Iranian Journal of Fisheries Sciences 19(2) 814-832 2020 DOI: 10.22092/ijfs.2018.120044. Use of otolithic morphometrics and ultrastructure for comparing between three (family: Mullidae) from the northern Red Sea, Hurghada, Egypt

Osman A.G.M.1*; Farrag M.M.1; Mehanna S.F.2; Osman Y.A.3

Received: December 2017 Accepted: July 2018 Abstract This work highlights the role of otolithic morphometrics, shape indices and ultrastructure in the identification of three Mullidae species from the Red Sea. Differences in otolithic measurements were detectable in all three-goatfish species. The statistical analysis of otolithic morphometric parameters showed that otolithic measurements are good indicators of fish size. For all three species, the correlation between fish length and different otolithic variables was statistically significant. The coefficient of determination (r2) ranged from 0.83 to 0.92 in the three species, being higher for M. vanicolensis in all cases. The otolith area of M. vanicolensis was most strongly related to fish length, with a high correlation between otolith area and fish length (r2=0.92) being observed in this species. The mean values of the six examined shape indices of the otoliths were considerably different among the three species, and the high degree of differentiation of these indices among species makes them useful for other researchers who wish to use otoliths in fish identification and classification. Remarkable variations in the morphological characteristics of fish otoliths were recorded among the studied species via scanning electron microscopy, including variations in the ornamentation of the ostium, cauda, and column of the otolith. These differences in otolithic characteristics and morphology might be useful for fisheries, biologists, archaeologists and geologists in discriminating Mulloidichthys flavolineatus, M. vanicolensis, and forsskali. This work contributes to the bioecological knowledge regarding commercially important fishes and provides key information for studying the trophic ecology of fish-eating species and fishery management.

Keywords: Goatfish species, Otoliths, Morphometrics, Scanning electron microscope, Red Sea

1-Department of Zoology, Faculty of Science, Al-Azhar University (Assiut Branch), 71524 Assiut, Egypt. 2-National Institute of Oceanography and Fisheries, Suez, Egypt 3-National Institute of Oceanography and Fisheries, Hurghada, Egypt *Corresponding author's Email: [email protected]

815 Osman et al., Use of otolithic morphometrics and ultrastructure for…

Introduction taxonomical and biological archives, as Family Mullidae () is widely they reflect species’ growth and distributed globally, especially in the development. Most importantly for the Pacific and Indo-Pacific regions, current study, otolithic morphology is representing important food-chain species specific (Popper et al., 2005). components in coastal ecosystems Otolithic morphology ranges from (Pavlov et al., 2015). The family ellipsoidal to more complex shapes, consists of six genera and with protrusions and invaginations approximately 62 species (Nelson, (Campana, 2004), and has been used in 2006). Goatfishes are one of the most many studies for species identification economically and commercially (Tuset et al., 2006; Lord et al., 2012; important groups inhabiting the Bani et al., 2013; Sadighzadeh et al., northern Egyptian Red Sea sector 2014). However, for accurate species (Sabrah, 2015). Three common goatfish identification, a reference collection of genera are present along the Egyptian otoliths from known species must be Red Sea coast: Mulloidichthys, produced to generate sufficient Parupeneus and Upeneus (Kuronuma comparative material to produce and Abe, 1986). accurate descriptions of morphological Otoliths are calcareous structures characteristics. found in the inner ear of fishes To our knowledge, there is no (Campana, 2004). There are three pairs available information concerning the of otoliths (sagittae, asterisci and lapilli) identification of goatfish from the in the otic sacs (Popper and Lu, 2000), Egyptian Red Sea using otolithic and they function as mechanoreceptors morphometrics and∕or ultrastructure. that are involved in balance and hearing Therefore, this study had three (Popper et al., 2005). In most species, objectives: 1) to determine the the sagittal otoliths (the largest of the possibility of readily discriminating three otolith pairs) are most commonly common goatfish species based on the used to estimate age and growth, shape and ultrastructure of their movement and habitat, population otoliths; 2) to quantify otolith length, structure, and trophic ecology width, and height relationships with (Campana and Casselman, 1993; respect to fish length; and 3) to identify Rooker et al., 2008). Otoliths are often characteristics and ultrastructural collected in routine fisheries features allowing otoliths from common investigations for age determination as Mullidae species to be distinguished well as from predator stomachs from each other. (Nielsen and Andersen, 2001), allowing precise information on the Materials and methods length, weight, age, and quantity of Sampling individual fish prey to be obtained in Three goatfish species (Family: many cases. Furthermore, otoliths are Mullidae) were collected (n=275) from generally considered to serve as the northern part of the Red Sea Iranian Journal of Fisheries Sciences 19(2) 2020 816

