Mussels Mytilus in Chile 2005

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    Journal of Shellfish Research, Vol. 24, No. 4, 1101-1115,2005.

    ALLOZYME IDENTIFICATION OF MUSSELS (BIVALVIA: MYTlLUS) ON THE PACIFICCOAST OF SOUTH AMERICA

    CLAUDIA CARCAMO,!'* ANGEL S. COMESANA,! FEDERICO M. WINKLER 2 ANDANDRES SANJUAN 1

    'Xenbica Evolutiva Molecular, Facultade de Bioloxfa, Universidade de Vigo, E-36200 Vigo, Spain;2Departamento de Biologfa Marina, Facultad de Ciencias del Mar, Universidad Cat6lica del Norte,Centro de Estudios Avanzados en Zonas Aridas (CEAZA), P.D. Box 117. Coquimbo Chile

    ABSTRACT The taxonomic identity of mussels in the southern hemisphere is still unclear, and the Mytilus that inhabit on the Pacificcoast 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 aswell as European control samples of M. edulis and M galloprovincialis for comparison. Thirty allozyme loci were studied and 9 loci(Aeo-i, Ap-i, Est-D, Gpi, Idh-I, Lap-i, 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 lociintermediate frequencies for typical alleles of M. edulis and M. galloprovincialisbetween those of the control samples, but they werecloser to those of M edul is for Ap-I and Mpi and to those of M. galloprovincialis for Aco-i and Est-D. The locus Lap-I showed allele

    frequencies similar to thoseof

    M. edulis, whereas the most frequent alleleof

    the loci Gpi, Idh-I and Odh was that typicalof M.

    galloprovincialis but at a higher frequency. Moreover, the partially diagnostic loci Me-2 and Lap-2, Pgm-2 and Pp showed importantdifferences with regard to the control populations. Genetic distances, dendrograms and multidimentional scaling as well as principalcomponent analysis on allele frequencies and factorial correspondence analysis on individual genotypes showed that South Americansamples were genetically closer to European M. galloprovincialis than to M. edulis but having particular and characteristic allelefrequencies.

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

    INTRODUCTION

    For many years, the taxonomy of individuals belonging to thegenus Mytilus (Mollusca: Bivalvia) has been subject to controversy, because the accurate establishment of the taxonomic statusof their members has proved to be difficult (Soot-Ryen 1955,McDonald et al. 1991, Gosling 1992a, 1992b). Initial taxonomicstudies on this group were based on shell characteristics, but thehigh phenotypic plasticity and the diversity of environments,where this group inhabits, have generated unclear identifications(Soot-Ryen 1955, Gosling & McGrath 1990, Koehn 1991, Gardner1992, Gosling 1992a, 1992b, Seed 1992).

    The Mytilus on the Pacific coast of South America is distributed from Latitude 40 South (Valdivia) to Latitude 60 South(Cape Horn, approximately), and it has been named in differentways (Osorio & Baltarnonde 1968). The name Mytilus chilensisHupe (1854) has been one of the most used and it was included asone 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 inSouth America as M. edulis chilensis (Pacific coast) and M. edulisplatensis (Atlantic coast).

    Molecular tools such as allozyme polymorphisms have beenuseful 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 (Koehn1991, Gardner 1992, Gosling1992a, 1992b, Seed 1992 and references therein). On the Pacific coast of South America, allozymeloci have been studied in few samples of Mytilus. The extensiveworldwide study of McDonald et al. (1991) included three samplesof about 25 individuals each from this area. They studied 18 mor-

    *Corresponding author. E-mail: [email protected]

    phologic characters and eight allozyme loci and suggested thatMytilus populations on the Atlantic (2 samples) and Pacific (3samples) coasts of South America could be tentatively included inM. edulis, because mussels from these samples were geneticallysimilar to the northern hemisphere M. edulis, although character

    istic allelesof

    the northern hemisphere M. galloprovincialis andM. trossulus were also found at high frequency at some allozymeloci. 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. Mostof works that have studied Chilean mussels have reported thatthese mussels are genetically closer to M. galloprovincialis than toM. edulis. Toro (1998) studied morphologic traits and one mitochondrial (COlIl) and two nuclear DNA markers (ITS, Glu5') fromone Chilean sample of 30 individuals, as well as several controlsamples of Mytilus taxa. He found the same Glu 5' pattern for theChilean sample and the M. galloprovincialis sample from NewZealand and different from those of the control samples of M.

    edulis and M. trossulus. He concluded, in line with the view ofSoot-Ryen (1955) and Gardner (1992), that the taxonomic status ofthe Chilean mussel corresponds to a subspecies of M. edulis, M.edulis chilensis. Daguin & Borsa (2000) analyzed 2 DNA markers(Glu5' and mac-i) from 1sample of 48 individuals from Chile andfound a high genetic similarity between these mussels and theMediterranean and North Pacific M. galloprovincialis samples.Hilbish et al. (2000) studied RFLPs and sequences of themito-chondrial 16S rRNA gene to elucidate routes and times of transequatorial migration of the Mytilus complex. Two samples fromthe Pacific coast of South America were included, which werecloser to M. galloprovincialis than to M. edulis, and they proposedthat these populations shared a common ancestor with M. galloprovincialis. Recently, Martfnez-Lage et al. (2005) using satelliteDNA sequences maintain that Chilean specimens are closer to M.

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    1102 CARCAMO ET AL.

    galloprovincialis than to M. edulis. However, Rego et al. (2002)using RAPDs have reported banding patterns of a Chilean samplecloser to those of the European M. edulis than M. galloprovincia

    lis. Moreover, the sequences of the HI histone coding region haveshown a closer relationship of Chilean mussels to M. californianusthan to northern M. edulis, M. galloprovincialis and M. trossulus(Eirfn-L6pez et al. 2002).

    Most of previous studies in Mytilus on the Pacific coast ofSouth America involved a low number of individuals per sample,from one to three samples and between two to three DNA markersand up to eight allozyme loci. In this work we tried to improve thetaxonomic identification of these mussels. The samples have beenconfined to the northern limit of the Mytilus distribution becauseimportant genetic differences between north a n 4 / s ~ ) U t hChileansamples at allozyme loci (McDonald et al. 1991) (and bandingpatterns of RAPDs (Toro et al. 2004) have been previously detected. However, a higher number of samples and individuals thanpreviously reported were studied (four samples, with a mean of 60individuals per sample). Moreover, a large number of allozymeloci (30 genes) were analyzed, which seems to be representative ofthe Mytilus genome.

    MATERIALS AND METHODS

    Sample Collections

    Mussel samples (genus Mytilus) from the Pacific coast of SouthAmerica, at the northern limit of their distribution, were collectedfrom four locations between Valdivia and Que1l6n (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 laboratoryand stored at -70C prior to electrophoresis analysis.

    Allozyme Electrophoresis

    Horizontal gel electrophoresis was carried out according toHarris & Hopkinson (1976) and Murphy et al. (1996). A piece ofdigestive gland or posterior adductor muscle was homogenized inan equal volume of 0.01 M dithiothreitol solution, prior to centrifugation at x12,000g for 7 min. Twenty-two enzymes, yielding30 presumptive enzyme coding loci with adequate activity andresolution to be genetically interpreted, were studied, including

    those partially diagnostic loci between M. edulis and M. galloprovincialis (see Gardner 1992, Gosling 1992a, 1992b and referencestherein). Electrophoresis was performed at 4C on 12% to 13%starch gels (Starch Art), except for the GPI enzyme, which wasanalyzed using a horizontal 7.8% polyacrylamide gel. The enzymesystems as well as the buffer systems and staining procedures areshown 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 glandand muscle for each individual, because both IDH electromorphsmigrate together and Idh-2 is expressed only in the muscle (Shaw& Prassad 1970). Allozyme nomenclature was according to Ahmad et al. (1977) for AP-I, EST-D, LAP-l 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 VainoUi & Hvilsom(1991) for ACO, ALD, ARK, GPI, MDH and ME. Cross-

    comparison among gels and samples were made to ensure scoringaccuracy.

