Allozyme identification of mussels (Bivalvia: Mytilus) on the pacific coast of South America.

ABSTRACT The taxonomic identity of mussels in the southern hemisphere is still unclear, and the Mytilus that inhabit on the Pacific coast of South America has been considered by different authors as M. chilensis, M. edulis, M. edulis chilensis and M. galloprovincialis. To clarify the taxonomic identity of these mussels four samples from the northern limit of the distribution of Mytilus were taken as well as European control samples of M. edulis and M. galloprovincialis for comparison. Thirty allozyme loci were studied and 9 loci (Aco-1, Ap-1, Est-D, Gpi, Idh-1, Lap-1, Mpi, Me-2 and Odh) were partially diagnostic between the European M. edulis and M. galloprovincialis control samples, as previously reported. Chilean samples showed for four of the above partially diagnostic loci intermediate frequencies for typical alleles of M. edulis and M. galloprovincialis between those of the control samples, but they were closer to those of M. edulis for Ap-1 and Mpi and to those of M. galloprovincialis for Aco-1 and Est-D. The locus Lap-1 showed allele frequencies similar to those of M. edulis, whereas the most frequent allele of the loci Gpi, Idh-1 and Odh was that typical of M. galloprovincialis but at a higher frequency. Moreover, the partially diagnostic loci Me-2 and Lap-2, Pgm-2 and Pp showed important differences with regard to the control populations. Genetic distances, dendrograms and multidimentional scaling as well as principal component analysis on allele frequencies and factorial correspondence analysis on individual genotypes showed that South American samples were genetically closer to European M. galloprovincialis than to M. edulis but having particular and characteristic allele frequencies.

KEY WORDS: allozyme polymorphisms, mussel, genetic structure, Mytilus, Chilean coast, taxonomic status

INTRODUCTION

For many years, the taxonomy of individuals belonging to the genus Mytilus (Mollusca: Bivalvia) has been subject to controversy, because the accurate establishment of the taxonomic status of their members has proved to be difficult (Soot-Ryen 1955, McDonald et al. 1991, Gosling 1992a, 1992b). Initial taxonomic studies on this group were based on shell characteristics, but the high phenotypic plasticity and the diversity of environments, where this group inhabits, have generated unclear identifications (Soot-Ryen 1955, Gosling & McGrath 1990, Koehn 1991, Gardner 1992, Gosling 1992a, 1992b, Seed 1992).

The Mytilus on the Pacific coast of South America is distributed from Latitude 40[degrees] South (Valdivia) to Latitude 60[degrees] South (Cape Horn, approximately), and it has been named in different ways (Osorio & Bahamonde 1968). The name Mytilus chilensis Hupe (1854) has been one of the most used and it was included as one of the distinct species of Mytilus in the Lamy's review (1936). On the other hand, Soot-Ryen (1955) considered most of Mytilus taxa to be subspecies of M. edulis, including those described in South America as M. edulis chilensis (Pacific coast) and M. edulis platensis (Atlantic coast).

Molecular tools such as allozyme polymorphisms have been useful in clarifying the systematics of the mussel genus Mytilus, mainly in the northern hemisphere where 3 taxa have been identified: M. edulis, M. galloprovincialis and M. trossulus (Koehn 1991, Gardner 1992, Gosling1992a, 1992b, Seed 1992 and references therein). On the Pacific coast of South America, allozyme loci have been studied in few samples of Mytilus. The extensive worldwide study of McDonald et al. (1991) included three samples of about 25 individuals each from this area. They studied 18 morphologic characters and eight allozyme loci and suggested that Mytilus populations on the Atlantic (2 samples) and Pacific (3 samples) coasts of South America could be tentatively included in M. edulis, because mussels from these samples were genetically similar to the northern hemisphere M. edulis, although characteristic alleles of the northern hemisphere M. galloprovincialis and M. trossulus were also found at high frequency at some allozyme loci. Moreover, these South American populations contained alleles that were rare or absent in the northern hemisphere.

Other molecular tools, such as DNA markers, have been applied to clarify several systematic aspects of the Mytilus taxa. Most of works that have studied Chilean mussels have reported that these mussels are genetically closer to M. galloprovincialis than to M. edulis. Toro (1998) studied morphologic traits and one mitochondrial (COIII) and two nuclear DNA markers (ITS, Glu5') from one Chilean sample of 30 individuals, as well as several control samples of Mytilus taxa. He found the same Glu 5' pattern for the Chilean sample and the M. galloprovincialis sample from New Zealand and different from those of the control samples of M. edulis and M. trossulus. He concluded, in line with the view of Soot-Ryen (1955) and Gardner (1992), that the taxonomic status of the Chilean mussel corresponds to a subspecies of M. edulis, M. edulis chilensis. Daguin & Borsa (2000) analyzed 2 DNA markers (Glu5' and mac-1) from 1 sample of 48 individuals from Chile and found a high genetic similarity between these mussels and the Mediterranean and North Pacific M. galloprovincialis samples. Hilbish et al. (2000) studied RFLPs and sequences of the mitochondrial 16S rRNA gene to elucidate routes and times of transequatorial migration of the Mytilus complex. Two samples from the Pacific coast of South America were included, which were closer to M. galloprovincialis than to M. edulis, and they proposed that these populations shared a common ancestor with M. galloprovincialis. Recently, Martinez-Lage et al. (2005) using satellite DNA sequences maintain that Chilean specimens are closer to M. galloprovincialis than to M. edulis. However, Rego et al. (2002) using RAPDs have reported banding patterns of a Chilean sample closer to those of the European M. edulis than M. galloprovincialis. Moreover, the sequences of the H1 histone coding region have shown a closer relationship of Chilean mussels to M. californianus than to northern M. edulis, M. galloprovincialis and M. trossulus (Eirin-Lopez et al. 2002).

Most of previous studies in Mytilus on the Pacific coast of South America involved a low number of individuals per sample, from one to three samples and between two to three DNA markers and up to eight allozyme loci. In this work we tried to improve the taxonomic identification of these mussels. The samples have been confined to the northern limit of the Mytilus distribution because important genetic differences between north and south Chilean samples at allozyme loci (McDonald et al. 1991) and banding patterns of RAPDs (Toro et al. 2004) have been previously detected. However, a higher number of samples and individuals than previously reported were studied (four samples, with a mean of 60 individuals per sample). Moreover, a large number of allozyme loci (30 genes) were analyzed, which seems to be representative of the Mytilus genome.

MATERIALS AND METHODS

Sample Collections

Mussel samples (genus Mytilus) from the Pacific coast of South America, at the northern limit of their distribution, were collected from four locations between Valdivia and Quellon (Chile) (Fig. 1). Individuals were collected from the intertidal and subtidal zones, during February 1997 and May 1998. Samples of pure M. edulis from The Netherlands (EH) and M. galloprovincialis from Vigo (NW Iberian Peninsula; GV) were also analyzed for comparison. Mussels were brought alive or frozen in dry ice to the laboratory and stored at -70[degrees]C prior to electrophoresis analysis.

[FIGURE 1 OMITTED]

Allozyme Electrophoresis

Horizontal gel electrophoresis was carried out according to Harris & Hopkinson (1976) and Murphy et al. (1996). A piece of digestive gland or posterior adductor muscle was homogenized in an equal volume of 0.01 M dithiothreitol solution, prior to centrifugation at x12,000g for 7 min. Twenty-two enzymes, yielding 30 presumptive enzyme coding loci with adequate activity and resolution to be genetically interpreted, were studied, including those partially diagnostic loci between M. edulis and M. galloprovincialis (see Gardner 1992, Gosling 1992a, 1992b and references therein). Electrophoresis was performed at 4[degrees]C on 12% to 13% starch gels (Starch Art), except for the GPI enzyme, which was analyzed using a horizontal 7.8% polyacrylamide gel. The enzyme systems as well as the buffer systems and staining procedures are shown in Table 1.

