.
Froese, R.; Pauly, D., (Eds) 2014: Species list: Alburnus FishBase.
World Wide Web electronic publication. Available at: www.fishbase.org. version (accessed on 14 February 2014).
Gauldie, R. W.; Crampton, J. S., 2002: An ecomorphological explication of individual variability in the shape of the fish otolith:
comparison of the otolith of Hoplostethus atlanticus with other
species by depth. J. Fish Biol. 60, 1221–1240.
_
Ilkyaz,
A. T.; Metin, G.; Kinacigil, H. T., 2010: The use of otolith
length and weight measurements in age estimations of three
Gobiidae species (Deltentosteus quadrimaculatus, Gobius niger,
and Lesueurigobius friesii). Turk J. Zool. 35, 819–827.
Iwata, H.; Ukai, Y., 2002: SHAPE: a computer program package
for quantitative evaluation of biological shapes based on elliptic
Fourier descriptors. J. Hered. 93, 384–385.
Kumar, K.; Chakraborty, S. K.; Jaiswar, A. K., 2012: Comparative
otolith morphology of sciaenids occurring along the north-west
coast of India. Indian J. Fish. 59, 19–27.
Lombarte, A.; Cruz, A., 2007: Otolith size trends in marine com-munities from different depth strata. J. Fish Biol. 71, 53–76.
Lombarte, A.; Rucabado, J.; Matallanas, J.; Lloris, D., 1991:
Numerical taxonomy of nototheniidae using otolith profiles. Sci.
Mar. 55, 413–418.
Lombarte, A.; Torres, G. J.; Morales-Nin, B., 2003: Specific Merluccius otolith growth patterns related to phylogenetics and environmental. J. Mar. Biol. Ass. U.K. 83, 277–281.
Lord, C.; Morat, F.; Lecomte-Finiger, R.; Keith, P., 2012: Otolith
shape analysis for three Sicyopterus (Teleostei: Gobioidei: Sicydiinae) species from New Caledonia and Vanuatu. Environ.
Biol. Fish. 93, 209–222.
Martınez, J.; Ch
avez, M.; Tello, L.; Morales, A., 2007: Utilizaci
on
de otolitos como herramienta en la determinaci
on de especies.
Rev. Zool. 18, 13–18.
Merigot, B.; Letourneur, Y.; Lecomte-Finiger, R., 2007: Characterization of local populations of the common sole Solea solea
(Pisces Soleidae) in the NW Mediterranean through otolith morphometrics and shape analysis. Mar. Biol. 151, 997–1008.
Mohadasi, M.; Eagderi, S.; Shabanipour, N.; Hosseinzadeh, M. S.;
AnvariFar, H.; Khaefi, K., 2014: Allometric body shape changes
and morphological differentiation of Shemaya, Alburnus chalcoides (Guldenstadi, 1772), populations in the southern part of
Caspian Sea using Elliptic Fourier analysis. Int. J. Aquat. Biol.
2, 164–171.
Monteiro, L.; Di Beneditto, A. P. M.; Guilhermo, L. H.; Rivera, L.
A., 2005: Allometric changes and shape differentiation of sagitta
otoliths in sciaenid fishes. Fish. Res. 74, 288–299.
Neves, A.; Sequeira1, V.; Farias, I.; Vieira, A.R.; Paiva, R.; Gordo,
L.S., 2011: Discriminating bluemouth, Helicolenus dactylopterus
(Pisces: Sebastidae), stocks in Portuguese waters by means of
otolith shape analysis. J. Mar. Biol. Assoc. U.K.. 91, 1237–
1242.
Parmentier, E.; Vandewalle, P.; Lagardere, F., 2001: Morpho-anatomy of the otic region in carapid fishes: eco-morphological
study of their otoliths. J. Fish Biol. 58, 1046–1061.
9
Pawson, M. G., 1990: Using otolith weight to age fish. J. Fish Biol.
36, 521–531.
Perrier, C.; Daverat, F.; Evanno, G.; Pecheyran, C.; Bagliniere, J.
L.; Roussel, J. M., 2011: Coupling genetic and otolith trace element analyses to identify river-born fish with hatchery pedigrees
in stocked Atlantic salmon (Salmo salar) populations. Can. J.
Fish Aquat. Sci. 68, 977–987.
Ponton, D., 2006: Is geometric morphometrics efficient for comparing otolith shape of different fish species? J. Morphol. 267, 750–
757.
Pothin, K.; Gonzales-Salas, C.; Chabanet, P.; Lecomte-Finiger, R.,
2006: Distinction between Mulloidichthys flavolineatus juveniles
from Reunion Island and Mauritius Island (south-west Indian
Ocean) based on otolith morphometrics. J. Fish Biol. 69, 38–53.
Poulet, N.; Berrebi, P.; Crivelli, A. J.; Lek, S.; Arguillier, C., 2004:
Genetic and morphometric variations in the pikeperch (Sander
lucioperca L.) of a fragmented delta. Arch. Hydrobiol. 159, 531–
554.
