Research Article Mediterranean Marine Science Indexed in WoS (Web of Science, ISI Thomson) and SCOPUS The journal is available on line at http://www.medit-mar-sc.net DOI: http://dx.doi.org/10.12681/mms.423 This work was presented at the “10th Hellenic Symposium on Oceanography and Fisheries” held in Athens on 7th-11th May 2012, organized by HCMR. Use of morphological differences for the identiication of two picarel species Spicara lexuosa and Spicara maena (Pisces: Centracanthidae) G. MINOS1, A. IMSIRIDOU1 and G. KATSELIS2 1 Alexander Technological Educational Institute of Thessaloniki, Department of Aquaculture & Fisheries Technology, P.O. Box: 157, GR-63200, Nea Moudania, Greece 2 Technological Educational Institute of Messolonghi, Department of Aquaculture & Fisheries Management, GR-30200, Messolonghi, Greece Corresponding author: gminos@otenet.gr Received: 9 April 2013; Accepted: 5 July 2013; Published on line: 17 September 2013 Abstract The recognition and identiication of the two species of Spicara genus (Spicara lexuosa, picarel and Spicara maena, blotched picarel) is dificult, due to a systematic confusion until now. In this work a number of external morphometric features (ten body ratios) are evaluated for their diagnostic possibilities. According to Principal Component Analysis results, the body ratios head length to standard length, head height to head length and the ratios of two body heights, indicated that these characters were not related to the maturity stage of the species. The discriminant analysis based on the above body ratios, indicated a rather high level of discrimination (83.2%) of the examined samples for two species. The results are discussed, and possibilities for improvement of the identiication methodology for the two species are proposed. Keywords: Centracanthidae, spicara, multivariate analysis, morphometric. Introduction Species that belong to the genus Spicara occur in shallow rocky and muddy bottoms throughout the Mediterranean and the Black Sea, in the Atlantic from Portugal to Morocco and around the Canary Islands (Froese & Pauly, 2013). These species contribute to isheries in inshore areas of the Greek seas (Mytilineou & Papaconstantinou, 1991; Stergiou et al., 2011), and represent a major proportion of the total catch for coastal isheries in Croatia (Dulcic et al., 2000). On the other hand, they are the common by-catch of coastal isheries in the Mediterranean (especially those operating bottom trawlers) with low commercial value (Ragonese et al., 2004). The genus Spicara has posed numerous identiication problems and consequently many different species have been described, leading to a variety of synonyms. This fact was attributed to marked variations in coloration related to the effects of sexual dimorphism (reinforced by the protogynous hermaphroditism sex inversion) and colour changes due to the state of sexual maturity (Pollard & Pichot, 1971). According to the review of Pollard & Pichot (1971), the old classiication distinguished two genera, Maena and Smaris that subsequently were distinguished into two genera Maena and Spicara and inally were fused into a single genus Spicara, which comprises three species: 26 Spicara maena (Linnaeus, 1758), Spicara smaris (Linnaeus, 1758) and Spicara chrysalis Valenciennes, 1830, which is a synonym of Spicara lexuosa Rainesque, 1810 (Tortonese, 1975). In literature, there was a systematic confusion about whether S. maena and S. lexuosa are the same (Tortonese, 1975; Eschmeyer, 2013; Froese & Pauly, 2013) or different species (Vasiliev, 1980; Papakonstantinou, 1988; Golani et al., 2006; Vasil’eva, 2007; Imsiridou et al., 2011). The above conlict is maintained till today; although karyological studies (Vasiliev, 1980) as well as genetic studies (Chiba et al., 2009; Imsiridou et al., 2011; Turan, 2011) undoubtedly prove that S. maena and S. lexuosa are two different species. Assuming that S. maena and S. lexuosa are two different species, identiication is possible using meristic and morphometric