Most enzymes were encoded by one locus, except AAT, ACO, IDH, MDH, ME, PGM and SOD, where two loci were detected. The two IDH isoenzymes were interpreted using digestive gland and muscle for each individual, because both IDH electromorphs migrate together and Idh-2 is expressed only in the muscle (Shaw & Prassad 1970). Allozyme nomenclature was according to Ahmad et al. (1977) for AP-1, EST-D, LAP-1 and LAP-2, Grant & Cherry (1985) for IDH, Sanjuan et al. (1990) for MPI and ODH, Skibinski et al. (1980) for AAT and PGM and Vainola & Hvilsom (1991) for ACO, ALD, ARK, GPI, MDH and ME. Cross-comparison among gels and samples were made to ensure scoring accuracy.

Data Analyses

Hardy-Weinberg equilibrium expectations for genotype frequencies at polymorphic loci were tested using a goodness-of-fit [chi square] test, and the probability of the null hypothesis was estimated using a Monte Carlo simulation. The genetic structure of populations was analyzed using F-statistics (Wright 1978, Nei 1987). F-statistics estimates were calculated according to Nei & Chesser (1983) and their statistical significance was tested using a homogeneity [chi square] test for homogeneity of allele frequencies across samples, and the probability of the null hypothesis was estimated using a Monte Carlo simulation. Tests were adjusted using the sequential Bonferroni method (Hochberg 1988); this method was applied to avoid the type I error resulting from multiple significance testing of the same null hypothesis (Rice 1989). Estimates of genetic variability were also calculated for each sample (Nei 1987).

Nei's (1978) unbiased genetic distances (D) and identities (I) among population pairs were estimated. The resulting pairwise genetic distance matrices were used to build UPGMA dendrograms (unweighted pair-group method with arithmetic averaging) (Sneath & Sokal 1973) and Neighbor-Joining (NJ) trees (Saitou & Nei 1987). To assess the confidence of the obtained trees, 1,000 bootstrap replicates of each data matrix were generated (Felsenstein 1985). The samples were also ordinated with a nonmetric multidimensional scaling of the genetic identity matrix, and the final stress was calculated (Dunn & Everitt 1982). A minimum length spanning tree (MST) from the identity matrix was also calculated and superimposed on the ordination diagram to graphically detect local distortions (Dunn & Everitt 1982, Rohlf 1995).

Data were reduced and displayed using other multivariate analyses. Samples were ordinated with principal component analysis on allele frequencies of the polymorphic loci (Pimentel 1979, Manly 1991). The major components might be expected to represented factors that have affected differentiation of several allele frequencies simultaneously. The analysis was carried out on the covariance matrix of arcsine-transformed allele frequencies. To reduce the effect of sampling error at low frequencies, frequencies smaller than 0.02 were replace by 0.02 before the transformation.

The individuals were also ordinated with factorial correspondence analysis (Benztcri 1982) of polymorphic loci. This method finds the orthogonal axes, which account for the greatest amount of variation in the multidimensional space. For each individual, each allele was treated as a separate variable, with the number of copies of the allele (0, 1 or 2) as the values of the variable. Only those individuals with genotypes for all polymorphic loci were considered. The "Selection AFC" procedure implemented in GENETIX v4.03 (Belkhir et al. 2002) was used.

The genetic data were analyzed using the BIOSYS-1 (Swofford & Selander 1981), FSTAT v2.9.3 (Goudet 2002), GENEPOP v3.2 (Raymond & Rousset 2000) and PHYLIP v3.5c (Felsenstein 1995) computer programs. Most ordinate multivariate analyses were performed with the NTSYS-pc (Rohlf 1995) package.

RESULTS

Four of the 30 analyzed enzyme loci ([beta]- Gal, Mdh-2, Sod-1 and Sod-2) were monomorphic for all samples, and the remainder 26 enzyme loci showed consistent polymorphism and their allele frequencies are shown in Table 2. M. edulis (EH) and M. galloprovincialis (GV) control populations showed important differences at the allele frequencies in 9 loci (Aco-1, Ap-1, Est-D, Gpi, Idh-1, Lap-1, Me-2, Mpi and Odh), which, in general, agreed with those previously reported for both mussel forms (Skibinski et al. 1983, Grant & Cherry 1985, Varvio et al. 1988, McDonald et al. 1990, McDonald et al. 1991, Vaintola & Hvilsom 1991, Gosling 1992b, Seed 1992, Sanjuan et al. 1994, Sanjuan et al. 1997 and references therein). Of the six most studied loci, Est-D, Gpi, Lap-1, Mpi and Odh showed important differences, and Ap-1 showed a lower differentiation. For example, [Gpi.sup.102] and [Gpi.sup.107] alleles together and also [Lap-1.sup.96] and [Lap-1.sup.100] together showed the highest frequencies in the control population of M. edulis (0.833 and 0.970, respectively), whereas in the M. galloprovincialis sample they were lower (0.151 and 0.000, respectively). Moreover, control populations also showed important differences for Aco-1 and Me-2 and at less degree for Idh-1. For example, the most frequent allele for M. edulis was [Aco-1.sup.110], with a frequency of 0.750 whereas it was 0.175 for M. galloprovincialis. This genetic differentiation was previously reported by Grant & Cherry (1985) for Me-2 and Idh-1 and for both and Aco-1 by Comesana (1997) and Trucco (2000).

To quantify the discrimination power of each locus for M. edulis and M. galloprovincialis samples, the diagnostic values (DVs) for all loci were calculated using the method of Ayala & Powell (1972). The diagnostic values represent the probability of making a correct diagnosis based on the genotype at each of the diagnostic loci, and a locus has been defined as diagnostic if an individual can be correctly assigned to one of two taxa with a probability of 99% or higher. Three loci (Est-D, Lap-1 and Mpi) shows DVs higher than 0.95, four (Aco-1, Gpi, Me-2 and Odh) were between 0.85 and 0.95, and two (Ap-1 and Idh-1) were between 0.70 and 0.85. The other polymorphic loci showed DVs lower than 0.70. However, these DVs obtained from two samples are indicative, and they must been taken with caution because of the genetic differentiation among samples of the same taxon. The locus Lap-1 showed DV > 0.99, and consequently it would be a diagnostic loci sensu Ayala & Powell (1972). However, in general, most of the earlier mentioned loci are considered as partially diagnostic loci for M. edulis and M. galloprovincialis forms.

Samples from Chile showed a different behavior in allele frequencies with respect to control populations (EH and GV) depending on loci (Table 2). The locus Lap-1 showed frequencies of the diagnostic alleles [Lap-1.sup.96] + [Lap-1.sup.100] in the Chilean populations (0.929-0.976) very close to that of M. edulis (EH, 0.970) (Table 2). For other 4 loci (Aco-1, Ap-1, Est-D and Mpi), Chilean samples showed intermediate allele frequencies for the typical alleles of M. edulis and M. galloprovincialis between those of both control samples. However, allele frequencies at 2 of these 4 loci (Ap-1 and Mpi) were more similar to those of M. edulis than those of M. galloprovincialis, whereas at the 2 other loci (Aco-1 and Est-D) were more similar to those of M. galloprovincialis. For example, for Mpi, the frequencies of the allele [Mpi.sup.200] for Chilean samples ranged from 0.600-0.838 (arithmetic mean: 0.700), which were closer to the high value of M. edulis (0.975) than to the low one of M. galloprovincialis (0.027). Alternatively, for Aco-1, the allele [Aco-1.sup.105] in Chilean samples ranged from 0.535-0.631 (arithmetic mean: 0.573), which were closer to that of M. galloprovincialis (0.800) than to that of M. edulis (0.112). At other 3 loci (Gpi, Idh-1 and Odh) the most frequent allele in the South American samples was a characteristic allele of M. galloprovincialis, but at a higher frequency. For example, for Odh the allele [Odh.sup.129] in Chilean samples ranged from 0.846-0.984 whereas in M. galloprovincialis was 0.329 and in M. edulis 0.039. At the Me-2 locus, the most frequent allele in Chilean samples was [Me-2.sup.90] that ranged from 0.293-0.514 (arithmetic mean: 0.436) and had a low frequency in M. edulis and M. galloprovincialis samples (< 0.015) (Table 2). The [Me-2.sup.100] allele, characteristic of M. galloprovincialis (0.716), was the second most common allele in Chilean samples (0.36-10.476; arithmetic mean: 0.397).

