Journal of Fish Biology (1996) 49, 1317–1372 Protein differences among the Mediterranean species of the genus Spicara M. A, A. M, G. S, S. L B, M. C  N. P Istituto di Zoologia, Via Archirafi 18, 90123—Palermo, Italy (Received 21 May 1996, Accepted 30 July 1996) Protein electrophoresis (PAGE) was used to study the three morphologically different species of Spicara (S. flexuosa, S. maena, S. smaris). Of the 28 enzymatic and additional myogenic loci, five monomorphic loci (LDH-1*, G6PD-1*, PGI-1* and two PMMs*) were species-specific markers of S. smaris with respect to S. flexuosa and S. maena. Four of the 28 enzymatic loci were polymorphic (EST-1*, GLDH*, PEPD*, PGI-2*). Discriminating genetic markers were not identified between S. flexuosa and S. maena. Genetic distance (D) as calculated by Nei’s index (1978), between S. smaris v. S. maena and S. flexuosa showed a value, respectively of D=0·137 and 0·141. Between S. flexuosa and S. maena the value was D=0·006. From the data it can be inferred that S. flexuosa and S. maena are conspecific, despite morphological differences. ? 1996 The Fisheries Society of the British Isles Key words: protein electrophoresis; Mediterranean Sea. species differentiation; genetic distance; Spicara; INTRODUCTION The genus Spicara is common in shallow rocky and mud bottoms all around the Mediterranean, Black Sea, Portugal to Morocco and the Canary Islands (Tortonese, 1975, 1986). This genus has posed numerous identification problems and consequently many species have been described, leading to a variety of synonyms; this was attributed to marked variations in coloration related to the effects of sexual dichromatism (sex inversion, state of sexual maturity) (Zei, 1941; Lozano Cabo, 1951, 1953; Lepori, 1959). Many Mediterranean fishes show notable chromatic and morphological modifications especially in the juvenile phase and during the reproductive period (Tortonese, 1975). The old classification distinguished the two genera Maena and Smaris that were subsequently fused in a single genus Spicara (Tortonese, 1975), which comprises three species: Spicara flexuosa (Rafinesque, 1810), S. maena (Linnaeus, 1758) and S. smaris (Linnaeus, 1758). Currently, Spicara smaris is a very characteristic species, whereas S. maena may be distinguished from S. flexuosa owing to the presence in the former of well-developed teeth on the vomer, to its head being shorter than the body depth, to sexual behaviour, and to variations in coloration (Tortonese, 1975, 1986). According to Tortonese (1975), S. flexuosa should be considered as a colour polymorphism of S. maena. Pollard & Pichot (1971), in reviewing this genus Tel.: +39 91 6621020; fax: +39 91 6172009 1317 0022–1112/96/121317+06 $25.00/0 ? 1996 The Fisheries Society of the British Isles 1318 .    . through electrophoretic studies of densitometric eye-lens proteins and morphometric analysis, proposed the presence of only two species: S. smaris and S. chryselis. The latter is a synonym of S. flexuosa (Tortonese, 1975). Since genetic differences between species can be evaluated through electrophoretic analysis of proteins (Altukhov, 1982; Ayala, 1983; Carvalho et al., 1991), in the present research, electrophoretic analysis of several enzymes and myogens were carried out, and the genetic distance between S. flexuosa, S. maena and S. smaris was determined using Nei’s (1978) index. MATERIALS AND METHODS SAMPLES Samples of S. flexuosa (n=58), S. maena (n=55) and S. smaris (n=55) were obtained from the Gulf of Palermo, Sicily, Italy. The individuals were identified according to Tortonese (1975). The frozen fishes were taken to the laboratory, and kept at "20) C