Province at Hurghada (latitudes 27° 10' total length (TL) was measured to the N–27° 33' N and longitudes 33° 70' E– nearest millimeter and weight (W) to 33° 85' E) (Fig. 1) using artisanal the nearest gram. The sagittal otoliths fishing gear (gill net and trammel net) (Fig. 2) of the fish were removed, from September 2015 to August 2016. washed and dried and then stored in These species included Mulloidichthys plastic vials until being examined and flavolineatus (n=152), Mulloidichthys photographed. vanicolensis (n=21), and Parupeneus forsskali (n=102). For all individuals,

Figure 1: Map showing the study area at Hurghada, Egypt.

Figure 2: Proximal view of the left sagitta of common goatfish species from the Red Sea, Egypt.

817 Osman et al., Use of otolithic morphometrics and ultrastructure for… Morphometric and shape analysis of (otolith variables=aTLb), followed by otoliths log transformation to estimate a and b For morphometric analysis, the otoliths via simple linear regression, in which a from the left side of the fish were is the angular coefficient characterizing oriented with the inner side (sulcus the otolith’s growth rate and b is a acusticus) upwards and the rostrum to constant specific to the species. the right, for digitization using a To describe otolith shape, six stereomicroscope linked to a digital dimensionless shape factors (aspect camera (Optica 2.1) (Fig. 2). Then, ratio (AS), compactness (CO), form otolith length (OL, mm), otolith width factor (FF), rectangularity (RE), (OWid, mm), otolith area (OA, mm2), roundness (RO), and ellipticity (EL) and otolith perimeter (OP, mm) were were obtained by combining size measured using ImageJ software. parameters (Russ, 1990; Tuset et al., Otolith weight (OW, mg) was measured 2003b; Pinkerton, 2015) (Table 1). using an AS220 kL-1 model balance. The relationships between fish total length (TL) and otolith variables were estimated using the power equation

Table 1: Otoliths shape indices. Index Formula Aspect ratio (AS) (OWid / OL) Compactness (CO) (OP2 / OA) Form factor (FF) 4 π OA / Op2 Rectangularity (RE) OA / (L * l) Roundness (RO) 4 OA / π L2 Ellipticity (EL) (OL - OWid) / (OL + OWid) OL, otolith length, OWid-otolith width, OA- otolith area, OP- otolith perimeter.

Scanning electron microscopy mode and on a Stereo Scan Cambridge The otoliths were physically cleaned by Mark 2A (15 KV) at the Assiut carefully removing any adhering tissue University Electron Microscope Center, and debris without damaging the scale Assiut, Egypt. The morphological surface. Then, they were immersed in a descriptions of the otoliths were based solution of sodium hypochlorite for on the terminology proposed by Tuset several minutes to soften adhering et al., (2008) (Fig. 2). tissues for further cleaning. For scanning electron microscopy (SEM) Statistical analysis examination, the otoliths were fixed on A Kolmogorov-Smirnov test was used a specimen holder using sticker tape to check the normality of the data and coated with a 30-nm layer of gold. distributions and variance homogeneity. Electron micrographs were produced on Statistical description of the weight, a GAOL, GSMS 400 LV scanning length and otolith size of goatfish from electron microscope in back-scattering the Red Sea was conducted using SPSS. Iranian Journal of Fisheries Sciences 19(2) 2020 818

A paired sample t-test was employed to descriptive statistics (minimum, compare the left and right otoliths maximum, mean, standard error and (SPSS, Version 18). Summaries of the standard deviation) and paired-t tests descriptive statistics for the otolith are shown in Table 2. An insignificant shape indices of common species of difference (p>0.05) in the weight, family Mullidae were performed using length, width, area, and perimeter of the SPSS. A power equation was applied to otoliths was observed between the left estimate the interaction between fish and right sagittal pairs for each goatfish length and otolithic measurements. species (Table 2). Therefore, the left otoliths were selected for the recording Results of other measurements and statistics. The summarized results for the left and right otolithic measurements as well as

Table 2: Summary of descriptive statistics and paired t-test results for left and right sagitta otoliths of three goatfish (Mulloidichthys flavolineatus, M. vanicolensis and Parupeneus forsskali) from the Red Sea, Egypt.

