The genetics and taxonomy of species in the genus Mytilus

Aquaculture, 94 (1991) 125-145 Elsevier Science Publishers B.V., Amsterdam

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The genetics and taxonomy of species in the genus Mytilus Richard K. Koehn Department of EcoIogy and Evolution, State Universityof New York, Stony Brook, NY 11794, USA

ABSTRACT Koehn, R.K., 199 1. The genetics and taxonomy of species in the genus Myth. 145.

Aquaculture, 94: I25-

It has been nearly 15 years since the genetics of Myths was the subject of a comprehensive review. In this period, our understanding of the nature of genetic variation in this group has been substantially altered. Studies on the extent of individual variation have demonstrated that genotype (i.e., multiple locus heterozygosity) has a significant effect on energy metabolism, producing significant statistical correlations between genotype and measures of both metabolic energy demand and productivity, particularly growth rate. These correlations can be mitigated by a number of factors, including age, reproductive state, the specific genes under study and ecological conditions. Studies of within-population variation have repeatedly noted marked deficiencies of heterozygotes, but no satisfactory explanation for this observation has yet been identified. Deficiencies are locusspecific and multilocus disequilibria have been described, suggesting a combination of larval mixing with additional forces such as selection, aneuploidy or molecular imprinting. Early work suggested substantial genetic differentiation between spatially proximate populations. Populations are now known to be relatively homogeneous, even over vast geographical distances. Genetic differences between proximate populations generally represent taxonomic differentiation among mytiliid species, not population differences. Studies of allozymes, morphology and (to some degree) mitochondrial genotype among worldwide samples have demonstrated the existence and geographic distributions of four species in the genus: Mytilus californianus, M. edulis, M. trossulusand M. galloprovincialis.Data for the three latter species are presented along with the geographic distribution of each.

INTRODUCTION

The genus M~~ilus is one of the most cosmopolitan of all marine genera, occurring in estuarine and ocean habitats, in both the subtidal and intertidal zones, occupying a diversity of substrates and distributed at higher latitudes in all the oceans and major seas of the world. The early taxonomy was based solely on morphological features and nearly exclusively characters of the shell. In the past century, many local morphological variants were given species and/or subspecies status, in the latter case, commonly subspecies of M. edulis. The environmental plasticity of the shell morphology, combined with what 0044-g486/91/%03.50

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we now know to be a complex interaction of several species, produced a confused and largely erroneous taxonomy. The availability of large samples of individuals has attracted the interest of population geneticists for the past 20 years, ever since the development of electrophoresis permitted the genetic study of organisms in which genetically simple visible polymorphisms did not naturally occur. The use of electrophoresis to characterize the genetic composition of samples has provided a means to more precisely estimate individual and population differences. These genetic differences, combined with multivariate statistical techniques applied to both enzyme and morphological phenotypes, have helped clarify the systematics and taxonomic status of species of the genus. Although several important reviews have been concerned with various aspects of the genetics or systematics of marine bivalve molluscs (cf., Koehn, 1983; Gosling, 1984; Zouros, 1987), the most recent overall review of the genetics of mussels was Levinton and Koehn ( 1976). At that time, geographic, patterns of genetic differentiation were being described, primarily among populations of Mytilus edulis along the Atlantic coast of North America. Correlations between allele frequency and shell size, genetic differences between various areas of the world, the existence of allele frequency clines, and (to a lesser degree) genetic differences between species of Mytilus had emerged from individual studies, but the generality, or not, of these findings was unknown. Since 1976, there has been a significant increase in our understanding of the genetics and taxonomic status of Mytilus species. The magnitude of genetic variation in Mytilus, at least as detected by enzyme polymorphism, is not atypical of most other marine and even non-marine organisms. Individual multilocus genotype is correlated with various measures of performance, or production, again as in several other studied organisms. The specilic mechanisms that form the basis of these correlations are still not precisely understood, but it is an area of current research activity. Though data available in 1976 suggested that mussel populations were much more highly genetically structured than would be supposed from the ability of larvae to be transported large distances, it is now apparent that populations of a species maintain similar genetic compositions over most of their ranges; many gene frequency clines and microgeographic differences that were thought to reflect genetic population structuring are now known to involve contact and hybridization between different species. While there are small differences in genetic composition over vast ranges in some mussel species, the very steep clines in the frequency of Lap alleles at the entrance to Long Island Sound and at Cape Cod (Koehn et al., 1976, 1980, 1984) are now the only well characterized large differences that occur within-species over relatively small geographic distances (see later). While there are some genetic differences among virtually world-wide pop-

