Zootaxa 4205 (1): 081–086 http://www.mapress.com/j/zt/ ISSN 1175-5326 (print edition) Article Copyright © 2016 Magnolia Press ZOOTAXA ISSN 1175-5334 (online edition) http://doi.org/10.11646/zootaxa.4205.1.7 http://zoobank.org/urn:lsid:zoobank.org:pub:F521B39B-2DD3-4880-A7E0-F403FC10A8F4 Identifying Neogobius species from the southern Caspian Sea by otolith shape (Teleostei: Gobiidae) PARISA DAVOODI & HASSAN RAHIMIAN Faculty of Biology, College of Science, University of Tehran, Tehran, Iran. E-mail: h.rahimian@ut.ac.ir Abstract Three Neogobius species are common in the southern Caspian Sea. Identification of these species, especially from remains, is not fully understood. The present study compares the morphological characteristics of sagittal otoliths of N. caspius, N. pallasi and N. melanostomus. Furthermore, it also provides a dichotomous identification key based on otoliths morphology. Key words: taxonomy, comparative morphology, sagittal otoliths Introduction Three species of the genus Neogobius are found in the Caspian Sea basin: N. caspius, N. pallasi and N. melanostomus. While, N. caspius and N. pallasi are endemic to the Caspian Sea, N. melanostomus is native in Eurasia, including the Black Sea, Caspian Sea, Sea of Azov and tributaries, being introduced into the Great Lakes and invasive in Central Europe (Kottelat & Freyhof 2007; Neilson & Stepien 2009, 2011). While complete and well preserved fishes of these species can be identified easily, the identification of these fishes in the remnant of piscivorous animal's diet is a challenge. In that case, gobies might be identified by their otoliths. Otoliths have been used for species identification in other fish group (Tuset et al. 2003; Tuset et al. 2008; Bani et al. 2013). Bani et al. (2013) have also demonstrated the usefulness of otoliths in the identification of other gobiidae species. The main objective for the present communication is to compare the otoliths of the three Caspian Neogobius species and explore their valence in species identification. Material and methods Fish were caught using a hand trawl net, applied at different depths (0–10 m), at four sites, along the southern coasts of the Caspian Sea (Anzali, 37°28'22"N 49°27'44"E; Nowshahr, 36°38'56"N 51°29'46"E; Babolsar, 36°42'09"N 52°39'27"E and Amirabad, 36°48'16"N 53°18'09"E; Fig. 1, Table 1). Species were identified following morphometric and meristic characteristics given by Berg (1949). Female and male individuals were sorted by their difference in the shape of the genital papilla (Berg 1949). Left and right otoliths were extracted and stored dry. For each otolith, length (OL), height (OH) and area (OA) were measured using tpsDig2 from digital image (Rohlf 2006). To precisely describe the otoliths shape, aspect ratio (OL/OH) and rectangularity (OA/ (OL*OH)) were also calculated (Tuset et al. 2006). The relationships between OA and total length (TL) were inferred from the slopes observed in regression equations and by distribution of data points in the scatter plot. The correction of morphometric variables by size was accounted for, using standard residuals from a linear regression of each of the variables on TL (Reist 1985; Stransky & MacLellan 2005). The size-corrected variables were screened for normality and homogeneity of variance using Kolmogorov–Smirnov normality test and Levene’s test, respectively (Zar 1999). The t test was used to examine the effects of fish sex and otoliths position (left or right) on morphometric variables in each species. The standardized values of different otolith parameters were compared Accepted by J. Freyhof: 1 Sept. 2016; published: 5 Dec. 2016 81 among species using one-way ANOVA. All statistical analyses were performed in SPSS version 19.00 (SPSS Inc. 2011). A dichotomous key was constructed based on otoliths morphology following the nomenclature provided by Tuset et al. (2008). TABLE 1. Rang of total length (in mm) and number of specimen examined (brackets) of Neogobius species from each locality. Species Anzali Nowshahr Babolsar Amirabad N. pallasi 80–116 (43) 83–127 (60) 81–122 (42) 80–102 (22) N. caspius 80–117 (42) 