until used. Tissue from eye, heart, liver and muscle were homogenized in one volume of NaCl 1% at 0) C. The extracts were centrifuged at 6000 g at 4) C for 30 min, and the supernatant was paper-filtered to remove the lipid layer. When not used immediately the homogenates were stored at "80) C. ELECTROPHORETIC ANALYSIS Polyacrylamide gel slab electrophoresis (PAGE) was carried out as described by Davis (1964). The sample, 1–5 ìl sample-buffer, was deposited into each well of the spacer gel (16#16 cm, 2 mm thick) and run vertically at a constant current of 40 mA. When examined for myogen patterns (PMM) the gel was stained with Coomassie Brilliant Blue (Merril, 1990). GEL STAINING FOR ISOENZYMES Nomenclature for protein-coding loci and alleles followed the recommendations by Shaklee et al. (1990) (Table I). Running buffers and staining procedures were those previously reported by Richardson et al. (1986) and Cammarata et al. (1991). Before staining, the gel slabs were incubated in the appropriate reaction mixture at 37) C until the bands were visualized; the reaction was stopped by rinsing with water and adding preservative solution (7% acetic acid). STATISTICS A locus was considered as polymorphic, when the frequency of the most common allele was <0·95 (Ayala, 1975). Genetic distance (D) was calculated from the formula proposed by Nei (1978) using the BIOSYS-1 program (Swofford & Selander, 1981). The obtained values were clustered in the UPGMA algorithm using the NTSYS (Rohlf, 1988) program which gives hierarchical levels according to genetic similarity. RESULTS Eighteen enzymes were resolved, and 28 loci scored in the three species (Table I). In addition, several loci encoding for myogens were examined. Of the 28 enzymatic and additional myogenic loci, five monomorphic loci (LDH-1*, G6PD*, PGI-1* and two PMMs*) were species-specific between Spicara smaris v. S. flexuosa and S. smaris v. S. maena, whereas no discriminating monomorphic locus was identified between S. flexuosa and S. maena       1319 T I. Enzymes stained, with E.C. No., abbreviation and loci scored Enzyme E.C. No. Abbreviation Loci scored Alcohol dehydrogenase Sorbitol dehydrogenase Lactate dehydrogenase Malate dehydrogenase Glucose dehydrogenase Glucose-6-phosphate dehydrogenase Isocitrate dehydrogenase Xanthine dehydrogenase Xanthine oxidase Superoxide dismutase Aspartate aminotransferase Adenylate Kinase Phosphoglucomutase Peptidase 1.1.1.1 1.1.1.14 1.1.1.27 1.1.1.37 1.1.1.47 1.1.1.49 1.1.1.42 1.2.1.37 1.2.3.2 1.15.1.1 2.6.1.1 2.7.4.3 2.7.5.1 3.4.– – 3.4.13.9 3.1.1.1 4.2.1.2 5.3.1.9 na ADH* L SDH* L LDH-1,2,3* E MDH-1,2* E GLDH* L G6PD* H IDH* L XDH* L XO* L SOD* L, M AAT* L AK-1,2* L PGM-1,2* M PEPB-1,2* L PEPD* L EST-1,2,3* L FUM-1,2* M PGI-1,2* E, L, M Several M Esterase Fumarate hydratase Glucose phosphate isomerase General muscle proteins ADH SDH LDH MDH GLDH G6PD IDH XDH XO SOD AAT AK PGM PEPB PEPD EST FUM PGI PMM Tissue na: not applicable; E: eye; H: heart; L: liver; M: muscle. (Table II) (Fig. 1). In particular, LDH-1*, G6PD-1* and PGI-1* monomorphic loci were fixed with different alleles in these species. Allele *93 of the LDH-1* locus was exclusive of S. smaris and allele *100 of the LDH-1* locus was present in S. flexuosa and S. maena; allele *71 of the G6PD-1* was found in S. smaris while allele *100 of the G6PD-1* was observed in S. flexuosa and S. maena; allele *78 of the PGI-1* locus was specific for S. smaris, and allele *100 of the PGI-1* locus for S. flexuosa and S. maena. Two markers were present in the myogenic electrophoretic patterns. Of the 28 enzymatic loci scored, five were polymorphic; the polymorphic locus EST-2* presented