Introduction

Understanding the relationship between genetic variation and the environment is of fundamental importance (Berry 1989). A group of animals that is used frequently to investigate that relationship are Gastropod mollusks, although their prevalence in such studies is perhaps unsurprising. Gastropod mollusks are one of the most diverse groups of animals alive today and live in marine, freshwater, and terrestrial habitats; moreover, they are usually conspicuous and generally easy to collect, they have a hard shell that has provided a good fossil record, and their biology and natural history are often well known due to a long tradition of both amateur and professional study.

The predatory marine dogwhelk Nucella lapillus (a common intertidal snail of the northern Atlantic) has been the focus of many studies concerning adaptation to life in the intertidal habitat (Crothers 1985). Genetic studies of N. lapillus have shown that this snail possesses two distinct nuclear haplotypes that are differentiated with respect to variations at several biochemically defined loci (for example, mitochondrial malate dehydrogenase [mMDH; EC 1.1.1.37], esterase-3 [Est-3; EC 1.1.1.31], leucine aminopeptidase-2 [Lap-2; EC 3.4.11], and peptidase-2 [Pep-2; EC 3.4.11]) (Day 1990, 1992), chromosomal fusion frequencies (Robertsonian translocations; centric fusions [Robertson 1916]), and inherited variations in phenotypic traits (shell shape and physiology) (Kirby et al. 1994a, b) (Fig. 1). In one intensively studied region, between the headlands of Peartree Point and Prawle Point (a distance of 6 km) on the southwestern peninsula of England, the two nuclear haplotypes appear as a multitrait step cline. Across this continuous shoreline the haplotypes vary clinally from fixation of one haplotype to near-fixation of the other and covary coincidentally with variation in air temperature and humidity on the shore during low tide (Kirby et al. 1997), which are both important environmental variables in life in the intertidal (Lewis 1964).

Figure 1
figure 1

The relationship between mMDH 10 frequency and the first principal component of shell shape measured using Raup’s (1966) three shell shape characteristics of whorl expansion rate, translation rate, and aperture circularity, in samples of juvenile N. lapillus collected from the shore. Similar correlations are observed for other biochemically defined loci that characterize the two haplotypes of N. lapillus and which vary clinally, but not for any other polymorphic loci that have been studied so far. Samples with a high frequency of mMDH 10 variants exhibit a more spherical shell shape, whereas samples with a low frequency of mMDH 10 variants (high frequency of mMDH 9 variants) exhibit a more elongate shell shape. Figure reproduced from Kirby et al. (1994a).

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Variations in shell shape, like those that characterise the haplotypes of N. lapillus, also occur in other species of interidal snails and are known to be important because they can confer an adaptive advantage in different environments (Newkirk and Doyle 1975; Crothers 1985; Grahame and Mill 1992; Vermeij 1993). The haplotype of N. lapillus occurring at Peartree Point (a low-temperature habitat) exhibits a spherical shell shape, whereas the haplotype found at Prawle Point (a high-temperature habitat) exhibits an elongate shell shape. In N. lapillus, the elongate shell shape retains a greater volume of fluid within the mantle cavity when out of water (Kirby et al. 1994a), which is related to temperature since intertidal snails use evaporative cooling during low tide (Vermeij 1973). The association between environmental variation and the haplotypes of N. lapillus is maintained beyond the region between Peartree Point and Prawle Point. Similar allozyme, chromosomal, and phenotypic variation covaries with habitat throughout the northeastern geographic range of N. lapillus (Staiger 1957; Hoxmark 1970, 1971; Bantock and Cockayne 1975; Day 1990), for example, which extends from the −1°C winter isotherm for oceanic waters to 37°N (Crothers 1985). N. lapillus with an elongate shell shape predominate in the more southern latitudes (Kitching 1977) and on shores where high temperature is an important environmental pressure (Crothers 1985), whereas individuals with a more spherical shell shape predominate in more northern latitudes and on cooler more wave-swept environments (Kitching 1986). I have therefore suggested that the haplotypes in N. lapillus may reflect what Dobzhansky (1937) envisaged as a coadapted gene complex, in this case involving multitrait coevolving genetic solutions to environmental variation that reflect “adaptive peaks” (Wright 1931) that enable N. lapillus to exploit its habitat successfully (Kirby 2000a).