    Data Analyses

    Hardy-Weinberg equilibrium expectations for genotype frequencies at polymorphic loci were tested using a goodness-of-fitX2 test, and the probability of the null hypothesis was estimatedusing 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 X2 test for homogeneity of allele frequencies acrosssamples, and the probability of the null hypothesis was estimatedusing a Monte Carlo simulation. Tests were adjusted using thesequential Bonferroni method (Hochberg 1988); this method wasapplied to avoid the type I error resulting from multiple signifi

    cance'testingof

    the same null hypothesis (Rice 1989). Estimatesof

    genetic variability were also calculated for each sample (Nei1987).

    Nei's (1978) unbiased genetic distances (D) and identities (l )among population pairs were estimated. The resulting pairwisegenetic distance matrices were used to build DPGMA dendrograms (unweighted pair-group method with arithmetic averaging)(Sneath & Soka11973) and Neighbor-Joining (NJ) trees (Saitou &Nei 1987). To assess the confidence of the obtained trees, 1,000bootstrap replicates of each data matrix were generated (Felsen"stein 1985). The samples were also ordinated with a nonmetricmultidimensional scaling of the genetic identity matrix, and thefinal stress was calculated (Dunn & Everitt 1982). A minimumlength 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 multivariateanalyses. 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 allelefrequencies simultaneously. The analysis was carried out on thecovariance matrix of arcsine-transformed allele frequencies. Toreduce the effect of sampling error at low frequencies, frequenciessmaller than 0.02 were replace by 0.02 before the transformation.

    The individuals were also ordinated with factorial correspondence analysis (Benzecri 1982) of polymorphic loci. This methodfinds the orthogonal axes, which account for the greatest amount ofvariation in the multidimensional space. For each individual, eachallele was treated as a separate variable, with the number of copiesof the allele (0, 1 or 2) as the values of the variable. Only thoseindividuals with genotypes for all polymorphic loci were considered. The "Selection AFC" procedure implemented in GENETIXv4.03 (Belkhir et al. 2002) was used.

    The genetic data were analyzed using the BIOSYS-l (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 ([3- Gal, Mdh-2, Sod-l andSod-2) were monomorphic for all samples, and the remainder 26enzyme loci showed consistent polymorphism and their allele fre-

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    1104 CARCAMO Er .AL.

    TABLE 1.

    Enzyme systems analyzed showing tbe Enzyme number (E.N.), the name abbreviation (Abbr.), a nd t he n um be r o f active loci (loci). Stainingprocedures were according to AB (Aebersold et al. 1987), AH (Ahmadet al. 1977), BS (Bulnheim & SchoH 1981), HH (Harr is & Hopkinson

    1976), MR (Murphy et al. 1996), GC (Gran t & Cherry 1985), SN (Sanjuan et al. 1990), SP (Shaw & Prasad 1970). Buffer systems usedwere: ACE 5.6 (Sodium acetate pH 5.6, Ahmad et al. 1977) TBE (Tris-borate-EDTA pH 8.7, McDonald 1985), TC 7 (Tr is CitratepH 7.0,

    Ahmad et al. 1977), TC 8 (Tr is Citrate pH 8.0, Ward & Beardmore 1977), TC 8.4 (Tris Citrate pH 8.417.0, Varvio-Aho & Pamilo 1980) an dTC E (Tris Citra teEDTA pH 7.0, Ayalaet al. 1974).

    Abbr. Enzyme System E.N. Loci Staining Buffer

    AAT Aspartate aminotransferase 2.6.1.1 2 SP TC 7ACO Acouitate hydratase 4.2.1.3 2 HH TCEALD Fructose"bisphosphate aldolase 4.1.2.13 1 AB TC 8.4AP"1 Glycil-Ieucine peptidase 3.4.13.18 1 AH TC 7ARK Arginine kinase 2.7.3.3 1 HH TC 8.4DIA Dihydrolipoamide dehydrogenase 1.8.1.4 1 MR TC 8.4EST Esterase 3.1.1.1 2 AH,MR ACE 5.613-GAL 13-galactosidase 3.2.1.23 1 MR TBE 8.7

    GAPDH Glyceraldehide-3-phosphate dehydrogenase 1.2.1.12 I MR TC 8.4GPI Glucose-6-phosphate isomerase 5.3.1.9 1 MR TC 8.4G3PDH Glycerol-3-phosphate dehydrogenase NAD+ 1.1.1.8 1 MR TBE 8.7IDH Isocitrate dehydrogenase NADP+ 1.1.1.42 2 SP TC 8LAP-l Leucine aminopeptidase-I 3.4.11.- I AH ACE 5.6LAP-2 Leucine aminopeptidase-2 3.4.11.- I AH TC 7MDH Malate dehydrogenase 1.1.1.37 2 AB TC 8.4ME Malate dehydrogenase NADP+ 1.1.1.40 2 AB TC 8.4MPI Mannose-6-phosphate isomerase 5.3.1.8 I SN TC 8.4ODH D-octopine dehydrogenase 1.5.1.11 I GC TC 8.4PGD 6-phosphogluconate dehydrogenase 1.1.1.44 1 HH TC 7PGM Phosphoglucomutase 5.4.2.2 2 HH TCEPP Phenyl"proline peptidase 3.4.13.9 I HH TBESOD Superoxide dismutase 1.15.1.1 2 MR TBE

    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 ofmaking a correct diagnosis based on the genotype at each of thediagnostic loci, and a locus has been defined as diagnostic if anindividual can be correctly assigned to one of two taxa with aprobability of 99% or higher. Three loci (Est-D, Lap-I and Mpi)shows DVs higher than 0.95, four (Aco-I, Gpi, Me-2 and Odh)were between 0.85 and 0.95, and two (Ap-I and Idh-l) were between 0.70 and 0.85. The other polymorphic loci showed DVslower than 0.70. However, these DVs obtained from two samplesare indicative, and they must been taken with caution because of

    the genetic differentiation among samples of the same taxon. Thelocus Lap-I showed DV > 0.99, and consequent ly i t would be adiagnostic loci sensu Ayala & Powell (1972). However, in general,most of the earlier mentioned loci are considered as partially di"agnostic loci for M. edulis and M. galloprovincialis forms.

    Samples from Chile showed a different behavior in allele frequencies with respect to control populations (Ell and GV) depending on loci (Table 2). The locus Lap-I showed frequencies of thediagnostic alleles Lap_I 96 + Lap_I lOO in the Chilean populations(0.929--0.976) very close to that of M. edulis (EH, 0.970) (Table2). For other4 loci (Aeo- I, Ap-I, Est-D and Mpi), Chilean samplesshowed intermediate allele frequencies for the typical alleles of M.edulis and M. galloprovineialis between those of both controlsamples. However, allele frequencies at 2 of these 4 loci (Ap-I and

    Mpi) were more similar to those of M. edulis than those of M.galloprovineialis, whereas at the 2 other loci (Aeo-I and Est-D)were more similar to those of M. galloprovincialis. For example,

    for Mpi, the frequencies of the allele Mpi 20 0 for Chilean samplesranged from 0.600-0.838 (arithmetic mean: 0.700), which werecloser to the high value of M. edulis (0.975) than to the low one ofM. galloprovincialis (0.027). Alternatively, for Aeo-I, the alleleAeo-I 10 5 in Chilean samples ranged from 0.535-0.631 (arithmeticmean: 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-Iand Odh) the most frequent allele in the South American sampleswas a characteristic allele of M. galloprovincialis, but at a higherfrequency. For example, for Odh the allele Odh J2 9 in Chileansamples ranged from 0.846-0.984 whereas in M. galloprovineialiswas 0.329 and in M. edulis 0.039. At the Me-2 locus, the most

    frequent allele in Chilean samples was Me_290

    that ranged from0.293-0.514 (arithmetic mean: 0.436) and had a low frequency inM. edulis and M. galloprovincialis samples 0.015) (Table 2).The Me_2 10 0 allele, characteristic of M. galloprovincialis (0.716),was the second most common allele in Chilean samples (0.3610.476; arithmetic mean: 0.397).

