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
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? 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. Financial support was given by Ministero dell’Università e Ricerca
Scientifica e Tecnologica 60%/1993.
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