Characteristics of Sagitta Morphology in Seven Goby Species from the Caspian Sea (Gobiiformes: Gobiidae)

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ORIGINAL ARTICLE

Characteristics of sagitta morphology in seven goby species from the Caspian Sea (Gobiiformes: Gobiidae)

Parisa DAVOODI

Department of Animal Science, Abstract Faculty of Science, University of In this study the sagitta morphology of seven species of Gobiidae including three species of the Mazandaran, Babolsar, Iran genera Neogobius and four Ponticola, inhabiting the southern part of the Caspian Sea was Correspondence described and analysed. The goal was to investigate the usefulness and efficiency of sagitta for [email protected] species identification. The morphological analysis was based on four shape indices, roundness (4S/πOL2), rectangularity (S/OL×OW), ellipticity (OL-OW/OL+OW), and aspect ratio Article history : Received 28 August 2020 (OL/OW). The results indicated that otolith morphology and morphometry can be used as Accepted 22 July 2021 diagnostic characters distinguishing the Caspian Sea gobies. While the overall shape of otoliths Online 2sss5 September 2021 differentiated the genera, the differences in the shape of the dorsal rim and development of posterior dorsal and anterior ventral lobes were particularly important for inter-specific distinction. The discriminant analysis could separate the specimens of genus Ponticola and Neogobius with 61% and 82% classification successes, respectively, proves that otolith shape indices were a more efficient tool for discrimination of otoliths in Neogobius than Ponticola species. Cluster analysis based on otoliths shape indices showed two main clusters, one cluster contains Neogobius species with higher roundness values, possessed more rounded shaped otoliths and the other includes Ponticola species with spindle and elongated shaped otoliths, except P. bathybius, recognized P. bathybius and Neogobius melanostomus as more similar taxa. These results were consistent with the phylogeny of these species proposed by recent molecular studies and indicated that otolith characteristics are partly genetically encoded and therefore have phylogenetic signals. The current research revealed a high degree of morphological variability of otolith shape in different species and confirmed its usefulness in Gobiidae taxonomy and phylogeny. Keywords: Taxonomy, Otolith, Neogobius, Ponticola.

INTRODUCTION food resources such as Clupeonella sp. and the Gobiidae is one of the most species-rich fish family considerable competitors for other species (Corkum et both in the Caspian Sea and among marine fishes more al. 2004). generally, with numbers of described gobies species The genus Neogobius Iljin, 1927 with three constantly increasing (Vasil'eva et al. 2016; Kovačić species, Caspian goby, N. caspius (Eichwald, 1831), et al. 2017, 2021; Esmaeili et al. 2017, 2018; Eagderi round goby, N. melanostomus affinis (Eichwald, et al. 2020; Zarei et al. 2021). Gobies play an 1831) and Caspian sand goby, N. pallasi (Berg, 1916), important ecological role in coastal ecosystems and the genus Ponticola Iljin, 1927 with seven species, because of their diversity and abundance (Zander Caspian bighead goby, P. gorlap (Iljin, 1949), syrman 2011), but their significance is often underestimated goby, P. syrman (Nordmann, 1840), ratan goby, because of their small size and low commercial P. ratan (Nordmann, 1840), Kura goby, Ponticola importance, making them easy to overlook (Patzner et cyrius (Kessler, 1874), P. bathybius (Kessler, 1877), al. 2011). The Gobies exhibit the main role in the Iranian goby, P. iranicus Vasil'eva, Mousavi-Sabet & general production of the Caspian Sea due to their Vasil'ev, 2015 and P. patimari Eagderi, Nikmehr & species diversity and unexploited stocks. In addition, Poorbagher, 2020 among other species of gobiidae, they are the main food sources for important species constitute the most percent caught of gobies in this such as sturgeon fish (Acipenseridae) and Caspian region. seal (Pusa caspica), known as the great consumers of The inner ear of bony fishes is involved in hearing,

Journal homepage: ijichthyol.org DOI: https://doi.org/10.22034/iji.v8i3.538 Iran. J. Ichthyol. (2021) 8(3): 170-180 171

Table 1. Details of samples studied. N: number of examined specimens. Total lengths (TL) are given as means±standard deviation. Species N TL (mm) Ponticola patimari 20 91.40±5.60 Ponticola gorlap 30 105.41±18.98 Ponticola syrman 15 214.60±15.10 Ponticola bathybius 21 257.66±29.40 Neogobius pallasi 196 101.60±10.24 Neogobius caspius 70 98.24±9.32 Neogobius melanostomus 50 102.04±18.46

to compare the otolith morphology of more species to determine discriminating characters of otolith in each species of this family. The aims of this study were (i) to compare otolith morphology of three and four species of the genera Neogobius and Ponticola, respectively, and (ii) to analyse the ability of otolith shape to classify and distinguish the studied fish at genus and species level.