    The South American samples also showed important differences with regard to M. edulis and M.galloprovincialis controlpopulations at allele frequencies for 3 other loci (Lap-2, Pgm-2 andPp). For two loci, the most common alleles for M. edulis and M.galloprovincialis samples Lap_2 10 0 and Pgm_2 JOo showed higherfrequencies for Chilean samples (arithmetic means: 0.742 and0.823, respectively) than for control samples (between 0.544 and0.583 for taxa and loci). Moreover, for Lap-2, the second most

    frequent allele in Chi lean samples was Lap_295

    (0.146-0.229;arithmetic mean: 0.195) instead of Lap_2 10 5 , which was commonin control samples (0.236 and 0.346). For Pp, the pp 90 allele had

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    ALLOZYME IDENTIFICATION OF MYTILUS

    TABLE 2.

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    Allele frequencies for 26 polymorphic enzyme loci for M. edulis (EH) from the Netherlands, M. galloprovincialis (GV) f ro m t he N W o f t heIberian Peninsula an d Mytilus sp. from South American Pacific coast . Populations codes as in Figure 1. N is the ..,ample size. He is t he

    unbia..,ed expected mean heterozygo..,ity (Nei, 1987) its s tandard er ror. P 95 an d P 99 ar e the percentage of the polymorphic loci with the 0.95an d 0.99 criteria with standard error, respectively. Four loci ((3-Gal, Mdh-:Z, Sod-] an d Sod-:Z) were monomorphic for all samples.

    Populations

    Locus EH GV PVA PAN PPM PQE

    Aat-l

    N 73 74 61 41 71 7294 0 0 0.082 0 0.007 0.035

    100 0.979 0.973 0.893 0.988 0.979 0.958108 0.021 0.020 0.025 0.012 0.007 0.007

    116 0 0.007 0 0 0.007 0Aat-2N 75 74 56 41 67 7094 0.007 0 0.009 0 0.015 0

    100 0.993 1 0.973 1 0.978 0.979108 0 0 0.018 0 0.007 0.021

    Aco-l

    N 58 20 61 41 67 71100 0 0 0 0.012 0 0.007105 0.112 0.800 0.631 0.573 0.552 0.535110 0.750 0.175 0.369 0.415 0.433 0.458ll 5 0.129 0.025 0 0 0.015 0120 0.009 0 0 0 0 0

    Aco-2N 51 62 50 33 67 43

    95 0.020 0.008 0.020 0.030 0.067 0100 0.157 0.121 0.040 0.045 0.022 0.011105 0.784 0.806 0.920 0.894 0.904 0.978107 0.039 0.056 0.010 0.020 0.007 0.011110 0 0 0.010 0 0 0112 0 0.008 0 0 0 0

    Ai d

    N 25 14 40 40 47 45100 0.920 0.964 0.988 1 1 1112 0.080 0.036 0.012 0 0 0

    Ap-l

    N 71 70 59 41 69 7293 0.015 0 0 0 0 096 0.007 0.007 0.007 0.008 0.012 0.007

    100 0.718 0.379 0.542 0.622 0.659 0.528

    104 0.007 0 0.008 0 0 0108 0.218 0.457 0.331 0.293 0.304 0.396114 0.035 0.128 0.093 0.061 0.029 0.069122 0 0.029 0.017 0 0.007 0128 0 0 0 0.012 0 0

    Ar kN 74 71 51 39 66 6992 0.014 0 0.216 0.141 0.061 0.159

    100 0.986 1 0.784 0.859 0.939 0.841Dia

    N 72 70 60 40 69 7195 I 0.104 0.064 0.042 0.112 0.058 0.049

    100 0.778 0.771 0.875 0.837 0.862 0.859102 0.021 0 0 0 0.007 0.021105 0.097 0.164 0.083 0.050 0.065 0.071

    llO 0 0 0 0 0.007 0

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    TABLE 2.

    continued

    Populations

    Locus EH GV PVA PAN PP M PQ E

    Est-DN 75 72 61 41 71 71

    82 0 0.035 0 0 0 090 0.013 0.910 0.566 0.622 0.451 0.627

    100 0.953 0.056 0.426 0.378 0.549 0.366107 0.020 0 0 0 0 0.007110 0.013 0 0.008 0 0 0

    Est-]N 60 74 59 41 70 6910 0 0.007 0.102 0.098 0.100 0.094

    100 0.525 0.473 0.593 0.561 0.536 0.486200 0.350 0.378 0.263 0.280 0.357 0.377300 0.108 0.135 0.042 0.061 0.007 0.043400 0.017 0.007 0 0 0 0

    GapdhN 56 31 55 36 62 47

    100 0.955 0.823 0.864 1 1 1120 0.036 0.129 0.136 0 0 0140 0.009 0.048 0 0 0 0

    GpiN 75 66 58 41 70 6993 0.007 0.008 0.009 0 0.007 096 0.013 0.015 0 0 0 0.01498 0.053 0.038 0.121 0,110 0.043 0.196

    100 0.020 0.545 0.759 0.768 0.871 0.674

    102 0.313 0.068 0.086 0.110 0.050 0.094105 0.047 0.235 0.026 0.012 0.029 0.022107 0.520 0.083 0 0 0 0110 0.020 0 0 0 0 0112 0.007 0.008 0 0 0 0

    G3pdhN 65 65 56 40 70 7290 0.031 0 0 0 0 0.007

    100 0.969 1 I 1 0.993 0.979115 0 0 0 0 0.007 0.014

    Idh-]N 54 47 58 39 60 71

    60 0 0.011 0 0 0 0.02180 0.315 0.638 0.862 0.833 0.800 0.845

    100 0.611 0.266 0.129 0.128 0.133 0.092

    150 0.065 0.085 0.009 0.038 0.067 0.042160 0.009 0 0 0 0 0

    Idh-2N 74 46 58 41 61 6996. 0.182 0.098 0.017 0 0.008 0.007

    100 0.811 0.891 0.966 0.976 0.992 0.993104 0.007 0 0.017 0.024 0 0108 0 0.011 0 0 0 0

    Lap-]N 65 38 60 41 71 71

    93 0 0 0.017 0.024 0.014 0.02896 0.208 0 0.250 0.317 0.246 0.211

    100 0.762 0 0.683 0.659 0.683 0.725102 0.008 0 0 0 0.021 0.014104 0.015 0.487 0.050 0 0.021 0.021108

    0.008 0.461 0 0 0.014 0110 0 0.026 0 0 0 0112 0 0.026 0 0 0 0

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    ALLOZYME IDENTIFICATION OF MYTlLUS 1107

    TABLE 2.

    continued

    Populations

    Locus EH GV PVA PAN PP M PQ E

    Lap-2N 72 68 59 41 71 7290 0.028 0.007 0.017 0.024 0.077 0.04295 0.139 0.088 0.229 0.146 0.204 0.201

