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Application of otolith shape analysis to species separation in Sebastes spp.

from the Bohai Sea and the Yellow Sea, northwest Pacific

Article  in  Environmental Biology of Fishes · February 2014

DOI: 10.1007/s10641-014-0286-z

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Environ Biol Fish
DOI 10.1007/s10641-014-0286-z
Application of otolith shape analysis to species separation
in Sebastes spp. from the Bohai Sea and the Yellow Sea,
northwest Pacific
Longchuan Zhuang & Zhenjiang Ye & Chi Zhang
Received: 27 September 2013 / Accepted: 2 June 2014
# Springer Science+Business Media Dordrecht 2014
Abstract Shape descriptors of sagittal otoliths were ap-
plied to the identification of four sympatric species of the
genus Sebastes (Sebastes schlegeli, S. hubbsi, S. nudus and
S. thompsoni) inhabiting the coastal reefs of the Bohai Sea
and the Yellow Sea, northwest Pacific. Canonical discrim-
inate analysis of standard length-corrected otolith shape
variables (a combination of shape indices and elliptic
Fourier coefficients) was demonstrated to be a useful tool
for species identification with a high overall classification
success of 96.6 %. An otolith morphological tree based on
hierarchical average linkage cluster analysis indicated the
closest proximity was between S. hubbsi and S. nudus. The
otolith morphological affinities coincide well with the
phylogenetic relationships reported among these species,
which indicates the potential to use otolith morphology to
establish phylogenetic relationships in this genus. In addi-
tion, ecomorphological comparisons suggest that the oto-
lith shapes might be influenced by several ecological and
environmental components such as depth, body color and
substrate type. Among the environmental factors, depth
could be the most comprehensive factor associated with
the inter-specific variations of otolith morphology.
Keywords Sagittae shape . Sebastes . Northwest
Pacific . Species separation . Ecomorphology
L. Zhuang : Z. Ye (*) : C. Zhang
College of Fisheries, Ocean University of China,
Qingdao 266003, China
e-mail: yechen@ouc.edu.cn
Introduction
The live-bearing genus Sebastes is the most speciose scor-
paenid genus with approximately 110 species worldwide
(Nelson 2006; Hyde and Vetter 2007), among which the
majority are distributed in temperate to Arctic waters
throughout the North Pacific. Seventy species are distrib-
uted within the northeast Pacific (NEP), and 33 species
occur in the northwest Pacific (NWP), extending in an arc
from Kyushu, Japan in the west to Baja California and the
Gulf of California, Mexico in the east (Love et al. 2002;
Kai et al. 2003). Within their range, many species are
important for commercial fisheries and recreational fisher-
ies (Lenarz 1986; Nagasawa 2001; Love et al. 2002), as
well as for basic research of viviparous teleost reproduction
(Wourms 1991).
Despite their abundance in the North Pacific, the genus
Sebastes remains the subject of taxonomic confusion due
to a high degree of overlap in their morphometric and
meristic characters and sympatry especially in the case of
closely related species. Several approaches have been used
for species identification and taxonomic analysis of
Sebastes. Traditionally, body morphometric and meristic
variations have been widely used for the species separation
of Sebastes and the genus has been divided into several
subgenera based on these variations in early taxonomic
studies (Matsubara 1936; Chen 1985, 1986; Kai et al.
2011). More recently, in addition to phenotype-based spe-
cies identification, various biochemical and genetic ap-
proaches, such as analysis of mitochondrial genes, nuclear
genes, AFLPs, microsatellites makers and allozymes have
been proposed to test the validity of several subgenera and

