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

ALLOZYME IDENTIFICATION OF MUSSELS (BIVALVIA: MYTlLUS) ON THE PACIFIC

COAST OF SOUTH AMERICA

CLAUDIA CARCAMO,!'* ANGEL S. COMESANA,! FEDERICO M. WINKLER2 AND

ANDRES 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 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
(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 loci
intermediate frequencies for typical alleles of M. edulis and M. galloprovincialisbetween those of the control samples, but they were
closer to those of M edulis 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 those of M. edulis, whereas the most frequent allele of the loci Gpi, Idh-I 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 contro-
versy, 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 distrib-
uted from Latitude 40° South (Valdivia) to Latitude 60° South
(Cape Horn, approximately), and it has been named in different
ways (Osorio & Baltarnonde 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 iden-
tified: M. edulis, M. galloprovincialis and M. trossulus (Koehn
1991, Gardner 1992, Gosling1992a, 1992b, Seed 1992 and refer-
ences 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 mor-
*Corresponding author. E-mail: tcoba@ccma.csic.es
phologic 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 character-
istic 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 al-
leles that were rare or absent in the northern hemisphere.
Other molecular tools, such as DNA markers, have been ap-
plied 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 mito-
chondrial (COlIl) 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-i) from 1sample 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 themito-
chondrial 16S rRNA gene to elucidate routes and times of trans-
equatorial 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. gallo-
provincialis. Recently, Martfnez-Lage et al. (2005) using satellite
DNA sequences maintain that Chilean specimens are closer to M.
1102 CARCAMO ET AL.

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. galloprovincia-
lis. Moreover, the sequences of the HI histone coding region have
shown a closer relationship of Chilean mussels to M. californianus
than to northern M. edulis, M. galloprovincialis and M. trossulus
(Eirfn-L6pez 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 an4/s~)Uth Chilean
samples at allozyme loci (McDonald et al. 1991) (and banding
patterns of RAPDs (Toro et al. 2004) have been previously de-
tected. 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 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 laboratory
and stored at -70°C prior to electrophoresis analysis.
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 cen-
trifugation 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. gallopro-
vincialis (see Gardner 1992, Gosling 1992a, 1992b and references
therein). Electrophoresis was performed at 4°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 Ah-
mad 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 scoring
accuracy.
Data Analyses
Hardy-Weinberg equilibrium expectations for genotype fre-
quencies at polymorphic loci were tested using a goodness-of-fit
X2 test, and the probability of the null hypothesis was estimated
using a Monte Carlo simulation. The genetic structure of popula-
tions 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 homo-
geneity X2 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 signifi-
cance'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 (l)
among population pairs were estimated. The resulting pairwise
genetic distance matrices were used to build DPGMA dendro-
grams (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, 1,000
bootstrap replicates of each data matrix were generated (Felsen"
stein 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 graphi-
cally detect local distortions (Dunn & Everitt 1982, Rohlf 1995).
Data were reduced and displayed using other multivariate
analyses. Samples were ordinated with principal component analy-
sis on allele frequencies of the polymorphic loci (Pimentel 1979,
Manly 1991). The major components might be expected to repre-
sented 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 correspon-
dence analysis (Benzecri 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 consid-
ered. The "Selection AFC" procedure implemented in GENETIX
v4.03 (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 per-
formed with the NTSYS"pc (Rohlf 1995) package.
RESULTS
Four of the 30 analyzed enzyme loci ([3- Gal, Mdh-2, Sod-l and
Sod-2) were monomorphic for all samples, and the remainder 26
enzyme loci showed consistent polymorphism and their allele fre-
 ALLOZYME IDENTIFICATION OF MYTILUS 1103

.........,
N
""'""----"'. - ...........,-. / ' ...
,,,
t
.........................IllF"""'"............................_ .............._ .......~~ . . s

50

CHilE
le Region
'100
Km

B)
A)

Figure 1. Sampling places for Mytilu$ sp. in Chile, in the northern limit of their distribution on Pacific coast of South America (Valdivia: PVA,
Puerto Montt: PPM, Ancud: PAN and Quell6n: PQE).