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ulations of each mussel species, these differences are not sufficiently large to obscure their clear systematic affmities with one another. Taxonomic groups (i.e., species) can be detected and defined on the basis of allozyme genotype; multivariate analysis of morphological variation is also capable of discriminating these groups and in all cases allozyme and morphological characters give concordant results. Hybridization occurs in virtually every known case where two species are in geographic contact, though in these areas individual populations of pure and hybrid populations are intermixed in a spatially complex patchiness. Lastly, mussels have only very recently been the subjects of studies at the DNA level and at that, exclusively mitochondrial DNA. These data tend to confirm the taxonomic judgments based on both allozymes and morphology. Nevertheless, such molecular approaches promise to give unique genetic information on mussels. For example, Myths edulis is the first known species where the mitochondrial genome is biparentally inherited (Hoeh et al., 199 I), rather than maternally inherited as has been found in every other studied animal and plant species. In the following sections, I detail the foregoing general points on the genetics and taxonomy of mussels, focusing upon areas of current genetic research and describing the existence and geographic distributions of three species of the genus: Mytilus edulis, M. galloprovincialis,and A4 trossulus. GENETIC VARIATION AT THE LEVEL OF THE INDIVIDUAL

Sexual reproduction, genetic recombination and extremely large effective population sizes are all conducive to the maintenance of large amounts of genetic variation within mussel populations. To date, estimates of levels of genetic variation have been made exclusively from electrophoretic studies of enzyme polymorphism. Although a large number of enzymes can be studied, in practice only a relatively few have sufficiently high levels of variation to be of use to population genetics. Of 22 loci studied in M. edulis (Ahmad et al., 1977 ), only 7 were polymorphic. These same polymorphic enzymes have been used by most investigators to characterize individual multilocus genotypes (see later), levels of population variation and/or differentiation and taxonomit status of mussel samples. They are: aminopeptidase-I (Lap), peptidaseII (Aap; these are the Lap-2 and Lap-l loci, respectively, of many European authors), esterase-D (Est-D), glucose phosphate isomerase (Gpi ), phosphoglucomutase (Pgm), aminopeptidase ( Ap ) , and octopine dehydrogenase (Odh). Mannose phosphate isomerase (Mpi), important in the differentiation among species (see later), is only weakly polymorphic within each species. A focus on the level of single locus enzyme polymorphisms dominated evolutionary genetics for nearly two decades. However, in 1978, Dr. E. Zouros and his colleagues, studying the American oyster, Crassostrea virginica, were

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the first to examine individual multilocus genotype. Each individual can be genetically characterized at several loci. Rather than analyzing these data locus by locus, Singh and Zouros ( 1978) examined the effect of individual heterozygosity (the proportion of studied loci of an individual that are heterozygous for any allele) and demonstrated a positive correlation between the level of individual multiple locus heterozygosity (MLH ) and rate of growth. This initial observation was repeated and confirmed (Zouros et al., 1980) and has subsequently been reported for a diversity of marine and non-marine plant and animal species (Mitton and Grant, 1984). These data are significant for several reasons. First, a strong correlation between genotype (in this case multiple locus genotype) and a measure of production (e.g., growth) suggests specific mechanisms that functionally connect genotype to an adaptively important phenotype. This would be of importance to evolutionary biology as a means of explaining or predicting evolutionary trajectories. Second, the data demonstrate a genetic component for energy balance (see later), challenging us to integrate information from genetics and physiological ecology. Third, if there are specific and detectable genetic elements that control growth, as these observations suggest, this might provide a strategy for enhancing commercial production of certain species by constructing a breeding program that exploits the higher rates of production of certain genotypes. In Mytilus edulis, Koehn and Gaffney ( 1984) reported the relationship between MLH at five loci and growth rate in a single cohort from Long Island Sound, NY (Fig. 1). The same cohort studied at 2 months of age by Koehn and Gaffney ( 1984) was re-sampled at 4 and 8 months after larval settlement (Diehl and Koehn, 1985). By 4 months, the relationship between MLH and growth changed significantly: selection removed slower growing and more homozygous individuals from the cohort, producing a small but statistically significant negative correlation. At 8 months, there was no correlation. The study suggested that natural selection, if and when it occurs, tends to act against .Z * 3.0 $