The morphometric measurements of the forsskali, respectively. Individual fish and otoliths are shown in Table 3, maximum otolith length (OL) ranged with statistical descriptions of each from 4.1 mm (P. forsskali) to 4.8 mm species. Fish total length (TL) ranged (M. vanicolensis). The otoliths of M. from 110–334 mm, 174–287 and 144– vanicolensis were the largest, reaching 271 for M. flavolineatus, M. up to 4.8 mm. The otoliths of this vanicolensis and P. forsskali, species also exhibited the greatest respectively. The weight of the fish perimeter (ranging from 11.03 to 18). ranged from 11.85–453.26 g, 52.8– The otoliths of P. forsskali presented 297.9 g and 32.13–237.37 g for M. the smallest values for all examined flavolineatus, M. vanicolensis and P. parameters. Overall individual otolith 819 Osman et al., Use of otolithic morphometrics and ultrastructure for… weight (OW) ranged from 0.005 (P. The overall otolithic measurements of forsskali) to 0.01 g (M. vanicolensis). M. flavolineatus occupied an M. vanicolensis displayed the heaviest intermediate position between those of otoliths, with an average OW of M. vanicolensis and P. forsskali. 0.0077±0.0007 g, as well as the widest otoliths and the greatest surface area.

Table 3: Statistical Descriptions of fish weight (FW), fish total length (TL), otolith weight (OW), otolith length (OL), otolith width (OWid), otolith perimeter (OP), and otolith area (OA) of three goatfish (Mulloidichthys flavolineatus, M. vanicolensis and Parupeneus forsskali) from the Red Sea, Egypt.

Species Variables Minimum Maximum Mean SE SD

FW 11.85 453.26 96.64 5.23 64.47 TL 110 334 207.4 2.75 33.92 OW 0.0012 0.0086 0.0039 0.000098 0.00122 OL 2.4 4.76 3.61 0.03 0.37 M. flavolineatus OWid 1.71 3.43 2.42 0.02 0.25 OP 6.55 14.98 10.58 0.12 1.45 OA 3.54 10.28 5.92 0.09 1.12 No. 152 FW 52.8 297.9 190.14 19.69 90.22 TL 174 287 246.91 9.06 41.50 OW 0.0035 0.015 0.008 0.00078 0.0034 OL 3.59 4.87 4.35 0.089 0.40 M. vanicolensis OWid 2.32 3.68 2.99 0.09 0.41 OP 11.03 18 14.49 0.43 1.96 OA 5.97 10.85 8.7493 0.38 1.73 No. 21 FW 32.13 237.37 76.94 3.94 39.75 TL 144 271 185.30 2.63 26.53 OW 0.0013 0.006 0.0025 0.00009 0.0009 OL 2.62 4.11 3.11 0.031 0.31 P. forsskali OWid 1.58 2.84 1.96 0.024 0.24 OP 7.14 12.15 8.94 0.097 0.98 OA 2.83 6.62 4.12 0.08 0.81 No. 102

According to the power equation length, weight, width, area and (otolith variables=aTLb), all perimeter of the otoliths were linearly morphometric measurements of the related to total fish length for the three selected species’ otoliths studied fish species. All regressions exhibited a good relationship with fish were highly significant, and the analysis total length (Table 4). The maximum of otolith morphometric parameters Iranian Journal of Fisheries Sciences 19(2) 2020 820 versus TL indicated that the regression vanicolensis was highly correlated with models explained nearly all of the fish length (r2=0.92) and was most variance that best fit TL. The strongly related to fish length. The coefficients of determination (r2) ranged statistical analysis of otolith from 0.78 to 0.81 in M. flavolineatus, morphometric parameters showed that from 0.83 to 0.92 in M. vanicolensis, the otolithic measurements were good and from 0.78 to 0.85 in P. forsskali, indicators of fish size. being higher for M. vanicolensis in all cases. The otolith area of M.

