]. mar, biol. Ass. U.K. (1994), 74,211-223 211 Printed in Great Britain GENETIC VARIATION, SYSTEMATICS AND DISTRIBUTION OF THE VENERID CLAM CHAMELEA GALLINA THIERRY BACKELJAU, PHILIPPE BOUCHET*, SERGE GOFAS* AND LUC DE BRUYN+ Koninklijk Belgisch Instituut voor Natuurwetenschappen, Afdeling Malacologie, Vautierstraat 29, B-1040 Brussels, Belgium. ‘Muséum National d'Histoire Naturelle, Laboratoire de Biologie des Invertébrés Marins et Malacologie, 55 Rue de Buffon, F-75005 Paris, France. TJniversiteit Antwerpen, Departement Biologie, Groenenborgerlaan 172, B-2020 Antwerpen, Belgium Two morphotypes of the venerid bivalve Chamelea gallina (L.), viz. C. gallina s.s. and C. striatula, were electrophoretically compared at seven polymorphic enzyme loci. In three populations from the Ría Formosa (southern Portugal), both morphotypes occurred sympatrically. Analyses of genotype frequencies in these mixed populations revealed departures from Hardy-Weinberg expectations at nearly all loci. These deviations were mainly attributable to a Wahlund effect, caused by mixing the two morphotypes. Nei's mean unbiased genetic distance between the two forms was D=1138, while the mean genetic distances between populations within morphotypes were D=0 083 in C.gallina s.s. and 0=0-229 in C.striatula. It is therefore concluded that C. gallina and C. striatula are reproductively isolated (biological) species, the geographical distribution of which is outlined. INTRODUCTION Chamelea gallina (Linnaeus, 1758) is an infaunal venerid bivalve, which is common in shallow water sand or mud habitats along the European coasts. Although the species is commercially exploited in the Mediterranean (e.g. Froglia, 1975), its taxonomy is still confused. There are three opinions about this issue. The first regards C. gallina as a single, widely distributed, polymorphic species (e.g. Dodge, 1952). The second assumes that C. gallina is a complex of two very similar, yet distinct, species, viz. the Mediterra­ nean C. gallina sensu stricto and the Atlantic C. striatula (Da Costa, 1778) (e.g. Spada & Maldonado Quiles, 1974). The third opinion relies on the apparent geographical separa­ tion of the two taxa to give them subspecific rank (e.g. Van Aartsen et al., 1984). In all cases the issue has been mostly a matter of opinion, the more recent papers quoting older ones without adding new evidence. The present study was prompted by the observation that C. gallina and C. striatula occur sympatrically in the Ría Formosa, southern Portugal. The two morphotypes differ by the outline of the shell (more pointed posteriorly in C. striatula), and by the shape and number of the concentric ridges (low, often bifurcated, 'fingerprint-like' ridges in C. gallina, versus acute, more numerous and rarely bifurcated ridges in C. 212 T. BACKELJAU, P. BOUCHET, S. GOFAS A N D L. DE BRUYN Figure 1. Left valves of (A-C) Chamelea gallina and (D-F) Chamelea striatula from the Ría Formosa. A Chamelea gallina 5 mm r \ B Chamelea striatula Ci/iîM WWW) Figure 2. Details of the siphons of (A) Chamelea gallina and (B)Chamelea striatula in the Ría Formosa. GENETIC VARIATION IN CHAMELEA GALLINA 213 striatula) (Figure 1). The lunula of C. gallina is heart-shaped and truncated towards the apex; that of C. striatula