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individuals of lowest heterozygosity and irrespective of the mechanisms that generate the initial correlation, the relationship can change with cohort age. Gosling ( 1989) attempted to repeat the observations of Koehn and Gaffney ( 1984) in a nearly identically designed experiment in Ireland, but there was no detectable effect of MLH on growth. Gaffney ( 1990) followed up the study of Koehn and Gaffney ( 1984) with studies on several subsequent annual cohorts in Long Island Sound. The relationship between MLH and growth was not consistently observed, suggesting that the effect of genotype may depend on additional factors. Indeed, this is now known to be the case (see later ) . The correlation of MLH with rates of mass specific oxygen consumption was demonstrated in M. eduh by Diehl et al. ( 1985), extending to that species a result first obtained with the American oyster (Koehn and Shumway, 1982 ). Higher individual heterozygosity was associated with lower energy demands for maintenance metabolism. This result has been obtained for other marine molluscs (Garton, 1984; Garton et al., 1984), as well as for a salamander (Mitton et al., 1986) and a fish (Danzmann et al., 1988), suggesting a general mechanism by which genotype can affect energy metabolism. The energy requirement for maintenance metabolism is a significant component of the total energy demands on an organism and ATP-dependent protein turnover contributes at least 25% to the energy requirements for maintenance in M. edulis (Hawkins, 1985 ) and other organisms (Waterlow and Jackson, 198 1). Hawkins et al. ( 1986) demonstrated that the decreased energy requirements in more heterozygous individuals of M. edulis were associated with greater efficiencies of protein synthesis and lower overall intensities of nitrogen metabolism. The reduced synthetic efficiencies of more homozygous mussels necessitated both elevated protein synthesis per unit deposition (i.e., growth) and greater proportional recycling of breakdown products. These results have been confirmed and extended (Hawkins et al., 1989). The correlation between genotype at protein synthesizing loci and general measures of protein metabolism may indicate that the correlation actually reflects cause, but this has not been established. Since MLH affects energy balance, any measure of production should correlate with MLH in the appropriate ecological circumstances (see later). For example, Rodhouse et al. ( 1986) studied the effects of MLH in M. edulis on reproduction which, like growth, is a measure of production. Using mantle weight as an index of reproduction and somatic tissue weight as a measure of somatic growth, the authors demonstrated a positive effect of MLH on reproductive growth in older individuals ( > 2.5 years), when virtually all available net energy is devoted to reproduction. [ Zouros et al. ( 1988 ) attempted a similar study using mantle glycogen as an index of reproduction, but the study was done immediately post-spawning when glycogen is virtually absent (Koehn, 1990) 1. Since energy to growth dominates production during early

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growth, MLH correlations with growth are demonstrative during this period of rapid growth. Over the developmental period in which energy is allocated to both growth and reproduction (much of the cohort life), correlation of MLH with either growth or reproduction will be most difficult to detect, since one is attempting to partition a single source of variance (energy balance) among MLH genotypes with respect to only one of two traits (i.e., growth or reproduction). At older ages, when the energy budget is dominated by reproduction (with respect to growth), MLH correlates with reproduction will be more readily measurable. Other energy-related traits, such as feeding rate (see Holley and Foltz, 1987) and viability (Zouros et al., 1983; Diehl and Koehn, 1985 ) have also been shown to correlate with MLH. It is easy to gain the impression from the foregoing studies that so long as genes are polymorphic, multiple locus genotype will invariably produce individual differences in production-related traits. However, while genotype can affect energy balance, the manifestation of a phenotypic effect on production appears to be dependent upon environmental conditions, specifically stressful environments that increase the energy demand of maintenance metabolism (Koehn and Bayne, 1989). This point has emerged from several studies (cf. Koehn and Shumway, 1982; Green et al., 1983; Ledig et al., 1983). Gentilli and Beaumont ( 1988 ) specifically tested the point in M. edulis and demonstrated that the phenotypic effects of MLH were enhanced by a higher density of individuals. Similar evidence that temperature and salinity stress can enhance the genetic effects of MLH on growth has been reported in another marine bivalve (Scott and Koehn, 1990). Lastly, the effect of MLH on production phenotypes is not independent of the specific genes that are used to characterize individual multiple locus genotype. In M. edulis, this issue has been investigated only anecdotally by a post hoc comparison of results with five loci from a Nova Scotia population (Zouros et al., 1987) with data in Koehn and Gaffney (1984). There was no relationship between the two studies in the magnitude of phenotypic effects by a specific enzyme polymorphism. However, a specific test of this hypothesis in another marine bivalve, Mulinia lateralis, employed 15 loci and demonstrated that there were very significant locus-specific effects of heterozygosity on both field and laboratory growth rates (Koehn et al., 1988). The particular phenotypic effect of a locus was independent of its level of heterozygosity, independent of linkage to other genes with a differing effect, the same in both natural and laboratory environments (relative to other gene effects), but apparently related to the function of the enzyme in the maintenance of energy balance and protein turnover. A typical study of a relationship between MLH and growth has employed five or six polymorphic loci (Fig. 2 ) . Since the relationship depends on locusspecific effects, the particular five or six genes that are sampled can signiticantly influence the experimental results, quite independent of the particular