species (Rocha-Olivares et al. 1999a; Rocha-Olivares et al.
1999b; Rocha-Olivares et al. 1999c; Kai et al. 2003; Hyde
and Vetter 2007; An et al. 2009; Kai et al. 2011; Bunke
et al. 2013; Venerus et al. 2013). Numerous revisions have
been made in their taxonomic status based on phylogenetic
analyses.
Similarly, with the development of digital techniques
and shape analysis methods (e.g., Fourier analysis, land-
marks and semi-landmarks), sagittal otoliths, the largest
of the three pairs of calcareous structures in the inner ear
of teleosts (Gauldie and Nelson 1988), are characterized
by their high degree of inter-specific variation, which
has proven to be a powerful time- and cost-efficient
means in species identification of marine fish species
(Monteiro et al. 2005; Stransky and MacLellan 2005;
Sadighzadeh et al. 2012; Tuset et al. 2013). It has been
suggested that the species-specific otolith morphology
is determined by synergistic influences of both genetic
and environmental components (Monteiro et al. 2005;
Vignon and Morat 2010). Vignon and Morat (2010)
analyzed the otolith shape of an introduced coral reef
snapper (Lutjanus kasmira) and confirmed the hypoth-
esis that environmental variation induces an overall
change in the otolith shape, while genetic variation
causes subtle local shape changes. For the genus
Sebastes, abundant phylogenetic analyses provide a po-
tential means for evaluating the relative importance of
genetic influence in inter-specific variation of otolith
shape especially among closely related species. On the
other hand, as mechanoreceptors that process acoustic
and postural information, otoliths bear a close relation-
ship to the ambient stimuli. The comparative study of
sagittal otolith morphology and its relation to environ-
mental variables provides a method for elucidating char-
acteristics of biological significance. Environmental fac-
tors such as water temperature, depth, feeding habits and
spatial niches are believed to be associated with otolith
shape, especially the morphological characteristics of
sagittae such as the sulcus area:otolith area ratio (S/O),
relative otolith size and rostrum (Campana and Neilson
1985; Gauldie and Nelson 1988; Lombarte and Lleonart
1993; Lombarte et al. 2010).
The species considered in this paper were four sym-
patric species of the genus Sebastes that belong to four
subgenera inhabiting rocky reefs in shallow coastal
waters: Sebastes schlegeli (Hilgendorf, 1880),
S. hubbsi (Matsubara, 1937), S. nudus (Matsubara,
1943) and S. thompsoni (Jordan & Hubbs, 1925). They
mainly dwell in demersal habitats at the adult stage and
Environ Biol Fish
display a bigger-deeper trend in fish size (Jin 2006). All
these species play important roles in energy and material
flows through coastal reef communities and commercial
fisheries although S. hubbsi catches are relatively limit-
ed because of its small size. In addition, they are known
to be closely related, as expressed by the small magni-
tude of meristic and morphometric differences (Chen
1986; Jin 2006) and close phylogenetic relationships
(Kai et al. 2003; Hyde and Vetter 2007). However,
systematic sagittal morphological descriptions of these
species are rare. Recently, some genetic studies have
been performed to confirm their phylogenetic relation-
ships (Kai et al. 2003; Hyde and Vetter 2007; Zhang
et al. 2013). Otolith shape analysis has been successfully
applied to the species separation of Sebastes in the
Atlantic and NEP by Stransky and MacLellan (2005)
and has achieved a very encouraging overall species
classification success of 91 % but has not been used
for Sebastes in the NWP. Moreover, Stransky and
MacLellan (2005) mainly investigated genetic influence
on otolith morphology variation of Sebastes, instead of
evaluating the dual regulation of both genetic and envi-
ronmental influence.
Accordingly, the primary aims of this paper were to
(i) describe the characteristics of sagittal otoliths of the
four species systematically, (ii) analyze otolith shape
variability and examine its potential in species separa-
tion and (iii) investigate the possible influence of genetic
and ecological and environmental variables on otolith
morphological features.
Material and methods
Sample collection
A total of 291 specimens of the four Sebastes species
were collected between October 2008 and June 2010 off
the coasts of Qingdao, Changshan Islands and
Cheniushan Island in the Bohai and Yellow Seas, China
(Table 1; Fig. 1). The individuals from Qingdao and
Cheniushan Island were taken randomly from the com-
mercial fishery, whereas specimens from Changshan
Islands were obtained from experimental fishing. Spe-
cies identification was performed by external identifica-
tion features. In particular, the taxonomy of S. nudus has