MATERIALS AND METHODS Sample collection: In total 402 specimens belong to Fig.1. Light micrograph of right otolith of Ponticola patimari and its features. Scale bar = 1mm. seven species were collected and examined from the mechanoreception, and equilibrium (Popper et al. south Caspian Sea (Table 1). Fish species were 2005). The saccular otolith (sagitta), one of three measured for total and standard lengths (TL in mm) paired otoliths found within the inner ear, displays and recognized based on morphological and meristic high interspecific variation in shape and size, making characteristics following Berg (1949) and Eagderi et it useful for taxonomy and phylogeny of various al. (2020). marine teleost groups (Lombarte & Castellon 1991; Otolith preparation and imaging: The right and left Tuset et al. 2003; Campana 2004; Ghanbarifardi et al. otoliths were removed from specimens. Otoliths were 2020; Purrafee Dizaj et al. 2020; Sadeghi et al. 2020). cleaned in distilled water, remaining tissue eliminated Studies of gobiid otoliths and their use for species with 1% potassium hydroxide solution for 6h, and identification are also valuable given the significant eventually washed with distilled water. The extracted role of gobies in coastal food webs (Bell & Harmelin- otoliths were stored dry in plastic tubes. A Vivien 1983; Kovačić & La Mesa 2008). Otoliths stereomicroscope fitted with a camera was used to morphology has been used to identify various prey capture the otolith images. Each otolith was species and their size classes in the diets of predators systematically placed with the sulcus acousticus based on recovered undigested otoliths from fecal facing up and anterior margin to the left (Fig. 1). The samples (Kovačić & La Mesa 2008). Concerning magnification was adjusted depending on the size of otolith morphology, Bani et al. (2013) and Davoodi & the otoliths. Rahimian (2016) proved that differences in otolith Otolith morphology and shape indices: To describe shape among studied Caspian gobies reflect the strong the shapes of otolith outlines and other morphological taxonomic distinctiveness of otolith in these species. features, we followed the terminology introduced by However, to confidently use otolith morphology to Tuset et al. (2008) and Schwarzhans (2014). To discriminate between Caspian goby species, we need analyse shape indices using the image of each otolith, 172 Davoodi/ Characteristics of sagitta morphology in seven goby species

Table 2. Shape description and shape indices of the studied fishes from the Caspian Sea, OL (otolith length), OW (otolith width), S (otolith surface).

Shape description Shape index Roundness 4S/πOL2 Rectangularity S/(OL×OW) Ellipticity (OL-OW)/(OL+OW) Aspect ratio OL/OW three parameters related to the otolith dimension were statistical analyses were carried out using SPSS 21.00. measured, which consisted of otolith length (OL), otolith width (OW), and otolith surface (S) (Tuset et RESULTS al. 2003). All measurements were taken using tpsDig2 Otolith morphology: The morphological software (Rohlf 2006). Four dimensionless shape characteristics of otolith for each species were indices were calculated (Ponton 2006): Roundness, described (Figs. 2-3). Ponticola patimari: Shape: Ellipticity, Aspect ratio, and Rectangularity (Table 2). Rectangular, with one anterior ventral and one Roundness is the ratio between the actual area and the posterior dorsal projection, dorsal and ventral margin area of a circle of the same length. This factor is larger flat. Anterior region: flat, with a pointed ventral when the shape of the otolith is more circular. projection and orthogonal predorsal angle. Posterior Ellipticity indicates the proportional change of the region: flattened, with long pointed dorsal projection short and long axes from zero (a perfectly round and orthogonal postventral angle (Fig. 2A-B). shape) to close to one (a spindle shape). The aspect Ponticola syrman: Shape: Rectangular, with one ratio is the ratio between the otolith length and otolith anterior ventral and one posterior dorsal projection, width and expresses the shape tendency of the otolith; dorsal margin with broad indentation, ventral margin the more elongated the otolith of the fish, the larger flat or slightly round and dentate. Anterior region: the aspect ratio is. Rectangularity describes the oblique, with round and large projection in the ventral variations of length and width for the area with one half. Posterior region: with a median notch separating being a perfect square and smaller than one being a a round ventral lobe, and a long, prominent pointed or nonsquare. round dorsal lobe that bent outward (Fig. 2C-D). All otolith shape indices were first examined for Ponticola bathybius: Shape: Approximately square to normality with Kolmogorov–Smirnov (K-S) test, slightly rectangular and robust, dorsal margin with P≤0.05. If this criterion was satisfied, analysis of broad and deep indentation, ventral margin nearly flat. variance (ANOVA) and t-test, P≤0.05, were used to Anterior region: Almost oblique and smooth. determine the statistically significant difference of Posterior region: with a median notch separating a shape indices among otoliths of studied fish. round ventral lobe, and a large and round dorsal lobe Discriminant function analysis (DFA) was carried out that bent outward (Fig. 2E-F). to determine the proportion of otoliths that could be Ponticola gorlap: Shape: Rectangular, with one correctly assigned to their corresponding fish species. anterior ventral and one posterior dorsal projection, The classification success was tested by jackknifed dorsal and ventral margin flat or slightly round and cross-validation. Additionally, a dendrogram was dentate. Anterior region: Almost oblique with pointed constructed based on the Euclidean distance as a ventral projection. Posterior region: with a median measure of dissimilarity. The between-groups linkage notch that separates round ventral lobe from sharply method based on the otolith shape indices was used as pointed dorsal lobe that bent outward (Fig. 2G-H). the clustering algorithm to show the phenotypic Neogobius pallasi: Shape: Discoidal, dorsal margin relationships among the studied species. The slightly rounder than the ventral. Anterior region: Iran. J. Ichthyol. (2021) 8(3): 170-180 173