Table 4: Power relationships between fish length and otolith variables for the three-goatfish species (Mulloidichthys flavolineatus, M. vanicolensis and Parupeneus forsskali) from the Red Sea. OW, otolith weight; TL, total length; OL, otolith length; OWid, otolith width; OA, otolith area; OP, otolith perimeter. Mulloidichthys flavolineatus Mulloidichthys vanicolensis Parupeneus forsskali

OL=0.1501TL0.597 r2= 0.84 OL=0.299TL0.486 r2= 0.85 OL=0.0996TL0.659 r2= 0.85 OWid=0.1058TL0.588 r2= 0.80 OWid=0.058TL0.716 r2= 0.83 OWid=0.0346TL0.773 r2= 0.83 OW=8E-07TL1.602 r2= 0.82 OW=1E-08TL2.431 r2= 0.87 OW=2E-08TL2.237 r2= 0.83 OA=0.0237TL1.033 r2= 0.78 OA=0.0168TL1.134 r2= 0.92 OA=0.00647TL1.236 r2= 0.83 OP=0.1646TL0.780 r2= 0.78 OP=0.8502TL0.717 r2= 0.85 OP=0.2275TL0.703 r2= 0.78

The mean values of the six examined gradually increased as fish otolith shape indices of the three species’ length increased. otoliths are shown in Table 5. They The relationship between otolith were considerably different among the length and the aspect ratio was close to three species. The highest mean values 1; thus, the otoliths of the three species of AS, CO, and RO were recorded in were elongated (Fig. 3). Roundness and M. vanicolensis. The lowest mean ellipticity values increased as otolith values of AS and RO were observed in length increased. Form factor and P. forsskali, whereas the lowest value rectangularity values decreased as of COM was recorded in M. otolith length decreased. Pearson’s flavolineatus. The highest FF was correlation coefficients between the recorded in M. flavolineatus and the examined shape factors and maximal lowest in M. vanicolensis. All three otolith length are presented in Table 6, species exhibited nearly the same value and all of the variables for M. of RE. The highest ellipticity was flavolineatus were significantly observed in P. forsskali (Table 5). The correlated with otolith length. Nearly all Kolmogorov-Smirnov Z test confirmed of the variables for M. vanicolensis a normal distribution (p>0.05; non- were also significantly correlated with significant) for nearly all measurements otolith length (excluding AS and EL). (excluding the weight and width of the For M. vanicolensis, only FF and RO otoliths of M. flavilineatus). According were significantly correlated with to the present results, the aspect ratio otolith length.

821 Osman et al., Use of otolithic morphometrics and ultrastructure for…

Table 5: Minimum, maximum, mean, standard error, and standard deviation of otoliths six shape descriptors (AS, CO, FF, RO, RE, and EL) of three goatfish species (Mulloidichthys flavolineatus, M. vanicolensis and Parupeneus forsskali) from the Red Sea, Egypt.

Variables N Minimum Maximum Mean SE SD

AS 152 0.58 0.77 0.67 0.003 0.036 CO 152 11.71 26.04 19.03 0.212 2.616 FF 152 0.48 1.07 0.67 0.008 0.100 M. flavolineatus RO 152 25.98 286.78 103.09 3.612 44.53 RE 152 0.57 0.90 0.67 0.004 0.049 EL 152 0.13 0.26 0.19 0.002 0.025 AS 21 0.62 0.80 0.69 0.011 0.049 CO 21 20.38 32.58 24.18 0.579 2.653 FF 21 0.39 0.62 0.52 0.011 0.052 M. vanicolensis RO 21 98.03 314.34 219.27 16.715 76.599 RE 21 0.60 0.72 0.67 0.006 0.029 EL 21 0.11 0.24 0.19 0.007 0.034 AS 102 0.56 0.72 0.63 0.003 0.035 CO 102 16.13 24.36 19.52 0.169 1.704 FF 102 0.52 0.78 0.65 0.006 0.057 P. forsskali RO 102 27.41 142.39 53.31 2.240 22.57 RE 102 0.57 0.79 0.68 0.0042 0.042 EL 102 0.16 0.28 0.23 0.003 0.026

Figure 3: Relationship between the shape indices (Aspect ratio, Compactness, Form factor, Rectangularity, Roundness and Ellipticity) of the left otolith and otolith length in three goatfish species (Mulloidichthys flavolineatus, M. vanicolensis and Parupeneus forsskali) from the Red Sea, Egypt.