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Fig. 2. The numbers of polymorphic enzyme loci used to characterize individual heterozygosity in 40 different studies. The distribution has a modal number of five and a mean of approximately six polymorphisms.

genetic and metabolic mechanisms involved. For example, in the study by Koehn et al. ( 1988 ), in a subsample of the five loci with weakest phenotypic effects, MLH was uncorrelated with individual growth rate variation. In a subsample of the five loci with strongest effects, MLH explained about 10% of the variation in growth rate. Until we understand the specific biochemical genetic mechanisms that functionally link genotype to phenotype, different studies can be expected to give variable results and the composition of a sample of genes cannot be functionally defined a priori. In summary, measured effects of MLH on production can be mitigated by a number of factors, including the age and reproductive state of sampled individuals, the specific polymorphic enzymes that are used to determine multilocus genotype, the ecological energetic conditions affecting the experimental organisms and the experimental circumstances employed. GENETIC VARIATION AT THE LEVEL OF THE POPULATION

Within-population variation Virtually every population genetic study of a marine mollusc has reported that genotypic distributions at several loci deviate from the expectations of the Hardy-Weinberg distribution because of a deficiency in the number of heterozygous individuals in studied samples. This has been especially true in studies of Mytilus populations. However, the origin of this widespread heterozygote deficiency has remained an enigmatic problem. Although some early studies were able to show the greater deficiencies were associated with large local spatial changes in genetic composition (cf.; Koehn et al., 1976), thus implicating a Wahlund effect from population mixing, in no instance were population differences sufficient to explain quantitatively the large observed deficiencies. Zouros and Foltz ( 1984) and Zouros ( 1987) have reviewed the pertinent early literature and the several explanations that have been offered to account for heterozygote deficiency, including self-fertilization, null alleles, mis-scoring of gels, population mixing and selection.

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In one of the first papers reporting the effects of multiple locus genotype on growth, Zouros et al. ( 1980) suggested the possibility that heterozygote deficiencies and the effect of MLH on growth might in some way be associated with one another. Two studied loci had neither an apparent contribution to growth rate variation nor deficit of heterozygotes. More recently, Zouros ( 1987 ) tabulated twelve studies of seven marine molluscan genera, concluding that in studies where a significant correlation between MLH and growth had been demonstrated, there was usually excess homozygosity, measured among all sampled loci. Although these reviews suggested that heterozygote deficiency might be associated with the effects of MLH, too few polymorphisms are available in most studied species (Fig. 2) to rigorously test this hypothesis. Utilizing the 15-10~~s data of Koehn et al. ( 1988) on Mulinia lateralis, Gaffney et al. ( 1990) reported dekiencies at 13 loci, expressed as D = (H, - H,) /H,, where H, and H, are the observed and expected frequencies of heterozygotes in the sample, respectively. The heterozygote deficiencies were highly significantly heterogeneous among loci and there was a significant positive correlation between D and the effects of heterozygosity on growth rate among individual loci (Fig. 3 ) . While allele frequencies did not differ among individual size classes (except at one locus), smaller size classes exhibited larger deficiencies of heterozygotes than faster growing individual size groups; this size dependency of D was evident only for those loci demonstrating effects of heterozygosity on growth rate. These observations unequivocally associate locus-specific heterozygote deficiency with locus-specific effects of MLH on growth, as suggested by Zouros and colleagues. Since several other studies of non-marine organisms have demonstrated similar effects of MLH on growth and other components of energy balance (cf., Mitton et al., 1986; Danzmann et al., 1988), wherein the genotypic distributions con-

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Fig. 3. Heterozygote deficiencies (D) in relation to the effect of locus-specific heterozygosity on growth rate in Mulinia lateralis. Average locus effect and D are significantly correlated (PC 0.0 1). Adapted from Gaffney et al. ( 1990).