quencies are shown in Table 2. M. edulis (EH) and M. gallopro-
vineildis (GV) control populations showed important differences at
the allele frequencies in 9 loci (Aeo-I, Ap-I, Est-D, Gpi, Idh-I,
Lap-I, 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 a1. 1991, VilinOla & Hvilsom 1991, Gosling 1992b,
Seed 1992, Sanjuan et al. 1994, Sanjuan et a1. 1997 and references
therein). Of the six most studied loci, Est-D, Gpi, Lap-I, Mpi and
Odh showed important differences, and Ap-I showed a lower dif-
ferentiation. For example, Gpi 102 and Gpi 107 alleles together and
also Lap-I 96 and Lap_I JOo together showed the highest frequencies
in the control population of M. edulis (0.833 and 0.970, respec"
tively), whereas in the M. galloprovincialis sample they were
lower (0.151 and 0.000, respectively). Moreover, control popula-
tions also showed important differences for Aco-I and Me-2 and at
less degree for Idh-I. For example, the most frequent allele for M.
edulis was Aeo-I llO , with a frequency of 0.750 whereas it was
0.175 for M. galloprovineialis. This genetic differentiation was
previously reported by Grant & Cherry (1985) for Me-2 and Idh-I
and for both and Aeo-I by Comesafia (1997) and Trucco (2000).
To quantify the discrimination power of each locus for M.
1104 CARCAMO Er .AL.
TABLE 1.
Enzyme systems analyzed showing tbe 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 (Ahmadet al. 1977), BS (Bulnheim & SchoH 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 CitratepH 7.0,
Ahmad et al. 1977), TC 8 (Tris Citrate pH 8.0, Ward & Beardmore 1977), TC 8.4 (Tris Citrate pH 8.417.0, Varvio-Aho & Pamilo 1980) and
TCE (Tris CitrateEDTA pH 7.0, Ayalaet al. 1974).

Abbr. Enzyme System E.N. Loci Staining Buffer

AAT Aspartate aminotransferase 2.6.1.1 2 SP TC7

ACO Acouitate hydratase 4.2.1.3 2 HH TCE
ALD Fructose"bisphosphate aldolase 4.1.2.13 1 AB TC 8.4
AP"1 Glycil-Ieucine peptidase 3.4.13.18 1 AH TC 7
ARK Arginine kinase 2.7.3.3 1 HH TC 8.4
DIA Dihydrolipoamide dehydrogenase 1.8.1.4 1 MR TC 8.4
EST Esterase 3.1.1.1 2 AH,MR ACE 5.6
13-GAL 13-galactosidase 3.2.1.23 1 MR TBE 8.7
GAPDH Glyceraldehide-3-phosphate dehydrogenase 1.2.1.12 I MR TC 8.4
GPI Glucose-6-phosphate isomerase 5.3.1.9 1 MR TC 8.4
G3PDH Glycerol-3-phosphate dehydrogenase NAD+ 1.1.1.8 1 MR TBE 8.7
IDH Isocitrate dehydrogenase NADP+ 1.1.1.42 2 SP TC 8
LAP-l Leucine aminopeptidase-I 3.4.11.- I AH ACE 5.6
LAP-2 Leucine aminopeptidase-2 3.4.11.- I AH TC 7
MDH Malate dehydrogenase 1.1.1.37 2 AB TC 8.4
ME Malate dehydrogenase NADP+ 1.1.1.40 2 AB TC 8.4
MPI Mannose-6-phosphate isomerase 5.3.1.8 I SN TC 8.4
ODH D-octopine dehydrogenase 1.5.1.11 I GC TC 8.4
PGD 6-phosphogluconate dehydrogenase 1.1.1.44 1 HH TC 7
PGM Phosphoglucomutase 5.4.2.2 2 HH TCE
PP Phenyl"proline peptidase 3.4.13.9 I HH TBE
SOD 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 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-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 be-
tween 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-I 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 di"
agnostic loci for M. edulis and M. galloprovincialis forms.
Samples from Chile showed a different behavior in allele fre-
quencies with respect to control populations (Ell and GV) depend-
ing on loci (Table 2). The locus Lap-I showed frequencies of the
diagnostic 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) (Table
2). For other 4 loci (Aeo- I, Ap-I, Est-D and Mpi), Chilean samples
showed intermediate allele frequencies for the typical alleles of M.
edulis and M. galloprovineialis between those of both control
samples. 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 Mpi200 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 Aeo-I, the allele
Aeo-I 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-I
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 J29 in Chilean
samples ranged from 0.846-0.984 whereas in M. galloprovineialis
was 0.329 and in M. edulis 0.039. At the Me-2 locus, the most
frequent allele in Chilean samples was Me_2 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_2 100 allele, characteristic of M. galloprovincialis (0.716),
was the second most common allele in Chilean samples (0.361-
0.476; arithmetic mean: 0.397).
The South American samples also showed important differ-
ences 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_2 100 and Pgm_2 JOo 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 95 (0.146-0.229;
arithmetic mean: 0.195) instead of Lap_2 105 , which was common
in control samples (0.236 and 0.346). For Pp, the pp 90 allele had
 ALLOZYME IDENTIFICATION OF MYTILUS 1105