Table 3. Values of otolith variables (mean±standard deviations) of the studied fish species. OL (otolith length), OW (otolith width), S (otolith surface).

Species OL OW S Ponticola patimari 5.67±0.87 3.49±0.44 14.81±4.16 Ponticola gorlap 7.99±2.00 4.94±1.03 32.41±18.85 Ponticola syrman 13.91±1.20 8.7±0.95 73.32±20.79 Ponticola bathybius 13.44±1.40 11.16±1.45 97.09±13.42 Neogobius pallasi 3.20±0.30 2.98±0.25 7.58±1.20 Neogobius caspius 3.39±0.26 2.93±0.22 10.45±0.84 Neogobius melanostomus 3.00±0.45 2.60±0.28 5.82±1.24

that bent outward (Fig. 3C-D). Neogobius melanostomus: Shape: Rhomboidal, with one anterior ventral and one posterior dorsal projection, dorsal and ventral margin round. Anterior region: oblique or flat. Posterior region: with two lobes separated by a median shallow notch and posterior dorsal projections that bent outward (Fig. 3E-F). Analysis of shapes indices: In the present study, four shape indices were calculated and analysed, in studied gobies specimens (Table 3, 4). There was no significant difference between right and left otoliths values (t-test, p≥0.05), so samples from both sides were pooled together. In studied gobies species, roundness index ranged from 0.47±0.03 in P. syrman to 0.93±0.07 in N. pallasi (Table 4). The roundness values were significantly lower in Ponticola (0.61±0.08) than Neogobius (0.99±0.16). All species, except P. patimari and P. gorlap, were significantly different (ANOVA, post hoc test, P≤0.05) based on roundness index (Table 4). In examined species, ellipticity values ranged from 0.04±0.03 in N. pallasi to 0.23±0.04 in P. gorlap Fig.2. Light microscopic photography of right otolith of the species of Ponticola. Ponticola patimari (A-B), P. syrman (Table 4). Ellipticity was significantly lower in (C-D), P.bathybius (E-F), P. gorlap (G-H). Scale P. patimari (0.23±0.03), P. gorlap (0.23±0.04), and bar=0.5mm. P. syrman (0.23±0.02) concerning P. bathybius round to flat. Posterior region: with two lobes (0.1±0.05), N. pallasi (0.04±0.03), N. caspius separated by a median shallow notch (Fig. 3A-B). (0.07±0.02), and N. melanostomus (0.06±0.04). So, in Neogobius caspius: Shape: Square and thin, with one the three mentioned Ponticola species, otolith with a anterior ventral and one posterior dorsal projection, higher ellipticity value, were more spindle than dorsal and ventral margin round. Anterior region: flat Neogobius species. There were no significant with a ventral projection that bent outward. Posterior differences in ellipticity values of P. bathybius otoliths region: with two lobes separated by a median shallow with those of N. caspius and N. melanostomus. notch and conspicuous posterior dorsal projections In studied species, the aspect ratio ranged from 174 Davoodi/ Characteristics of sagitta morphology in seven goby species