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Table 6: Correlation coefficients of Pearson r between shape indices of the left otoliths and otoliths length in the three-goatfish species (Mulloidichthys flavolineatus, M. vanicolensis and Parupeneus forsskali) from the Red Sea, Egypt. Index M. flavolineatus M. vaniclensis M. forsskali

r p r p r p AS -0.243** 0.003 0.401 0.072 0.098 0.325 Com 0.513** 0.000 0.433 0.50 0.239* 0.015 FF -0.517** 0.000 -0.480* 0.028 -0.235* 0.018 RO 0.953** 0.000 0.992** 0.000 0.972** 0.000 RE -0.402** 0.000 -0.302 0.183 -0.432** 0.000 ELL 0.242** 0.003 -0.409 0.066 -0.095 0.341 Correlation is significant at the 0.01 level

Scanning electron microscopy analysis lobed and sinuate (Fig. 4 A). In the case of goatfish otolithic topography of M. vanicolensis and P. forsskali, the Images of the otoliths of the three dorsal rim was sinuate and emarginated examined goatfish species are presented (Fig. 4 B, C). The ventral rim of M. in Fig. 4. The sagittal shape was oval in flavolineatus presented regular lobes M. flavolineatus (Fig. 4 A and D) and only in the most dorsal portion (Fig. 4 oblong in M. vanicolensis (Fig. 4 B and A). In M. vanicolensis, the ventral lobes E) and P. forsskali (Fig. 4 C and F). occurred in a single row and were The rostrum occupied the anterior irregular, extending from the rostrum margin in all three goatfish species. The anteriorly to the post rostrum posterior margin was rounded in all posteriorly (Fig. 4 B). P. forsskali three species but was also equipped exhibited many rows of irregular dense with projections in M. vanicolensis and lobes covering the entire ventral rim, P. forsskali (Fig. 4 B, C). All three extending from the rostrum anteriorly sagittae exhibited scalloped or to the post rostrum posteriorly (Fig. 4 crenulated ventral and dorsal rims. The C). dorsal rim of M. flavolineatus was

Figure 4: The scanning electron microscope photographs of the proximal (A, B, C) and distal (D, E, F) views of the left sagitta of the three-goatfish species from the Red Sea (A and D, Mulloidichthys flavolineatus; B and E, Mulloidichthys vanicolensis; C and F, Parupeneus forsskali). 823 Osman et al., Use of otolithic morphometrics and ultrastructure for… The rostrum of M. flavolineatus was vanicolensis and P. forsskali were short elongated, free of projections and did and shallow, with curved crista superior not exhibit an antirostrum (Fig. 4 A). In and inferior (Fig. 4 B, C). contrast, the rostra of M. vanicolensis and P. forsskali were nearly round, with Ornamentations in the sulcus acusticus projections and antirostra (Fig. 4 B, C). At low magnification, the surface The proximal face of the sagittal otolith appeared relatively smooth, but a displayed a sulcus acusticus lined remarkable difference was observed in dorsally by crista superior and ventrally the ornamentation of the ostium, cauda, by crista inferior. The sulcus was ostial and column. and homosulcoid. The crista superior of M. flavolineatus (Fig. 4 A) was smooth, Ostium while those of M. vanicolensis and P. Two types of ornamentation were forsskali were rough and crushed (Fig. observed on the ostium of each species 4 B, C). The sagittae presented (Fig. 5). Fine ornamentation was depressions in dorsal and ventral areas, observed toward the edge of the ostium, oriented parallel to the sulcus acusticus. transforming into a more coarsely These depressions were shallow in the textured surface or a coarser crystalline case of M. flavolineatus (Fig. 4 A) and structure toward the inner side of the deep in the case of M. vanicolensis and ostium in both M. flavolineatus and M. P. forsskali (Fig. 4 B, C). The sulcus vanicolensis (Fig. 5 A, B). In contrast, acusticus was open anteriorly and the ostium of P. forsskali exhibited posteriorly and divided into the ostium, nearly the same textured surface on the column, and cauda (Fig. 4). The ostium edge and the inner side (Fig. 5 C). In M. was provided with a pore in all three flavolineatus, the ornamentation of the species. This pore was small and edge of the ostium had a sand-like rounded in M. flavolineatus (Fig. 4 A), appearance, whereas that of the inner while it was large and irregularly side of the ostium had the appearance shaped in both M. vanicolensis and P. of rectangular stones (Fig. 5 A). The forsskali (Fig. 4 B, C). The cauda of M. ostium of M. vanicolensis presented flavolineatus was funnel shaped (Fig. 4 fine sandy ornamentation externally and A), while those of both M. vanicolensis projecting lath-like crystals internally and P. forsskali were crucible shaped (Fig. 5 B). Both the outer and the inner and were initially straight, with curves projections of P. forsskali consisted of then occurring distally toward the inner projecting lath-like crystals (Fig. 5 C). rim (Fig. 4 B, C). The column of the M. flavolineatus was long, narrow, and deep, with straight crista superior and inferior (Fig. 4 A). The columns of M.