The South American samples also showed important differences with regard to M. edulis and M. galloprovincialis control populations at allele frequencies for 3 other loci (Lap-2, Pgm-2 and Pp). For two loci, the most common alleles for M. edulis and M. galloprovincialis samples [Lap-2.sup.100] and [Pgm-2.sup.100] showed higher frequencies for Chilean samples (arithmetic means: 0.742 and 0.823, respectively) than for control samples (between 0.544 and 0.583 for taxa and loci). Moreover, for Lap-2, the second most frequent allele in Chilean samples was [Lap-2.sup.95] (0.146-0.229; arithmetic mean: 0.195) instead of [Lap-2.sup.105], which was common in control samples (0.236 and 0.346). For Pp, the [Pp.sup.90] allele had higher frequencies in Chilean samples (0.427-0.543; arithmetic mean: 0.477) than in M. edulis and M. galloprovincialis samples (<0.080), whereas the most common allele [Pp.sup.100] was lower (0.436-0.524; arithmetic mean: 0.487) than in control samples (0.710, 0.912) (Table 2).

Unbiased expected mean heterozygosity ([H.sub.e]), the mean number of alleles and the polymorphism at the 95% and 99% criteria are also shown in Table 2. All populations showed similar genetic variability, being the Ancud sample (PAN) that with the lowest expected heterozygosity (0.220 [+ or -] 0.039) and the highest the M. edulis control sample (0.262 [+ or -] 0.045). However, no significant differences among all pair of values were found with the t-test (Nei 1987) (data not shown). Puerto Montt (PPM) sample showed the lowest number of alleles per locus (2.5 [+ or -] 0.2) and M. galloprovincialis (GV) the highest (3.5 [+ or -] 0.3).

Twenty-seven of the 142 [chi square] tests were significant (Table 3), showing departure of genotype frequencies from Hardy-Weinberg expected proportions (P < 0.05). Of these, only 13 showed significant deviations after Bonferroni correction. These 13 significative values showed positive values of F (deficiencies of heterozygotes) and involved only 5 loci: Est-1 in EH, PPM and PQE samples, Gpi in GV, PVA, PAN and PQE samples, Lap-1 in GV sample, Me-1 in PVA, PPM and PQE samples and Me-2 in GV and EH samples (Table 3). Note that for Chilean samples only 1 partially diagnostic locus (Gpi) showed significant positive F values and a possible Wahlund effect can be disregarded. Moreover, heterozygote deficiency at allozyme loci in bivalves has been described previously although its causes are still unclear (Gosling 1992b, Raymond et al. 1997).

The mean value of [F.sub.ST] over all analyzed samples, including Chilean and European samples, was high and significant ([F.sub.ST] = 0.180) (Table 4, Group A) but the [F.sub.ST] value for Chilean samples was low ([F.sub.ST] = 0.012) (Table 4, Group B). Significant interpopulation genetic differentiation was detected for most loci (20) when European and South American Pacific samples were included in the analysis (Table 4, Group A), but only six loci showed significant genetic differentiation among South American samples after Bonferroni correction (Table 4, Group B).

Unbiased genetic distance (Nei 1978) between M. edulis (EH) and M. galloprovincialis (GV) control samples was the highest (D = 0.190) (data not shown). The average genetic distances between the South American samples and M. edulis (D = 0.125) and M. galloprovincialis populations (D = 0.101) were lower than that between control Mytilus samples, and the average genetic distance among South American samples was of D = 0.011 (data not shown).

The UPGMA dendrogram (Fig. 2a) and the Neighbor-Joining tree (Fig. 2b) constructed from the unbiased genetic distances matrix (Nei 1978) showed a first cluster that included the four South American populations. This Chilean cluster was grouped with the M. galloprovincialis (GV) control sample. The bootstrap values were high for the UPGMA tree (82% for Chilean samples and 100% for Chilean and M. galloprovincialis sample) but not so much for the Neighbor-joining. The multidimensional scaling of the genetic identity matrix (Fig. 2c) showed the Chilean populations in an intermediate position between M. edulis and M. galloprovincialis, but closer to M. galloprovincialis.

[FIGURE 2 OMITTED]

Considering the allele frequencies of samples, a principal component analysis (PCA) based on the transformed arcsine of allele frequencies was carried out and projections of samples on first two principal components are shown in Figure 3. The first component (explains 40.79% of the variation among allele frequencies of samples) clearly separated Chilean samples from both Mytilus control samples, but closer to M. galloprovincialis (GV) than to M. edulis (EH). The second component (28.09%), showed Chilean samples in an intermediate position between M. edulis and M. galloprovincialis.

[FIGURE 3 OMITTED]

A more detailed analysis at individual level was carried out. A factorial correspondence analysis (FCA) from a matrix of 153 individuals (110 from South America, 23 M. edulis and 20 M. galloprovincialis) and 107 alleles from 23 polymorphic loci (the 26 polymorphic loci except Ald, Gapdh and Pgm-1, which showed low number of individuals for a control sample) was carried out. The projection of individuals on the first two axes (Fig. 4), showed the separation of the 3 groups of individuals, the M. edulis, the M. galloprovincialis and South American individuals. The Chilean individuals were most similar in their axes scores to M. galloprovincialis. The first axis showed Chilean mussels in an extreme position closer to M. galloprovincialis than to M. edulis, in a similar way that the first principal component of the above PCA (Fig. 3). The second axis showed Chilean individuals in an intermediate position between M. edulis and M. galloprovincialis individuals.

[FIGURE 4 OMITTED]

DISCUSSION

The allele frequencies of the most extensively studied partially diagnostic loci Ap-1, Est-D, Gpi, Lap-1, Mpi and Odh for the European control samples of M. edulis (EH) and M. galloprovincialis (GV) in general were in agreement with those previously reported (Skibinski et al. 1983, Grant & Cherry 1985, Varvio et al. 1988, McDonald et al. 1990, McDonald et al. 1991, Vainola & Hvilsom 1991, Sanjuan et al. 1994, Sanjuan et al. 1997, Comesana 1997, Comesana et al. 1998, Trucco 2000). Other 3 less studied loci (Aco-1, ldh-1 and Me-2) were found to be partially diagnostic between these two Mytilus forms in accordance with Grant & Cherry (1985) for Idh-1 and Me-2 and Comesana (1997) and Trucco (2000) for the three loci. Moreover, the Nei's (1978) genetic distance between both European control samples using 30 loci was D = 0.190, which was at the level of that found by Skibinski et al. (1980) (D = 0.172 using 16 enzyme loci) and Grant & Cherry (1985) (D = 0.162 using 23 loci) for the same Mytilus taxa.

