J. Exp. Mar. Biol. Ecal., 1990, Vol. 143, pp. l-14 Elsevier

JEMBE 01489

On the occurrence of ~y~Z~s gffZZu~~~vi~ci~Zi~ Lmk. on the N.W. coast of the Iberian Peninsula

A. Sanjuan, H. Quesada, C. Zapata and G. Alvarez Departamento de Biologia Fundamental, Facultad de Biologia, Universidad de Santiago de Compostela,

Santiago de Compostela, Spain

(Received 1 March 1990; revision received 4 June 1990; accepted 23 June 1990)

Abstract: The blue mussel Mytilus edulis L. and its southern relative, M. galloprovincialir Lmk., are two forms of mussel that live and are cultivated on the coasts of Europe. Up to date, the type or types of mussel that exist on the Atlantic coast of the Iberian Peninsula have not been clearly determined even though the N.W. comer of this region (Galician Rias) has the highest mussel production in the world. Diagnostic morpho- logical characters (length of anterior adductor muscle scar and size and shape of the hinge plate) and diagnostic enzyme-gene polymorphisms (esterase-D, ieucine aminopeptidase-1, octopine dehydrogenase and mannose phosphate Lsomerase) for M. edul& and M. gai~oprovincial~ were investigated in samples of mussel populations (wild and cultivated) collected from N.W. Iberian Peninsula coastal sites. Evidence from both morphological variation and enzyme polymorphisms indicates that iU. gaZlopro~ncja2~ commonly occurs on the N.W. Iberian Peninsula. On the other hand, no evidence for the occurrence of M. edulir was found. The results are discussed in relation to the geographical dist~bution of both types of Myt#as on European coasts.

Key words: Biogeography; Iberian Peninsula; My~~~~; Population genetics; Protein ~l~o~hism

INTRODUCTION

Myth edulis L. and Myths galloprovincialis Lmk. are two forms of mussels that live on the coasts of Europe. At present, these two mussels are recognized on morphological and genetical criteria, although the systematic status of iU. galloprovincialis is uncertain since it could be considered either as a different biological species or a subspecies or race of M. edulis (see Seed, 1978; Skibinski et al., 1980, 1983; Gosling, 1984). On the other hand, the distribution of these two mussels on the coasts of Europe has been controversial and, at the moment, is not completely known. M, edulis, has traditionally been considered as an Atlantic form and M. galloprovincialis as a Mediterranean form, but the separation of the two types of mussels based on shell morphology allowed the identification of M. ga~loprovinc~~is coexisting with M. edulis on large areas of the Atlantic coasts of Europe: SW. England (Hepper, 1957; Lewis & Seed, 1969), France (Seed, 1972) and Ireland (Seed, 1974). The analysis of several protein loci confumed the presence of iw: ga~~oprov~~ia~~ on the coasts of the British Isles (Ahmad &

Correspondence address: G. Alvarez, Departamento de Biologla Fundamental, Facultad de Biologia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

0022-0981/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

1 A. SANJUAN ET AL.

Beardmore, 1976; Skibinski et al., 1978, 1983) as well as the varying intermixing between n-S. edufis and M. gaffo~~#ui~~jaf~s (Skibinski et a%., 1978, 1983 ; Skibinski & Beardmore, 1979; Gosling & Wilkins, 1981; Skibinski, 1983). Recently, the existence of a genetically distinct form of M. edulis has been found in the Baltic Sea and some localities on the Canadian coast (Koehn et al., 1984; Varvio et al., 1988; Bulnheim & Gosling, 1988; Johannesson et al., 1990) and a taxonomic status similar to that of iw. edufis and M. g~if~~ruvjnciafi~ has been demanded for this mussel which has been termed M. tmssufus (Koehn et al., 1984; McDonald & Koehn, 1988; Varvio et al., 1988).