been confused and subject to several revisions (Kai et al.
2011; Kai and Nakabo 2013). Thus, the individuals of
S. nudus analyzed were identified according to the most
Environ Biol Fish
recent taxonomic study by Kai and Nakabo (2013).
After measurement of standard fish length (SL, in mm)
and eye diameter (ED, in mm) (Table 1), the sex of each
individual was determined by visual observation of the
gonads. All sagittal otoliths were dissected, organic
tissues removed, air-dried and stored in plastic tubes.
Image and otolith shape analysis
Each sagitta, systematically positioned with the sulcus
acusticus upwards and the rostrum to the right horizon-
tally, was imaged using a Nikon SMZ800 biological
dissection microscope equipped with a video camera at
10X magnification. Prior to shape analysis, we proc-
essed all digital images in Adobe Photoshop CS3 by
generating a contrast of 100 % along the otolith contour
and drawing the outline of the sulcus acusticus (Fig. 2).
The otoliths were described morphologically according
to the terminology proposed by Tuset et al. (2008).
Image-Pro Plus (V.6.0) was used for image measure-
ment and preliminary analysis. The shape variables
measured were: otolith length (OL, in mm), otolith
height (OH, in mm), length and height of the sulcus
acusticus (LS and HS, in mm), perimeters of the otolith
(OP, in mm) and sulcus acusticus (PS, in mm), areas of
the otolith (OA, in mm2) and sulcus acusticus (AS, in
mm2), perimeter ratio (ratio of convex perimeter to
perimeter) of the otolith and sulcus acusticus, aspect
ratio (ratio between major axis and minor axis of ellipse
equivalent to otolith) of the otolith and sulcus acusticus,
radius ratio (ratio between Max. radius and Min. radius)
of the otolith and sulcus acusticus, rectangularity (OA/
(OH×OL)), roundness (perimeter2/(4×pi×area)) and
S/O (the ratio between the sulcus acusticus area and
otolith area).
The first twenty elliptic Fourier harmonics of each
otolith contour which was normalized for size and ori-
entation were calculated using the software SHAPE
(V.1.3) (Iwata and Ukai 2002). Each harmonic was
composed of four elliptic Fourier coefficients (eFcs),
resulting in 80 eFcs per individual. To normalize the
contour to the first harmonic, the software package
automatically generated the first three eFcs of each
individual to fixed values: a1=1, b1=c1=0 (all har-
monics were marked a1, b1, c1, d1, a2, etc.). Hence,
only 77 eFcs were selected for the subsequent analyses.
Average otolith shapes were drawn for each species
using principal components analysis (PCA) of eFcs for
visual comparison of the differences among species.
Paired-samples t-tests revealed no significant differ-
ence (p>0.05) on all the shape variables between left
otoliths and mathematically mirrored right otoliths.
Thus, we used the left otolith when it was available
and the right when the left was not utilizable or present.
Statistical analyses
A Kolmogorov-Smirnov (K-S) two sample test was
performed to test the differences in the distributions of
SL and all the otolith morphological variables between
females and males for each species.
Each shape indice and eFc was examined for normal-
ity and homogeneity of variance by K-S test and
Levene’s test, respectively. When necessary, data were
log10- transformed or square-root transformed to satisfy
the assumptions. The transformed variables that did not
meet the criteria were excluded from the subsequent
analysis.
To remove allometric effects of body size in shape
analysis, the theoretical equations outlined below were
used to scale data (Lleonart et al. 2000):
 b
 X0
Yi ¼ Yi ð1Þ
Xi
LgY i ¼ LgðaÞ þ b  LgðX i Þ þ ε ð2Þ
Where in Eq (1), Xi is the SL of individual i; X0 is the
reference SL value; Yi is the measured value of otolith
morphological variables of individual i, and Yi* is the
theoretical value that would measure Yi if the SL was X0,
considering its allometric growth and residual. The al-
lometric parameters b could be estimated in Eq (2) using
linear regression procedures. In the present study, the
reference SL was 130 mm based on the mean value for
SL of the specimens studied.
Analysis of covariance (ANCOVA) was performed
to determine the effect of SL on variables. Morpholog-
ical variables were treated as variates, with ‘Species’ as
main factor and the length as the covariate. If the
“SL*species” interaction effect in the ANCOVA was
not significant, a common slope was used to scale data.
If not, the data were adjusted separately for each group
using the group-specific slope. Variables were excluded
from the further analyses if they were not significantly
correlated with lengths by using Pearson correlation
analysis. Relative otolith size (OR) was calculated for
 Environ Biol Fish