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form to Hardy-Weinberg expectations, the co-occurrence in marine molluscs suggests a potential influence of their unique life histories and population biology on the population genetic data of these species. This suggestion gains support from a further analysis of the Mulinia data discussed above. These data can be used to generate an expected multiple locus heterozygosity distribution that predicts the number of individuals with MLH values 0, 1, 2.....15 in a sample of n individuals (Gaffney et al., 1990). When the observed MLH distribution is compared to the expected distribution, there are highly significant excesses of both the most highly homozygous and the most highly heterozygous individuals, with a deficiency of individuals of intermediate levels of heterozygosity (Fig. 4). Significantly, the MLH deviations in Fig. 4 are contributed only by those loci having individually significant effects on growth; loci without individual effects on growth collectively conform to the expected MLH distribution. Thus, each locus exhibits heterozygote deficiency in proportion to its effect on growth, and genotypes at these loci do not occur randomly with respect to genotypes at other loci with similar effects. Explanations for heterozygote deficiency must take account of these two observations, one at the level of the single locus, the other a multiple locus disequilibrium in heterozygosity. Gaffney et al. ( 1990) have statistically analyzed each classical explanation for heterozygote deficiency with regard to these two observations, examining such mechanisms as inbreeding, null alleles, aneuploidy, molecular imprinting (Chakraborty, 1989), population mixing (Wahlund effect) and various forms of selection for and against parts of the MLH distribution. The authors conclude that inbreeding, null alleles, aneuploidy and molecular mimicry cannot individually explain the observed genetic data. While very strong selection against heterozygotes could produce the observed single-locus deliciencies, no form of selection alone can simultaneously produce both singlelocus heterozygote deficiency and the MLH disequilibrium. Interestingly, population mixing can produce a pattern of both deficiencies and MLH dis-

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equilibrium, but known population differences are too small to produce the observed values, nor could mixing produce the relationship between MLH and size. The authors also considered more than a single mechanism, acting together, concluding that population mixing of larval subpopulations combined with varying degrees of selection, aneuploidy or imprinting in the subpopulations are required to account for these data. The correlations between MLH and energy balance (see above) presently exist as a phenomenon that challenges us to understand the relationship between genotype and metabolism. The association of this phenomenon with population genetic parameters, like heterozygote deficiency and multiple locus disequilibrium, demonstrates a complexity that necessitates future genetic studies of larval populations, a technically difficult but seemingly necessary task. These problems represent important areas of future research; they relate to fundamental properties of natural mussel populations that need to be understood, both in terms of the biology and the exploitation of mussels by mariculture. Between-population variation

Over the past 20 years, there has been both geographically extensive and local intensive study of genetic differences among natural populations of Myths. Results of these studies have produced a large body of information demonstrating very substantial genetic differences between populations; in many cases these differences have involved spatially proximal samples. To many investigators, such differences have implied an important role for natural selection in maintaining genetic differentiation, because of a passively distributed planktonic larval dispersal stage that may last up to several weeks (see Widdows, 199 1). In virtually every case, natural selection has been supposed to originate from the diverse marine habitats (primarily temperature and salinity) occupied by natural mussel populations. For example, Gartner-Kepkay et al. ( 1980, 1983) described genetic differences between samples throughout embayments, as well as between different geographic areas, in mussel populations of Nova Scotia, Canada. The differences were attributed to habitat-specific differences in temperature and salinity. Theisen ( 1978) described very steep clines at several loci in mussel populations of putative M. edulis in the Danish Belt Sea, which connects the North and Baltic seas. Baltic mussels were studied further by Bulnheim and Gosling ( 1988) and their genetic differences from North Sea populations were attributed to the low salinity of the Baltic Sea. The very substantial geographic differentiation among mussel populations of the British Isles has been shown to result from hybridization between M. edulis and M. galloprovincialis (Skibinski and Beardmore, 1979; Gosling and Wilkins, 198 1; Skibinski, 1983; Skibinski et al., 1978, 1980, 1983). Steep clines in Lap allele frequency are present in M. edulis at Cape Cod

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(Koehn et al., 1976) and the entrance to Long Island Sound, and similar but smaller differences in Lap allele frequency are present in A4. trossulusin Oregon estuaries (McDonald and Siebenaller, 1989). All other described instances of differentiation over short distances, including those in the preceding paragraph, can be attributed to interspecific hybridization (see next section). As a consequence, mussel populations exhibit similar genetic composition over broad geographic ranges of both continuous and discontinuous spatial distribution, as expected from their life history. The long-standing impression that mussel populations are greatly differentiated has been solely the consequence of an inadequate taxonomy for the genus. TAXONOMY OF THE GENUS