    100 0.583 0.544 0.746 0.817 0.676 0.729102 0.007 0 0 0 0.007 0.007105 0.236 0.346 0.008 0.012 0.028 0.021110 0.007 0.015 0 0 0.007 0

    Mdh-l

    N 75 69 56 41 71 6970 0.013 0.007 0 0.024 0.014 0.007

    100 0.987 0.986 1 0.976 0.972 0.986130 0 0.007 0 0 0.014 0.007

    Me-l

    N 64 33 57 41 65 7270 0.039 0 0 0 0 090 0.008 0 0 0 0 0

    100 0.914 0.939 0.860 0.951 0.946 0.972100 0.039 0.061 0.140 0.049 0.054 0.028120 0 0 0 0 0 0

    Me-2N 65 58 61 41 66 7170 0 0 0.008 0.037 0.008 090 0.015 0 0.475 0.293 0.462 0.514

    100 0.100 0.716 0.361 0.476 0.371 0.380110 0.831 0.276 0.156 0.159 0.152 0.106ll 5 0.023 0 0 0.037 0.008 0120 0.023 0.009 0 0 0 0

    MpiN 59 56 59 40 68 7025 0 0 0.042 0 0.022 0.086

    100 0.017 0.973 0.322 0.262 0.140 0.314200 0.975 0.027 0.636 0.725 0.838 0.600300 0.008 0 0 0.013 0 0

    OdhN 64 35 58 31 68 3780 0 0 0 0 0 0.014

    100 0.055 0.486 0.034 0.016 0.125 0112 0.008 0 0 0 0 0115 0.891 0.186 0.026 0 0.015 0.014120 0.008 0 0 0 0 0129 0.039 0.329 0.940 0.984 0.846 0.973140 0 0 0 0 0.015 0

    PgdN 72 56 56 40 71 7295 0.021 0.009 0.018 0.025 0.021 0.007

    100 0.938 0.964 0.982 0.962 0.958 0.986105 0.035 0.018 0 0.013 0.021 0.007110 0.007 0 0 0 0 0115 0 0.009 0 0 0 0

    Pgm-l

    N 14 19 36 28 15 3590 0 0.026 0 0 0 095 0 0.132 0 0.036 0 0

    100 0.964 0.789 0.667 0.768 1 0.957105 0 0.053 0.333 0.196 0 0.043115 0.036 0 0 0 0 0

    Pgm-2

    N 74 73 55 37 71 7290 0.007 0 0 0 0 092 0.041 0.007 0.009 0 0.007 096 0.142 0.144 0.018 0 0.049 0.014

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    TABLE 2.

    continued

    Populations

    Locus EH GV PVA PAN PPM PQE

    100 0.574 0.548 0.818 0.838 0.796 0.840102 0 0.007 0.009 0 0 0.007104 0.189 0.240 0.145 0.162 0.148 0.132107 0.041 0.048 0 0 0 0.007110 0.007 0.007 0 0 0 0

    PpN 69 68 60 41 70 7080 0 0 0.017 0.037 0.014 0.02990 0.072 0.029 0.458 0.427 0.543 0.479

    100 0.710 0.912 0.508 0.524 0.436 0.479110 0.210 0.044 0.017 0.012 0.007 0.014120 0.007 0 0 0 0 0

    125 0 0.015 0 0 0 0He 0.262 0.045 0.236 0.040 0.259 0.039 0.220 0.039 0.237 0.041 0.228 0.042AlIe1esllocus 3.2 0.3 3.5 0.3 2.8 0.3 3.1 0.3 2.5 0.2 2.9 0.2P9 5 60.0 56.7 66.7 56.7 53.3 50.0P 99 76.7 86.7 80.0 76.7 73.3 80,8

    higher frequencies in Chilean samples (0.427-0.543; arithmeticmean: 0.477) than in M. edulis and M. galloprovincialis samples0.080), whereas the most common allele PpJOo was lower(0.436-0.524; arithmetic mean: 0.487) than in control samples(0.710,0.912) (Table 2).

    Unbiased expected mean heterozygosity (He)' the mean number of alleles and the polymorphism at the 95% and 99% criteria

    are also shown in Table 2. All populations showed similar geneticvariability, being the Ancud sample (PAN) that with the lowestexpected heterozygosity (0.220 : t 0.039) and the highest the M.edulis control sample (0.262 :t 0.045). However, no significantdifferences among all pair of values Were found with the Hest (Nei1987) (data not shown). Puerto Montt (PPM) sample showed thelowest number of alleles per locus (2.5 :t 0.2) and M. galloprovincialis (GV) the highest (3.5 :t 0.3).

    Twenty-seven of the 142 X 2 tests were significant (Table 3),showing departure of genotype frequencies from Hardy-Weinbergexpected 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-l in EH, PPM and PQE

    samples, Gpi in GV, PVA, PAN and PQE samples, Lap-l in GVsample, Me-l in PVA, PPM and PQE samples and Me-2 in GV andEH samples (Table 3). Note that for Chilean samples only I partially diagnostic locus (Gp!) showed significant positive F valuesand 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 (Gosling1992b, Raymond et al. 1997).

    The mean value of F ST over all analyzed samples, includingChilean and European samples, was high and significant (FST =0.180) (Table 4, Group A) but the F ST value for Chilean sampleswas low ( F S T = 0.012) (Table 4, Group B). Significant interpopulation genetic differentiation Was detected for most loci (20) whenEuropean 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 afterBonferroni 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 betweenthe South American samples and M. edulis (D = 0.125) and M.galloprovincialis populations (D = 0.101) were lower than thatbetween control Mytilus samples, and the average genetic distanceamong South American samples was of D = 0.011 (data not

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

    Considering the allele frequencies of samples, a principal com

    ponent analysis (PCA) based on the transformed arcsine of allelefrequencies was carried out and projections of samples on first twoprincipal components are shown in Figure 3. The first component(explains 40.79% of the variation among allele frequencies ofsamples) 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 Chileansamples in an intermediate position between M. edulis and M.galloprovincialis.

    A more detailed analysis at individual level was carried out. Afactorial correspondence analysis (FCA) from a matrix of 153individuals (110 from South America, 23 M. edulis and 20 M.galloprovincialis) and 107 alleles from 23 polymorphic loci (the26 polymorphic loci except Aid, Gapdh and Pgm-l, which showed

    low number of individuals for a control sample) was carried out.The projection of individuals on the first two axes (Fig. 4), showedthe separation of the 3 groups of individuals, the M. edulis, the M.

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    ALLOZYME IDENTIFICATION OF MYTILUS

    TABLE 3.

    1109

    F estimates by locus in populations from Europe, M. edulis (Ell) and M. galloprovincialis (GV), and populations on Pacific coast of SouthAmerica. X 2 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 PAN PPM PQE

    Aat-l F -0.021 -0.022 -0.012 -0.012 -0.014 -0.037X 0.021 0.043 2.863 0.000 0.022 0.113

    Aat-2 F -0.007 -0.021 -0.023 -0.022

    X 0.000 0.028 0.046 0.022Aeo-l F 0.282 -0.217 -0.021 -0.026 0.273 0.078

    X 17.724** 1.073 0.010 0.737 5.877* 1.696Aco-2 F 0.234 0.173 0.207 0.491 0.004 -0.017

    X 9.898 14.398 15.610 37.241 * 3.791 0.011Aid F -0.087 -0.037 -0.013

    x2 0.139 0.000 0.000Ap-l F 0.125 0.205 0.019 -0.118 0.094 0.107

    X 10.218 15.669* 6.375 4.115 3.255 4.464Ark F -0.014 0.305 0.259 0.202 0.135

    X 0.007 5.142* 3.063 3.229 1.430Dia F 0.147 0.121 0.262 0.030 0.042 0.002

    X 6.729 5.289 9.633* 1.343 18.588 2.742Est-D F -0.032 0.091 -0.085 0.015 -0.012 -0.042