Iranian Journal of Fisheries Sciences 19(2) 2020 824

Figure 5: Scanning electron microscope photographs of the ostium of the left sagitta of the three- goatfish species from the Red Sea (A, Mulloidichthys flavolineatus; B, Mulloidichthys vanicolensis; C, Parupeneus forsskali).

Cauda vanicolensis and P. forsskali presented Each species exhibited only one type of the same type of ornamentation, in the ornamentation on the cauda. M. form of projecting lath-like crystals flavolineatus presented sand-like (Fig. 6 B, C). crystals (Fig. 6 A), whereas M.

Figure 6: Scanning Electron Microscope photographs of the cauda of the left sagitta of the three- goatfish species from the Red Sea (A, Mulloidichthys flavolineatus; B, Mulloidichthys vanicolensis; C, Parupeneus forsskali).

825 Osman et al., Use of otolithic morphometrics and ultrastructure for… Column of M. vanicolensis took the form of The column’s ornamentation differed irregular projections. In the case of P. among the three species. The forsskali, the ornamentation was ornamentation of M. flavolineatus rectangular and stone-like (Fig. 7 B, C). consisted of triangular and square stone-like shapes (Fig. 7 A), while that

Figure 7: Scanning Electron Microscope photographs of the column of the left sagitta of the three- goatfish species from the Red Sea (A, Mulloidichthys flavolineatus; B, Mulloidichthys vanicolensis; C, Parupeneus forsskali).