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 ..,ample size. He is the
unbia..,ed expected mean heterozygo..,ity (Nei, 1987) ± its standard error. P 95 and P 99 are the percentage of the polymorphic loci with the 0.95
and 0.99 criteria with standard error, respectively. Four loci ((3-Gal, Mdh-:Z, Sod-] and Sod-:Z) were monomorphic for all samples.

Populations
Locus EH GV PVA PAN PPM PQE
Aat-l
N 73 74 61 41 71 72
94 0 0 0.082 0 0.007 0.035
100 0.979 0.973 0.893 0.988 0.979 0.958
108 0.021 0.020 0.025 0.012 0.007 0.007
116 0 0.007 0 0 0.007 0
Aat-2
N 75 74 56 41 67 70
94 0.007 0 0.009 0 0.015 0
100 0.993 1 0.973 1 0.978 0.979
108 0 0 0.018 0 0.007 0.021
Aco-l
N 58 20 61 41 67 71
100 0 0 0 0.012 0 0.007
105 0.112 0.800 0.631 0.573 0.552 0.535
110 0.750 0.175 0.369 0.415 0.433 0.458
ll5 0.129 0.025 0 0 0.015 0
120 0.009 0 0 0 0 0
Aco-2
N 51 62 50 33 67 43
95 0.020 0.008 0.020 0.030 0.067 0
100 0.157 0.121 0.040 0.045 0.022 0.011
105 0.784 0.806 0.920 0.894 0.904 0.978
107 0.039 0.056 0.010 0.020 0.007 0.011
110 0 0 0.010 0 0 0
112 0 0.008 0 0 0 0
Aid
N 25 14 40 40 47 45
100 0.920 0.964 0.988 1 1 1
112 0.080 0.036 0.012 0 0 0
Ap-l
N 71 70 59 41 69 72
93 0.015 0 0 0 0 0
96 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 0
108 0.218 0.457 0.331 0.293 0.304 0.396
114 0.035 0.128 0.093 0.061 0.029 0.069
122 0 0.029 0.017 0 0.007 0
128 0 0 0 0.012 0 0
Ark
N 74 71 51 39 66 69
92 0.014 0 0.216 0.141 0.061 0.159
100 0.986 1 0.784 0.859 0.939 0.841
Dia
N 72 70 60 40 69 71
95 I 0.104 0.064 0.042 0.112 0.058 0.049
100 0.778 0.771 0.875 0.837 0.862 0.859
102 0.021 0 0 0 0.007 0.021
105 0.097 0.164 0.083 0.050 0.065 0.071
llO 0 0 0 0 0.007 0