    X 0.153 13.456 1.123 0.030 0.002 0.665Est-l F 0.350 0.209 0.103 0.200 0.357 0.442

    X 35.210*** 12.291 14.411 14.179 28.292*** 55.220***Gapdh F -0.039 0.046 0.305

    X 0.097 20.399 5.677*Gpi F 0.062 0.452 0.391 0.614 0.212 0.553

    X 46.780 68.506*** 45.267*** 37.826*** 33.068 108.406***G3pdh F -0.032 -0.007 -0.016

    X 0.049 0.000 0.022ldh-l F 0.292 0.173 -0.149 0.019 0.112 0.079

    X 11.945** 10.659 1.382 0.834 4.350 5.416ldh-2 F 0.082 0.334 -0.027 -0.025 -0.008 -0.007

    X 4.832 7.871 0.055 0.013 0.000 0.000Lap-l F 0.142 0.569 0.216 0.109 -0.100 0.045

    X 6.497 90.764*** 11.115 0.953 2.788 1.583Lap-2 F 0.144 0.209 0.047 -0.022 0.149 0.217

    X 13.770 10.735 1.383 0.636 28.468 13.315**Mdh-l F -0.014 -O.01! -0.025 -0.021 -0.011

    X 0.007 0.007 0.013 0.044 0.007Me-l F 0.129 0.468 0.564 0.474 0.604 0.785

    X 11.740 9.818 19.443*** 12.488 42.190*** 49.175***Me-2 F 0.406 0.498 0.259 0.114 0.069 0.223

    X 43.302*** 17.815*** 15.950* 14.407 14.722 6.828Mpi F -0.020 -0.028 -0.106 0.383 0.154 -0.017

    X 0.027 0.028 1.102 7.687* 45.600 0.582Odh F 0.227 0.311 -0.048 -0.016 0.001 -0.021

    X2 16.276 7.511* 0.203 0.000 14.539 0.014

    Pgd F -0.047 -0.025 -0.018 -0.030 -0.032 -0.011X 0.283 0.057 0.009 0.040 0.113 0.007

    Pgm-l F -0.037 0.113 0.125 0.133 0.652X 0.000 2.868 0.702 1.858 22.328*

    Pgm-2 F 0.093 0.270 0.058 -0.194 0.130 0.246X 13.208 26.881* 1.636 1.256 147.080 8.962

    Pp F 0.270 0.201 0.134 0.099 0.054 0.049X

    2 11.462 23.324 42.074 5.477 3.225 3.541

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

    galloprovincialis and South American individuals. The Chilean similar way that the first principal component of the above PCA

    individuals were most similar in their axes scores to M. gallopro- (Fig. 3). The second axis showed Chilean individuals in an inter-vincialis. The first axis showed Chilean mussels in an ext reme mediate position between M. edulis and M. galloprovincialis in-position closer to M. galloprovincialis than to M. edulis, in a dividuals.

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    1110 CARCAMO ET AL.

    TABLE 4.

    Wright's F-statistics (FIs> F m F ST) for 26 polymorphic loci f or t he South American Pacif ic coast samples an d control populations of M.edulis an d M. galwpr()vincialis from Europe (Group A) an d f or t he four South American populations (Group B). Underlined X 2 homogeneity

    values were sigulficant after Bonferroni's correction (P < 0.05). Populations codes as in Figure 1.

    A) South American an d control populations B) South American populations

    Locus F IS FI T F ST F IS FI T F ST

    Aat-l -0.0119 0.0108 0.0225*** -0.0107 0.0161 0.0265***Aat-2 -0.0110 -0.0082 0.0028 -0.0117 -0.0135 -0.0018Aeo-l 0.1162 0.2328 0.1319*** 0.0982 0.0961 -0.0022Aco-2 0.1726 0.2000 0.0031 *** 0.1195 0.1249 0.0062Ai d ~ . 0 5 1 O -0.0038 0.0449** -0.0009 0.0003 0.0012Ap-l 0.1058 0.1383 0.0364*** 0.0547 0.0595 0.0051Ar k 0.2215 0.2863 0.0833*** 0.2307 0.2503 0.0255**Dia 0.0979 0.1043 0.0072 0.0595 0.0558 -0.0040Est-D -0.0192 0.3413 0.3537*** -0.0304 -0.0074 0.0223*Est-l 0.3019 0.3056 0.0053*** 0.3160 0.3152 -0.0011

    Gapdh 0.1648 0.2334 0.0822*** 0.3144 0.3982 0.1222***Gpi 0.3578 0.5292 0.2669*** 0.4619 0.4735 0.0214**G3pdh -0.0147 -0.0055 0.0090 -0.0057 -0.0045 0.0012ldh-l 0.1316 0.2925 0.1854*** 0.0389 0.0363 -0.0026Idh-2 0.1276 0.2058 0.0897*** -0.0126 -0.0106 0.0020Lap-l 0.1451 0.3170 0.2012*** 0.0621 0.0598 -0.0025Lap-2 0.1542 0.2051 0.0602*** 0.1316 0.1332 0.0019Mdh-l -0.0093 -0.0115 -0.0021 -0.0112 -0.0109 0.0004Me-l 0.4381 0.4470 0.0158** 0.5430 0.5557 0.0279**Me-2 0.2363 0.4241 0.2459*** 0.1733 0.1796 0.0076*Mpi 0.0575 0.4424 0.4084*** 0.0616 0.1000 0.0410***Odh 0.1476 0.6768 0.6208*** -0.0286 0.0176 0.0449**Pgd -0.0250 -0.0245 0.0004 -0.0179 -0.0195 ~ 0 . 0 0 1 5Pgm-l 0.1818 0.2838 0.1246*** 0.1966 0.3106 0.1419***Pgm-2 0.1540 0.1968 0.0505*** 0.1102 0.1048 -0.0060

    Pp 0.1140 0.2634 0.1686*** 0.0707 0.0692 -0.0016Mean 0.1622 0.3134 0.1804*** 0.1339 0.1445 0.0122

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

    DISCUSSION

    The allele frequencies of the most extensively studied partiallydiagnostic loci Ap-I, Est-D, Gpi, Lap-I , Mpi and Odh for theEuropean control samples of M. edulis (EH) and M. galloprovincialis (GV) in general were in agreement with those previouslyreported (Skibinski et al. 1983, Grant & Cherry 1985, Varvio et a1.1988, McDonald et al. 1990, McDonald et a1. 1991, Vainola &Hvilsom 1991, Sanjuan et a1. 1994, Sanjuan et a1. 1997, Comesafia1997, Comesafia et al. 1998, Trucco 2000) . Other 3 less s tudied

    loci (Aeo-I, Idh-I and Me-2) were found to be partially diagnosticbetween these two Mytilus forms in accordance with Grant &Cherry (1985) for Idh-I and Me-2 and Comesafia (1997) andTrucco (2000) for the three loci . Moreover, the Nei 's (1978) genetic distance between both European control samples using 30loci was D= = 0.190, Which was at the level of that found bySkibinski et al. (1980) (D == 0.172 using 16 enzyme loci) andGrant & Cherry (1985) (D == 0.162 using 23 loci) for the sameMytilus taxa.