Discussion fisheries and biology of goatfishes in Goatfish represent one of the most Egypt (Golani and Ritte, 1999; Sabrah, economically important fisheries in 2015), with little attention being paid to Egyptian waters, in both the Red and goatfish and phylogeny. Mediterranean Seas. The fishery status Recently, stock identification was of any fish stock needs to be identified achieved using otolithic features correctly. The fishes of this group (Lombarte et al., 2006; Mehanna et al., (family Mullidae) are teleost fishes 2016). The morphological comprising many species with some characteristics of fish otoliths are the shape similarities; hence, these species most widely used tools in species need to be clearly distinguished. Many identification and comparative studies have been performed on the taxonomy of fishes, due to the large Iranian Journal of Fisheries Sciences 19(2) 2020 826 size and inter-specific variability of fish Harvey et al., (2000) observed no otoliths (Battaglia et al., 2010). To our significant differences between the left knowledge, the otolithic shape indices and right otoliths of 63 species and measurements of goatfish from the collected from the Eastern North Red Sea have never been estimated Pacific Ocean. However, significant previously. Thus, the present study may differences between left and right be the first to discriminate goatfish otolithic variables have been recorded species using otolithic indices and their for some other fish species (Mérigot et relationships with fish size and otolithic al., 2007). ultrastructure. In the present work, The relationships between the sagittal otoliths were employed because morphological parameters (length, they are easily accessible structures and width, perimeter, and area) of the exhibit a distinctive degree of otoliths and fish body length (TL) in the interspecific variation in their form, three goatfish species were examined weight, and growth, in accordance with using power equations. The Nolf (1985). The morphometric relationships between otolith size and parameters of sagittal otoliths have body length (TL) have also been been used in earlier studies to identify estimated in other fish species using species in other groups of teleosts linear functions (Morat et al., 2008; (Tuset et al., 2003a; Ponton, 2006; Pavlov, 2016). Previous studies have Short et al., 2006; Tuset et al., 2006). focused mainly on the relationship Our results suggest that differences in between otolith length and width and otolithic measurements are detectable in fish size (Jawad et al., 2011b). This all three goatfish species examined in work supplies additional information this work. (compared with other similar studies) The results of the present study by considering six otolithic indicate no significant morphometric measurements (OL, OH, OW, Owid, differences between the measurements OA, and OP). Thus, our findings are of the left and right otoliths. more reliable than those relying on a Remarkable differences are not usually single equation, since the tip of the observed between left and right otolithic rostrum may be damaged, otolithic measurements (for many fish making it impossible to measure OL or species) (Morat et al., 2008; Jawad et OW (Jawad et al., 2011b; Yilmaz et al., al., 2011a; Yilmaz et al., 2014). This 2014), influencing the reliability of finding is in agreement with previous subsequent calculations. Presenting the work (Valinassab et al., 2012) on six models (OL=aTLb, OH=aTLb, clupeids from the Persian Gulf. In OW=aTLb, OWid=aTLb, OA=aTLb and another previous study, Hunt, (1992) OP=aTLb) for each species helps studied eight species from the Atlantic mitigate this potential problem. It is Ocean, and no statistically significant appropriate to use the functions difference was observed between the indicated in this paper within the range left and right otoliths. Similarly, of the fish size examined in the present 827 Osman et al., Use of otolithic morphometrics and ultrastructure for… work. Further studies on fish size– length and different otolithic variables otolithic variable relationships was statistically significant (0.005), involving a larger sample size, a wider with otolith length showing the highest range of fish lengths, and different (0.70–0.96) Pearson’s correlation growth phases would help to support coefficient, compromised by high the results presented herein. The ability variability in otolith shape. This result to identify species using morphometric could be explained by the fact that parameters of the sagittal otoliths has otolithic length is most sensitive to been demonstrated for other groups of variations in the growth rate and most teleosts (Tuset et al., 2003b; Ponton, closely related to changes in fish 2006; Short et al., 2006; Tuset et al., metabolism (Pawson, 1990; Flecher, 2006). The differences in the 1991). In the present study, the morphological characteristics of the coefficient of determination (r2) ranged sagittal otoliths among these species from 0.83 to 0.92 in the three species, may be associated with the size of the being higher for M. vanicolensis in all skull (Bani et al., 2013). Although cases. The otolith area of M. relative skull size was not recorded in vanicolensis was most strongly related this study, personal observations to fish length, with a high correlation indicate that the greatest head length between otolith area and fish length belongs to M. vanicolensis, which also (r2=0.92) being observed in this species. exhibits the longest otoliths. Concerning findings of this study, the The power relationship between observed variability of otolithic shape sagittal otoliths and fish length was encourages further research to verify described by a negative allometric the potential role of otolithic relationship (b less than 3). Similar morphometric measurements in fish results were previously recorded in the identification. freckled goatfish, Upeneus tragula Otolithic shape indices such as the (Pavlov et al., 2015). Several studies aspect ratio, compactness or circularity, have shown a linear relationship form factor, roundness, rectangularity, between body length and otolith length and ellipticity have been described for in fish (Harvey et al., 2000; Fossen et many fish species (Tuset et al., 2003b; al., 2003; Lychakov et al., 2006). Pavlov et al., 2015), confirming Linear otolith growth might represent a identifications performed based on common relationship between otoliths morphometric variables (Tuset et al., and body length in the juvenile stage 