continued on next page

1106 CARCAMO ET AL.

TABLE 2.
continued

Populations

Locus EH GV PVA PAN PPM PQE

Est-D
N 75 72 61 41 71 71
82 0 0.035 0 0 0 0
90 0.013 0.910 0.566 0.622 0.451 0.627
100 0.953 0.056 0.426 0.378 0.549 0.366
107 0.020 0 0 0 0 0.007
110 0.013 0 0.008 0 0 0
Est-]
N 60 74 59 41 70 69
10 0 0.007 0.102 0.098 0.100 0.094
100 0.525 0.473 0.593 0.561 0.536 0.486
200 0.350 0.378 0.263 0.280 0.357 0.377
300 0.108 0.135 0.042 0.061 0.007 0.043
400 0.017 0.007 0 0 0 0
Gapdh
N 56 31 55 36 62 47
100 0.955 0.823 0.864 1 1 1
120 0.036 0.129 0.136 0 0 0
140 0.009 0.048 0 0 0 0
Gpi
N 75 66 58 41 70 69
93 0.007 0.008 0.009 0 0.007 0
96 0.013 0.015 0 0 0 0.014
98 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 0.094
105 0.047 0.235 0.026 0.012 0.029 0.022
107 0.520 0.083 0 0 0 0
110 0.020 0 0 0 0 0
112 0.007 0.008 0 0 0 0
G3pdh
N 65 65 56 40 70 72
90 0.031 0 0 0 0 0.007
100 0.969 1 I 1 0.993 0.979
115 0 0 0 0 0.007 0.014
Idh-]
N 54 47 58 39 60 71
60 0 0.011 0 0 0 0.021
80 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 0.042
160 0.009 0 0 0 0 0
Idh-2
N 74 46 58 41 61 69
96. 0.182 0.098 0.017 0 0.008 0.007
100 0.811 0.891 0.966 0.976 0.992 0.993
104 0.007 0 0.017 0.024 0 0
108 0 0.011 0 0 0 0
Lap-]
N 65 38 60 41 71 71
93 0 0 0.017 0.024 0.014 0.028
96 0.208 0 0.250 0.317 0.246 0.211
100 0.762 0 0.683 0.659 0.683 0.725
102 0.008 0 0 0 0.021 0.014
104 0.015 0.487 0.050 0 0.021 0.021
108 0.008 0.461 0 0 0.014 0
110 0 0.026 0 0 0 0
112 0 0.026 0 0 0 0

continued on next page

 ALLOZYME IDENTIFICATION OF MYTlLUS 1107

TABLE 2.
continued

Populations

Locus EH GV PVA PAN PPM PQE

Lap-2
N 72 68 59 41 71 72
90 0.028 0.007 0.017 0.024 0.077 0.042
95 0.139 0.088 0.229 0.146 0.204 0.201
100 0.583 0.544 0.746 0.817 0.676 0.729
102 0.007 0 0 0 0.007 0.007
105 0.236 0.346 0.008 0.012 0.028 0.021
110 0.007 0.015 0 0 0.007 0
Mdh-l
N 75 69 56 41 71 69
70 0.013 0.007 0 0.024 0.014 0.007
100 0.987 0.986 1 0.976 0.972 0.986
130 0 0.007 0 0 0.014 0.007
Me-l
N 64 33 57 41 65 72
70 0.039 0 0 0 0 0
90 0.008 0 0 0 0 0
100 0.914 0.939 0.860 0.951 0.946 0.972
100 0.039 0.061 0.140 0.049 0.054 0.028
120 0 0 0 0 0 0
Me-2
N 65 58 61 41 66 71
70 0 0 0.008 0.037 0.008 0
90 0.015 0 0.475 0.293 0.462 0.514
100 0.100 0.716 0.361 0.476 0.371 0.380
110 0.831 0.276 0.156 0.159 0.152 0.106
ll5 0.023 0 0 0.037 0.008 0
120 0.023 0.009 0 0 0 0
Mpi
N 59 56 59 40 68 70
25 0 0 0.042 0 0.022 0.086
100 0.017 0.973 0.322 0.262 0.140 0.314
200 0.975 0.027 0.636 0.725 0.838 0.600
300 0.008 0 0 0.013 0 0
Odh
N 64 35 58 31 68 37
80 0 0 0 0 0 0.014
100 0.055 0.486 0.034 0.016 0.125 0
112 0.008 0 0 0 0 0
115 0.891 0.186 0.026 0 0.015 0.014
120 0.008 0 0 0 0 0
129 0.039 0.329 0.940 0.984 0.846 0.973
140 0 0 0 0 0.015 0
Pgd
N 72 56 56 40 71 72
95 0.021 0.009 0.018 0.025 0.021 0.007
100 0.938 0.964 0.982 0.962 0.958 0.986
105 0.035 0.018 0 0.013 0.021 0.007
110 0.007 0 0 0 0 0
115 0 0.009 0 0 0 0
Pgm-l
N 14 19 36 28 15 35
90 0 0.026 0 0 0 0
95 0 0.132 0 0.036 0 0
100 0.964 0.789 0.667 0.768 1 0.957
105 0 0.053 0.333 0.196 0 0.043
115 0.036 0 0 0 0 0
Pgm-2
N 74 73 55 37 71 72
90 0.007 0 0 0 0 0
92 0.041 0.007 0.009 0 0.007 0
96 0.142 0.144 0.018 0 0.049 0.014