Table 1 List of the studied species of Sebastes spp. in the NWP, including valid subgenera (Kendall 2000), sampling locations, number of
individuals, and descriptive statistics of SL (standard fish length) and ED (eye diameter)

Species Subgenus Sampling Mean SL Mean ED No. of

locations (and range) (and range) samples
(mm) (mm)

S. schlegeli Neohispaniscus Changshan Islands, Qingdao 158.57(137–182) 13.85(12–16) 53

S. hubbsi Sebastocles Cheniushan Island, Qingdao 99.34(71–127) 9.08(7–11) 132
S. nudus Murasoius Cheniushan Island, Qingdao 110.08(76–167) 11.00(8–17) 36
S. thompsoni Mebarus Qingdao 174.46(112–344) 17.76(12–33) 70

each otolith according to OR =10,000×OA×LT− 2
(Lombarte and Cruz 2007), where LT =fish total length
(in mm). Once the data were standardized, a Student-
Newman-Keuls (S-N-K) test was used for comparisons
of the standardized shape indices, eye diameter:standard
fish length ratio (ED:SL) and OR among species.
Before proceeding with discriminant function analy-
sis, we tested for homogeneity of the within-group
covariance matrices using a Bartlett’s modification of
the likelihood ratio test. We built a canonical discrimi-
nate analysis (CDA) for species discrimination because
the covariance matrices were heterogeneous. CDA was
applied using only the previously selected standardized
shape indices and eFcs. A stepwise discriminate method
was performed to determine selection of the variable
sets utilized in each function. The classification
accuracy was estimated by using leave-one-out cross-
validation. A hierarchical average linkage cluster anal-
ysis based on Euclidean distances was used to investi-
gate interspecific morphological similarities.
All tests were conducted with a significance level of
0.05 using SPSS (V.19.0) and PAST (V.2.17).
Results
Otolith anatomical descriptions
Sebastes schlegeli Shape: elliptic to rhombus, margin
dorsal and ventral markedly crenate, with posterior mar-
gin irregular. Sulcus acusticus: heterosulcoid, ostial,
median. Ostium: funnel-like, as long as the cauda.

Fig. 1 Map of sample locations showing the area from where the specimens of the genus Sebastes were collected
Environ Biol Fish