MYTZLUS

Species differences defined withallozymes The taxonomy of mussels has been greatly hampered by a paucity of reliable morphological characters; the overall shell morphology and most specific measurements from it are notoriously responsive to local environmental conditions. As a consequence, allozyme variation has served as important additional information for estimating differentiation among natural mussel populations and for assessing the possible taxonomic status of differentiated groups of samples. Before the use of electrophoresis, eight Mytilus taxa were commonly recognized: M. edulis, M, galloprovincialis,M. planulatus, M. platensis, M. chilensis, M. desolationis,M. coruscus and M. californianus. The last species is found on the Pacific coast of North America, is easily distinguished by the radiating ribs on the shell ( Soot-Ryen, 1955 ), and it will not be considered further here. M. coruscus has been reported from the Pacific coast of Asia (Scarlatto, 198 1)) but since no one has collected allozyme data from this species, its taxonomic status remains obscure. The remaining taxa have been considered races, varieties or subspecies of M. edulis by some authors, while others have considered them full species. For example, M. desolationiswas described by Lamy ( 1936) as an apparent endemic of the Kerguelen archipelago in the southern Indian Ocean. It has only recently been taxonomically evaluated, exclusively by use of allozyme variation (Blot et al., 1988). The authors suggested that M. desolationisis a “semi-species in the super-species Mytilus edulis”. Our results (see later) suggest that M. desolationis=M. edulis and that Kergeulen mussels, whatever their status, do not differ from other populations of putative M. edulis from sites throughout the southern hemisphere. Koehn et al. ( 1984) studied populations of putative M. edulis from sites throughout eastern North America, including samples from the Canadian Maritimes and Hudson Bay. They separated samples into three distinguishable groups on the basis of five allozyme loci. One of these involved differentiation at only a single locus (Lap) and was not thought to represent a tax-

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onomic group. The other two groups (Groups II and III) were very different from one another at several allozyme loci and the authors suggested that the Group III mussels might constitute a hitherto unrecognized species. This suggestion was given additional support by Varvio et al. ( 1988) who showed that the Group III type mussels from North America were most similar to mussels from the Baltic Sea, but distinguishable from Group II (M. e&/is) and M. galloprovincialis from Venice, Italy and Padstow, UK. The latter site is where Hepper ( 1957 ) first described M. galloprovincialis from outside the Mediterranean Sea. The Group III mussels from North America and mussels from the Baltic are now recognized as Mytilus trossulus, a species first described from the Pacific coast of Oregon (McDonald and Koehn, 1988). The extensive papers on electrophoresis by Skibinski and Beardmore and their colleagues (see above) have convincingly demonstrated hybridization between two species, A4. edulis and M. galloprovincialis, throughout the British Isles. However, Gosling ( 1984) reviewed available evidence, predominantly electrophoretic data, and concluded that M. galloprovincialis was an ecotype, subspecies, or variety of the “larger Mytilus edulis complex”. Indeed, virtually all concern with taxonomy in the genus has focused on the validity of M. galloprovincialis. It has not simplified matters that most of the studies bearing on this point have involved populations nearly exclusively in the British Isles where a complex interaction, including hybridization, is widespread between species. Allozyme characters have greatly clarified the taxonomy of the genus. While many geographic areas will require further, more detailed sampling, enough locations have been sampled to sketch an overall picture of the taxonomy and distribution of mussel species in the genus Mytilus. McDonald et al. ( 1990) have concluded an extensive world-wide sampling of Mytilus and have used a series of multivariate statistical techniques to discriminate groups on the basis of both allozyme and morphological characters. The following paragraphs summarize their results, which demonstrate that ( 1) populations of M. edulis, M. galloprovincialis and M. trossulus each constitute a homogenous group, ( 2 ) each group maintains a unique genetic and morphological phenotype over a distribution that is in each case virtually world-wide, (3) because of these unique phenotypes, species status for each is justified, and (4) hybridization is the rule where two species come into geographic contact with one another. The study by McDonald et al. ( 1990) employed eight loci (see above) and 45 world-wide samples, but because of the cross-matching with Varvio et al. ( 1988), and in association with the study by Koehn et al. ( 1984), the results can be shown to be consistent with a much larger body of data. Allozyme data define three groups of individuals in the northern and two groups in the southern hemispheres, but with some individuals falling in intermediate multivariate spaces (Fig. 5A, B). Intermediate individuals are from samples in geographic areas where individuals of two clusters are in con-