continued on next page

1108 CARCAMO ET AL.

TABLE 2.
continued

Populations

Locus EH GV PVA PAN PPM PQE

100 0.574 0.548 0.818 0.838 0.796 0.840
102 0 0.007 0.009 0 0 0.007
104 0.189 0.240 0.145 0.162 0.148 0.132
107 0.041 0.048 0 0 0 0.007
110 0.007 0.007 0 0 0 0
Pp
N 69 68 60 41 70 70
80 0 0 0.017 0.037 0.014 0.029
90 0.072 0.029 0.458 0.427 0.543 0.479
100 0.710 0.912 0.508 0.524 0.436 0.479
110 0.210 0.044 0.017 0.012 0.007 0.014
120 0.007 0 0 0 0 0
125 0 0.015 0 0 0 0
He 0.262 ± 0.045 0.236 ± 0.040 0.259 ± 0.039 0.220 ± 0.039 0.237 ± 0.041 0.228 ± 0.042
AlIe1esllocus 3.2 ± 0.3 3.5 ±0.3 2.8 ± 0.3 3.1 ± 0.3 2.5 ± 0.2 2.9 ±0.2
P95 60.0 56.7 66.7 56.7 53.3 50.0
P 99 76.7 86.7 80.0 76.7 73.3 80,8

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 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 num-
ber 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 :t 0.039) and the highest the M.
edulis control sample (0.262 :t 0.045). However, no significant
differences among all pair of values Were found with the Hest (Nei
1987) (data not shown). Puerto Montt (PPM) sample showed the
lowest number of alleles per locus (2.5 :t 0.2) and M. gallopro-
vincialis (GV) the highest (3.5 :t 0.3).
Twenty-seven of the 142 X2 tests were significant (Table 3),
showing departure of genotype frequencies from Hardy-Weinberg
expected proportions (P < 0.05). Of these, only 13 showed sig-
nificant deviations after Bonferroni correction. These 13 signifi-
cative values showed positive values of F (deficiencies of het-
erozygotes) and involved only 5 loci: Est-l in EH, PPM and PQE
samples, Gpi in GV, PVA, PAN and PQE samples, Lap-l in GV
sample, Me-l in PVA, PPM and PQE samples and Me-2 in GV and
EH samples (Table 3). Note that for Chilean samples only I par-
tially diagnostic locus (Gp!) showed significant positive F values
and a possible Wahlund effect can be disregarded. Moreover, het-
erozygote deficiency at allozyme loci in bivalves has been de-
scribed previously although its causes are still unclear (Gosling
1992b, Raymond et al. 1997).
The mean value of FST over all analyzed samples, including
Chilean and European samples, was high and significant (FST =
0.180) (Table 4, Group A) but the FST value for Chilean samples
was low (FST= 0.012) (Table 4, Group B). Significant interpopu-
lation 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 signifi-
cant 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 multidi-
mensional 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. galloprovin-
cialis.
Considering the allele frequencies of samples, a principal com-
ponent 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 con-
trol 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.
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 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), showed
the separation of the 3 groups of individuals, the M. edulis, the M.
 ALLOZYME IDENTIFICATION OF MYTILUS 1109