Presently, the fberian Peninsula is the only area on the !&antic coast of Europe where the distribution of 1w. eduf~s and M. guff~~r~vin~iaffs is not clearly es~blished. Thus, whiIe some authbrities have suggested that 1Lp. g~fl~~~~vj~cj~l~~ is the predominant mussel on the iberian Peninsula Attantic coast flubet, 1973, 1976), others are of the opinion that M. edulis extends over the whole of the Atlantic littoral of Europe as far as N. Af~cainclusive (Seed, 19761978; Suchanek, 1985). However, a study of several morphological characters (not including the best diagnostic morphological traits) in mussel populations from the N.W. Atlantic coast of Spain indicated that these mussels present intermediate forms between the typical eduiis and gaff~~ravjnc~ufjs form (Figueras & Figueras, 1983). Therefore, no clear conclusion has been obtained to date with regard to the identity of the mussels living on the coasts of the Iberian Peninsula.

To determine the biologicat identity of the Iberian Pe~~suia mussels is ofinterest for several reasons. The Atlantic coast of the Iberian Peninsula is a critical area for understanding the occurrence of M. gaff5~r~vi~~~~fi~ on the N.W Atlantic coast of Europe (British Isles and Atlantic coast of France), since it is a geographically inter- mediate area between N.W. coasts and the Mediterranean Sea, Secondly, the Iberian Peninsula is a very interesting area from the point of view of mussel production. Spain is the world’s leading producer of mussels with a current annual production of =: ~00000-200000 t, almost ail from the Galician Rias in the N.W. of the Iberian Peninsula (Korringa, 1976; Mason, 1976; Figueras, 1976; FROM, 1987; FAG, 1988). The aim of this work is, therefore, to identify the type or types of musseis that live and are cuhivated on the N. W. coast of Iberian Peninsula. For this purpose, both diagnostic mvrpho~o~cal characters and atbzyme loci will be studied in mussel populations from this geographicd area.

MATERIALS AND METHODS

Mussel populations were sampled from the intertidal region at four sites along the N. W. coast of the Iberian Peninsula (Gahcian Rias, N.W. Spain) during April to August 1987 (Fig. 1). The samples were collected as close as possible to the low level of the intertidal vertical range of the mussel bed. In March 1988, a fifth sample (Aidan) was collected on submerged ropes hung from the mussef culture rafts (Fig. 1). Mussels were

~YTIL~S ~ALLOPRO~~~~~A~~S IN THE XBERIAN PENINSULA 3

Fig. 1. Location of sampling sites on N.W. coast (B)of Iberian Peninsula (A): (1) Sada; (2) Laxe; (3) Carril; (4) Pontevedra and (5) Aldan.

returned alive to the laboratory where they were dissected. The digestive gland was removed from each ~~vidu~ and stored at - 40 “C until required. For comparative purposes, pure populations of M. galloprovincialis from the Mediterranean coast of the Iberian Peninsula and M. edulis from the Atlantic coast of Europe were also studied. Samples of M. galloprovincialis populations from Benicasim, Cullera and Almeria were studied for morphological characters, and populations from Marbella and Tarragona were sampled for electrophoretic analyses. Populations of 1M. edulis from Helsingor and Blavands (Denmark), Anglesey (British Isles) and Dieppe (France) were sampled for

4 A. SANJUAN ETAL

the study of morphologic~ traits and a population from The Netherlands was sampled

for electrophoretic analyses.

Several morphological traits used to distinguish typical M. gulfoprovinciulis from

typical M. edulis (Lewis & Seed, 1969; Seed, 1974, 1978; Verduin. 1979; Beaumont

et al., 1989) were studied. The length of the anterior adductor muscle scar was measured

in dry shells using a pair of dividers and the ratio of the scar length to the shell length

was calculated (AAS x lOOO/,). The length of the hinge plate is a difficult character to

measure accurately, but the hinge plate ratio (HP x 1000/Z) is a useful diagnostic index

(Lewis & Seed, 1969; Seed, 1972). The internal radius of the hinge plate was measured

to compute the radius ratio (R x 10/r) according to Verduin (1979). We have also

calculated a mixed index proposed by Verduin (1979) that uses a combination of

characters (length adductor plate/height shell + f.5 x internal radius of the hinge

plate/height shell) which tends to be co.25 in M. gal~oprovincialjs and > 0.25 in