Fig. 2 View of medial surface of

the sagittal otoliths of Sebastes
spp. illustrating various features

Cauda: tubular, slightly curved, ending far from the
posterior margin. Anterior region: peaked; rostrum
short, broad; antirostrum absent; excisura wide without
a notch. Posterior region: peaked to irregular.
Sebastes hubbsi Shape: elliptic to pentagonal, dorsal
and ventral margin smooth or slightly crenate. Sulcus
acusticus: heterosulcoid, ostial, median. Ostium: funnel-
like, as long as the cauda. Cauda: tubular, slightly
curved, ending far from the posterior margin. Anterior
region: peaked; rostrum short, broad, pointed;
antirostrum absent or short, broad, round; excisura wide
without or with a shallow notch. Posterior region: two-
peaked to flattened.
The average shape outlines of the otoliths and sulcus
acusticus reproduced by PCA analysis of eFcs within
each species, scaled to the same size, displayed detailed
differences (Fig. 3). S. schlegeli displayed hardly any
notch in the anterior side, whereas the others possessed
shallow notches and more pointed rostrums. Margins in
the dorsal and ventral sides appeared straighter in
S. nudus, whereas they curved slightly in S. hubbsi
and S. thompsoni and remarkably in S. schlegeli. The
posterior region differed most among the average
shapes. The region was a little peaked in S. schlegeli,
round and flattened in S. hubbsi and somewhat concave
in the posterior-ventral side for both S. nudus and
S. thompsoni.

Sebastes nudus Shape: elliptic to pentagonal, dorsal and Shape variables analysis
ventral margin smooth, nearly parallel. Sulcus acusticus:
heterosulcoid, ostial, median. Ostium: funnel-like, lon- There was no significant difference between females
ger than the cauda. Cauda: tubular, slightly curved, and males in the distributions of SL and all otolith
ending far from the posterior margin. Anterior region:
peaked; rostrum short, broad, pointed; antirostrum ab-
sent or very short; excisura wide without or with a
shallow notch. Posterior region: flattened to irregular.

Sebastes thompsoni Shape: elliptic to pentagonal, dor-

sal and ventral margin crenate. Sulcus acusticus:
heterosulcoid, ostial, median. Ostium: funnel-like, sim-
ilar in size to the cauda. Cauda: tubular, curved slightly,
ending far from the posterior margin. Anterior region:
peaked; rostrum short, broad, pointed; antirostrum
absent or very short; broad, pointed; excisura wide
without or with a shallow notch. Posterior region: Fig. 3 The average shape outlines of Sebastes otoliths reproduced
flattened to irregular. by PCA analysis of eFcs within each species
 Environ Biol Fish

Table 2 Result of ANCOVA

showing the shape indices re- Variable SL×species SL
markably correlated with SL and
allometric parameters b were used F d.f. P F d.f. P b
to scale data for reference SL
OA 3.627 3 0.013 _ _ _ group-specific
AS 1.294 3 0.277 253.481 1 0.000 1.010
Aspect ratio 1.466 3 0.224 35.668 1 0.000 0.118
OP 2.853 3 0.038 _ _ _ group-specific
PS 0.518 3 0.671 318.552 1 0.000 0.547
Radius ratio 1.593 3 0.191 33.126 1 0.000 0.117
Roundness 2.369 3 0.071 18.660 1 0.000 0.123
Note: Significant values are in
bold. group-specific: Shape vari- LS 0.502 3 0.681 352.885 1 0.000 0.559
ables were adjusted separately for S/O 0.823 3 0.482 6.679 1 0.010 −0.188
each group using the group-spe- OH 1.187 3 0.315 597.282 1 0.000 0.543
cific slope

morphological variables on the basis of the K–S two
sample test (p>0.05). Thus, the morphological data
were pooled across sex for the subsequent analyses.
After being screened by the K-S normality test,
Levene’s test and Pearson correlation analysis with
lengths, 10 shape indices and 12 eFcs were selected
for normalization using reference SL. Two shape
variables (OA (p = 0.013) and OP (p = 0.038)) re-
vealed significant interactions between species.
Therefore, they were standardized using the group-
specific slopes with others using common within-
group slopes from the ANCOVA (Table 2).
The fitted variables were then tested for significant
multicollinearity using Pearson’s correlation coefficient
test. When two variables correlated significantly, only
the one showing a higher F statistic from ANOVA was
retained for CDA. Ten SL-corrected shape indices and
eFcs (roundness, radius ratio, S/O, OH, d2, a6, c7, d7,
d16 and d19) met the assumptions finally and corrected
shape indices displayed differences among the four
species (Table 3). Roundness displayed distinctly higher
adjusted mean values in S. schlegeli. The mean value of
the radius ratio was higher in S. hubbsi and S. nudus,
whereas that of OH was greater in S. schlegeli and
S. thompsoni.
The ED:SL ratio varied significantly across all spe-
cies (Fig. 4), with the highest mean value (mean and
standard deviation, 0.1021±0.0336) with S. thompsoni
and the lowest value (0.0874±0.0021) with S. schlegeli.
S. nudus displayed higher values (0.1000±0.0030) than
S. hubbsi (0.0916±0.0037).
The adjusted mean S/O value was significantly
higher in S. hubbsi than in the other three species. The
mean S/O value of S. nudus was intermediate between
S. schlegeli and S. thompsoni, though not significantly
different from S. thompsoni. The smallest value was for
S. thompsoni (Fig. 4).
The S-N-K test for multiple comparisons displayed
significant differences among the mean OR values of the
four species (Fig. 4). S. hubbsi had larger otoliths than