TABLE 3.
F estimates by locus in populations from Europe, M. edulis (Ell) and M. galloprovincialis (GV), and populations on Pacific coast of South
America. X2 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 -0.037
2
X 0.021 0.043 2.863 0.000 0.022 0.113
Aat-2 F -0.007 -0.021 -0.023 -0.022
2
X 0.000 0.028 0.046 0.022
Aeo-l F 0.282 -0.217 -0.021 -0.026 0.273 0.078
2
X 17.724** 1.073 0.010 0.737 5.877* 1.696
Aco-2 F 0.234 0.173 0.207 0.491 0.004 -0.017
2
X 9.898 14.398 15.610 37.241 * 3.791 0.011
Aid F -0.087 -0.037 -0.013
x2 0.139 0.000 0.000
Ap-l F 0.125 0.205 0.019 -0.118 0.094 0.107
2
X 10.218 15.669* 6.375 4.115 3.255 4.464
Ark F -0.014 0.305 0.259 0.202 0.135
2
X 0.007 5.142* 3.063 3.229 1.430
Dia F 0.147 0.121 0.262 0.030 0.042 0.002
2
X 6.729 5.289 9.633* 1.343 18.588 2.742
Est-D F -0.032 0.091 -0.085 0.015 -0.012 -0.042
2
X 0.153 13.456 1.123 0.030 0.002 0.665
Est-l F 0.350 0.209 0.103 0.200 0.357 0.442
2
X 35.210*** 12.291 14.411 14.179 28.292*** 55.220***
Gapdh F -0.039 0.046 0.305
2
X 0.097 20.399 5.677*
Gpi F 0.062 0.452 0.391 0.614 0.212 0.553
2
X 46.780 68.506*** 45.267*** 37.826*** 33.068 108.406***
G3pdh F -0.032 -0.007 -0.016
2
X 0.049 0.000 0.022
ldh-l F 0.292 0.173 -0.149 0.019 0.112 0.079
2
X 11.945** 10.659 1.382 0.834 4.350 5.416
ldh-2 F 0.082 0.334 -0.027 -0.025 -0.008 -0.007
2
X 4.832 7.871 0.055 0.013 0.000 0.000
Lap-l F 0.142 0.569 0.216 0.109 -0.100 0.045
2
X 6.497 90.764*** 11.115 0.953 2.788 1.583
Lap-2 F 0.144 0.209 0.047 -0.022 0.149 0.217
2
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
2
X 0.007 0.007 0.013 0.044 0.007
Me-l F 0.129 0.468 0.564 0.474 0.604 0.785
2
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
2
X 43.302*** 17.815*** 15.950* 14.407 14.722 6.828
Mpi F -0.020 -0.028 -0.106 0.383 0.154 -0.017
2
X 0.027 0.028 1.102 7.687* 45.600 0.582
Odh F 0.227 0.311 -0.048 -0.016 0.001 -0.021
2
X 16.276 7.511* 0.203 0.000 14.539 0.014
Pgd F -0.047 -0.025 -0.018 -0.030 -0.032 -0.011
2
X 0.283 0.057 0.009 0.040 0.113 0.007
Pgm-l F -0.037 0.113 0.125 0.133 0.652
2
X 0.000 2.868 0.702 1.858 22.328*
Pgm-2 F 0.093 0.270 0.058 -0.194 0.130 0.246
2
X 13.208 26.881* 1.636 1.256 147.080 8.962
Pp F 0.270 0.201 0.134 0.099 0.054 0.049
2
X 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 extreme mediate position between M. edulis and M. galloprovincialis in-
position closer to M. galloprovincialis than to M. edulis, in a dividuals.
1110 CARCAMO ET AL.

TABLE 4.
Wright's F-statistics (FIs> Fm F ST) for 26 polymorphic loci for the South American Pacific coast samples and control populations of M.
edulis and M. galwpr()vincialis from Europe (Group A) and for the four South American populations (Group B). Underlined X2 homogeneity
values were sigulficant after Bonferroni's correction (P < 0.05). Populations codes as in Figure 1.

A) South American and control populations B) South American populations

Locus F IS FIT F ST F IS FIT 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 -0.0018
Aeo-l 0.1162 0.2328 0.1319*** 0.0982 0.0961 -0.0022
Aco-2 0.1726 0.2000 0.0031 *** 0.1195 0.1249 0.0062
Aid ~.051O -0.0038 0.0449** -0.0009 0.0003 0.0012
Ap-l 0.1058 0.1383 0.0364*** 0.0547 0.0595 0.0051
Ark 0.2215 0.2863 0.0833*** 0.2307 0.2503 0.0255**
Dia 0.0979 0.1043 0.0072 0.0595 0.0558 -0.0040
Est-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 0.0012
ldh-l 0.1316 0.2925 0.1854*** 0.0389 0.0363 -0.0026
Idh-2 0.1276 0.2058 0.0897*** -0.0126 -0.0106 0.0020
Lap-l 0.1451 0.3170 0.2012*** 0.0621 0.0598 -0.0025
Lap-2 0.1542 0.2051 0.0602*** 0.1316 0.1332 0.0019
Mdh-l -0.0093 -0.0115 -0.0021 -0.0112 -0.0109 0.0004
Me-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 ~0.0015
Pgm-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 -0.0016
Mean 0.1622 0.3134 0.1804*** 0.1339 0.1445 0.0122