M. cd&s. Four allozyme polymorphisms effective in identifying mussel forms (diagnostic loci)

were investigated by starch-gel electrophoresis (see Ahmad & Beardmore, 1976;

Skibinski et al., 1978, 1983; Grant & Cherry, 1985; Beaumont et al., 1989). Tissue

samples were prepared from the digestive gland and the homogenization was carried

out in an equal volume of 0.01 M dithiothreitol solution. Prior to electrophoresis, all the

samples were centrifuged at 9000 rpm for 7 min. The supernatant was used as the

enzyme source for e~ectrophoresis, which was performed at 4 “C in horizontal

starch-gel. Gels consisted of lo-12 “/;, hydrolysed potato starch (Sigma Chemical).

Enzymatic systems were esterase-D (Est-D), leucine a~i~opept~da.~e-~ (Lap-I), octopine dehydrogenase (Odh) and mannose phosphate isomerase (Mpi). Electrophoretic proce-

dures and terminology for Est-D and Lap-l allozymes were essentially those used by

Ahmad et al. ( 1977), and for Odh were those described by Grant & Cherry (1985). For

IMpi, the buffers were as follow: electrode buffer: 100 mM Tris. 100 mM malic acid.

100 mM EDTA, 20 mM Mg Cl, and the pH adjusted to 7.4 with NaOH; gel buffer was

a 1 : 9 dilution of electrode buffer.

The traditional measures of genetic structure of populations, Wright’s F statistics

(I;;,, Fss and FIT), were used to analyse allozyme genotypic frequencies of mussel

populations. F statistics were calculated by the method of Nei (1977, 1986; see also Nei

& Chesser, 1983). Estimation of F in each mussel population was carried out by a

statistic developed by Robertson & Hill (1984). This estimate is unbiased for F = 0 and

a significance test for F = 0 exists which has a statistical power higher than the usual

x2 test. Estimation of Nei’s unbiased genetic distance (Nei 8c Roychoudhury, 1974; Nei,

1978) was carried out among pairs of populations. The genetic distance matrix was used

to perform an hierarchical cluster analysis using the unweighted pair-group method with

arithmetic averaging (UPGMA). F statistics and genetic distance analyses were carried

out using the Biosys- 1 computer program of Swofford & Selander (198 1).

MYl’fLUS GALLOPRG~~~~IALZS IN THE IBERIAN PENINSULA 5

RESULTS

Frequency distributions of different morphological characters in Galician mussel populations (N.W. Iberian Peninsula) and A4. gulloprovincialis and M. edulis control samples are shown in Fig. 2, and a summary of the descriptive statistics corresponding to these distributions is presented in Table I. In Fig. 2, the control distributions of

TABLE I

Means and standard errors of anterior adductor scar (AAS x 1000/Z), hinge plate (HP x lOOO,V), hinge plate interior radius (R x IO/Z) ratios and IV for Galician mussel ~pulations (N.W. Iberian Peninsula) and

1M. gaZ~oprovincjaZ~ and hi. eduiis control samples. n, number of indi~d~s scored.

Mussel populations n AAS x 1000/I HP x 1000/l R x IO/l IV

M. edub Helsingar 43 102.2 + 3.3(**) 74.7 f 2.2 0.452 f 0.022 0.339 + 0.009 B&and 60 100.2 + 2.1 73.8 + 1.2 0.404 * 0.014 0.320 & 0.006 Anglesey 17 87.2 f 3.3 77.4 + 3.6 0.354 f 0.012 0.293 + 0.009 Dieppe 29 99.9 f 2.2 70.9 & 1.7 0.414 f 0.018 0.337 + 0.009

M. galloprovincialis Benicasim 100 36.4 f 1.0 53.4 * 0.7 0.164 + 0.004 0.112 + 0.002 Cullera 74 49.8 f 1.8 64.8 & 1.1 0.194 f. 0.006(**) 0.129 + 0.003 Almeria 93 51.0 + 1.5 58.6 + 0.8 0.231 + 0.007 0.159 + 0.004

Galieian populations Sada 130 61.4% 1.2 63.3 & 0.7 0.243 + O.~q*) 0.174 + 0.003 Laxe 108 63.9 f 1.3 58.5 f 0.9 0.185 + 0.005 0.160 if: 0.003 Carril 112 64.9 f 1.1 61.3 + 0.8 0.211 f o.OOs(*) 0.171 * 0.002 Pontevedra 110 82.4+ 1.3 64.1 + 0.9 0.255 + 0.006 0.224 rt: 0.003(*) Aldan 137 48.0 f 1.1 56.3 + 0.6 0.189 & 0.004 0.149 f 0.002

*p < 0.05 **p < 0.01 (Kolmogorov-Smimov test of goodness of tit to normal distribution).