is more leaf-shaped. The two morphotypes can also be distinguished by their siphons. In C. gallina they are short, stout and mottled with yellow and violet dots, while the blunt tentacles are covered by orange spots which are lacking elsewhere (Figure 2). This type of siphon is similar to that figured by Amouroux (1980) for specimens from Banyuls, French Medi­ terranean. The siphons of C. striatula, on the contrary, are longer, more slender, and completely covered by yellow and orange spots. The tentacles are colourless. The palliai sinus reflects the differences in siphonal morphology for it is deeper in shells of C. striatula. We conducted an electrophoretic analysis of C. gallina sensu lato in the Ría Formosa to test whether the two morphotypes described above are reproductively isolated and may represent biological species sensu Mayr (1969). MATERIAL AND METHODS Live Chamelea gallina s.Z. were collected by hand-picking and diving in the Parque Natural da Ría Formosa, southern Portugal (Figure 3) in May-June 1988 (mission ALGARVE 88). Locality data and morphotype composition of the populations are provided in Table 1. Each sampling site covered only a few m2. Morphotypes were identified using the shell and siphon characteristics outlined above. Specimens were transported in liquid nitrogen and stored at -80°C. Total body homogenates of 154 clams were prepared by homogenizing each animal in a 20% (w/v) aqueous sucrose solution (5 pi mg'1 tissue). Crude homogenates were centrifuged during 45 min at -27000# (15000 rpm) and at ~4°C. Vertical Polyacrylamide Gel Electrophoresis (PAGE) was performed as described by Backeljau (1989), using two buffer systems: a discontinuous one with tris/glycine (pH 9-0) in the tray and tris/HCl (pH 9-0) in the gels, and a continuous one with tris/citric acid (pH 8-0) in both tray and gels. Seven polymorphic enzymes were surveyed. The discontinuous buffer system was employed to resolve superoxide dismutase (SOD, E.C. 1.15.1.1). The tris/citric acid buffer was used to resolve glucose-6-phosphate isomerase (GPI, E.C. 5.3.1.9), glycerol-3-phosphate dehydrogenase (GPD, E.C. 1.1.1.8), malate dehydrogenase (MDH, E.C. 1.1.1.37), malic enzyme (ME, E.C. 1.1.1.40), 6- phosphogluconate dehydrogenase (PGD, E.C. 1.1.1.44) and xanthine dehydrogenase (XDH, E.C. 1.2.3.2). All enzymes were stained according to Harris & Hopkinson (1976). Alleles were designated alphabetically according to decreasing electrophoretic mobilities (A=fastest allele or most anodal position). Previously typed specimens were included with each run in order to compare different gels. Data were analysed with the BIOSYS-1 computer package (Swofford & Selander, 1981). For each population we determined allele frequencies, mean observed heterozy­ gosities (H0), mean Nei's (1978) unbiased expected heterozygosities (He) and heterozy­ gote deviations [DH=(H0-He)/H J. Genotype frequencies were tested for departures from Hardy-Weinberg (HW) equi­ librium expectations with the exact probability test implemented by BIOSYS-1. For loci 214 T. BACKELJAU, P. BOUCHET, S. GOFAS A N D L. DE BRUYN FARO J V ILHA /DA CULATRA tLHA D A ^ -~ BARRETA 10 km Figure 3. Map of the Iberian Peninsula showing the sampling localities ofChamelea gallina s.l. Figures