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

DISCUSSION allele of M. galloprovincialis but at a higher frequency. The

The allele frequencies of the most extensively studied partially
diagnostic loci Ap-I, Est-D, Gpi, Lap-I, Mpi and Odh for the
European control samples of M. edulis (EH) and M. galloprovin-
cialis (GV) in general were in agreement with those previously
reported (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, Comesafia
1997, Comesafia et al. 1998, Trucco 2000). Other 3 less studied
loci (Aeo-I, Idh-I and Me-2) were found to be partially diagnostic
between these two Mytilus forms in accordance with Grant &
Cherry (1985) for Idh-I and Me-2 and Comesafia (1997) and
Trucco (2000) for the three loci. Moreover, the Nei's (1978) ge-
netic 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-I and Mpi) the allele frequencies were
more similar to those of M. edulis and for Aeo-I and Est-D to those
of M. galloprovincialis. The locus Lap-I showed similar allele
frequencies to those of M. edulis, whereas the most common allele
for Gpi, Idh-Iand Odh in Chilean samples was one of the typical
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 analy-
sis, 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. gallo-
provincialis 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. galloprovineialis was lower
(D == 0.101) than that with M. edulis (D == 0.125).
Before the present work, only McDonald et a1. (1991) had
analyzed by allozyme electrophoresis Mytilus individuals from the
Pacific coast of South America. They analyzed the 8 allozyme loci
Ap-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 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 individu-
als per sample. To establish reasonably allele homologies between
McDonald et a1. (1991) paper and the present study several criteria
as those reported by Sanjuan et a1. (1997) were used. The present
allele frequencies at 6 of these loci (Ap-I, Lap-I, Lap-2, Mpi, Odh
 ALLQZYME IDENTIFICATION OF MYTILUS 1111

8\oo;1...
6 ;::===0;:12=======III::,o.e========0:;04=======0:;Il1O
----:..[---...:;11O~""'1 -=-~~~

-PPIl'l

..... - - - GY

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

o.os
(c)
1.6.....- - - - _ _- ,

o.

J::I
J1 0.0
~
-0.8

.
-1·~±.,...-----_o-:l. ,--------:0T:".0-------,0:r:.8------;;l1.6
DDoumo..... 1
Figure 2. UPGMA dendrogram (a) and Neighbor-Joining tree (b), both based on genetic distances (Nei 1978) and non metrical multidimentional
scaling plot (MDS) (c) on genetic identities among populations (Nei 1978), for 30 aJlozyme loci ofthe South American samples (PAN, PpM, PQE,
and PVA) and European M. edulis (EH) and M. galloprovincialis (GV) control samples. A minimum spanning tree is superimposed on the MDS
plot. Stress of the MDS was S = 0.0086. Numbers above main branches represent the bootstrap percentage above 40% obtained after 1,000
replications. Populations codes as in Figure 1.

I
and Pgm-2) were close to those reported by McDonald et a1.
(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 com-
mon allele for the four Chilean samples (Gpi JOo ) showed frequen-
cies (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 a1.
(1991) the most common allele (Gpi 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 Vain6Hi & Hvilsom (1991) and Varvio et a1. (1988) and that of
McDonald et a1. (1990, 1991) for M. galloprovincialis, but not for
M. edulis. The most frequent allele for M. edulis from Denmark
and White Sea is Gpi J07 (frequencies 44% and 58%) (McDonald et
a1. 1990, see also Gosling, 1992b, pp. 319), as in the present work
and also in Vain61a & Hvilsom (1991) and Varvio et a1. (1988), but
at 2nd and 3rd positions were Gpi JOO (14% and 18%) and Gpi96
(16% and 10%), instead of Gpi lO:2 (and perhaps Gpi98 ) as in the
present work and in Vain61a & Hvilsom (1991) and Varvio et a1.
(1988). These results on M. edulis and Chilean lTIussels are con-
tradictories with present data, and the explanation is not easy.
1112 CARCAMO ET AL.

~:t;'Ir---~------------------------""""

-J}.2'+-------""1""--------r------.. . -,.-------""""
'0.2 ·0.1 -0.0
Component 1 (40.79%)
Figure 3. Projectious of samples of South America (PAN, PPM, PQE, aud PVA) aud M. edulis (EH) aud M. galloprovincialis (GV) control
samples on first two principal components of arcsine-transformed allele frequencies. A minimum spanning tree is superimposed. Population
codes as in Figure 1.