M. edulis and A4. galloprovincialis are obtained by pooling the Helsingor, Blavands, Anglesey and Dieppe, and the Benicasim, Cullera and Almeria samples, respectively. The mean ratios of the anterior adductor muscle scar to shell length (AAS x 1000/l), the hinge plate length to shell length (HP x 1000/l), the hinge plate internal radius ratio (R x IO/l) and the index of Verduin (IV = AAS/H + 1.5 ~~~) are usually lower in &6 gallopr~v~n~iulis than M. edulik as has been shown by Lewis & Seed (1969), Seed ( 1974,1978) and Verduin ( 1979) and the same occurs in our control samples of A4. edulis and M. galloprovincialis (Table I, Fig. 2). Frequency distributions of the four ratios in Galician mussel populations sampled appear to be unimodal and there is no evidence of bimodality (Fig. 2), as would happen if both types of mussel, M. edulis and M. gullo- provincialis, occur together. Thus, Kolmogorov & Smimov tests for goodness of fit to the normal distribution (Sokal & Rohlf, 1981) did not show significant deviations in most Galician samples. Mean ratios of the four indices of Galician mussel samples are located in the range of typical M. galloprovinciuIis (see Seed, 1978). The Pontevedra sample is the only population that presents a certain proportion of individuals with

A. SANJUAN ET AL

1. v. HPx 1000/L -

J!i-

AASxl 000, L

I

1 L 0 }Al_L._

c

I

i’ / t

I c 0.4 0.8

Ll 0

% 33

20

10

0

% _JJi

$ 0 0.25 0.4

A AS x lOOO/ L n P x1coo/ L R x 101 I. I. v.

Fig. 1. Histograms showing distributions of ratios of anterior adductor muscle scar/shell length fAAS x I~/~); hinge plate ie~h~sbell length (HP x 1~~~); hinge plate internal radio/shell length (R x lOi/) and IV in Galician mussel populations (2nd-5th row). Distributions of typical M. edulis

( :-T*,+*:*: ) and M. galloprovincialis ( A\\$ ) are shown in first row.

MYTILUS GALLOPROVINCIALIS IN THE IBERIAN PENINSULA 1

values of typical M. edulis (10.9% individuals > 100 for the anterior adductor scar ratio, 25.6% > 0.30 for the radius ratio; and 20.9% individuals > 0.25 for IV). This site was chosen deliberately because previous morphological evidence showed similar results (unpubl. data).

The results of the electrophoretic analyses are presented in Table II. The four allo- zyme loci included in these analyses (i.e., Es&D, Lap-l, Odh and Mpi) are partially diagnostic for M. edulis and A4. galloprovincialis (Ahmad & Beardmore, 1976; Skibinski et al., 1978, 1983; Grant & Cherry, 1985; Varvio et al., 1988). The allele frequency distributions at the four loci in our control samples of M. edulis (The Netherlands) and M. gulloprovincialis (Marbella and Tarragona on S. and S.E. Iberian coasts) agree with those shown by Ahmad & Beardmore (1976), Skibinski et al. (1978,1980,1983), Grant & Cherry (1985), Varvio et al. (1988) and Bulnheim & Gosling (1989) for both types of mussels. It is interesting to note that alleles Odh’” and 0dhlz9 present geographical variation between Marbella and Tarragona samples (Mediterranean coast) but this does not modify the diagnostic value of Odh, i.e., the Odh II5 allele is associated with A4. edulis while the Odh”’ and 0dh’29 alleles are associated with A4. galloprovincialis. On the whole, the Galician mussel populations show allele frequencies for the four allozyme loci that correspond essentially to M. galloprovincialis (Table II). The genetic similarities of Galician mussel populations and M. gulloprovinciulis populations from the Medi- terranean coast can be easily observed in the dendogram (UPGMA) of Fig. 3, built from