Radhakrishnan, K. V.; Yuxuan, L.; Jayalakshmy, K. V.; Liu, M.;
Murphy, B. R.; Xie, S. G., 2012: Application of otolith shape
analysis in identifying different ecotypes of Coilia ectenes in the
Yangtze Basin. China. Fish. Res. 125, 156–160.
Reichenbacher, B.; Kamrani, E.; Esmaeili, H. R.; Teimori, A., 2009:
The endangered cyprinodont Aphanius ginaonis (Holly, 1929)
from southern Iran is a valid species: evidence from otolith morphology. Environ. Biol. Fishes 86, 504–521.
Reznick, D.; Lindbeck, E.; Bryga, H., 1989: Slower growth results in
larger otoliths: an experimental test with guppies (Poecilia reticulata). Can. J. Fish Aquat. Sci. 46, 108–112.
Russello, M. A.; Kirk, S. L.; Frazer, K. K.; Askey, P. J., 2011: Detection of outlier loci and their utility for fisheries management.
Evol. Appl. 5, 39–52.
Shafee, Z.; Dorafshan, S.; Keivany, Y.; Qasemi, S. A., 2013: Genetic
structure of Mosul bleak (Alburnus mossulensis Heckel, 1843)
using microsatellite marker in Tigris basin. Taxon. Biosyst. 17,
9–22. (in Persian).
Skeljo, F.; Ferri, J., 2012: The use of otolith shape and morphometry
for identification and size-estimation of five wrasse species in
predator-prey studies. J. Appl. Ichthyol. 28, 524–530.
Smale, M. J.; Watson, G.; Hetch, T., 1995: Otolith Atlas of Southern African Marine Fishes. J. L. B. Smith Institute of Ichthyology, Grahamstown, South Africa. pp. 253.
Stransky, C.; Murta, A. G.; Schlickeisen, J.; Zimmermann, C., 2008:
Otolith shape analysis as a tool for stock separation of horse
mackerel (Trachurus trachurus) in the Northeast Atlantic and
Mediterranean. Fish. Res. 89, 159–166.
Torres, G. J.; Lombarte, A.; Morales-Nin, B., 2000: Variability of
the sulcus acusticus in the sagittal otolith of the genus Merluccius (Merluciidae). Fish. Res. 46, 5–13.
Tracey, S. R.; Lyle, J. M.; Duhamel, G., 2006: Application of elliptical Fourier analysis of otolith form as a tool for stock identification. Fish. Res. 77, 138–147.
Tuset, V. M.; Lozano, I. J.; Gonzalez, J. A.; Pertusa, J. F.; GarcıaDıaz, M. M., 2003: Shape indices to identify regional differences
in otolith morphology of scomber Serranus cabrilla (L., 1758). J.
Appl. Ichthyol. 19, 88–93.
Tuset, V. M.; Rosin, P. L.; Lombarte, A., 2006: Sagittal otolith
shape used in the identification of fishes of the genus Serranus.
Fish. Res. 81, 316–325.
Tuset, V. M.; Lombarte, A.; Assis, C. A., 2008: Otolith atlas for the
western Mediterranean, north and central eastern Atlantic. Scientia Mar. 72, 7–198.
Vignon, M., 2012: Ontogenetic trajectories of otolith shape during
shift in habitat use: interaction between otolith growth and environment. J. Exp. Mar. Biol. Ecol. 420–421, 26–32.
Volpedo, A. V.; Fuchs, D. V., 2010: Ecomorphological patterns of
the lapilli of Paranoplatense Siluriforms (South America). Fish.
Res. 102, 160–165.
Ye, Z. J.; Zhang, C.; Panhwar, S. K.; Li, Z. G.; Wan, R., 2015: Ageing Belanger’s croaker, Johnius belangerii (Cuvier, 1830), based
on otolith shape analysis. J. Appl. Ichthyol. 31, 27–31.
10
Yilmaz, S.; Yazicioglu, O.; Saygin, S. A.; Polat, N., 2014: Relationships of otolith dimensions with body length of European perch,
Perca fluviatilis L., 1758 From Lake Ladik, Turkey Pakistan. J.
Zool. 46, 1231–1238.
Yu, X.; Cao, L.; Liu, J.; Zhao, B.; Shan, X.; Dou, S., 2014: Application of otolith shape analysis for stock discrimination and species identification of five goby species (Perciformes: Gobiidae) in
the northern Chinese coastal waters. Chin. J. Oceanol. Limnol.
32, 1060–1073.
D. Bostanci et al.
Zorica, B.; Sinovcic, G.; Cikes, K. V., 2010: Preliminary data on the
study of otolith morphology of five pelagic fish species from the
Adriatic Sea (Croatia). Act. Adr. 51, 89–96.
Author’s address: Derya Bostanci, Department of Biology, Ordu
University, Ordu, Turkey.
E-mail: deryabostanci@gmail.com