Table 3 Student–Newman–Keuls mean comparisons for SL-corrected otolith shape indices, ED:SL (eye diameter:standard fish length
ratio) and OR (relative otolith size) of Sebastes spp.

Species Roundness Radius ratio S/O OH ED:SL OR No. of samples

S. schlegeli 1.826a 2.081a 0.318a 3.371a 0.087a 5.031a 53

b b b b b
S. hubbsi 1.505 2.381 0.332 3.172 0.092 7.459b 132
S. nudus 1.545b 2.339b 0.297c 2.808c 0.100c 5.621c 36
S. thompsoni 1.507b 2.131a 0.287c 3.650d 0.102d 6.575d 70

Note: Values in rows with different superscript lower case letters are significantly different (p <0.05)
Environ Biol Fish
the remaining species. S. thompsoni had a mean OR of
6.57, S. nudus had a value of 5.62, whereas S. schlegeli
had the smallest values, 5.03.
Canonical discriminate analysis and cluster analyses
The scatter-plots of discriminant scores (Fig. 5) and
classification matrix (Table 4) were generated to visual-
ize the results of the CDA. The CDA standardizing in
relation to SL was constructed by using four shape
indices (roundness, radius ratio, S/O and OH) and six
eFcs (d2, a6, c7, d7, d16 and d19). All three discriminate
Fig. 4 Box-whisker plots (means±SE) of (a) the eye diameter: standard fish length ratio (ED:SL), (b) the S/O standardized to 130 mm SL
and (c) relative otolith size (OR) for the Sebastes spp.
functions were significant. The first two discriminate
functions explained 57.0 and 36.6 % of the total varia-
tion, whereas the third function explained the remainder.
Roundness and radius ratio explained most of the vari-
ation in the first discriminant function, whereas OH
explained most in the second discriminant function.
The classification success of samples through leave-
one-out cross-validation averaged 96.6 %, ranging from
93.9 to 100 %.
The hierarchical cluster analysis performed on the
morphological characters divided the four species into
two clusters (Fig. 6). S. hubbsi and S. nudus were
 Environ Biol Fish