GENETIC DISTANCE

2.0 1.5 1.0 0.5

SADA

LAXE

MARBELLA

CARRIL

ALDAN

PONTEVEDRA

L TARRAGONA

HOLLAND

DENMARK

Fig. 3. UPGMA cluster analysis of genetic distance from allozyme frequencies at four loci for Galician mussel populations and M. gulZoprovinciab (Tarragona, Marbella) and M. edulis (The Netherlands,

Denmark) control samples.

Nei’s unbiased genetic distance for the four diagnostic loci, where two clusters are detected: one with M. edulis populations and the other with Mediterranean (M. gallopro- Vinci&is) and N.W. Iberian samples. Note that genetic distances are not used in our

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MYTILUS GALLOPROVINCIALIS IN THE IBERIAN PENINSULA 9

for phylogenetic inferences, given that we did not use a random sample of loci, but instead used loci known to be useful in discriminating h4. galloprovincialis from M. edulis.

It is worth noting that, in spite of the fact that four allozyme loci considered in our study being only partial diagnostic, the probability of misclassification at the individual level is very low. Individuals of a population can be assigned to one of the two types of mussels by using the allozyme data of pure populations of M. galloprovincialis and M. edulis in accordance with the methods of Ayala & Powell (1972) and Ward & Janson (1985). Thus, in the sample of Pontevedra, 20.9% individuals exceed the discriminant value of the morphological Index of Verduin (IV = 0.25). In the extreme supposition that this sample had 20.9% individuals of A4. edulis, the probability of misclassification by locus (R) are: REst_D = 0.01, R,,_, = 0.01, R,, = 0.05 and RMPi = 0.03. That is, z 99% of individuals can be assigned to the correct types simply from Es&D genotype. If all loci are considered together, the probability of misclassifying an individual is R, = 1.5 x 10 - 7. On this basis, all specimens of the Pontevedra sample can be assigned to the M. galloprovincialis form. This is in accordance with the observation of the allozyme multilocus genotypes corresponding to the individuals with morphological scores close to A4. edulis (IV > 0.25): only two individuals are heterozygotes “e/g” for two loci (“e” and “g” denoting diagnostic alleles typical of M. edulis and A4. galloprovin-

cialis, respectively), nine are heterozygotes “e/g” for one locus and only one individual is a homozygote “e/e” for one locus.

An analysis of genetic structure of Galician mussel populations has been carried out by computing F statistics (Nei, 1977, 1986) from genotype frequencies at the four allozyme loci. In Table III, measures of deviation of genotypic frequencies from Hardy-Weinberg expectation within populations (F,,) and the total population (FIT),

and estimates of the degree of genetic differentiation of populations (F,,) are calculated by Nei’s method (Nei, 1977,1986; Nei & Chesser, 1983) and Festimates in each mussel population are computed according to Robertson & Hill (1984). In Galician mussel populations, F estimates present a wide range of variation among both loci and popu- lations, but two particular results should be noted. Positive F values appear to be associated with the Laxe population and the Odh locus (Table III). In the Laxe popula- tion F estimates per locus range from 0.084 to 0.202, and a significant homozygote excess is detected for Odh (0.186 f 0.0668) and Lap-l (0.202 k 0.049). In relation to Odh, this locus presents an excess of homozygotes in different populations ranging from 0.106 to 0.186. Heterozygote deficiency is statistically significant for Odh in Laxe (0.186 f 0.068), Carril (0.154 + 0.067) and Pontevedra (0.241 k 0.073). Note that different significant levels are attained by x2 and F tests (Table III) due to the higher statistical power of the latter (Robertson & Hill, 1984). On the other hand, a slight but significant genetic differentiation of Galician populations is detected for Est-D

(FST = 0.010) and Odh (FST = 0.006).