characteristics (Tortonese, 1986; Fischer et al., 1987; Rizkalla, 1996). Multivariate analysis, on a set of phenotypic characters, is regarded as a powerful technique for the determination of morphological relationships among different populations of a species (Claytor & MacCrimmon, 1988; Corti & Crosetti, 1996; Vidalis et al., 1997) and for investigating taxonomic problems among species (Spain et al., 1980; Karakousis et al., 1991; Iliadou et al., 1996; Katselis et al., 2006). It also creates the mathematical foundation for the development and support of new tools. The development of specialized software tools such as Medit. Mar. Sci., 14/3, 2013, SPECIAL ISSUE, 26-31 TPS-dig software, version 1.37 (Rohlf, 2003) offers the possibility of taking measurements of an item, on a digital photo. These tools are predominantly used in ichthyology nowadays. Despite the beneits, they have limitations associated with the quality of the extracted information. If a ish photo is to be used for determining the species, the recognition characters should be clearly identiiable and measurable. The aim of this study is to evaluate the similarity/ dissimilarity of S. lexuosa and S. maena species, using easily distinguishable morphometric characters. A second aim is to develop a mathematical background for the diagnosis of the two species, using the multivariate analysis of morphometric characters. Materials and Methods Fish Samples Specimens were collected by professional ishermen in the Thermaikos Gulf (40o 23’ N; 22o 50’ E), Northern Aegean Sea. Individuals of S. maena were collected with purse seine nets (20 mm mesh-size stretched), in 25-35 m depth on sandy bottom with Posidonia oceanica beds, while those of S. lexuosa were collected with pots (metal frame lined with a wire mesh, box shaped 80x60x30 cm, mesh size 20 mm). Pilchards were used as bait and the pots were set at 25-35 m depth, on sandy or muddy bottoms, with parts covered with hard substrate. The advantages of these ishing gears (purse seines and traps) are that they capture a wide range of ish length from a speciic ish population and area, either encircling shoals of ish or baiting individuals that are seeking shelter or food. In contrast, other ishing gears such as static gill nets and bottom trawls are selective on the range of ish length caught, and sweep a broader area. Individuals of S. maena and S. lexuosa were randomly collected from March to August 2008, a period which coincides with their spawning season (Tortonese, 1986; Karidas et al., 2009, 2011). Specimens were identiied according to the characters of the identiication keys (Tortonese, 1986; Fischer et al., 1987). These characters are easily distinguishable and visible on a ish photo, and some of these have been used to identify the two species (Fischer et al., 1987; Lythgoe & Lythgoe, 1992; Golani et al., 2006). All measurements were made to the nearest 0.1 cm with electronic vernier caliper; for paired structures only the left structure value was taken. After measuring, ish were dissected and the gonads were removed and observed for sex identiication. Sex and maturity stage of gonads were determined macroscopically according to the scale of Nikolsky (1963), slightly modiied to it the characteristics of the gonad stages of maturity of the picarels (Vidalis, 1994). Total weight of body (W) and weight of gonads (Wg) were measured to the nearest 0.1g, and the Gonadosomatic Index (GSI=100*Wg/W) was calculated. To minimize any variation resulting from allometric growth, all morphometric measurements were standardized according to Reist (1985):X’i,j = logXi – b . (logTLj – logTLi) where X’i,j is the standardized measurement of the i morphometric character; logXi is the mean logarithm of i morphometric character measurement; TLj is the total length of the individual j; logTL is the logarithm of the mean total length of pooled individuals and b is the slope of the logX against logTL plot. Also, the anti-logarithms of standardized measurements and their ratios