    The Chilean samples showed for four of the nine partiallydiagnostic loci, intermediate allele frequencies between those oftypical alleles of European M. edulis and M. galloprovincialissamples; for 2 loci (Ap-I and Mpi) the allele frequencies weremore similar to those of M. edulis and for Aeo-I and Est-D to those

    of M. galloprovincialis. The locus Lap-I showed similar allelefrequencies to those of M. edulis, whereas the most common allelefor Gpi, Idh-Iand Odh in Chilean samples was one of the typical

    allele of M. galloprovincialis bu t a t a high er frequency. TheUPGMA dendrogram and the Neighbor-joining tree (Fig. 2a, b) aswell as the multidimensional scaling (Fig. 2c) grouped the fourChilean mussel populations, closer to M. galloprovincialis than toM. edulis. Moreover, ordination analyses of the samples based onthe transformed allele frequencies (the principal component analysis, Fig . 3) , as well as the factoria l correspondence analys is ongenotypes of individuals (Fig. 4), showed the Chilean samples andindividuals clearly separated from both M. edulis and M. gallo

    provincialis control samples by the first component and in anintermediate position between M. edulis and M. galloprovincialisby the second component, but closer to M. galloprovincialis thanto M. edulis (Fig. 3, 4). Moreover, the genetic distance betweenChilean samples and the European M. galloprovineialis was lower(D == 0.101) than that with M. edulis (D == 0.125).

    Before the present work, only McDonald et a1. (1991) hadanalyzed by allozyme electrophoresis Mytilus individuals from thePacific coast of South America. They analyzed the 8 allozyme lociAp-I, Est-D, Gpi, Lap-I(Aat), Lap-2 (Lap), Mpi, Odh and Pgm-2,all of them included in this study and included 2 samples (40 and41) from the same area of the present samples (the sample 42 wasfrom the southern Chile), with a mean sample size of 25 individuals per sample. To establish reasonably allele homologies between

    McDonald et a1. (1991) paper and the present study several criteriaas those reported by Sanjuan et a1. (1997) were used. The presentallele frequencies at 6 of these loci (Ap-I, Lap-I, Lap-2, Mpi, Odh

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    (c)

    ALLQZYME IDENTIFICATION OF MYTILUS

    8\oo;1... ;::===0;:12=======III::,o.e========0:;04=======0:- - - - : . . [ - - - . . . : ; 1 1 O ~ " " ' 1- = - ~ ~ ~

    -PPIl ' l

    ..... - -- GY

    &.....---- ...... , . . . . .-----------I:H!I

    o.os

    1.6 . . . . .- - -

    - __ - ,

    o.

    1111

    J::I

    J1 0.0~

    -0.8

    - 1 ~ . , . . . - - - - - _ o - : l. , - - - - - - - -: 0 T : " . 0 - - - - - - - , 0 : r : .8 - - - - - - ; ; l1 . 6

    DDoumo ..... 1Figure 2. UPGMA dendrogram (a) an d Neighbor-Joining tree (b), both based on genetic distances (Nei 1978) an d non metrical multidimentionalscaling plot (MDS) (c) on genetic identities among populations (Nei 1978), for 30 aJlozyme loci ofthe South American samples (PAN, PpM, PQE,and PVA) an d European M. edulis (EH) an d M. galloprovincialis (GV) control samples. A minimum spanning tree is superimposed on the MDSplot. St ress of the MDS was S =0.0086. Numbers above main branches represent the bootstrap percentage above 40% obtained after 1,000replications. Populations codes as in Figure 1.

    Iand Pgm-2) were close to those reported by McDonald e t a1.(1991) and for Est-D they were similar to those of one of thesamples (sample 41). The case of Gpi is peculiar. The most common allele for the four Chilean samples (Gpi JOo ) showed frequencies (67.4% to 87.1%) closer to those of M. galloprovincialiscontrol sample (frequency of 54.5%) than to those of M. edulis(2.0%), whereas for the two Chilean samples of McDonald et a1.

    (1991) the most common allele (Gpi98

    ) (frequency of 85% to 92%)was that typical of M. trossulus (frequency of 56%). Note thatpublished data of samples of M. edulis and M. galloprovincialisfrom Europe have similar frequencies that present results, as those

    of Vain6Hi & Hvilsom (1991) and Varvio et a1. (1988) and that ofMcDonald et a1. (1990, 1991) for M. galloprovincialis, but not forM. edulis. The most frequent allele for M. edulis from Denmarkand White Sea is Gpi J0 7 (frequencies 44% and 58%) (McDonald eta1. 1990, see also Gosling, 1992b, pp. 319), as in the present workand also in Vain61a & Hvilsom (1991) and Varvio et a1. (1988), butat 2nd and 3rd posi tions were Gpi JOO (14% and 18%) and Gpi

    96

    (16% and 10%), instead of GpilO

    :2 (and perhaps Gpi98

    ) as in thepresent work and in Vain61a & Hvilsom (1991) and Varvio et a1.(1988). These results on M. edulis and Chilean lTIussels are contradictories with present data, and the explanation is not easy.

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    1112 CARCAMO ET AL.

    ~ : t ; ' I r - - - ~ - - - - - - - - - - - - - - - - - - - - - - - - " " " "

    -0.0J}.2'+-------""1""--------r------..... -,.-------""""0.2 0.1Component 1 (40.79%)

    Figure 3. Pro ject ious of samples of South America (PAN, PPM, PQE, au d PV A) au d M. edulis (EH) au d M. galloprovincialis (GV) controlsamples on first two principal components of arcsine-transformed allele frequencies. A minimum spanning tree is superimposed. Populationcodes as in Figure 1.

    However, the principal component analysis (PCA) of McDonald etal. (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 byMcDonald et al. (1991) showed an UPGMA dendrogram with thecluster of the Chilean samples (bootstrap value of 91 %) groupedwith the M. edulis sample (bootstrap value of 100%). Conse"quently, it could be that the similarity of Pacific South Americanmussels to M. edulis, reported by McDonald et al. (1991), could bean effect of the low number of loci used. Present results using 30allozyme 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 2nuclear DNA markers (ITS and Glu5'; Heath et al. 1995, Rawson

    B ~ Eo ,., B ~

    ' t" u B~ 0

    DD D D

    0 00 0

    0 0 0(l) o ~ o'l>

    0 00

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    ALLOZYME IDENTIFICATION OF MYTILUS 11 13

    quences of the HI histone coding region have shown that Chileanmussels were closer to M. californianus than to the northern M.edulis, M. galloprovincialis and M. trossulus (Eirfn-L6pezet aI.2002). These results could be a consequence of the peculiarity ofthe Chilean mussels (and South American samples), as previouslyconsidered by McDonald et al. (1991). The mussel samples fromthe 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 theordination analyses (Fig. 2c, 3, 4). They showed characteristic andconsistent patterns at allele frequencies for several loci, manlyAco-I, Ap-I, Est-D, Gpi, Idh-I , Lap-2, Me-2, Mpi , Odh, Pgm-2and Pp, different from those of M. eduli s and M. galloprovincialissamples (Table 2). Of these loci, only Gpi and Odh showed significant degree of genetic interpopulation differentiation with F STvalues of 0.0214 and 0.0449, respectively (Table 4, Group B).

    Moreover, theF

    ST mean for the South American samples was low( F S T = 0.012), which suggests a high level of gene flow amongpopulations (Nei 1987). This is in accordance with the generalstatement that within each mussel form and for a particular distinctgeographic region the allele frequencies are generally homogenous(Gosling 1992b and references therein). Moreover, the diagnosticvalues (DVs) between Chilean mussels (using the mean of theallele frequencies across the foufsamples) and the control sampleswere calculated. The DVs were higher than 0.80 at Aco-I (0.81),Est-D (0.89), Gpi (0.97), Idh-I (0.82), Me-2 (0.91), Odh (0.98) andPp (0.82) for M. edulis and at Lap-I (0.999), Me-2 (0.85), Mpi(0.94) and Odh (0.89) for M. galloprovincialis. None of these locidiscriminated unequivocally among the three groups, althougheach locus made some contribution. Consequently, if three Mytilus

    taxa were considered, individuals with a given genotype for theearlier mentioned loci (or combinations of only several loci) can beassigned to a taxon with one probability of erroneous assignmentusing the method of Ayala & Powell (1972). This means that thethree different gene pools could be genetically identified. In resume, the Chilean mussels seem to be a Mytilus taxon different toboth M. edulis and M. galloprovincialis from the Atlantic Euro"pean waters.