10 A. SANJUAN ET AL.

TABLE 111

Estimates of F statistics for Galician mussel populations (N.W. Iberian Peninsula). F,,, F,, and F,, are Wright’s fixation indices and F statistic in each population is Robertson & Hill’s (1984) estimate with its

standard error (fi SE)

LOCUS

ESI-D

s,.

%’ Lq- I

\F

z’ Odh

sr

x1 Mpi

Sf

%”

Sada

- 0.042 0.084

& 0.099 + 0.068 0.84 0.34

- 0.010 * 0.057

7.70

0.106 0.186**

i 0.064 + 0.068 9.27 11.62*

- 0.053 0.174

2 0.098 i 0.098

0.54 2.96

Laxe

0.202*** * 0.049 22.63**

Carril Pontev.

0.09 1 - 0.023 0.049

+ 0.098 + 0.103 + 0.060 0.90 0.23 1.99

0.058 - 0.089 + 0.099 5 0.068

9.66 3.57

0.154* 0.241*** 0.110

i 0.067 + 0.073 i 0.060 9.38 15.51** 7.17

- 0.080 - 0.035 + 0.106 f 0.126

1.29 0.22

Aldan

- 0.023 + 0.060

1.80

- 0.066 + 0.085

0.83

F I.\ F II Fs 7

0.00 I 0.010 0.010

16.12*

0.026 0.03 I 0.005

18.3X

0.151 0.156 0.006

22.51*

0.008 - 0.002 0.006

4.33

*P < 0.05 **P < 0.01 ***P < 0.001 (significance levels for y2 test and for test of F = 0; Robertson & Hill,

19x4 ).

DISCUSSION

The analyses of morphological variation and enzyme polymorphisms in live Galician

mussel populations of the N.W. of the Iberian Peninsula indicate that these populations

belong to the M. galloprovinciah form (Tables I and II). The results of protein variation

are especially clear since the allozyme frequencies at diagnostic loci are very similar in

mussel populations from Galician Rias and M. galbprovinciulis from the Mediterranean

Sea. as shown in the dendrogram (UPGMA) of genetic distances where all these

samples constitute a cluster clearly separated from M. edulis populations (Fig. 3). The

M. galfoprovincialis mussel form is widespread in the world in relatively disjunct areas

such as Japan (Wilkins et al., 1983), South Africa (Grant & Cherry, 1985), Hong Kong

(Lee & Morton, 1985) and California (McDonald & Koehn. 1988) where it was

probably introduced as a result of human activity. M. galloprovincialis is believed to be

a recent derivative of M. edulis that arose allopatrically in the Mediterranean Sea

(Barssoti & Meluzzi, 1968) and its occurrence on the N.W. coasts of Europe (British

Isles and Atlantic coast of France) has been attributed to several causes. Migration from

the Mediterranean Sea or evolution from an edulis-like ancestral population, as a result

of selective forces operating within Britain, are two possibilities that have been

considered (see Skibinski et al., 1978, 1983; Gosling. 1984). The presence of M. gallo-

provincialis on the N.W. coast of the Iberian Peninsula suggests the possibility of a

continuous distribution of this mussel all along the Atlantic littoral from the British Isles

MY’Z’ZLUS GALLOFROVZNCZALZS IN THE IBERIAN PENINSULA 11

and Atlantic coast of France to the Mediterranean Sea. From this galloprovincialis populations living on the N.W. coasts of Europe could have

originated from Medite~~e~ populations. On the other hand, it is necessary to consider whether M. edulis occurs on the N.W.