Genetic variations in the biochemical loci that differentiate the two haplotypes of N. lapillus behave like alleles when visualized by electrophoresis and they conform to Hardy–Weinberg frequencies in samples of N. lapillus collected from the shore (Day and Bayne 1988). Recent molecular studies of genetic variation in the mMDH locus of N. lapillus have shown that differentiation between the mMDH variants that represent one or the other of the haplotypes (MDH 9 and mMDH 10) is extreme; mMDH 9 and mMDH 10 alleles differ by 23 and 20% at the nucleotide (233 substitutions) and amino acid (70 changes) levels, respectively (Kirby 2000a). The extent of variation between mMDH 9 and mMDH 10 in N. lapillus (compared to that seen between orthologous loci from less related taxa) (Kirby 2000a) suggests strongly that their divergence is either extremely ancient and probably much older than the genus Nucella, which arose during the Early Miocene about 25 MY (million years) ago (Kool 1993a, b; Collins et al. 1996), or the result of strong disruptive selection. Here, the age and distribution of the mMDH alleles of N. lapillus are determined using a combination of molecular data and the fossil record, in order to help understand the evolution of the genetic variations found in these snails.

Materials and Methods

Variation in mMDH in the Marine Gastropoda

The general distribution of variation in mMDH in the marine Gastropoda was first examined in a number of taxa using a 91-bp nucleotide sequence of mMDH that occurs from nucleotide position 266 to position 357 in the mature mMDH cDNA. In N. lapillus this 91-bp fragment exhibits similar differentiation between the mMDH 9 and the mMDH 10 alleles (22% at the nucleotide level) to that observed between the complete open reading frame cDNAs (Kirby 2000a). The 91-bp cDNA sequences were obtained from the following representative marine Gastropoda: Patella vulgata (Patellogastropoda, which is considered a basal gastropod group [Ponder and Lindberg 1997]), Monodonta lineata (Archaeogastropoda), Calyptraea chinensis, Littorina littorea, Natica alderi, Hinia reticulata, Buccinum undatum, Plicopurpura patula, N. lima, N. freycineti, and N. lapillus (members of the Caenogastropoda). The sequence from the terrestrial snail, Cepaea nemoralis (Pulmonata), was also included. The cuttlefish, Sepia officinalis (Cephalopoda), was used as the outgroup taxon in this and all subsequent analyses; the Gastropoda and Cephalopoda are considered to be sister taxa (Ponder and Lindberg 1997), which arose abruptly from the Monoplacophora (Runnegar and Pojeta 1985). cDNA sequences were obtained by RT-PCR using a proofreading polymerase (Pfu; Stratagene) and amplification protocols described previously (Kirby 2000b). Amplified sequences were cloned and five clones were sequenced manually for each species. The mMDH sequences of N. lapillus and N. freycineti were obtained previously (Kirby 2000a, b). With the exception of P. patula, N. lima, and N. freycineti, which were collected from Barbados, Alaska, and Japan, respectively, all other species were collected in and around Plymouth Sound, Devon, UK. Intertidal species were collected by hand and the subtidal species by dredging. C. nemoralis was collected from a south Devon hedgerow. Trawling in Whitsand bay, Cornwall, collected S. officinalis.

Divergence Time of mMDH 9 and mMDH 10 of N. lapillus

The approach of a molecular clock was adopted to determine a date for the divergence of mMDH 9 and mMDH 10. A contiguous 679-bp mMDH cDNA sequence comprising the 91-bp fragment and the 3′ cDNA end from a subset of the above taxa was obtained for this purpose; this cDNA represents 72% of the mature mMDH cDNA (approximately 227 amino acids depending on the taxon). The 3′ cDNA end of mMDH was obtained by RACE reactions (Frohman et al. 1988) using species specific oligonucleotide PCR primers designed to the 91-bp fragments and methods described previously (Kirby 2000b). Based upon the phylogenetic tree determined from the 91-bp sequences, the 3′ cDNA ends of mMDH were obtained from S. officinalis, M. lineata, C. chinensis, L. littorea, B. undatum, P. patula, N. freycineti mMDH 9 (N. freycineti is the closest living relative of N. lapillus [Amano et al. 1993]) and N. lapillus mMDH 9 and mMDH 10 (N. lapillus cDNAs were obtained previously [Kirby 2000a, b]).