The Chilean samples showed for four of the nine partially diagnostic loci, intermediate allele frequencies between those of typical alleles of European M. edulis and M. galloprovincialis samples; for 2 loci (Ap-1 and Mpi) the allele frequencies were more similar to those of M. edulis and for Aco-1 and Est-D to those of M. galloprovincialis. The locus Lap-1 showed similar allele frequencies to those of M. edulis, whereas the most common allele for Gpi, Idh-1 and Odh in Chilean samples was one of the typical allele of M. galloprovincialis but at a higher frequency. The UPGMA dendrogram and the Neighbor-joining tree (Fig. 2a, b) as well as the multidimensional scaling (Fig. 2c) grouped the four Chilean mussel populations, closer to M. galloprovincialis than to M. edulis. Moreover, ordination analyses of the samples based on the transformed allele frequencies (the principal component analysis, Fig. 3), as well as the factorial correspondence analysis on genotypes of individuals (Fig. 4), showed the Chilean samples and individuals clearly separated from both M. edulis and M. galloprovincialis control samples by the first component and in an intermediate position between M. edulis and M. galloprovincialis by the second component, but closer to M. galloprovincialis than to M. edulis (Fig. 3, 4). Moreover, the genetic distance between Chilean samples and the European M. galloprovincialis was lower (D = 0.101) than that with M. edulis (D = 0.125).

Before the present work, only McDonald et al. (1991) had analyzed by allozyme electrophoresis Mytilus individuals from the Pacific coast of South America. They analyzed the 8 allozyme loci Ap-1, Est-D, Gpi, Lap-1(Aat), Lap-2 (Lap), Mpi, Odh and Pgm-2, all of them included in this study and included 2 samples (40 and 41) from the same area of the present samples (the sample 42 was from the southern Chile), with a mean sample size of 25 individuals per sample. To establish reasonably allele homologies between McDonald et al. (1991) paper and the present study several criteria as those reported by Sanjuan et al. (1997) were used. The present allele frequencies at 6 of these loci (Ap-1, Lap-1, Lap-2, Mpi, Odh and Pgm-2) were close to those reported by McDonald et al. (1991) and for Est-D they were similar to those of one of the samples (sample 41). The case of Gpi is peculiar. The most common allele for the four Chilean samples ([Gpi.sup.100]) showed frequencies (67.4% to 87.1%) closer to those of M. galloprovincialis control sample (frequency of 54.5%) than to those of M. edulis (2.0%), whereas for the two Chilean samples of McDonald et al. (1991) the most common allele ([Gpi.sup.98]) (frequency of 85% to 92%) was that typical of M. trossulus (frequency of 56%). Note that published data of samples of M. edulis and M. galloprovincialis from Europe have similar frequencies that present results, as those of Vainola & Hvilsom (1991) and Varvio et al. (1988) and that of McDonald et al. (1990, 1991) for M. galloprovincialis, but not for M. edulis. The most frequent allele for M. edulis from Denmark and White Sea is [Gpi.sup.107] (frequencies 44% and 58%) (McDonald et al. 1990, see also Gosling, 1992b, pp. 319), as in the present work and also in Vainola & Hvilsom (1991) and Varvio et al. (1988), but at 2nd and 3rd positions were [Gpi.sup.100] (14% and 18%) and [Gpi.sup.96] (16% and 10%), instead of [Gpi.sup.102] (and perhaps [Gpi.sup.98]) as in the present work and in Vainola & Hvilsom (1991) and Varvio et al. (1988). These results on M. edulis and Chilean mussels are contradictories with present data, and the explanation is not easy. However, the principal component analysis (PCA) of McDonald et al. (1991) suggests that Chilean mussels were more similar to M. edulis from the northern hemisphere than to M. galloprovincialis. A reanalysis of present data using only the eight loci analyzed by McDonald et al. (1991) showed an UPGMA dendrogram with the cluster of the Chilean samples (bootstrap value of 91%) grouped with the M. edulis sample (bootstrap value of 100%). Consequently, it could be that the similarity of Pacific South American mussels to M. edulis, reported by McDonald et al. (1991), could be an effect of the low number of loci used. Present results using 30 allozyme loci seem to be more representative of the genetic relationship among Mytilus taxa than other works using a lower number of genes.

With regard to other molecular markers, Toro (1998) used 2 nuclear DNA markers (ITS and Glu5'; Heath et al. 1995, Rawson et al. 1996) and one mitochondrial DNA marker (COIII), to analyze a sample of 30 mussels from Bahia Corral, located within the geographic range of the present study. Data from ITS and COIII showed the same pattern for Chilean individuals, M. edulis from Newfoundland and M. galloprovincialis from New Zealand, but, the Glu5' marker showed the same banding pattern for Chilean Mytilus and M. galloprovincialis from New Zealand and different from M. edulis control sample from Newfoundland. Moreover, Daguin & Borsa (2000) analyzed the two diagnostic nuclear DNA markers, Glu 5' and the polymorphic mac-1. In a sample of cultivated mussels from Dichato, about 400 km to the north of the present sample of Valdivia (PVA) (see Fig. 1), they found for Glu 5' the typical M. galloprovincialis allele fixed (Rawson et al. 1996) and for mac-1 locus (Ohresser et al. 1997) a high frequency of the [b.sub.1] and [c.sub.2] characteristic alleles of M. galloprovincialis. Moreover, Hilbish et al. (2000) studied RFLPs and sequences of the mitochondrial 16S rRNA gene, and they found that all RFLP haplotypes obtained in two northern Chilean samples were 100% type D, the typical haplotype of M. galloprovincialis, and the phylogenetic analysis of the 16S rRNA gene grouped Chilean sequences within the cluster of the northern hemisphere M. galloprovincialis. In the same line, Martinez-Lage et al. (2005) using satellite DNA sequences maintain that Chilean specimens are closer to M. galloprovincialis than to M. edulis.

In resume, taking into account the data based on 30 enzyme loci, an important representation of the genome and the earlier mentioned genetic results with nuclear DNA markers, RFLPs and sequences of the mitochondrial 16S DNA, it seems that mussels from the northern limit of distribution on Pacific coast of South America were genetically closer to M. galloprovincialis than to M. edulis.

However, Rego et al. (2002) have reported banding patterns of RAPDs for a Chilean sample closer to those of the European M. edulis than those of M. galloprovincialis. Moreover, DNA sequences of the H1 histone coding region have shown that Chilean mussels were closer to M. californianus than to the northern M. edulis, M. galloprovincialis and M. trossulus (Eirin-Lopez et al. 2002). These results could be a consequence of the peculiarity of the Chilean mussels (and South American samples), as previously considered by McDonald et al. (1991). The mussel samples from the northern Chile seem to constitute a group relatively homogeneous and different from the European M. edulis and M. galloprovincialis samples, as shown in the dendrograms (Fig. 2a, b) and the ordination analyses (Fig. 2c, 3, 4). They showed characteristic and consistent patterns at allele frequencies for several loci, manly Aco-1, Ap-1, Est-D, Gpi, Idh-1, Lap-2, Me-2, Mpi, Odh, Pgm-2 and Pp, different from those of M. edulis and M. galloprovincialis samples (Table 2). Of these loci, only Gpi and Odh showed significant degree of genetic interpopulation differentiation with [F.sub.ST] values of 0.0214 and 0.0449, respectively (Table 4, Group B). Moreover, the [F.sub.ST] mean for the South American samples was low ([F.sub.ST] = 0.012), which suggests a high level of gene flow among populations (Nei 1987). This is in accordance with the general statement that within each mussel form and for a particular distinct geographic region the allele frequencies are generally homogenous (Gosling 1992b and references therein). Moreover, the diagnostic values (DVs) between Chilean mussels (using the mean of the allele frequencies across the four samples) and the control samples were calculated. The DVs were higher than 0.80 at Aco-1 (0.81), Est-D (0.89), Gpi (0.97), Idh-1 (0.82), Me-2 (0.91), Odh (0.98) and Pp (0.82) for M. edulis and at Lap-1 (0.999), Me-2 (0.85), Mpi (0.94) and Odh (0.89) for M. galloprovincialis. None of these loci discriminated unequivocally among the three groups, although each locus made some contribution. Consequently, if three Mytilus taxa were considered, individuals with a given genotype for the earlier mentioned loci (or combinations of only several loci) can be assigned to a taxon with one probability of erroneous assignment using the method of Ayala & Powell (1972). This means that the three different gene pools could be genetically identified. In resume, the Chilean mussels seem to be a Mytilus taxon different to both M. edulis and M. galloprovincialis from the Atlantic European waters.