Fig. 5 Discriminant function

scores for the otoliths from
Sebastes spp. and plots showing
separation

grouped in cluster 1, whereas the other two species were (Table 4), which are known to be closely related
grouped in cluster 2. A comparatively closer morpho- with low interspecific morphologic and genetic var-
logical relationship was found in cluster 1 rather than iation (Chen 1986; Kai et al. 2003; Kai and Nakabo
cluster 2. 2013). According to the phylogenetic trees con-
structed by Kai et al. (2003) and Hyde and Vetter
(2007), all the four species were incorporated into
Discussion “clade NWP” and S. hubbsi and S. nudus displayed
the closest phylogenetic affinity to be separated into
The species separation of the four sympatric one subclade. More recently, Zhang et al. (2013)
Sebastes from the Bohai Sea and the Yellow Sea reconfirmed the interspecific phylogenetic relation-
was studied by otolith shape analysis in the present ship among these Sebastes and demonstrated the
study. CDA provided a higher overall species clas- closest affinity between S. hubbsi and S. nudus,
sification success rate within the four NWP Sebastes whereas S. schlegeli was closer to S. thompsoni in
species than that within the NEP species (94 %) and genetic distance. In light of the previous genetic
the North Atlantic (NA) samples (88 %) (Stransky evidences and our discriminant and cluster analyses,
and MacLellan 2005). The largest misclassification otolith morphological affinities among these species
rates occurred between S. hubbsi and S. nudus coincided with their phylogenetic relationships. This
implied some genetically programmed control over
Table 4 Leave-one-out cross-validated classification matrix for otolith shape.
the discriminate function analysis of Sebastes spp.
S. thompsoni prefers a low relief reef/soft bottom
S. schlegeli S. hubbsi S. nudus S. thompsoni lifestyle and is present at comparatively greater
depths, whereas the others prefer a high-relief reef
S. schlegeli 100.0 (53) 0 (0) 0 (0) 0 (0) lifestyle and shallower depth (Hyde and Vetter
S. hubbsi 0 (0) 93.9 (124) 3.8 (5) 2.3 (3) 2007). Although only the depth distributions of
S. nudus 0 (0) 0 (0) 100.0 (36) 0 (0) S. schlegeli (3–100 m) (Boehlert et al. 1986; Parin
S. thompsoni 1.4 (1) 1.4 (1) 0 (0) 97.1 (68) 2003) and S. thompsoni (40–150 m) (Yamada et al.
1995) have been demonstrated, the following premise
Note: The percentage of individuals classified in each species and
the number of samples (in parentheses) are given. The percentages
could apply: larger eyes (relative to body size) are
of correctly classified species are in bold. Total correct classifica- related to a deeper depth distribution. This relation-
tion=96.6 %, Wilks’λ=0.028 ship has been recognized and widely applied to
Environ Biol Fish
Fig. 6 Otolith morphological tree
inferred from the SL-corrected
shape indices and eFcs using
average linkage
marine fishes (Denton 1990; Warrant 2000; Herler
2007). Ingram and Shurin (2009) confirmed that in-
creased eye size relative to body size is a common
adaptation to decreased light availability within the
depth range occupied by Sebastes. More recently, the
premise has been applied in otolith ecomorphology
by Tuset et al. (2010) to indicate the ecological seg-
regation and depth distribution of Aphanopus carbo
and A. intermedius. In the present study, according to
the comparison of ED:SL values (Fig. 4), it could be
hypothesized that both S. hubbsi and S. nudus inhabit
greater depths than S. schlegeli, whereas S. thompsoni
prefers the deepest depth distribution.
The depth gradient is known to be important in
determining environmental conditions such as tem-
perature and light and its association with otolith has
been investigated (Lombarte and Cruz 2007). In the
Fig. 7 Fresh body color patterns of (a) S. schlegeli, (b) S. nudus, (c) S. thompsoni and (d) S. hubbsi
present study, relationships between sagittae size,
S/O, rostrum of the sagittae and depth were found.
The species inhabiting greater depth tended to pos-
sess larger relative sagitta size, with the exception of
S. hubbsi, which had considerably higher mean
values (Fig. 4). This overall tendency is in
accordance with Lombarte and Cruz (2007) in the
demersal communities. Sebastes is notable for diver-
sity in body color patterns (Orr et al. 2000), which
are a qualitative criterion related to depth and sub-
stratum (Aleev 1969). Rocha and Bowen (2008)
proposed that the segregation of body color may
follow sympatric speciation in reef fishes. The
closely related species S. hubbsi and S. nudus
differ barely in morphology but most strikingly in
color patterns. Cruz and Lombarte (2004) indicated
that there was a relationship between color patterns