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Fig. 5. (A) First and second principal component allozyme data for eight loci for individuals from 45 locations in the not-them hemisphere, illustrating the discrimination among Myths edulis, M. trossuh and M. galloprovincialis. The assignment of a specific taxon to each cluster is described in the text. Individuals in the intermediate multivariate spaces are from locations where contact and hybridization occurs between two species (see text). Outlines of clusters are drawn only to aid visual comparison between Figs. 5A and SB. (B) Same as A, but only individuals from nine southern hemisphere locations are shown, illustrating clustering of M. edulis and M. galloprovincialis. M. trossulus has not been found in the southern hemisphere.

tact and hybridization occurs (see later). Each of the main groups can be assigned to an extant species, based upon previously designated species status for mussels in a particular geographic area. For example, all the mussels from Venice, Italy, are in a single cluster; this cluster is therefore identified as M. gufloprovincialis.This cluster is also occupied by samples from other regions of the northern hemisphere where M. galloprovincialisis known to occur, either naturally or by introduction, including samples from Portugal, Spain, southern California and Japan. Southern hemisphere samples from Western Australia, Tasmania and New Zealand also cluster with known samples of this species and must therefore be judged to represent M. gulloprovinciaiis,unless additional contradictory evidence becomes available. Similarly, a second cluster is defined by a sample from Tillamook, Oregon, the type locality for M. trossufus (McDonald and Koehn, 1988 ), Alaska, Siberian USSR, and the Baltic; all can be taken to represent this species. The results of Varvio et al. ( 1988 ) allow placement of samples from the Canadian Maritimes (Koehn et al., 1984) in the M. trossulusgroup. A4. edulis represents a third group, consisting of samples from the eastern USA and northern Europe (Denmark, White Sea, USSR). Southern hemisphere samples from Argentina, Chile, Falkland Islands and Kerguelens are homogeneous and only slightly different from A4. edulis in the northern hem-

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Fig. 5. (C) First and second canonical variables for 19 morphometric shell characters from locations in the northern hemisphere showing multivariate discrimination among the three Mytifus species. The lines that separate each cluster from one another are drawn subjectively to aid visual comparison with Fig. 5D. (D) Same as C, but for morphometric characters from samples in the southern hemisphere. Adapted from McDonald et al. ( 1990).

isphere. The shift towards both the M. gafloprovincialis and M. trossulxs clusters is not caused by intermediate allele frequencies that might suggest gene flow from these species into the southern hemisphere M. edulis type. Rather, the southern hemisphere populations possess M. gulloprovincialis-type alleles at some loci and M. trossulus-type alleles at other loci. Until contradictory information is forthcoming, these samples from the southern hemisphere are taken to represent M. edulis. We consider M. edulis, M. galloprovincialis, and M. trossulus to be distinct species because each maintains a distinct set of alleles, with fairly homogeneous allele frequencies across vast distances, in the face of massive migration of planktonic larvae. Species differences defined with morphology

A canonical correlation analysis employed 19 different morphological traits, virtually all of which have been used in more traditional analyses (c.f., Seed, 1974; Beaumont et al., 1989). Detailed descriptions of the morphological characters used, as well as the statistical analyses, are given in McDonald et al. ( 1990)) but the more important characters emerging from our analyses included lengths of the anterior adductor muscle scar and the hinge plate. The multivariate analysis of 19 morphological characters in samples from the northern hemisphere resulted in three clusters (Fig. K), corresponding to M. galloprovincialis, M. edulis and M. trossulus.