The identification of the Chilean mussels as a different taxon of the other Mytilus forms does not resolve the problem of the appropriate taxonomic category of this mussel group. The main problem is the controversy on the taxonomic status of the main smooth Mytilus lineages, M. edulis, M. galloprovincialis and M. trossulus (see Varvio et al. 1988, McDonald et al. 1991, Gardner 1992, Gosling 1992a, 1992b, Seed 1992). For example, Toro (1998) found the same Glu 5' pattern for a Chilean sample and a M. galloprovincialis sample from New Zealand and different from those of the control samples of M. edulis and M. trossulus. Based upon these results and those reported by McDonald et al. (1991) for Est-[D.sup.90] allele frequency ("the most common esterase allele in M. galloprovincialis and the rarest in M. edulis was the most common allele in the two mussel samples from Chile" (?), Toro 1998, pp. 352), he considered that the similarity between M. chilensis and M. galloprovincialis gives them the same taxonomic status. According to the hypothesis that M. galloprovincialis is a subspecies of M. edulis (Gardner 1992); Toro (1998) proposed that the taxonomic status of the Chilean mussel is M. edulis chilensis. Other authors recognize the smooth Mytilus lineages, M. edulis, M. galloprovincialis and M. trossulus as distinct species based on different reasons (Koehn 1991, McDonald et al. 1991; see for a review Gosling 1992a, 1992b and references therein). The results of the present work, using a large number of allozyme loci (30 loci) and four samples from the northern distribution of Mytilus on the South American Pacific coasts, showed that (1) mussels from this area form a relatively homogenous group with characteristic allele frequencies for several loci and different from those of the European M. edulis and M. galloprovincialis control populations. Moreover, (2) Chilean mussels were genetically closer to European M. galloprovincialis than M. edulis using 30 enzyme loci, as well as for most of the reported DNA markers. Moreover, (3) the genetic distances among Chilean mussels and European M. edulis and M. galloprovincialis were lower than that between these European taxa. Taken these results into account, we propose that the taxonomic status for mussels from the Pacific of South America, at least in the north limit, could be Mytilus galloprovincialis chilensis.

ACKNOWLEDGMENTS

The authors thank Dr. Annie Machordom for their careful reading of manuscript, useful comments and suggestions. This research was supported by grants PB98-1093 (Direccion General de Ensenanza Superior e Investigacion Cientifica, MEC, Spain) and PGIDT00PXI30117PN (Xunta de Galicia, Spain) to AS. CC was supported by a research fellowship from AECI (Agencia Espanola de Cooperacion Internacional, Spain) and University of Vigo (Spain).

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CLAUDIA CARCAMO, (1) * ANGEL S. COMESANA, (1) FEDERICO M. WINKLER (2) AND ANDRES SANJUAN (1)

(1) Xenetica Evolutiva Molecular, Facultade de Bioloxia, Universidade de Vigo, E-36200 Vigo, Spain;

(2) Departamento de Biolog{a Marina, Facultad de Ciencias del Mar, Universidad Catolica del Norte, Centro de Estudios Avanzados en Zonas Aridas (CEAZA), P.O. Box 117. Coquimbo Chile

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Enzyme systems analyzed showing the Enzyme number (E.N.), the name
abbreviation (Abbr.), and the number of active loci (loci). Staining
procedures were according to AB (Aebersold et al. 1987), AH (Ahmad et
al. 1977), BS (Bulnheim & Scholl 1981), HH (Harris & Hopkinson 1976),
MR (Murphy et al. 1996), GC (Grant & Cherry 1985), SN (Sanjuan et al.
1990), SP (Shaw & Prasad 1970). Buffer systems used were: ACE 5.6
(Sodium acetate pH 5.6, Ahmad et al. 1977) TBE (Tris-borate-EDTA pH
8.7, McDonald 1985), TC 7 (Tris Citrate pH 7.0, Ahmad et al. 1977),
TC 8 (Tris Citrate pH 8.0, Ward & Beardmore 1977), TC 8.4 (Tris
Citrate pH 8.4/7.0, Varvio-Aho & Pamilo 1980) and TCE (Tris Citrate
EDTA pH 7.0, Ayala et al. 1974).

Abbr.                  Enzyme System

AAT          Aspartate aminotransferase
ACO          Aconitate hydratase
ALD          Fructose-bisphosphate aldolase
AP-1         Glycil-leucine peptidase
ARK          Arginine kinase
DIA          Dihydrolipoamide dehydrogenase
EST          Esterase
[beta]-GAL   [beta]-galactosidase
GAPDH        Glyceraldehide-3-phosphate dehydrogenase
GPI          Glucose-6-phosphate isomerase
G3PDH        Glycerol-3-phosphate dehydrogenase NAD (+)
IDH          Isocitrate dehydrogenase NADP (+)
LAP-1        Leucine aminopeptidase-1
LAP-2        Leucine aminopeptidase-2
MDH          Malate dehydrogenase
ME           Malate dehydrogenase NADP (+)
MPI          Mannose-6-phosphate isomerase
ODH          D-octopine dehydrogenase
PGD          6-phosphogluconate dehydrogenase
PGM          Phosphoglucomutase
PP           Phenyl-proline peptidase
SOD          Superoxide dismutase

Abbr.           E.N.      Loci   Staining   Buffer

AAT          2.6.1.1       2     SP         TC 7
ACO          4.2.1.3       2     HH         TCE
ALD          4.1.2.13      1     AB         TC 8.4
AP-1         3.4.13.18     l     AH         TC 7
ARK          2.7.3.3       l     HH         TC 8.4
DIA          1.8.1.4       1     MR         TC 8.4
EST          3.1.1.1       2     AH, MR     ACE 5.6
[beta]-GAL   3.2.1.23      1     MR         TBE 8.7
GAPDH        1.2.1.12      1     MR         TC 8.4
GPI          5.3.1.9       1     MR         TC 8.4
G3PDH        1.1.1.8       1     MR         TBE 8.7
IDH          1.1.1.42      2     SP         TC 8
LAP-1        3.4.11.-      I     AH         ACE 5.6
LAP-2        3.4.1l.-      1     AH         TC 7
MDH          1.1.1.37      2     AB         TC 8.4
ME           1.1.1.40      2     AB         TC 8.4
MPI          5.3.1.8       1     SN         TC 8.4
ODH          1.5.1.11      1     GC         TC 8.4
PGD          1.1.1.44      1     HH         TC 7
PGM          5.4.2.2       2     HH         TCE
PP           3.4.13.9      1     HH         TBE
SOD          1.15.1.1      2     MR         TBE

TABLE 2.
Allele frequencies for 26 polymorphic enzyme loci for M. edulis (EH)
from the Netherlands, M. galloprovincialis (GV) from the NW of the
Iberian Peninsula and Mytilus sp. from South American Pacific coast.
Populations codes as in Figure 1. N is the sample size. [H.sub.e] is
the unbiased expected mean heterozygosity (Nei, 1987) [+ or -] its
standard error. [P.sub.95] and [P.sub.99] are the percentage of the
polymorphic loci with the 0.95 and 0.99 criteria with standard error,
respectively. Four loci ([beta]-Gal, Mdh-2, Sod-1 and Sod-2) were
monomorphic for all samples.