coast of the Iberian Peninsula in small numbers. In this respect, mo~holo~c~ variation in some of the studied Galician mussel populations reveals some individuals with morphological scores relatively close to M. edulis. Thus, in the Pontevedra population, 20.9% of sampled individuals present an IV value > 0.25 (see Fig. 2 and Table I), and it is possible to suppose that these individuals located in the tail of the distribution of morphological variation could correspond to M. edulis. Several approaches can be attempted to answer this question. First of all, the allele frequencies for the four diagnostic loci in the Pontevedra population agree with those shown by it4. galloprovin- cialis pure populations. Moreover, subsamples of the Pontevedra population with IV values < or >0.25 (the discriminant value) do not present important differences in allelic frequencies and, in addition, allozyme multilocus genotypes corresponding to individuals with morphological scores close to M. edulis assign all these individuals to the M. gulloprovinciulis form. In this context, it is also necessary to consider that mixing of M. edulis and iM. galloprovincialis individuals would have to produce both genetic associations among loci and deficits of heterozygotes with respect to Hardy-Weinberg expectations, for all those loci in which allele frequencies are different in the two forms of mussels. In fact, samples of M. edulis and M. ga~~oprov~ncia~~ populations that live s~pa~ic~ly in S.W. England and other localities in the British Isles show large deficits of heterozygotes and strong association of genotypes among loci (Skibinski et al., 1978; Skibinski & Beardmore, 1979; Gosling & Wilkins, 1981). Associations of genotypes for pairs of loci using diagnostic alleles were examined in the five Galician mussel samples by the x2 test of association. Not one of the loci pairs presented a statistically significant association except Odh-Lap-l in the Carril sample (P < 0.01). Moreover, the analysis of genetic structure of the Galician mussel populations for the four diagnostic allozyme loci shows significant deficiencies of heterozygotes in only a few cases which are mainly associated with the Odh locus. The remaining loci (&t-D, Lap-l and Mpi) do not present significant deviations of Hardy-Weinberg proportions except the Lap-l locus in the Laxe population. Therefore, the analysis of F statistics does not provide any evidence in favour of the occurrence of two different forms of mussels coexisting in our population samples. In addition, many species of marine molluscs exhibit significant deficits of heterozygotes (Zouros & Foltz, 1984a,b; Alvarez et al., 1989; Volckaert 8c Zouros, 1989) so that heterozygote deficiencies at the Odh locus in M. galioprovincialis Galician mussel populations cannot be considered an unusual result. In our case, the Odh locus presents a slight genetic di~erentiation among populations (Table III) for which the existence of a Wahlund effect could be invoked to explain deficits of heterozygotes at this locus, although other factors such as natural selection can also explain this result.

The evidence that m. eduiis ~dividu~s do not occur in the live mussel population samples from the N.W. Iberian Peninsula leads us to suppose that M. edulis is probably

I2 A. SANJUAN ET AL

absent on these coasts. This is in contradiction with the affirmation that on European coasts M. edulis range from the Arctic to N. Africa (see Seed, 1978; Suchanek, 1985). The ecological conditions of Galician Rias make the N.W. Iberian coast an important Pleistocene refuge of northern seaweeds, crustacean and mollusc species (Lewis, 1964: Margalef, 1980; Flowerdew, 1983) but, in spite of this, no evidence of the occurrence of&f. edulis has been found. It seems that the southern limit of the distribution area of M. edufis in Europe is more to the north than had been thought, and probably the Iberian Peninsula is not included in this area (Sanjuan et al., unpubl. data).

Finally, the determination of Galician mussel as M. guli~rov~~ciu~~ presents some implications with regard to mussel production. From the point of view of mussel cultivation on the coasts of Europe, it is necessary to emphasize that M. edufis has been considered traditionally as a much more important mussel than M. galluprovirzciulis. According to FAO (1988), mussel production on the European coasts during 1986 was 508 727 t, corresponding 4 16 726 t to M. eduh and 92 00 1 t to M. gallopruvinciulis. How- ever, 118 342 t included in the total production of M. edulis, correspond to mussels cultured in Spain which are grown in the Galician Rias, N.W. Iberian Peninsula. It has been shown here that these mussels belong to the M. g~~~pr~vi~cjalis form. Thus, this reveals that the production ofM. gull5~rovi~ciufis mussels on the European coasts is very similar in quantitative terms to that of M. e&iis.

ACKNOWLEDGEMENTS

We are grateful to J. M. Gabeiras and X. Perez Pintos for assistance in the collection of the mussel samples from The Netherlands and Aldan, respectively, and to N, Somaza for technical assistance. This research was supported by grant PAC84-0035 from the Comision Asesora de Investigation Cientifica y Tecnica (CAICYT) of the Ministerio de Education y Ciencia (Spain) to G. Alvarez.

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