unclear patterns and was not considered further. In order to estimate the differences among genotypes of the three species (S. smaris, S. flexuosa and S. maena), the genetic distances (D) were calculated using Nei’s (1978) index. The distance values between S. smaris and S. flexuosa (D=0·141) and between S. smaris and S. maena (D=0·137) were rather large, whereas the distance between S. flexuosa and S. maena were only D=0·006 (Fig. 2). DISCUSSION Although the genetic variation limited to protein encoding loci may lead to an underestimate of genetic diversity, our findings reveal that they were useful to discriminate among the Spicara species group. Allozyme and myogen diversity did not reflect the systematic relationships reported through analyses of morphological characters and species biology (Tortonese, 1975). Spicara smaris appeared to diverge from S. flexuosa and S. maena in the expression of five monomorphic loci of the 28 loci scored. The alleles of the LDH-1*, G6PD-1*, 1320 .    . T II. Allele frequencies in Spicara flexuosa, Spicara maena and Spicara smaris Loci ADH* SDH* LDH-1* LDH-2* LDH-3* MDH-1* MDH-2* GLDH* G6PD-1* IDH* XDH* XO* SOD* AAT* AK-1* AK-2* PGM-1* PGM-2* PEPB-1* PEPB-2* PEPD* EST-1* EST-3* FUM-1* FUM-2* PGI-1* PGI-2* Alleles S. flexuosa S. maena S. smaris *100 *100 *93 *100 *100 *100 *100 *100 *95 *100 *135 *156 *71 *100 *100 *100 *100 *100 *100 *100 *100 *100 *100 *100 *100 *87 *100 *104 *83 *90 *92 *100 *110 *100 *100 *100 *78 *100 *94 *100 *109 1 1 0 1 1 1 1 1 0 0·36 0·13 0·51 0 1 1 1 1 1 1 1 1 1 1 1 1 0·21 0·63 0·16 0·12 0·03 0·03 0·56 0·25 1 1 1 0 1 0·00 0·97 0·03 1 1 0 1 1 1 1 1 0·14 0·23 0·45 0·18 0 1 1 1 1 1 1 1 1 1 1 1 1 0·29 0·52 0·19 0·00 0·15 0·15 0·58 0·12 1 1 1 0 1 0·04 0·96 0·00 1 1 1 0 1 1 1 1 0·15 0·77 0·04 0·04 1 0 1 1 1 1 1 1 1 1 1 1 1 0·05 0·74 0·21 0·00 0·07 0·00 0·61 0·32 1 1 1 1 0 0·11 0·89 0·00 PGI-1* monomorphic loci and two PMMs*, were species-specific genetic markers for S. smaris. The level of genetic diversity expressed by Nei’s index showed a value of D=0·141 for S. flexuosa v. S. smaris and D=0·137 for S. maena v. S. smaris which are in agreement with the values for congeneric fish species reported by Thorpe (1982). The genetic distance between S. flexuosa v. 1321       1 LDH-1 2 3 1 G6PD 2 3 1 PGl-1 2 3 1 PMM 2 3 F. 1. Species-specific markers of monomorphic loci in the genus Spicara: 1, S. maena; 2, S. flexuosa; 3, S. smaris. 0.20 0.15 0.10 0.05 0.00 1 2 3 F. 2. UPGMA dendrogram based on Nei’s (1978) index (D). 1, Spicara flexuosa; 2, S. maena; 3, S. smaris. S. maena showed a value of D=0·006 which parallels their morphological similarity. According to Pollard & Pichot (1971), who studied the eye-lens proteins by densitometry and morphological characters, and Tortonese (1975), who described the weak discriminatory power of specific morphological characters (chromatic variability, presence or absence of teeth on the vomer), our results suggest that in the genus Spicara, S. maena and S. flexuosa can be distinguished easily from S. smaris. Therefore, it is necessary to investigate specifically the relationship between S. flexuosa and S. maena to verify the hypothesis of Pollard & Pichot (1971) and Tortonese (1975) who considered S. flexuosa to be a chromatic ecophenotype or habitat-coloration of S. maena: our data support such an hypothesis. Further research by means of additional protein loci and DNA analysis, will contribute to evaluate the degree of variability between S. flexuosa and S. maena. The authors are grateful to Y. Altukhov, Moscow (Russia), for his valuable comments on the manuscript. 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