                            Populations

Locus                   EH                     GV

Aat-1
   N              73                     74
   94              0                      0
  100              0.979                  0.973
  108              0.021                  0.020
  116              0                      0.007
Aat-2
   N              75                     74
   94              0.007                  0
  100              0.993                  1
  108              0                      0
Aco-1
   N              58                     20
  100              0                      0
  105              0.112                  0.800
  110              0.750                  0.175
  115              0.129                  0.025
  120              0.009                  0
Aco-2
   N              51                     62
   95              0.020                  0.008
  100              0.157                  0.121
  105              0.784                  0.806
  107              0.039                  0.056
  110              0                      0
  112              0                      0.008
Ald
   N              25                     14
 l00               0.920                  0.964
  112              0.080                  0.036
Ap-1
   N              71                     70
   93              0.015                  0
   96              0.007                  0.007
  100              0.718                  0.379
  104              0.007                  0
  108              0.218                  0.457
  114              0.035                  0.128
  122              0                      0.029
  128              0                      0
Ark
   N              74                     71
   92              0.014                  0
  100              0.986                  1
Dia
   N              72                     70
   95              0.104                  0.064
  100              0.778                  0.771
  102              0.021                  0
  105              0.097                  0.164
  110              0                      0
Est-D
   N              75                     72
   82              0                      0.035
   90              0.013                  0.910
  100              0.953                  0.056
  107              0.020                  0
  110              0.013                  0
Est-1
   N              60                     74
   10              0                      0.007
  100              0.525                  0.473
  200              0.350                  0.378
  300              0.108                  0.135
  400              0.017                  0.007
Gapdh
   N              56                     31
  100              0.955                  0.823
  120              0.036                  0.129
  140              0.009                  0.048
Gpi
   N              75                     66
   93              0.007                  0.008
   96              0.013                  0.015
   98              0.053                  0.038
  100              0.020                  0.545
  102              0.313                  0.068
  105              0.047                  0.235
  107              0.520                  0.083
  110              0.020                  0
  112              0.007                  0.008
Gapdh
   N              65                     65
   90              0.031                  0
  100              0.969                  1
  115              0                      0
Idh-1
   N              54                     47
   60              0                      0.011
   80              0.315                  0.638
  100              0.611                  0.266
  150              0.065                  0.085
  160              0.009                  0
Idh-2
   N              74                     46
   96              0.182                  0.098
  100              0.811                  0.891
  104              0.007                  0
  108              0                      0.011
Lap-1
   N              65                     38
   93              0                      0
   96              0.208                  0
  100              0.762                  0
  102              0.008                  0
  104              0.015                  0.487
  108              0.008                  0.461
  110              0                      0.026
  112              0                      0.026
Lap-2
   N              72                     68
   90              0.028                  0.007
   95              0.139                  0.088
  100              0.583                  0.544
  102              0.007                  0
  105              0.236                  0.346
  110              0.007                  0.015
Mdh-1
   N              75                     69
   70              0.013                  0.007
  100              0.987                  0.986
  130              0                      0.007
Me-1
   N              64                     33
   70              0.039                  0
   90              0.008                  0
  100              0.914                  0.939
  100              0.039                  0.061
  120              0                      0
Me-2
   N              65                     58
   70              0                      0
   90              0.015                  0
  100              0.100                  0.716
  110              0.831                  0.276
  115              0.023                  0
  120              0.023                  0.009
Mpi
   N              59                     56
   25              0                      0
  100              0.017                  0.973
  200              0.975                  0.027
  300              0.008                  0
Odh
   N              64                     35
   80              0                      0
  100              0.055                  0.486
  112              0.008                  0
  115              0.891                  0.186
  120              0.008                  0
  129              0.039                  0.329
  140              0                      0
Pgd
   N              72                     56
   95              0.021                  0.009
  100              0.938                  0.964
  105              0.035                  0.018
  110              0.007                  0
  115              0                      0.009
Pgm-1
   N              14                     19
   90              0                      0.026
   95              0                      0.132
  100              0.964                  0.789
  105              0                      0.053
  115              0.036                  0
Pgm-2
   N              74                     73
   90              0.007                  0
   92              0.041                  0.007
   96              0.142                  0.144
  100              0.574                  0.548
  102              0                      0.007
  104              0.189                  0.240
  107              0.041                  0.048
  110              0.007                  0.007
Pp
   N              69                     68
   80              0                      0
   90              0.072                  0.029
  100              0.710                  0.912
  110              0.210                  0.044
  120              0.007                  0
  125              0                      0.015
[H.sub.e]      0.262 [+ or -] 0.045   0.236 [+ or -] 0.040
Alleles/locus    3.2 [+ or -]0.3        3.5 [+ or -] 0.3
[P.sib.95]      60.0                   56.7
[P.sub.99]      76.7                   86.7

                            Populations

Locus                  PVA                    PAN

Aat-1
   N              61                     41
   94              0.082                  0
  100              0.893                  0.988
  108              0.025                  0.012
  116              0                      0
Aat-2
   N              56                     41
   94              0.009                  0
  100              0.973                  1
  108              0.018                  0
Aco-1
   N              61                     41
  100              0                      0.012
  105              0.631                  0.573
  110              0.369                  0.415
  115              0                      0
  120              0                      0
Aco-2
   N              50                     33
   95              0.020                  0.030
  100              0.040                  0.045
  105              0.920                  0.894
  107              0.010                  0.020
  110              0.010                  0
  112              0                      0
Ald
   N              40                     40
 l00               0.988                  1
  112              0.012                  0
Ap-1
   N              59                     41
   93              0                      0
   96              0.007                  0.008
  100              0.542                  0.622
  104              0.008                  0
  108              0.331                  0.293
  114              0.093                  0.061
  122              0.017                  0
  128              0                      0.012
Ark
   N              51                     39
   92              0.216                  0.141
  100              0.784                  0.859
Dia
   N              60                     40
   95              0.042                  0.112
  100              0.875                  0.837
  102              0                      0
  105              0.083                  0.050
  110              0                      0
Est-D
   N              61                     41
   82              0                      0
   90              0.566                  0.622
  100              0.426                  0.378
  107              0                      0
  110              0.008                  0
Est-1
   N              59                     41
   10              0.102                  0.098
  100              0.593                  0.561
  200              0.263                  0.280
  300              0.042                  0.061
  400              0                      0
Gapdh
   N              55                     36
  100              0.864                  1
  120              0.136                  0
  140              0                      0
Gpi
   N              58                     41
   93              0.009                  0
   96              0                      0
   98              0.121                  0.110
  100              0.759                  0.768
  102              0.086                  0.110
  105              0.026                  0.012
  107              0                      0
  110              0                      0
  112              0                      0
Gapdh
   N              56                     40
   90              0                      0
  100              1                      1
  115              0                      0
Idh-1
   N              58                     39
   60              0                      0
   80              0.862                  0.833
  100              0.129                  0.128
  150              0.009                  0.038
  160              0                      0
Idh-2
   N              58                     41
   96              0.017                  0
  100              0.966                  0.976
  104              0.017                  0.024
  108              0                      0
Lap-1
   N              60                     41
   93              0.017                  0.024
   96              0.250                  0.317
  100              0.683                  0.659
  102              0                      0
  104              0.050                  0
  108              0                      0
  110              0                      0
  112              0                      0
Lap-2
   N              59                     41
   90              0.017                  0.024
   95              0.229                  0.146
  100              0.746                  0.817
  102              0                      0
  105              0.008                  0.012
  110              0                      0
Mdh-1
   N              56                     41
   70              0                      0.024
  100              1                      0.976
  130              0                      0
Me-1
   N              57                     41
   70              0                      0
   90              0                      0
  100              0.860                  0.951
  100              0.140                  0.049
  120              0                      0
Me-2
   N              61                     41
   70              0.008                  0.037
   90              0.475                  0.293
  100              0.361                  0.476
  110              0.156                  0.159
  115              0                      0.037
  120              0                      0
Mpi
   N              59                     40
   25              0.042                  0
  100              0.322                  0.262
  200              0.636                  0.725
  300              0                      0.013
Odh
   N              58                     31
   80              0                      0
  100              0.034                  0.016
  112              0                      0
  115              0.026                  0
  120              0                      0
  129          0.940                  0.984
  140              0                      0
Pgd
   N              56                     40
   95              0.018                  0.025
  100              0.982                  0.962
  105              0                      0.013
  110              0                      0
  115              0                      0
Pgm-1
   N              36                     28
   90              0                      0
   95              0                      0.036
  100              0.667                  0.768
  105              0.333                  0.196
  115              0                      0
Pgm-2
   N              55                     37
   90              0                      0
   92              0.009                  0
   96              0.018                  0
  100              0.818                  0.838
  102              0.009                  0
  104              0.145                  0.162
  107              0                      0
  110              0                      0
Pp
   N              60                     41
   80              0.017                  0.037
   90              0.458                  0.427
  100              0.508                  0.524
  110              0.017                  0.012
  120              0                      0
  125              0                      0
[H.sub.e]      0.259 [+ or -] 0.039   0.220 [+ or -] 0.039
Alleles/locus    2.8 [+ or -] 0.3       3.1 [+ or -] 0.3
[P.sib.95]      66.7                   56.7
[P.sub.99]      80.0                   76.7