were estimated. Finally, the t-test was used (Zar, 1999) to identify whether there were any statistically signiicant differences between the species for each character ratio. Principal component analysis (PCA) was used to test the contribution of the morphometric characters ratios and logarithm of GSI, to the coniguration of ish shape variance (Hair et al., 1998). To elucidate the differentiation of the species, forward stepwise discriminant analysis (DA), on the characters based on the generalized Mahalanobis distance, was used, to determine the similarity between groups and the ability of these variables to identify the specimens correctly (Hair et al., 1998). In order to identify any signiicant differences among the sex per species groups (immature, males and females) on the scores of each extracted canonical vari- Morphometric Analysis A series of measurements was recorded on each specimen, for six (6) distance characters (Fig. 1). Speciically, Total Length (TL) is deined as the distance from the tip of the nose to the longest caudal in ray, Standard Length (SL) as the distance from the tip of the nose to the end of the vertebral column, Head Length (HL) as the distance from the tip of the nose to the posterior margin of the opercula, Head Height (HH) as the distance from the lower end to the upper end of the head, Maximum Body Height 1 (MBH1) as the vertical distance from the anterior part of the dorsal in and ventral part of the body and Maximum Body Height 2 (MBH2) as the vertical distance from the dorsal in and ventral part of the body. Medit. Mar. Sci., 14/3, 2013, SPECIAL ISSUE, 26-31 Fig. 1: Measurements that were taken on picarel and blotched picarel individuals. TL: total length; SL: standard length; HL: head length; HH: head height; MBH1: body height in the origin of dorsal in; MBH2: maximum body height. 27 ables (CaV), analysis of variance (ANOVA) was applied. Furthermore, the Tukey HSD test was applied, to check which sex per species differs from each other. Also, to minimize any variation resulting from the maturity stage of individuals, the morphometric ratios that related with the GSI were excluded from the discriminant analysis. All statistical analyses were performed with the SPSS PC ver. 10 software package. Results Eight hundred and eighty eight (888) individuals were examined for morphometric analysis (9.9-84.3 g total body weight; 9.6-18.1 cm total length). From the above, two hundred and ninety nine (299) individuals of S. maena (11.6-18.1 cm total length) and ive hundred and eighty nine (589) individuals of S. lexuosa (9.6-18.1 cm total length). Summary statistics and t-test results are shown in Table 1. Signiicant differences for all exam- Table 1. Summary statistics for total length (TL), Gonadosomatic index (GSI) and ratios among the morphometric characters, estimated after the Reist (1985) standardization. X: mean value (cm); SD: standard deviation; Min-max: minimum and maximum value (cm); N: number of individuals; IM: immature individuals; M: males and F: females. Character Spicara maena Spicara lexuosa N=299 (IM=0; M=78; F=221) N=589 (IM=174; M=269; F=146) X SD min-max X SD min-max TL 15.37 1.09 11.6-18.1 GSI 0.08 0.05 0-0.37 HL/SL 0.28 0.01 HH/SL 0.24 0.01 MBH1/SL 0.30 MBH2/SL HH/HL 14.10 1.80 9.6-18.1 13.1 <0.05 0.01 0.02 0-0.24 23.2 <0.05 0.25-0.31 0.29 0.01 0.27-0.33 18.4 <0.05 0.21-0.29 0.24 0.01 0.19-0.31 5.1 <0.05 0.02 0.25-0.34 0.27 0.01 0.24-0.36 20.3 <0.05 0.30 0.02 0.26-0.35 0.27 0.01 0.22-0.32 24.8 <0.05 0.86 0.04 0.75-1.02 0.81 0.04 0.67-1.00 17.9 <0.05 P MBH1/HL 1.05 0.06 0.87-1.21 0.93 0.04 0.79-1.11 29.0 <0.05 MBH2/HL 1.06 0.06 0.93-1.22 0.93 0.04 0.73-1.08 33.0 <0.05 MBH1/HH 1.22 0.07 0.97-1.45 1.15 0.05 0.90-1.40 16.2 <0.05 MBH2/HH MBH2/MBH1 1.24 1.01 0.07 0.04 1.03-1.49 0.93-1.17 1.15 1.01 0.06 0.04 0.92-1.34 0.72-1.23 17.8 4.5 <0.05 <0.05 Table 2. Results of principal components analysis (PCA) and factor loadings for each morphometric variable on the four extracted PCA factors (Fi), after varimax normalized