However, the principal component analysis (PCA) of McDonald et et al. 1996) and one mitochondrial DNA marker (COllI), to ana-
al. (1991) suggests that Chilean mussels were more similar to M. Iyze a sample of 30 mussels from Bahia Corral, located within the
edulis from the northern hemisphere than to M. galloprovincialis. geographic range of the present study. Data from ITS and COIII
A reanalysis of present data using only the eight loci analyzed by showed the same pattern for Chilean individuals, M. edulis from
McDonald et al. (1991) showed an UPGMA dendrogram with the Newfoundland and M. galloprovincialis from New Zealand, but,
cluster of the Chilean samples (bootstrap value of 91 %) grouped the Glu5' marker showed the same banding pattern for Chilean
with the M. edulis sample (bootstrap value of 100%). Conse" Mytilus and M. galloprovincialis from New Zealand and different
quently, it could be that the similarity of Pacific South American from M. edulis control sample from Newfoundland. Moreover,
mussels to M. edulis, reported by McDonald et al. (1991), could be Daguin & Borsa (2000) analyzed the two diagnostic nuclear DNA
an effect of the low number of loci used. Present results using 30 markers, Glu 5' and the polymorphic mac-I. In a sample of cul-
allozyme loci Seem to be more representative of the genetic rela- tivated mussels from Dichato, about 400 km to the north of the
tionship among Mytilus taxa than other works using a lower num- present sample of Valdivia (PVA) (see Fig. 1), they found for Glu
ber of genes. 5' the typical M. galloprovincialis allele fixed (Rawson et al.
With regard to other molecular markers, Toro (1998) used 2 1996) and for mac- I locus (Ohresser et al. 1997) a high frequency
nuclear DNA markers (ITS and Glu5'; Heath et al. 1995, Rawson of the hI and c2 characteristic alleles of M. galloprovincialis.
Moreover, Hilbish et al. (2000) studied RFLPs and sequences of
the mitochondrial l6S 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 l6S rRNA gene grouped Chilean
B
• sequences within the cluster of the northern hemisphere M. gallo-
~
.,., Eo
B • • provincialis. In the same line, Martinez-Lage et al. (2005) using
~ satellite DNA sequences maintain that Chilean specimens are
'" • 't uB
closer to M. galloprovincialis than to M. edulis.
~
0
D D
DD In resume, taking into account the data based on 30 enzyme
loci, an important representation of the genome and the earlier
0 0
0 0 mentioned genetic results with nuclear DNA markers, RFLPs and
0 0
(l) o ~o 'l> sequences of the mitochondrial l6S DNA, it seems that mussels
0 0
0
0 <IJ from the northern limit of distribution on Pacific coast of South
Axis 1 (6.94%) America were genetically closer to M. galloprovincialis than to M.
Figure 4. Factorial correspondence analysis (FCA) based on the ma· edulis.
trix of 153 individuals, including 23 Mytilus edulis (0), 20 M. gallo· However, Rego et al. (2002) have reported banding patterns of
provincialis (Ill) and 110 Chilean mussels (D), using 107 alleles from 23 RAPDs for a Chilean sample closer to those of the European M.
allozyme loci (the 26 polymorphic loci except Aid, Gapdh and Pgm-l). edulis than those of M. galloprovincialis. Moreover, DNA se-
 ALLOZYME IDENTIFICATION OF MYTILUS
quences of the HI histone coding region have shown that Chilean
mussels 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 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 homoge-
neous and different from the European M. edulis and M. gallopro-
vincialis 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-I, Ap-I, Est-D, Gpi, Idh-I, 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 sig-
nificant degree of genetic interpopulation differentiation with FST
values of 0.0214 and 0.0449, respectively (Table 4, Group B).
Moreover, the F ST mean for the South American samples was low
(FST = 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 foufsamples) and the control samples
were 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) and
Pp (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 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 re-
sume, the Chilean mussels seem to be a Mytilus taxon different to
both M. edulis and M. galloprovincialis from the Atlantic Euro"
pean waters.
The identification of the Chilean mussels as a different taxon of
the other Mytilus forms does not resolve the problem of the ap-
propriate taxonomic category of this mussel group. The main prob-
lem is the controversy on the taxonomic status of the main smooth
Mytilus lineages, M. edulis, M. galloprovincialis and M. trossulus
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ACKNOWLEDGMENTS
The authors thank Dr. Annie Machordom for their careful read"
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was supported by grants PB98-1093 (Direcci6n General de
Ensefianza Superior e Investigaci6n Cientffica, MEC, Spain) and
PGIDTOOPXI30117PN (Xunta de Galicia, Spain) to AS. CC was
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