and otolith size in fishes inhabiting reef environ-
ments, which are characterized by their wide range
of light wavelengths that allow intraspecific commu-
nication to be based on color. Thus, differences in
color patterns (related to visual communication) may
induce specific differences in otolith size (related to
hearing ability) among these Sebastes. Body color
variations (dusky to striking color patterns) of the
four species studied are as follows (Fig. 7):
S. schlegeli: body black, somewhat paler below;
S. nudus: body dark or gray, somewhat paler below;
S. thompsoni: body brownish yellow with 5 dark
brown transverse bands on body, somewhat paler
below; and S. hubbsi: body dark red, reddish orange
below. Based on our results, the species with
brighter color patterns displayed a larger relative
otolith size. These findings could explain the high
mean OR of S. hubbsi, and the clear difference in the
value from that of S. nudus.
In addition, there is a clear relationship between S/O
and depth that species inhabiting shallow waters such as
S. schlegeli and S. hubbsi tended to present large S/O
values, whereas a deeper dweller, such as S. thompsoni
displayed the opposite tendency. Sulcus acusticus and
sensory macula have been reported as organs of equi-
librium and hearing (Gauldie and Nelson 1988;
Lombarte and Popper 1994; Arellano et al. 1995). As
an approximation of the ratio of macula to otolith area,
S/O is related to the frequency response and auditory
threshold (Gauldie and Nelson 1988). Rogers and Cox
(1988) found that fishes are most sensitive to sound
where the ambient noise is high, such as coastal reefs.
A comparative study of otolith morphology between
Mullus barbatus and M. surmuletus, which live on the
coastal continental shelf, revealed that fish associated
with rocky reefs have larger S/O ratios than fish associ-
ated with soft bottom (Aguirre and Lombarte 1999). Our
data are in accordance with these results. Sebastes asso-
ciated with high rocky reefs where the ambient noise is
higher (S. hubbsi, S. schlegeli and S. nudus) displayed
significantly higher S/O ratios than the low relief
reef/soft bottom dweller (S. thompsoni) (Fig. 4).
The rostrum of the sagittae has been demonstrated to
be another morphologic feature related to environment
(Volpedo and Echeverrı́a 2003; Volpedo et al. 2008).
Comparing the interspecific differences among the
mean shapes (Fig. 3), we found there was some corre-
lation between the development degrees of otolith ros-
trum and depth. The most developed and pointed
Environ Biol Fish
rostrum was found in the deepest dweller
S. thompsoni, whereas relatively less developed and
pointless ones were found in the remainder species,
especially in the shallowest dweller S. schlegeli, which
displayed no well-defined rostrum.
Collectively, this study demonstrated clear interspe-
cific differences in otolith shapes among the studied
species and confirmed its potential for using otolith
shape analysis for species separation. Given the very
high classification success among the four NWP
Sebastes species in the present study and that among
the NEP species and the NA samples by Stransky and
MacLellan (2005), we found otolith shape analysis pro-
vide a powerful tool for species separation of Sebastes
spp. In addition, the otolith morphological affinities
coincided well with the phylogenetic relationships
among these species, which implies the potential to
use otolith morphology to establish phylogenetic rela-
tionships in this genus. The inter-specific otolith mor-
phology variations were found to be correlated with
ecological and environmental variations in depth, body
color and substrate type, but the analysis suggested
depth could be the environmental factor most compre-
hensively associated with otolith morphology.
Acknowledgments We greatly acknowledge the financial sup-
port of the Public Science and Technology Research Funds Pro-
jects of Ocean (Grant No. 201305030) and the Specialized Re-
search Fund for the Doctoral Program of Higher Education (Grant
No. 20120132130001). We thank Professor X. Q. Zeng for his
great assistance in taking photographs of otoliths. All the four
Sebastes species in this study are economic edible fish and not
evaluated in the IUCN red list of threatened species. Although
animal ethics approval is not required, all Sebastes experimental
procedures were performed following the general animal ethical
guidelines of Institutional Animal Ethical Committee (IAEC).
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