Ponticola species, the classification success was 61% (jackknifed). While all P. syrman otoliths were classified into its group, individual of P. patimari and P. gorlap were classified to each other group up to 50 and 40% (jackknifed), respectively. The classification success in P. bathybius was 82%. Among Neogobius species, the classification success was 82%. The classification successes were 85% in N. pallasi, 96% in N. caspius, and 60% in N. melanostomus. In examined gobies species, otolith of P. syrman and N. caspius significantly different from the other species and showed the lowest (0.59±0.05) and highest (0.98±0.05) values of rectangularity index, respectively (Table 4). No remaining species were distinguished based on this index. The Discriminant Function Analysis (DFA) based on four shape indices indicated 94.4% (jackknifed) correct classification at the genus level. Among Ponticola species, the classification success was 61% (jackknifed). While all P. syrman otoliths were classified into its group, individual of P. patimari and Fig.3. Light microscopic photography of right otolith for the P. gorlap were classified to each other group up to studied species of Neogobius. Neogobius pallasi (A-B), 50% and 40% (jackknifed) respectively. The N. caspius (C-D), N. melanostomus (E-F). Scale bar=1mm. classification success in P. bathybius was 82%. 1.07±0.05 in N. pallasi to 1.62±0.1 in P. patimari Among Neogobius species, the classification success (Table 4). It was significantly lower in P. patimari was 82%. The classification successes were 85% in N. (1.62±0.1), P. gorlap (1.61±0.11), and P. syrman pallasi, 96% in N. caspius, and 60% in (1.61±0.04) for P. bathybius (1.21±0.1), N. pallasi N. melanostomus. (1.07±0.05), N. caspius (1.10±0.07), and The cluster analysis based on otolith shape indices N. melanostomus (1.15±0.11). So, all Ponticola showed that there were two main clusters, one cluster species except P. bathybius with a higher aspect ratio included all species of the genus Neogobius with a were more elongated than Neogobius species. There more rounded shape and the other cluster contained all were no significant differences in aspect ratio values species of genus Ponticola except P. bathybius with a of P. bathybius with those of N. caspius and more elongated shape (Fig. 4). Individuals of N. melanostomus. P. bathybius mostly clustered with N. melanostomus In examined gobies species, otolith of P. syrman individuals. Cluster analysis recognized N. pallasi and and N. caspius significantly different from the other N. melanostomus as more similar taxa based on otolith species and showed the lowest (0.59±0.05) and indices. In addition, P. gorlap and P. patimari were highest (0.98±0.05) values of rectangularity index, identified as more similar taxa while P. syrman was respectively (Table 4). No remaining species were recognized as a separate clade. distinguished based on this index. The Discriminant Function Analysis (DFA) based DISCUSSIONS on four shape indices indicated 94.4% (jackknifed) The morphology of the sagitta has been generally correct classification at the genus level. Among considered species-specific. Since over 130 years, Iran. J. Ichthyol. (2021) 8(3): 170-180 175

Table 4. Sagitta shape indices (mean±standard deviations) of the studied fish species.

Species Roundness Ellipticity Aspect ratio Rectangularity Ponticola patimari 0.59±0.05 0.23±0.03 1.62±0.10 0.72±0.04 Ponticola gorlap 0.60±0.06 0.23±0.04 1.61±0.11 0.74±0.05 Ponticola syrman 0.47±0.03 0.23±0.02 1.61±0.04 0.59±0.05 Ponticola bathybius 0.73±0.09 0.1±0.05 1.21±0.1 0.69±0.04 Neogobius pallasi 0.93±0.07 0.04±0.03 1.07±0.05 0.75±0.03 Neogobius caspius 0.80±0.09 0.07±0.02 1.10±0.07 0.98±0.05 Neogobius melanostomus 0.84±0.10 0.06±0.04 1.15±0.11 0.70±0.06