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The results are virtually identical to those from the allozymes for both southern (Fig. 5B and 5D) and northern hemisphere (Fig. 5A and 5C) samples, though there is a slightly greater degree of overlap in the morphological character analysis, especially between iV. trossulus and AI. galloprovincialis. Somewhat surprisingly, the best discrimination is between M. edulis and M. galloprovincialis, a long-standing problem in the taxonomy of the genus (Gosling, 1984). Although many previous studies of morphological variation in Mytilus have been able successfully to discriminate individual samples of putative species, none has been able to do so with consistency over broad geographic sampling ranges. Local environments are known to influence shell morphology (cf., Seed, 1968) so it has often been possible to demonstrate morphological differences between samples from different sites. Such differences, however, have not been consistent and have not therefore been practical in the identification or definition of individual species. McDonald et al. ( 1990) have shown that multivariate analyses can discriminate quite successfully among these three species of the genus, on a world-wide basis. However, it is still impossible to identify with certainty any particular individual from a specific location, at least on the basis of morphological characters alone. Taken singly, morphological traits are both unreliable and without discriminatory power. For example, of the 19 characters used here, the best and second-best discrimination between M. galloprovincialis and M. edulis is afforded by the anterior adductor muscle scar length and the hinge plate length, respectively. The adductor muscle scar alone gives only modest discrimination while the hinge plate alone discriminates very poorly. However, a linear combination of all characters in the canonical variable gives total separation of the two species. Dr. Wesley Brown and his colleagues have initiated studies of the mitochondrial DNA sequence variation in the same 45 samples presented here. While these data are still preliminary, they are so far consistent with the taxonomic relationships indicated by both the allozyme and morphology data. As yet, however, samples from the southern hemisphere have not been studied in detail, and these data will be critical in establishing the relationship between M. edulis in the northern and southern hemisphere. Where two species are in geographical contact, there is in every case hybridization between them. The most geographically widespread hybridization occurs between M. edulis and M. galloprovincialis, ranging from northern Spain to Brittany in northwest France and northwards to include virtually all of the British Isles. The precise region of transition from hybrid populations to pure M. edulis in northern-eastern France has not been identified. Hybridization between M. edulis and M. trossulus occurs over a relatively short distance in the Danish Belt Sea. The contact between M. edulis and M. trossulus in North America is poorly studied because of difficult accessibility to areas of the Gulf of St. Lawrence, Labrador, and so forth, but from available samples, hybridization occurs at several sites in the extreme upper reaches of the Gulf of St.

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Fig. 6. World-wide distribution of Myths edulis ( 0). M. trossulus (m) and M. galloprovincia/is (0 ). Areas of known hybridization are shaded, though the precise distribution of hybrid populations is not known. Some sample points in northern Europe, from Varvio et al. ( 1988), have been matched with the samples from McDonald et al. ( 1990).

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Lawrence. Indeed, hybridization between species does not occur as simple introgression, with monotonic clines connecting pure populations of each species. Rather, hybrid zones are spatially complex with pure, mixed and hybrid populations occurring in a patchwork pattern (cf., Skibinski and Beardmore, 1979; see Fig. 1 in McDonald and Koehn, 1988 ). The only contact area where hybridization has not been found is in the region near the North Korean and USSR border where there is contact between M. trossulus and M. gulloprovincialis, though this may be only because the region has not yet been well studied. Fig. 6 illustrates the world-wide distribution of the species of Mytilus recognized by McDonald et al. ( 1990), showing areas where hybridization is known to occur. Several points deserve emphasis. With the exception of Chile, there is no evidence that M. edulis occurs anywhere in the Pacific Ocean. The most extensively studied populations in western North America consist of M. trossulus in the north and M. gulloprovincialis in the south (McDonald and Koehn, 1988). The latter species has been introduced into various sites in the Pacific, including southern California, Japan (Wilkins et al., 1983), and Hong Kong (see McDonald and Koehn, 1988 ). On the other hand, this species has been previously reported in the southern hemisphere only in South Africa (Grant and Cherry, 1985), where it is thought to have been introduced because of the absence of edulis-like mussels from aboriginal middens. The widespread occurrence of M. galloprovincialis in the south Pacific may also be a consequence of introduction, but this seems unlikely (though the mtDNA data will unequivocally test this point ) , since fossils and subfossils are known from these areas. Also, the species has not been found in the southern oceans around South America or the Kerguelens. Both areas have had historically frequent trade with Mediterranean countries, where M. gulloprovincialis is found exclusively. As M. gulloprovinciulis is probably native to large areas of the south Pacific, introductions into northern Pacific sites like Japan, Hong Kong and/or southern California may not have originated in Europe. M. trossulus has a disjunct distribution throughout the colder waters of the north Pacific Ocean, the northwest Atlantic Ocean and the Baltic Sea, where it may represent a zoogeographical remnant. The species has not been found in the southern hemisphere, which is also consistent with the absence of historical introductions of these mussels from the northern to the southern hemisphere. ACKNOWLEDGEMENTS

Much of the work reported here was supported by National Science Foundation grants BSR-8415060 and BSR-8614380 to R.K. Koehn and BSR86 13902 to Wesley M. Brown. John McDonald suggested manuscript changes.

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This is contribution 746 from the Program in Ecology and Evolution, State University of New York at Stony Brook.

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