rotation. Factors Variance F1 F2 F3 F4 Eigenvalues 5.668 2.416 1.382 1.094 % of Variance 43.25 23.09 15.15 14.52 Cumulative % 43.25 66.34 81.49 96.00 Character GSI HL/SL 0.67 0.22 0.35 0.07 -0.23 0.08 -0.96 -0.05 HH/SL 0.07 0.95 -0.29 -0.08 MBH1/SL 0.86 0.45 -0.08 -0.23 MBH2/SL 0.83 0.38 -0.05 0.39 HH/HL 0.24 0.89 0.38 -0.04 MBH1/HL 0.83 0.34 0.39 -0.16 MBH2/HL 0.80 0.28 0.40 0.34 MBH1/HH 0.90 -0.34 0.18 -0.18 MBH2/HH 0.79 -0.35 0.18 0.46 MBH2/MBH1 0.00 -0.09 0.04 0.99 28 t-test ined ratios of morphometric characters among the species, were shown (t-test>5; df= 887; P<0.05). The PCA analysis extracted four factors with eigenvalues>1, explaining 96% of the variance (Table 2). Using a cut-off value of 0.6 for the factor loadings, factor 1 (F1) expressed characters associated with the maximum body height to length ratios and gonadosomatic index, factor 2 (F2) expressed variables associated with the head height to head length and to standard length ratios (HH/HL and HH/ SL respectively). Finally, factor 3 (F3) expressed variables associated with the head length to standard length ratio (HL/SL), while factor 4 (F4) expressed variables associated with the maximum body height ratio (MBH2/MBH1). The discriminant analysis (DA) on the HL/SL, HH/SL, HH/HL and MBH2/MBH1 presented one canonical variable (CaV) which contributed overall to the variance (the expressed characters by factor 1 were excluded, due to their relation to the maturity stage of each ish). The characters of primary importance in distinguishing species were HL/ SL and HH/HL. The discriminant function (DF) was: DF = –134.03 – 539.8 . (HH/SL) + 384.4 . (HL/SL) + 175.2 . (HH/HL) + 6.96 . (MBH2 / MBH1) Medit. Mar. Sci., 14/3, 2013, SPECIAL ISSUE, 26-31 The discriminant function identiied the membership (classiication) of individual ish in the data to one of the two species, with a success rate of 83.19%. The percentage of correctly identiied S. maena and S. lexuosa individuals was 64.5% and 92.7%, respectively (Table 3). The frequency distribution of discriminant function scores for two species is shown in Figure 2. The 77% of S. lexuosa individuals were given DF<0, while the 83% of S. maena were given DF>0 (Fig. 2). The DF scores differ signiicantly between sexes per species (ANOVA; df=4.804; F=162; P<0.05). On the other hand, no signiicant differences appeared among sexes on the same species (Tukey HSD test; P>0.05) (Fig. 3). Discussion The morphometric characters (phenetic characters) derive from the composite effect of genotype and environmental factors, and they are under the inluence of natural selection (Dobzansky, 1970). It is certain that parameters related to the allometric growth of ish and the timing of the sampling (feeding activity and maturation), could pose some major limitations for the study of morphological relationships between species. This study attempted to minimize variances caused by these parameters through the common sampling period, as well as the transformation of the original measurements (Reist, Table 3. Results of discriminant analysis classiication showing the percentage of specimens classiied in each species. Predicted species Spicara maena Spicara lexuosa Total individuals Spicara maena 64.5 35.5 299 Spicara lexuosa 7.3 92.7 589 Species Fig. 2: Frequency distribution of discriminant function scores of the irst extracted canonical variable for S. lexuosa and S. maena. Fig. 3: Box and Whisker plot (min-max, 95% values. Mean, SD) of the discriminant function scores of the irst extracted canonical variable for male, female and immature individuals of S. lexuosa and S. maena. Groups that they do not differ statistically (Tukey HSD test; P>0.05) are indicated with the same letter, a or b. Medit. Mar. Sci., 14/3, 2013, SPECIAL ISSUE, 26-31 29 1985). It also attempted to remove the characters which are related to the maturity stage of ish, from the analysis. The analysis of the morphological variability presented here, indicated signiicant differences in external shape between the two species (Table 1). The ratios of body measurements indicated that the individuals of S. maena are characterized by shorter/higher head and deeper body than those of S. lexuosa. These indings are in agreement with those of other authors (Pollard & Pichot, 1971; Tortonese, 1986; Fischer et al., 1987; Lythgoe & Lythgoe, 1992; Miller & Loates, 1997; Golani et al., 2006). Some authors have used the relation between head length and maximum body height, to identify the two species. The head length is equal or greater than the body height in S. lexuosa, while the head length is shorter than the body height in S. maena (Fischer et al., 1987; Lythgoe & Lythgoe, 1992; Golani et al., 2006). Although our results are conirmed by the above indings, the fact that the ratios of the body height (MBH1/HL, MBH2/HL, MBH1/HH, MBH2/HH, MBH1/SL, MBH2/SL) apart from the ratio of MBH2/MBH1, are related to the maturity stage of species (Table 2), decreases the discrimination ability of these characters. In substitution of these recognizing characters, our results proposed that the ratios of a) head length to standard length (HL/SL) as F3, b) head height to head length (HH/HL) as F2, c) head height to standard length (HH/SL) as F2 and d) the ratio of the two body heights (MBH2/MBH1) as F4, show low association (<0.35) to the species maturity stage (GSI), since the factor loadings of F2, F3 & F4 are 0.22, 0.35 and 0.07 respectively (Table 2). The discriminant analysis based on these characters indicated a rather high level of species identiication. Indeed, 83.2% of the examined specimens could be distinguished and classiied correctly in the two species. Moreover, the loadings of discriminant function (linear results of these characters) were not related to the sexual dimorphism of the species (Fig. 3) and could thus constitute a more reliable tool for identiication of the two species than the ones used today. Consequently, they can be a reliable tool for the identiication of the two species. The sharpness of the characters as well as the high discriminative values (>83%), provide a great possibility to be used for identiication of the two species from the new technologies. The relatively small percentage of non-discrimination (16.8%) of the above characters, can be overcome by more general information about the species such as differences in certain biological aspects i.e. spawning period and sexual behaviour (Tortonese, 1986; Fischer et al., 1987; Lythgoe & Lythgoe, 1992; Dulčić et al., 2000; length of sex reversal: Lepori, 1960; Relini et al., 1999; Dulčić et al., 2000; Karakulak et al., 2007; Karidas et al., 2009, 2011), body colour pattern (Lythgoe & Lythgoe, 1992; Louisy, 2002; Karidas et al., 2009), and external morphology (Fischer et al., 1987; Lythgoe & 30 Lythgoe, 1992; Louisy, 2002; Golani et al., 2006). Also, the fact that S. maena and S. lexuosa are two different species is reinforced by an anatomical character, which is present in S. maena: well-developed teeth on the vomer (Tortonese, 1986; Lythgoe & Lythgoe, 1992). Additionally, each species prefers different habitats, with S. maena inhabiting sandy - muddy Posidonia beds down to about 100 m., while S. lexuosa appears on sand or muddy bottoms, with rocky particles down to about 130 m (Tortonese, 1986). In conclusion, the results from the morphometric analysis revealed discrimination between S. maena and S. lexuosa, based on four body proportions that derive from ive easily identiiable morphological characters (HH, HL, SL MBH1 and MBH2). The above characters could be used by information technology for developing new species recognition tools. In any case, these indings contribute to the improvement of the identiication methods for the two species, based on external features. Acknowledgements We would like to thank Theofanis Karidas and Nikos Argiridis for their valuable help in ish collection and measurements, and Alexandra Dara for her help as far as the English language is concerned. 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