98–147 (12) 80–157 (14) 79–122 (23) N. melanostomus - - 75–130(28) - FIGURE 1. Location of sampling sites. Results The morphometric variable did not show any significant difference between sex and otoliths position (i.e. left and right otoliths) (t test; P≥ 0.05), so samples from both sexes and positions were pooled together. This study revealed great differences in the structures of the sagitta amongst the studied Neogobius species (Figs 2 and 3). Below is a brief description of the otoliths of the three Neogobius species: Neogobius pallasi: Otolith discoid; dorsal margin round, ventral margin round to approximately flat; anterior region of otolith round without any lobe and projection, posterior region of otolith round with two lobes separated by a median shallow notch, without projection (Fig. 3A–B). Neogobius caspius: Otolith squared; dorsal margin round, ventral margin round to approximately flat; anterior region of otolith flat without lobe, with a conspicuous pointed ventral projection, posterior region of otolith flat with two lobes separated by a median shallow notch, with conspicuous pointed dorsal projection (Fig. 3C–D). Neogobius melanostomus: Otolith approximately squared to rhomboidal; dorsal margin round, ventral margin round to approximately flat; anterior region of otolith oblique or flat without lobe, with a small ventral projection, posterior region of otolith oblique with two lobes separated by a median shallow notch, with a small dorsal projection (Fig. 3E–F). OL, OH, OA and Aspect Ratio of N. pallasi and N. caspius were significantly different from those of N. melanostomus. Rectangularity was not different significantly among species (Table 2). 82 · Zootaxa 4205 (1) © 2016 Magnolia Press DAVOODI & RAHIMIAN FIGURE 2. SEM micrograph of sagittal otolith of a 60 mm specimen of Neogobius pallasi and its features. Otolith areas (OA) were linearly correlated with the TL in all three species (Fig. 4). The slope of the regression line was lowest in N. melanostomus (OA=0.067TL —1.014) in comparison with that of N. caspius (OA=0.09TL — 2.40) and N. pallasi (OA=0.09TL —2.33). Also, the OA mean was smaller in N. melanostomus than in N. caspius and N. pallasi. TABLE 2. Otoliths morphometric variables of three Neogobius species. Values are mean±SE. * Significantly different values (P <0.05) Species N. pallasi N. caspius N. melanostomus OL (mm) 2.66±0.02 2.67±0.03 2.46±0.05* OH (mm) 2.74±0.05 2.68±0.06 2.4±0.05* OA (mm2) 6.09±0.06 6.15±0.06 4.80± 0.05* Aspect ratio 0.97±0.007 0.99±0.0076 1.027±0.005* Rectangularity 0.82±0.004 0.84±0.004 0.83±0.007 Key to the Caspian species of Neogobius based on otoliths morphology 1a 1b 2a 2b Otoliths discoidal without anterioventral and posteriodorsal projections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. pallasi Otoliths squared or rhomboidal, with anterioventral and posteriodorsal projections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Otoliths squared, with conspicuous anterioventral and posteriodorsal projections . . . . . . . . . . . . . . . . . . . . . . . . . . . .N. caspius Otoliths squared to rhomboid with small anterioventral and posteriodorsal projections . . . . . . . . . . . . . . . . . . N. melanostomus Discussion In both taxonomic and ecological, and paleontological studies, there are urgent needs for reliable characters that allow determining the species correctly. Otoliths of N. caspius, N. pallasi and N. melanostomus can be identified by the dichotomous key compiled here. Otoliths of N. melanostomus were more elongated rather than those in the other two species. Growth rate of sagitta relative to the fish total length was different in different species, being smallest in N. melanostomus and overlapping, but different in the other two. OTOLITH MORPHOLOGY IN NEOGOBIUS Zootaxa 4205 (1) © 2016 Magnolia Press · 83 FIGURE 3. Sagittal otoliths of: A–B: N. pallasi (TL: 95 mm); A, male; B, female. C–D: N. caspius (TL: 95 mm); C, male; D, female. E–F: N. melanostomus (TL: 95 mm); E, male; F, female. 