refer to the population numbers in Table 1. Table 1. Locality data of the Chamelea gallina s.l. populations sampled in the Ría Formosa. Stn Date Locality Nt n g N s Habitat 1 30-05-1988 Canai de Olhâo 34 23 11 Sand, depth: 5m 2 02-06-1988 Ilha da Barreta 31 31 - Sand, at low tide 3 06-06-1988 Canai de Olhâo, Culatra 28 19 9 Muddy sand, depth: 7m 4 09-06-1988 Canai de Olhâo, Hangares 61 19 42 Fine sand, depth: 5m Nt/ total numbers of individuals; NG, numbers ofChamelea gallina; with more than two alleles, data were pooled in three genotype classes: (1) homozygotes for the most common allele, (2) all heterozygotes involving the most common allele, and (3) all other genotypes. Finally, Nei's (1978) unbiased genetic identities (I) and distances (D) between popula­ tions were calculated. The I values were used to construct a UPGMA dendrogram. The shells of the specimens studied are deposited in the collections of the Koninklijk Belgisch Instituut voor Natuurwetenschappen (I.G. no. 27353), Brussels. GENETIC VARIATION IN CHAMELEA GALLINA 215 RESULTS The seven enzymes surveyed were assumed to be coded by single loci. Allele frequencies at these loci are presented in Table 2. Table 2. Allele frequencies in the two morphotypes of Chamelea gallina s.l. in the Ría Formosa. Locus 1G ÍS 2G 3G 3S 4G 4S MDH (N) 18 11 31 19 9 19 42 A 0.028 0.000 0.000 0.026 0.000 0.000 0.000 B 0.000 1.000 0.000 0.000 1.000 0.000 0.988 C 0.972 0.000 1.000 0.948 0.000 1.000 0.012 D 0.000 0.000 0.000 0.026 0.000 0.000 0.000 ME (N) 11 10 30 19 9 17 42 A 0.000 0.000 0.000 0.000 0.111 0.000 0.000 B 0.000 0.000 0.000 0.000 0.444 0.000 0.500 C 0.091 0.900 0.033 0.105 0.222 0.000 0.214 D 0.909 0.100 0.467 0.368 0.000 0.235 0.262 E 0.000 0.000 0.100 0.053 0.000 0.000 0.000 F 0.000 0.000 0.400 0.474 0.222 0.706 0.024 G 0.000 0.000 0.000 0.000 0.000 0.059 0.000 XDH (N) 18 10 29 19 9 19 42 A 0.000 0.100 0.000 0.000 0.278 0.000 0.155 B 0.167 0.900 0.000 0.000 0.111 0.000 0.250 C 0.777 0.000 0.414 0.474 0.555 0.053 0.571 D 0.056 0.000 0.552 0.526 0.056 0.894 0.024 E 0.000 0.000 0.034 0.000 0.000 0.053 0.000 PGD (N) 23 11 26 19 9 19 42 A 0.000 0.000 0.000 0.000 0.000 0.000 0.012 B 0.000 0.000 0.000 0.000 0.000 0.026 0.012 C 0.000 0.000 0.019 0.000 0.000 0.000 0.000 D 0.044 0.182 0.096 0.132 0.333 0.053 0.202 E 0.000 0.000 0.000 0.000 0.000 0.000 0.012 F 0.891 0.773 0.846 0.868 0.556 0.921 0.702 G 0.065 0.045 0.039 0.000 0.111 0.000 0.060 GPI (N) 18 11 31 19 9 19 39 A 0.000 0.000 0.000 0.026 0.000 0.000 0.038 B 0.043 0.000 0.000 0.026 0.000 0.053 0.026 C 0.043 0.000 0.161 0.184 0.056 0.158 0.077 D 0.065 0.228 0.177 0.133 0.056 0.053 0.090 E 0.174 0.228 0.097 0.105 0.222 0.235 0.231 F 0.261 0.045 0.145 0.158 0.277 0.079 0.064 G 0.239 0.136 0.242 0.263 0.111 0.211 0.256 H 0.022 0.182 0.048 0.000 0.111 0.132 0.064 I 0.131 0.045 0.113 0.079 0.167 0.053 0.090 I 0.022 0.091 0.017 0.026 0.000 0.026 0.038 K 0.000 0.045 0.000 0.000 0.000 0.000 0.026 SOD (N) 19 9 31 18 1 19 28 A 0.132 0.000 0.113 0.167 0.000 0.079 0.018 B 0.842 0.444 0.839 0.805 0.000 0.895 0.268 C 0.000 0.232 0.000 0.000 0.000 0.000 0.143 D 0.026 0.222 0.048 0.028 1.000 0.026 0.285 E 0.000 0.000 0.000 0.000 0.000 0.000 0.143 F 0.000 0.112 0.000 0.000 0.000 0.000 0.107 G 0.000 0.000 0.000 0.000 0.000 0.000 0.036 216 T.