                            Populations

Locus                  PPM                    PQE

Aat-1
   N              71                     72
   94              0.007                  0.035
  100              0.979                  0.958
  108              0.007                  0.007
  116              0.007                  0
Aat-2
   N              67                     70
   94              0.015                  0
  100              0.978                  0.979
  108              0.007                  0.021
Aco-1
   N              67                     71
  100              0                      0.007
  105              0.552                  0.535
  110              0.433                  0.458
  115              0.015                  0
  120              0                      0
Aco-2
   N              67                     43
   95              0.067                  0
  100              0.022                  0.011
  105              0.904                  0.978
  107              0.007                  0.011
  110              0                      0
  112              0                      0
Ald
   N              47                     45
 l00               1                      1
  112              0                      0
Ap-1
   N              69                     72
   93              0                      0
   96              0.012                  0.007
  100              0.659                  0.528
  104              0                      0
  108              0.304                  0.396
  114              0.029                  0.069
  122              0.007                  0
  128              0                      0
Ark
   N              66                     69
   92              0.061                  0.159
  100              0.939                  0.841
Dia
   N              69                     71
   95              0.058                  0.049
  100              0.862                  0.859
  102              0.007                  0.021
  105              0.065                  0.071
  110              0.007                  0
Est-D
   N              71                     71
   82              0                      0
   90              0.451                  0.627
  100              0.549                  0.366
  107              0                      0.007
  110              0                      0
Est-1
   N              70                     69
   10              0.100                  0.094
  100              0.536                  0.486
  200              0.357                  0.377
  300              0.007                  0.043
  400              0                      0
Gapdh
   N              62                     47
  100              1                      1
  120              0                      0
  140              0                      0
Gpi
   N              70                     69
   93              0.007                  0
   96              0                      0.014
   98              0.043                  0.196
  100              0.871                  0.674
  102              0.050                  0.094
  105              0.029                  0.022
  107              0                      0
  110              0                      0
  112              0                      0
Gapdh
   N              70                     72
   90              0                      0.007
  100              0.993                  0.979
  115              0.007                  0.014
Idh-1
   N              60                     71
   60              0                      0.021
   80              0.800                  0.845
  100              0.133                  0.092
  150              0.067                  0.042
  160              0                      0
Idh-2
   N              61                     69
   96              0.008                  0.007
  100              0.992                  0.993
  104              0                      0
  108              0                      0
Lap-1
   N              71                     71
   93              0.014                  0.028
   96              0.246                  0.211
  100              0.683                  0.725
  102              0.021                  0.014
  104              0.021                  0.021
  108              0.014                  0
  110              0                      0
  112              0                      0
Lap-2
   N              71                     72
   90              0.077                  0.042
   95              0.204                  0.201
  100              0.676                  0.729
  102              0.007                  0.007
  105              0.028                  0.021
  110              0.007                  0
Mdh-1
   N              71                     69
   70              0.014                  0.007
  100              0.972                  0.986
  130              0.014                  0.007
Me-1
   N              65                     72
   70              0                      0
   90              0                      0
  100              0.946                  0.972
  100              0.054                  0.028
  120              0                      0
Me-2
   N              66                     71
   70              0.008                  0
   90              0.462                  0.514
  100              0.371                  0.380
  110              0.152                  0.106
  115              0.008                  0
  120              0                      0
Mpi
   N              68                     70
   25              0.022                  0.086
  100              0.140                  0.314
  200              0.838                  0.600
  300              0                      0
Odh
   N              68                     37
   80              0                      0.014
  100              0.125                  0
  112              0                      0
  115              0.015                  0.014
  120              0                      0
  129              0.846                  0.973
  140              0.015                  0
Pgd
   N              71                     72
   95              0.021                  0.007
  100              0.958                  0.986
  105              0.021                  0.007
  110              0                      0
  115              0                      0
Pgm-1
   N              15                     35
   90              0                      0
   95              0                      0
  100              1                      0.957
  105              0                      0.043
  115              0                      0
Pgm-2
   N              71                     72
   90              0                      0
   92              0.007                  0
   96              0.049                  0.014
  100              0.796                  0.840
  102              0                      0.007
  104              0.148                  0.132
  107              0                      0.007
  110              0                      0
Pp
   N              70                     70
   80              0.014                  0.029
   90              0.543                  0.479
  100              0.436                  0.479
  110              0.007                  0.014
  120              0                      0
  125              0                      0
[H.sub.e]      0.237 [+ or -] 0.041   0.228 [+ or -] 0.042
Alleles/locus    2.5 [+ or -] 0.2       2.9 [+ or -] 0.2
[P.sib.95]      53.3                   50.0
[P.sub.99]      73.3                   80.8

TABLE 3.
F estimates by locus in populations from Europe, M. edulis (EH) and
M. galloprovincialis (GV), and populations on Pacific coast of South
America. [chi square] is the goodness-of-fit test to Hardy-Weinberg
equilibrium expected proportions by locus. Significant tests after
Bonferroni correction are underlined. Populations codes as in
Figure 1.