Fig.4. Average linkage dendrogram (based on Euclidean distance) showing the phenotypic relations between otoliths of the studied species. The dendrogram is based on four otolith shape indices. differences in otolith shape have been used to identify gobies, Yu et al. 2014 in five goby species from teleost species (Nolf 1985; Campana 2004; Tuset et al. northern China, Lombarte et al. (2018) and Gut et al. 2008; Sanjarani Vahed 2018, 2019; Ghanbarifardi et (2020) in Mediterranean goby species. Our analyses al. 2020; Purrafee Dizaj et al. 2020; Sadeghi et al. extend the application of otolith morphology in the 2020). The results of the present study showed that identification of the Caspian Sea gobies. otolith morphology is an efficient tool for The main differences among studied the Caspian identification of the Caspian Sea goby genera and Sea gobies were related to general morphology of the species. High species-specificity of the sagitta in otolith, the development and shape of posterior dorsal gobiidae had also considered in previous work; Lord and anterior ventral projections, present or absence of et al. (2012) in three sym¬patric species of dorsal margins depression and anterior and posterior Sicyopterus, Bani et al. (2013) in three Caspian margin shape. The similar differences noted among 176 Davoodi/ Characteristics of sagitta morphology in seven goby species species in Mediterranean goby lineages (Lombarte et 2014; Lombarte et al. 2018; Teimori et al. 2019; Zarei al. 2018; Gut et al. 2020), may be due to et al. 2021), and therefore have phylogenetic signals. phylogenetically relationship of Mediterranean goby In case of genus Neogobius, the cluster analysis of as sister taxon of Ponto-Caspian goby lineages otolith shape indices are in consistent with (Thacker & Roje 2011). phylogenetic classification of these species based on Although studying shape indices is complicated, it molecular studies (Medvedev et al. 2013). In case of confirms identifications made by general morphology genus Ponticola, P. syrman separated from P. patimari (Tuset et al. 2003). The calculated shape indices and P. gorlap and P. bathybius was clearly separated revealed that the values of ellipticity and aspect ratio from all others, clustered with N. melanostomus. Zarei were larger for the members of genus Ponticola than et al. (2021) based on mitochondrial-based phylogeny Neogobius species. This indicated that Ponticola demonstrated that, P. bathybius should be reassigned species have spindle and elongated shaped otoliths, from genus Ponticola (Neilson & Stepien 2009) to the while those species in genus Neogobius with larger genus Neogobius, as it is the sister group of that roundness value possessed more rounded shaped N. melanostomus. The results of mentioned study are otoliths. In this study, roundness worked better than in consistent with this study and recognize other indices in species separation. The ellipticity and P. bathybius and N. melanostomus as more similar aspect ratio indices showed little or no separation taxa and isolated P. bathybius from other species in power among members of genus Ponticola except genus Ponticola. However, recognition of P. patimari P. bathybius, while they worked more efficient for and P. gorlap as more closely related taxa is different Neogobius. The results of this study, showed modest with previous phylogenetic analysis of these species classification success for Ponticola species with 61% (Eagderi et al. 2020) and use of other methods of shape (jackknifed) respect to those of Neogobius species analysis may help to resolve this ambiguity. 82% (jackknifed). These findings suggested that Otoliths in bony fishes play an important role in the otolith morphology analysis only using shape indices senses of balance and hearing. Otolith mass and shape might not always effectively describe the actual shape are likely to be decisive factors influencing otolith contours of otoliths for all species and their motion and thus ear functioning. Until now, the discrimination power appears to be related to the kind selective forces and/or constraints driving the of otolith shape differences between species. The evolution of otoliths and their diversity in shape in shape indices are calculated from several teleosts are largely unknown (Schulz-Mirbach et al. measurements which characterize some aspects of the 2018). Although otolith shapes may have evolved otolith contours and, in some instances, provide a along with the considerable diversity of, and powerful tool for discrimination (Tuset et al. 2003, improvements in, auditory abilities in teleost fishes Bani et al. 2013; Xin et al. 2014). Other methods such (Schulz-Mirbach et al. 2018). Experimental studies as Elliptic Fourier shape analysis and wavelet have revealed that several species of gobies including functions (Lombarte et al. 2006; Bani et al. 2013; Ponto-Caspian gobies are capable of producing Lombarte et al. 2018; Gut et al. 2020) explain the multiple sounds, were demonstrated to be relevant for otolith contours in more detail and extract finer mating success, and interestingly acoustic features morphological information from the image analysis proved to be a species-specific trait and used in than do shape indices. So, analyzing otolith shape reconstruction of phylogenetic relationships (Horvatić using other methods may be useful for species et al. 2015 & 2019). Interestingly, otolith shapes in discrimination in genus Ponticola. those species were distinct, and therefore the possible The recent studies are emphasized that at least reason for the production of different sounds may be some otolith characteristics are genetically encoded related to the different shapes of otoliths. But our (Medvedev et al. 2013; Reichenbacher & Reichard knowledge regarding the role of fish otoliths in inner Iran. J. Ichthyol. (2021) 8(3): 170-180 177 ear function remains limited because experiments Esmaeili, H.R., Sayyadzadeh, G., Eagderi, S., & Abbasi, K. visualizing and quantifying otolith motion are still 2018. Checklist of freshwater fishes of Iran. FishTaxa, methodologically challenging (Platt & Popper 1981; 3(3), 1-95. Popper et al. 2005; Schulz-Mirbach et al. 2018). Ghanbarifardi, M.; Gut, C.; Gholami, Z.; Esmaeili, H.R.; Similar comparative studies on the Caspian Sea gobies Gierl, C. & Reichenbacher, B. 2020. Possible link may help clarify the mystery of the relationship between the structure of otoliths and amphibious mode between acoustic communication and otolith of life of three mudskipper species (Teleostei: morphology. Gobioidei) from the Persian Gulf. Zoology in the In conclusion, the results of present study showed Middle East 66(4): 311-320. that the Caspian Sea goby otoliths can be used to Gierl, C.; Liebl, D.; Šanda, R.; Vukić, J.; Esmaeili, H.R. & identification of specimens at genus and species level. Reichenbacher, B. 2018. What can goby otolith Moreover, we have detected an apparent phylogenetic morphology tell us? Cybium 42(4): 349-363. clustering in the studied gobies otolith that recently Gut, C.; Vukić, J.; Šanda, R.; Moritz, T. & Reichenbacher, mentioned in molecular studies. B. 2020. 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مقاله پژوهشی ویژگیهای ریختی ساژیتا در هفت گونه گوبی ماهی)ماهیان استخوانی عالی: گوبی ماهیان( از دریای خزر