84 · Zootaxa 4205 (1) © 2016 Magnolia Press DAVOODI & RAHIMIAN FIGURE 4. Linear regression between total length and otolith area in three species of Neogobius. The morphology of the otoliths is determined by genetic characteristics of the species, and is influenced by ontogenical and environmental factors (Vignon & Morat 2010; Vignon 2012). Neogobius species live sympatrically along the southern coasts of the Caspian Sea, all found in inshore waters. Therefore, genetic factors should have dictated those differences observed in the otolith morphology among Neogobius species. These findings are in congruence with results of the study by Bani et al. (2013), in which the authors have studied otoliths of the gobies Ponticola gorlap, P. bathybius and Neogobius caspius from the Southern Caspian Sea and found pronounced differences between those. Our study demonstrated that sagittal otoliths can be considered as useful tools in species identification in gobies. Acknowledgment We thank S. Abdolmaleki and K. Abbasi (Bony Fish Research Centre, Anzali) and A. Nazaran (Fisheries Research Centre, Nowshahr) for their help during field work. References Bani, A., Poursaeid, S. & Tuset, V.M. (2013) Comparative morphology of the sagittal otolith in three species of south Caspian gobies. Journal of Fish Biology, 82, 1321–1332. http://dx.doi.org/10.1111/jfb.12073. Berg, L.S. (1949) Freshwater Fishes of the U.S.S.R. and Adjacent Countries. Izdatel'vesto Akademii Nauk SSSR, Moscow. Kottelat, M. & Freyhof, J. (2007) Handbook of European freshwater fishes. Publication Kottelat, Cornol and Freyhof, Berlin, 646 pp. http://dx.doi.org/10.1007/s10228˗007˗0012˗13. OTOLITH MORPHOLOGY IN NEOGOBIUS Zootaxa 4205 (1) © 2016 Magnolia Press · 85 Neilson, M.E. & Stepien, C.A. (2009) Escape from the Ponto-Caspian: evolution and biogeography of an endemic goby species flock (Benthophilinae: Gobiidae: Teleostei). Molecular Phylogenetics and Evolution, 52, 84–102. http://dx.doi.org/10.1016/j.ympev.2008.12.023. Neilson, M.E. & Stepien, C.A. (2011) Historic cryptic speciation and recent colonization of Eurasian monkey gobies (Neogobius fluviatilis and N. pallasi) revealed by DNA sequences, microsatellites and morphology. Diversity and Distributions, 17, 688–702. http://dx.doi.org/10.1111/j.1472-4642.2011.00762.x. Reist, J.D. (1985) An empirical evaluation of several univariate methods that adjust for size variation in morphometric data. Canadian Journal of Zoology, 63, 1429–1439. http://dx.doi.org/10.1139/z85-213. Rohlf, F.J. (2006) tpsDig2, digitize landmarks and outlines, version 2.10. Department of Ecology and Evolution, State University of New York at Stony Brook. SPSS Inc. (2011) SPSS. Ver. 19.0. Base. Chicago, IL: SPSS, Inc. Stransky, C. & MacLellan, S.E. (2005) Species separation and zoogeography of redfish and rockfish (genus Sebastes) by otoliths shape analysis. Canadian Journal of Fisheries and Aquatic Sciences, 62, 2265–2276. http://dx.doi.org/10.1139/f05-143. Tuset, V.M., Lozano, I.J., Gonzalez, J.A., Pertusa, J.F. & Garcia-Diaz, M.M. (2003) Shape indices to identify regional differences in otolith morphology of comber, Serranus cabrilla. Journal of Applied Ichthyology, 19, 88–93. http://dx.doi.org/10.1046/j.1439-0426.2003.00344.x. Tuset, V.M., Rosin, P.L. & Lombarte, A. (2006) Sagittal otoliths shape as useful tool for the identification of fishes. Fisheries Research, 81, 316–325. http://dx.doi.org/10.1016/j.fishres.2006.06.020. Tuset, V.M., Lombarte, A. & Assis, C. (2008) Otolith atlas for the western Mediterranean, north and central eastern Atlantic. Scientia Marina, 72, 1–198. http://dx.doi.org/10.3989/scimar.2008.72s17. Vignon, M. (2012) Ontogenetic trajectories of otolith shape during shift in habitat use: Interaction between otolith growth and environment. Journal of Experimental Marine Biology and Ecology, 420, 26–32. http://dx.doi.org/10.1016/j.jembe.2012.03.021 Vignon, M. & Morat, F. (2010) Environmental and genetic determinant of otolith shape revealed by a nonindigenous tropical fish. Marine Ecology Progress Series, 411, 231–241. http://dx.doi.org/10.3354/meps08651 Zar, J.H. (1999) Biostatistical Analysis, 4th ed. Upper Saddle River, NJ: Prentice-Hal 86 · Zootaxa 4205 (1) © 2016 Magnolia Press DAVOODI & RAHIMIAN