                              Populations

Locus                      EH           GV           PVA

Aat-1   F              -0.021       -0.022       -0.012
        [chi square]    0.021        0.043        2.863
Aat-2   F              -0.007                    -0.021
        [chi square]    0.000                     0.028
Aco-1   F               0.282       -0.217       -0.021
        [chi square]   17.724 **     1.073        0.010
Aco-2   F               0.234        0.173        0.207
        [chi square]    9.898       14.398       15.610
Ald     F              -0.087       -0.037       -0.013
        [chi square]    0.139        0.000        0.000
Ap-1    F               0.125        0.205        0.019
        [chi square]   10.218       15.669 *      6.375
Ark     F              -0.014                     0.305
        [chi square]    0.007                     5.142 *
Dia     F               0.147        0.121        0.262
        [chi square]    6.729        5.289        9.633 *
Est-D   F              -0.032        0.091       -0.085
        [chi square]    0.153       13.456        1.123
Est-1   F               0.350        0.209        0.103
        [chi square]   35.210 ***   12.291       14.411
Gapdh   F              -0.039        0.046        0.305
        [chi square]    0.097       20.399        5.677 *
Gpi     F               0.062        0.452        0.391
        [chi square]   46.780       68.506 ***   45.267 ***
Gapdh   F              -0.032
        [chi square]    0.049
Idh-1   F               0.292        0.173       -0.149
        [chi square]   11.945 **    10.659        1.382
Idh-2   F               0.082        0.334       -0.027
        [chi square]    4.832        7.871        0.055
Lap-1   F               0.142        0.569        0.216
        [chi square]    6.497       90.764 ***   11.115
Lap-2   F               0.144        0.209        0.047
        [chi square]   13.770       10.735        1.383
Mdh-1   F              -0.014       -0.011
        [chi square]    0.007        0.007
Me-1    F               0.129        0.468        0.564
        [chi square]   11.740        9.818       19.443 ***
Me-2    F               0.406        0.498        0.259
        [chi square]   43.302 ***   17.815 ***   15.950 *
Mpi     F              -0.02        -0.028       -0.106
        [chi square]    0.027        0.028        1.102
Odh     F               0.227        0.311       -0.048
        [chi square]   16.276        7.511 *      0.203
Pgd     F              -0.047       -0.025       -0.018
        [chi square]    0.283        0.057        0.009
Pgm-1   F              -0.037        0.113        0.125
        [chi square]    0.000        2.868        0.702
Pgm-2   F               0.093        0.270        0.058
        [chi square]   13.208       26.881 *      1.636
Pp      F               0.270        0.201        0.134
        [chi square]   11.462       23.324       42.074

                               Populations

Locus                     PAN           PPM           PQE

Aat-1   F              -0.012        -0.014        -0.037
        [chi square]    0.000         0.022         0.113
Aat-2   F                            -0.023        -0.022
        [chi square]                  0.046         0.022
Aco-1   F              -0.026         0.273         0.078
        [chi square]    0.737         5.877 *       1.696
Aco-2   F               0.491         0.004        -0.017
        [chi square]   37.241 *       3.791         0.011
Ald     F
        [chi square]
Ap-1    F              -0.118         0.094         0.107
        [chi square]    4.115         3.255         4.464
Ark     F               0.259         0.202         0.135
        [chi square]    3.063         3.229         1.430
Dia     F               0.030         0.042         0.002
        [chi square]    1.343        18.588         2.742
Est-D   F               0.015        -0.012        -0.042
        [chi square]    0.030         0.002         0.665
Est-1   F               0.200         0.357         0.442
        [chi square]   14.179        28.292 ***    55.220 ***
Gapdh   F
        [chi square]
Gpi     F               0.614         0.212         0.553
        [chi square]   37.826 ***    33.068       108.406 ***
Gapdh   F                            -0.007        -0.016
        [chi square]                  0.000         0.022
Idh-1   F               0.019         0.112         0.079
        [chi square]    0.834         4.350         5.416
Idh-2   F              -0.025        -0.008        -0.007
        [chi square]    0.013         0.000         0.000
Lap-1   F               0.109        -0.100         0.045
        [chi square]    0.953         2.788         1.583
Lap-2   F              -0.022         0.149         0.217
        [chi square]    0.636        28.468        13.315 **
Mdh-1   F              -0.025        -0.021        -0.011
        [chi square]    0.013         0.044         0.007
Me-1    F               0.474         0.604         0.785
        [chi square]   12.488        42.190 ***    49.175 ***
Me-2    F               0.114         0.069         0.223
        [chi square]   14.407        14.722         6.828
Mpi     F               0.383         0.154        -0.017
        [chi square]    7.687 *      45.600         0.582
Odh     F              -0.016         0.001        -0.021
        [chi square]    0.000        14.539         0.014
Pgd     F              -0.030        -0.032        -0.011
        [chi square]    0.040         0.113         0.007
Pgm-1   F               0.133                       0.652
        [chi square]    1.858                      22.328 *
Pgm-2   F              -0.194         0.130         0.246
        [chi square]    1.256       147.080         8.962
Pp      F               0.099         0.054         0.049
        [chi square]    5.477         3.225         3.541

(* P < 0.05; ** P < 0.01 and *** P < 0.001).

TABLE 4.
Wright's F-statistics ([F.sub.IS], [F.sub.IT], [F.sub.ST]) for 26
polymorphic loci for the South American Pacific coast samples and
control populations of M. edulis and M. galloprovincialis from Europe
(Group A) and for the four South American populations (Group B).
Underlined [chi square] homogeneity values were significant after
Bonferroni's correction (P < 0.05). Populations codes as in Figure 1.

               A) South American and
                control populations

Locus   [F.sub.IS]   [F.sub.IT]   [F.sub.ST]

Aat-1      -0.0119       0.0108    0.0225 ***
Aat-2       -0.011      -0.0082    0.0028
Aco-1       0.1162       0.2328    0.1319 ***
Aco-2       0.1726       0.2000    0.0031 ***
Ald         -0.051      -0.0038    0.0449 **
Ap-1        0.1058       0.1383    0.0364 ***
Ark         0.2215       0.2863    0.0833 ***
Dia         0.0979       0.1043    0.0072
Est-D      -0.0192       0.3413    0.3537 ***
Est-1       0.3019       0.3056    0.0053 ***
Gapdh       0.1648       0.2334    0.0822 ***
Gpi         0.3578       0.5292    0.2669 ***
G3pdh      -0.0147      -0.0055    0.0090
Idh-1       0.1316       0.2925    0.1854 ***
Idh-2       0.1276       0.2058    0.0897 ***
Lap-1       0.1451       0.3170    0.2012 ***
Lap-2       0.1542       0.2051    0.0602 ***
Mdh-1      -0.0093      -0.0115   -0.0021
Me-1        0.4381       0.4470    0.0158 **
Me-2        0.2363       0.4241    0.2459 ***
Mpi         0.0575       0.4424    0.4084 ***
Odh         0.1476       0.6768    0.6208 ***
Pgd        -0.0250      -0.0245    0.0004
Pgm-1       0.1818       0.2838    0.1246 ***
Pgm-2       0.1540       0.1968    0.0505 ***
Pp          0.1140       0.2634    0.1686 ***
Mean        0.1622       0.3134    0.1804 ***

            B) South American populations

Locus   [F.sub.IS]   [F.sub.IT]   [F.sub.ST]

Aat-1      -0.0107       0.0161    0.0265 ***
Aat-2      -0.0117      -0.0135   -0.0018
Aco-1       0.0982       0.0961   -0.0022
Aco-2       0.1195       0.1249    0.0062
Ald        -0.0009       0.0003    0.0012
Ap-1        0.0547       0.0595    0.0051
Ark         0.2307       0.2503    0.0255 **
Dia         0.0595       0.0558   -0.004
Est-D      -0.0304      -0.0074    0.0223 *
Est-1       0.3160       0.3152   -0.0011
Gapdh       0.3144       0.3982    0.1222 ***
Gpi         0.4619       0.4735    0.0214 **
G3pdh      -0.0057      -0.0045    0.0012
Idh-1       0.0389       0.0363   -0.0026
Idh-2      -0.0126      -0.0106    0.0020
Lap-1       0.0621       0.0598   -0.0025
Lap-2       0.1316       0.1332    0.0019
Mdh-1      -0.0112      -0.0109    0.0004
Me-1        0.5430       0.5557    0.0279 **
Me-2        0.1733       0.1796    0.0076 *
Mpi         0.0616       0.1000    0.0410 ***
Odh        -0.0286       0.0176    0.0449 **
Pgd        -0.0179      -0.0195   -0.0015
Pgm-1       0.1966       0.3106    0.1419 ***
Pgm-2       0.1102       0.1048   -0.0060
Pp          0.0707       0.0692   -0.0016
Mean        0.1339       0.1445    0.0122

(* P < 0.05, ** P < 0.01 and *** P < 0.0001).