2003a; Tuset et al., 2003b). The results (Huang and Chiu, 1997). In the present of this study show that the shape study, power equations were applied indices differ significantly in the and showed a strong correlation analyzed species. Such differences were between the variables, indicating that observed for almost all shape indices of otolithic measurements are good the three selected goatfish species. indicators of fish size. For all three Among the shape indices, AS, COM, species, the correlation between fish RO, and FF were found to be more Iranian Journal of Fisheries Sciences 19(2) 2020 828 efficient than other factors in the value being found in M. vanicolensis studied species. The highest mean and the highest in P. forsskali. values of AS, COM, and RO were According to the Kolmogorov-Smirnov recorded in M. vanicolensis, whereas Z test, a normal distribution was the lowest mean values of AS and RO confirmed for nearly all otolithic were found in P. forsskali, although the measurements in each of the examined lowest value of COM was recorded in fish species. Otolith area, perimeter and M. flavolineatus. Thus, in the last shape indices have been suggested as an species, otolith shape is more circular easier means of discriminating stocks than in M. vanicolensis, indicating that compared with other methods (Bolles all of the otoliths of M. flavolineatus and Begg, 2000; Tuset et al., 2003b). grow equally in terms of length and The aspect ratio and ellipticity were height. The numeric values of the FF found to be directionally proportional to show that the sagittal shape is otolith length, while the form factor geometrically irregular in the three (FF) and roundness (RD) were species. The highest FF was recorded in inversely proportional to this parameter. M. flavolineatus, and the lowest was Such similarity in otolithic shape observed in M. vanicolensis, which indices may be derived from the fact presented the most irregular otolithic that all of the studied goatfish species shape among the studied species. The occupy the same ecological niche. Fish shape analyses revealed rectangular occupying the same ecological niche shapes in the otoliths of the three show similarities in otolithic shape species, as they exhibited nearly the variables (Parmentier et al., 2001). The same correlation between shape factors and Although the study of shape indices otolith length were strong and is complicated, it can confirm significant in the case of M. identifications made based on the flavolineatus. Excluding AS and El, all differentiation of fish species (Špiranec other shape factors in M. forsskali and Banek Zorica, 2010; Sadighzadeh exhibited significant correlations with et al., 2012) and populations (Mérigot otolith length. In the case of M. et al., 2007; Duarte-Neto et al., 2008; vanicolensis, only FF and RO were Pavlov, 2016). In the present study, the significantly correlated with otolith shape indices were compromised by length. The results of this study show their high differentiation; thus, the that the shape indices significantly results of this study will be useful for differ from species to species, although other researchers in verifying the role of they indicate a similar otolithic pattern. the otolith in fish identification and These results correlate with those of classification. These findings were Tuset et al. (2008), who postulated that supported by previous results (Tuset et otoliths are the most widely employed al., 2008). The obtained ellipticity tool for the discrimination of fish values show that the shape of the three species because of their form, weight, species is irregular, with the lowest 829 Osman et al., Use of otolithic morphometrics and ultrastructure for… growth, consistency and chemical and fish size in some mesopelagic composition. and bathypelagic species from the The current study also compared Mediterranean Sea (Strait of otolithic shape among the three goatfish Messina, Italy). Scientia Marina, species using SEM, to observe 74(3), 605-612. variations in otolithic morphology. In Bolles, K.L. and Begg, G.A., 2000. M. flavolineatus, the sagittal shape was Distinction between silver hake found to be oval, with regular lobes on (Merluccius bilinearis) stocks in US the dorsal and ventral rims. In M. waters of the northwest Atlantic vanicolensis and P. forsskali, the based on whole otolith otolithic shape is oblong, with morphometrics. Fishery Bulletin, irregularly lobed dorsal and ventral 98(3), 451-451. rims. Remarkable variations in the Campana, S.E., 2004. Photographic morphological characteristics of fish atlas of fish otoliths of the Northwest otoliths were recorded between the Atlantic Ocean Canadian special studied species, including variations in publication of fisheries and aquatic the rostrum, sulcus acusticus, ostium, sciences No. 133. NRC Research column and cauda. Remarkable Press, 284P. variations were also recorded in the Campana, S.E. and Casselman, J.M., ornamentation of the ostium, cauda, and 1993. Stock discrimination using column. These differences in otolithic otolith shape analysis. Canadian characteristics might be important to Journal of Fisheries and Aquatic fishery biologists, archaeologists and Sciences., 50(5), 1062. geologists, who can use them to Duarte-Neto, P., Lessa, R., Stosic, B. distinguish M. flavolineatus, M. and Morize, E., 2008. The use of vanicolensis and P. forsskali. This work sagittal otoliths in discriminating contributes to the bioecological stocks of common dolphinfish knowledge regarding commercially (Coryphaena hippurus) off important fishes and provides key northeastern Brazil using multishape information for studying the trophic descriptors. ICES Journal of Marine ecology of fish-eating species and Science: Journal du Conseil, 65(7), fisheries management. 1144-1152. Flecher, W., 1991. A test of the References relationship between otolith weight Bani, A., Poursaeid, S. and Tuset, V. and age for the pilchard Sardinops M., 2013. Comparative morphology neopilchardus. Canadian Journal of of the sagittal otolith in three species Fisheries and Aquatic Sciences, of south Caspian gobies. Journal of 48(1), 35-38. Fish Biology, 82(4), 1321-1332. Fossen, I., Albert, O.T. and Nilssen, Battaglia, P., Malara, D., Romeo, T. E.M., 2003. Improving the precision and Andaloro, F., 2010. of ageing assessments for long rough Relationships between otolith size dab by using digitised pictures and Iranian Journal of Fisheries Sciences 19(2) 2020 830

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