    The identification of the Chilean mussels as a different taxon ofthe 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 smoothMytilus lineages, M. edulis, M. galloprovincialis and M. trossulus

    (see Varvio et aI. 1988, McDonald et al. 1991, Gardner 1992,Gosling I992a, 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 fromthose of the control samples of M. edulis and M. trossulus. Basedupon these results and those reported by McDonald et al. (1991)for Est-D 9 allele frequency ("the most common esterase allele inM. galloprovincialis and the rarest in M. edulis was the mostcommon allele in the two mussel samples from Chile" (7), Toro1998, pp. 352), he considered that the similarity between M. chilensis and M. galloprovincialis gives them the same taxonomicstatus. According to the hypothesis that M. galloprovincialis is asubspecies of M. edulis(Gardner 1992); Toro (1998) proposed thatthe 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 aI. 1991; see for areview Gosling 1992a, 1992b and references therein). The resultsof the present work, using a large number of allozyme loci (30loci) and four samples from the northern distribution of Mytilus onthe South American Pacific coasts, showed that (1) mussels fromthis area form a relatively homogenous group with characteristicallele frequencies for several loci and different from those of theEuropean 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, aswell as for most of the reported DNA markers. Moreover, (3) thegenetic distances among Chilean mussels and European M. edulisand 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, atleast in the north limit, could be Mytilus galloprovincialis chilensis.

    ACKNOWLEDGMENTS

    The authors thank Dr. Annie Machordom for their careful read"ing of manuscript, useful comments and suggestions. This researchwas suppor ted by grants PB98-1093 (Direcci6n General deEnsefianza Superior e Investigaci6n Cientffica, MEC, Spain) andPGIDTOOPXI30117PN (Xunta de Galicia, Spain) to AS. CC wassupported by a research fellowship from AECI (Agencia Espafiolade Cooperaci6n Internacional, Spain) and University of Vigo(Spain).

    LITERATURE CITED

    Aebersold, P. B., G. A. Winans, D. J. Teel, G. B. Milner & F. M. Utter.1987. Manual for starch gel electrophoresis: a method for the detectionof genetic variation. NOAA Technical Report NMFS 61. pp. 1-19.

    Ahmad, M., D. O. F. Skibinski & J. A. Beardmore. 1977. An estimate of

    the amount of genetic variation in the common mussel Mytilus edulis.Biocnem. Genet. 15:833-846.

    Ayala, F. J., J. W. Valentine, L. G. Barr & G. S. Zumwalt. 1974. Geneticvariability in a temperate intertidal phoronid, Phoronopsis viridis. Biochem. Genet. 11:413-427.

    Ayala, F. J. & J. R. Powell. 1972. Allozymes as diagnostic characters ofsibling species of Drosophila. Proc. Nat!' Acad. Sci. USA 69:10941096.

    Belkhir, K., P. Borsa, J. Goudet, L. Chikhi & F. Bonhomme. 2002.GENETIX, logiciel sous Windows pour la genetique des populations v3.0. Universite Montpellier, Montpellier, France, 2.

    Benzecri, J. P. 1982. L'Analyse des Donnees. 2. L' Analyse des Correspondances. Dunod, Paris.

    Bulnheim, H. P. & A. Scholl. 1981. Genetic variation between geographicpopulations of the amphipods Gammarus zaddachi and G. salinus.Mar. Bio!. 64:105-115.

    Comesafia, A. S. 1997. Diferenciaci6n genetica en Mytilus galloprovincialis Lmk. del SO. del Mediterraneoy en la zona hfbrida con M. edulisL. Doctoral Thesis (PhD). University of Vigo, Spain.

    Comesafia, A. S., D. Posada & A. Sanjuan. 1998. Mytilus galloprovincialisLmk. in northern Africa. J. Exp. Mar. Bio!. Eco!. 223:271-283.

    Daguin, C. & P. Borsa. 2000. Genetic relationships of Mytilus gallopro"vincialis Lamarck populations worldwide: evidence from nuclear

    DNA markers. In the evolutionary biologyof

    the bivalvia. GeologicalSociety, London, Special Publications 177:389-397.Dunn, G. & D. S. Everitt. 1982. An introduction to mathematical tax

    onomy. Cambridge University Press, Cambridge.

  • 8/9/2019 Mussels Mytilus in Chile 2005

    14/15

    1114 CARCAMO ET AL.

    Eirin-L6pez, J. M., A. M. Gonzalez-Tiz6n, A. Martinez & J. Mendez.2002. Molecular and evolutionary analysis of mussel histone genes(Mytilus spp.): Possible evidence of an "Orphon Origin" for HI histonegenes. J. Mol. Evol. 55:272-283.

    Felsenstein, J. 1985. Confidence limits on phylogenies: an approach usingthe bootstrap. EvoI.30:783-791.

    Felsenstein, J. 1995. PHYLIP (phylogeny inference package) version3.57c. University of Washington. USA.

    Gardner, J . P. A. 1992. Mytilus galloprovincialis (Lmk) (Bivalvia, Mollusca): the taxonomic status of the Mediterranean mussel. Ophelia35:219-243.

    Gran t, W. S. & M. 1. Cherry. 1985. Mytilus galloprovincialis Lrnk. Insouthern Africa. J. Exp. Mar. Bioi. Ecol. 90:179-191.

    Gosling, E. M. 1992a. Systematics and geographic distribution of Mytilus.In: E . M. Gosling, editor. The mussel Mytilus: ecology, physiology,genetics and culture. Amsterdam: Elsevier. pp. 1-20.

    Gosling, E. M. 1992b. Genetics of Mytilus. The mussel Mytilus: ecology,physiology, genetics and cultive. In: E. M. Gosling, editor. Elsevier,Amsterdam. pp. 309-382.

    Gosling, E. M. & D. McGrath. 1990. Genetic variability in exposed-shoremussels, Mytilus spp., along an environmental gradient. Mar. BioI.104:413-418.

    Goudet, J. 2002. FSTAT, a program to est imate and test gene diversi tiesand fixation indices version 2.9.3. Univ. Lausanne, Switzerland.

    Harris, H. & D. A. Hopkinson.1976. Handbook of enzyme electrophoresisin human genetics. North-Holland, Amsterdam.

    Heath, D. D., P. D. Rawson & T. 1. Hilbish. 1995. PCR-based nuclearmarkers identify alien blue mussel (Mytilus spp.) genotypes on the westcoast of Canada. Can. J. Fish. Aquat. Sci. 52:2621-2627.

    Hilbish, T. J., A. Mullinax, S. L. Dolven, A. Meyer, R. K. Koehn & P. D.Rawson. 2000. Origin of the antitropical distribution pattern in marinemussels (Mytilus spp.): routes and timing of transequatorial migration.Mar. BioI. 136:69-77.

    Hochberg, Y. 1988. A sharper Bonferroni procedure for multiple tests ofsignificance. Biometrika 75:800-802.

    Koehn, R. K. 1991. The genetics and taxonomy of species in the genusMytilus. Aquaculture 94:125-145.

    Lamy, E. 1936 . Rev is ion des Myt il idae v ivant s du Museum Nationald'Histoire Naturelle de Paris. J. Conch, Paris 80:107-198.