پریسا داودی گروه علوم جانوری، دانشکده علوم ، دانشگاه مازندران، بابلسر، ایران.

چکیده: در این مطالعه ریختشناسی اتولیت ساژیتا در هفت گونه از گوبی ماهیان، شامل سه گونه از جنس Neogobius و چهار گونه از جنس Ponticola، ساکن در بخش جنوبی دریای خزر، توصیف و مورد بررسی قرار گرفت. هدف ما بررسی کاربرد و کارایی ساژیتا در شناسایی گونهها است. تحلیلهای ریخت شناسی بر اساس چهار شاخص شکلی، میزان گردی )4S/πOL2(، مستطیل شکل بودن )S/OL×OW(، بیضی شکل بودن )OL-OW/OL+OW( و نسبت تصویر )OL/OW( بود. نتایج نشان داد ریختشناسی اتولیت و ریختسنجی میتواند به عنوان ابزاری مفید در شناسایی گوبیهای دریای خزر مورد استفاده قرار گیرد. درحالیکه شکل کلی اتولیت جنسها را متمایز میسازد، تفاوت در شکل حاشیه پشتی و میزان رشد لوبهای پشتی خلفی و قدامی شکمی در تمایز بین گونهای اهمیت دارد. تحلیل ممیزی گونههای جنس Ponticola و Neogobius را به ترتیب با 66% و 28% با موفقیت در گروههای خود طبقه بندی نمود و این ثابت مینماید شاخصهای شکلی ابزار کارآمدتری در تمایز گونههای جنس Neogobius نسبت به Ponticola است. تحلیل خوشهای بر اساس شاخصهای شکلی دو خوشه اصلی را نشان میدهد، یک خوشه شامل گونههای جنس Neogobius با مقادیر باالتر شاخص میزان گردی، دارای اتولیتهای گردتر و خوشه دیگر شامل اعضای جنس Ponticola دارای اتولیتهای کشیدهتر و بیضوی، به جز P. bathybius که P. bathybius و N. melanostomus را به عنوان گونههای مشابه در نظر میگیرد. این نتایج با فیلوژنی گونههای مورد بررسی در مطالعات مولکولی قبلی مطابقت دارد و اثبات مینماید بخشی از ویژگیهای اتولیت مبنای ژنتیکی داشته و از این رو شواهد فیلوژنتیکی را نمایان میسازند. مطالعه حاضر درجه باالی تنوع ریختی در شکل اتولیت را نشان میدهد و بر کارایی این روش در تاکسونومی و فیلوژنی گاوماهیان تاکید مینماید. کلماتکلیدی: تاکسونومی، اتولیت، Ponticola ،Neogobius.

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