    Manly, B. F. J. 1991. Multivariate statistical methods: a primer. London:Chapman & Hall.

    Martfnez-Lage, A., F. Rodrfguez-Farifia, A. Gonzalez-Tiz6n & J. Mendez.2005. Origin and evolution of Mytilus mussel satellite DNAs. Genome48:247-256.

    McDonald, J. H. 1985. No bad ge ls . S ta rch gel e lect rophoresi s for themasses. Department of Ecology and Evolution. State University NewYork, Stony Brook, New York.

    McDonald, J. H., R. K. Koehn, E. S. Balakirev, G. P. Manchenko, A. I.

    Pudovkin, S. O. Sergievskii'& K. V. Krutovskii. 1990. Species identityof the "common mussel" inhabiting the Asiatic coasts of the PacificOcean. SOy. J. Mar. Bioi. 16(1):10-18. English translation from BioI.Morya. Vladivostok 1990(1):13-22. (in Russian)

    McDonald, J. H., R. Seed & R. K. Koehn. 1991. Allozymes and morphometric characters of three species of Mytilus in the Northern andSouth"ern hemispheres. Mar. BioI. 111:323-333.

    Murphy, R. W., C. W. Sites, Jr., D. G. Buth & C. H. Haufler. 1996.Proteins I: isozyme electrophoresis. In: D.M. Hillis & C. Moritz, editors. Molecular systematics. Massachusetts: Sinauer Associates. pp.45-126.

    Nei, M. 1978. Estimation of average heterozygosity and genetic distancefrom a small number of individuals. Genetics 89:83-590.

    Nei, M. 1987. Molecular evolutionary genet ics. Columbia Univ. Press ,New York, USA.

    Nei , M. & R. K. Chesser. 1983. Estimation of fixation indices and genediversities. Ann. Hum. Genet. 47:253-259.

    Ohresser, M., P. Borsa & C. Delsert. 1997. Intron length polymorphism atthe actin gene locus mac-]: a genetic marker for population studies inthe marine mussels Mytilus galloprovincialis Lrnk. and Mo edulis L.Mol. Mar. BioI. Biotechnol. 6:123-130.

    Osorio, C. & N. Bahamonde. 1968. Moluscos bivalvos en las pesquerfaschilenas. Bio!. Pesq. 3:69-128.

    Pimentel, R. A. 1979. Morphometrics. The multivariate analysis of biological data. Dubuque lA: Kendal/Hunt Publication Co. 276 pp

    Rawson, P. D., K. L. Joyner, K. Meetze & T. J. Hilbish. 1996. Evidence forintragenic recombination within a novel genet ic marker that dis tinguishes mussels in the Mytilus edulis species complex. Heredity 77:599-607.

    Raymond, M. R. L., R. L. Vaantii , F. Thomas, F. Rousset , T. Meeiis & F.Renaud. 1997. Heterozygote deficiency in the mussel Mytilus edulisspecies complex revisited. Mar. Ecol. Prog. Ser. 1 5 6 : 2 2 5 ~ 2 3 7 .

    Raymond, M. R. L. & F. Rousset . 2000. GENEPOP v3.2 populat ion genetics software for exact tests and ecumenicism. Universite de Montpellier 11, France.

    Rego, 1., A. Martinez, A. Gonzalez-Tiz6n, J. Vieites, F. Leira & J. Mendez.2002. PCR technique for identification of mussel species. J. Agric.Food Chem. 50:1780-1784.

    Rice, W. R. 1989. Analyzing tables of statistical tests. Evol. 43:223-225.

    Rohlf, F. J. 1995. NTSYS-pc numerical taxonomy and multivariate analysis system, version 1.70, Exeter Software, New York.

    Saitou, N. & A. Nei. 1987. The Neighbor-Joining method: a new methodfor reconstructing phylogenetic trees. Mol. Bioi. Evol. 4:406-425.

    Sanjuan, A., H. Quesada, C. Zapata & G. Alvarez. 1990. On the occurrenceof Mytilus galloprovincialis on the NW coast of the Iberian Peninsula.J. Exp. Mar. Bioi. Ecol. 143:1-14.

    Sanjuan, A., C. Zapata & G. Alvarez. 1994. Mytilus galloprovincialis andMo edulis on the coasts of the Iberian Peninsula. Mar. Eco!. Prog. Ser.13:131-146.

    Sanjuan, A., C. Zapata & G. Alvarez. 1997. Genetic differentiation inMytilus galloprovincialis Lrnk. throughout the world. Ophelia 47(1):13-31.

    Seed, R. 1992. Systematics evolution and distribution of mussels belongingto the genus Mytilus: an overview. Amer. Malacol. Bull. 9 : 1 2 3 ~ 1 3 7 .

    Shaw, C. R. & R. Prassad. 1970. Starch gel electrophoresis of enzymes-acompilation of recipes. Biochem. Genet. 4:297-320.

    Skibinski, D. O. F., T. D. Cross & M. Ahmad. 1980. Electrophoreticinvestigation of systematic relationships in the marine mussels Modiolus modiolus L., Mytilusedulis L. a nd Mytilus galloprovincialis Lrnk(Mytilidae; Mollusca). BioI. J. Linn. Soc. 13:65-73.

    Skibinski, D. O. F., J. A. Beardmore & T. F. Cross. 1983. Aspects of thepopulation genetics of Mytilus (Myti lidae: Mollusca) in the Bri tishIsles. Bioi. J. Linn. Soc. 19:137-183.

    Sneath, P. H. A. & R. R. Sokal. 1973. Numerical taxonomy. San Francisco,California: Freeman.

    Soot-Ryen, T. 1955. A report on the family Mytilidae (Pelecypoda), AllanHancock pacif. exped. Los Angeles, California: California Press, LosAngeles. pp. 1-175.

    Swofford, D. L. .& R. B. Selander. 1981. BIOSYS-l version 1.7. A computer program for the analysis of allelic variation in genetics. Department of genetics and development, Univeristy of Illinois, Urbana, Illinois, USA.

    Toro, J. 1998. PCR-based nuclear and mtDNA markers and shell morphology as an approaCh to study the taxonomic status of the Chilean bluemussel, Mytilus chilensis (Bivalvia). Aquat. Living Resour. 11(5):347353.

    Toro, J. E., J. A. Ojeda & A. M. Vergara. 2004. The genetic structure of

    Mytilus chilensis (Hupe 1854) populat ions along the Chilean coastbased on RAPDs analysis. Aqua. Res. 35:1466-1471.

  • 8/9/2019 Mussels Mytilus in Chile 2005

    15/15

    ALLOZYME IDENTIFICATION OF MYTILUS 1115

    Trucco, M. I. 2000. Diferenciaci6n genetica con polimorfismos alozimicosde Mytilus spp. del Athintico Sudoccidental, PhD thesis. University ofVigo, Spain.

    Vliinolii, R. & M. M. Bvilsom. 1991. Genetic divergence and a hybrid zonebetween Baltic and North Sea Mytilus populations(Mytilidae: Mollusca). Bioi. J. Linn. Soc. 43:127-148.

    Varvio-Aho, S. L. & P. Pamilo. 1980. A new buffe r sys tem with wideapplicability. Isozyme Bull. 13:14.

    Varvio, S. L., R. K. Koehn & R. VliinOlii. 1988. Evolutionary genetics ofthe Mytilus edulis complex in the North Atlantic region. Mar. Bioi.98:51-60.

    Ward, R. D. & J. A. Beardmore . 1977. Protein var ia tion in the p la icePleuronectes platessa. Gen(!t. Res. 30:45-62.

    Wright, S. 1978. Evolution and the genetics of populations. Variabilitywithin and among natural populations, vol. 4. Chicago: University of

    Chicago Press.