Data Analysis

All DNA sequences were aligned using Clustal W (Thompson et al. 1994). Neighbor joining on Kimura (1980) two-parameter distances determined the phylogenetic tree of the 91-bp mMDH cDNA sequences. To estimate the divergence time of mMDH 9 and mMDH 10 from the 3′cDNA sequences using a molecular clock, third-position codons (242 sites) and first-position Leu codons, where Leu was conserved across all sequences (16 sites), were first deleted from the data set; these sites were removed because the extent of divergence among sequences suggested that they were likely to be saturated. A particularly good example of the extent of saturation between mMDH 9 and mMDH 10 that justifies this approach is shown in the full mMDH ORF sequences (GenBank accession Nos. AF280052 and AF218064, respectively) (Kirby 2000a) by the terminal eight amino acids of the mtDNA leader peptide, which forms the protease cleavage recognition site in mMDH. Apart from the conserved amino acids of phenylalanine and serine (residue positions −8, −7) that are required for cleavage (Gavel and von Heijne 1990), the only evolutionary constraint on this region is that it is eight amino acids long; the nucleotide and amino acid substitution in this region between mMDH 9 and mMDH 10 is 55 and 100%, respectively.

The phylogenetic tree of the mMDH 3′ cDNA ends (nucleotide positions 266–945; 421 sites) was determined by neighbor joining on Kimura two-parameter gamma distances using a gamma shape parameter a = 0.432, which was determined by the method of Gu and Zhang (1997). The two-cluster test (Takezaki et al. 1995) was used to examine the heterogeneity of evolutionary rates among different lineages of the mMDH 3′ cDNA end sequences in order to determine the validity of applying a molecular clock to the sequence data. A Z-test was used to examine whether positive selection might be operating on mMDH variants in N. lapillus presently. Values for the number of synonymous substitutions per synonymous site and the number of nonsynonymous substitutions per nonsynonymous site (d S and d N, respectively) that were used in the Z-test were estimated from the full-length ORF sequences of mMDH variants in N. lapillus using the method of Nei and Gojobori (1986) with a Jukes–Cantor correction to account for multiple substitutions per site, SEM values were calculated by the method of Nei and Jin (1989). To determine the minimum divergence time of the mMDH 9 and mMDH 10 allelic lineages a linearized tree was constructed using the computer program LINTRE (Takezaki et al. 1995). To calibrate the molecular clock a divergence time of 55 MY ago was assumed for the lineages leading to Nucella and the Neotropical intertidal snail, P. patula; this date was obtained from the fossil record (Kool 1989). P. patula is a member of the Rapaninae subfamily of the Muricidae (Kool 1993a, 1993b), which is a sister clade to the Ocenebrinae (the subfamily that includes Nucella). Phylogenetic and molecular evolutionary analyses were conducted using the computer program, MEGA version 2.1 (Kumar et al. 2001).

Results

Variation in mMDH in the Marine Gastropoda

The bootstrap support for many of the phylogenetic relationships among the 91-bp fragments of mMDH was low, as might be expected for small regions of such divergent sequences. However, with the exception of the relationships within the Neogastropoda, the phylogenetic relationships conformed to the expected evolutionary relationships among the taxa, with the patellogastropod, P. vulgata, representing the most basal gastropod taxon (Fig. 2). The mMDH cDNA from P. patula appeared most closely related to mMDH 10. The gene tree therefore tentatively suggests that the divergence of the mMDH 9 and mMDH 10 occurred before the divergence of the lineages leading to the Muricidae (comprising Plicopurpura and Nucella) and the Buccinidae. Figure 3 shows the phylogenetic relationships among the mMDH 3′ cDNA end sequences that were subsequently used to estimate the divergence time of mMDH 9 and mMDH 10. Although the position of C. chinensis and L. littorina mMDH sequences remains unresolved, there is strong bootstrap support for the divergence of mMDH 9 and mMDH 10 at least prior to the divergence of the Buccinidae and the Muricidae.

Figure 2
figure 2

Neighbor-joining tree on Kimura two-parameter distances using the 91 bp cDNA fragment of mMDH. Numbers by interior nodes represent bootstrap values determined from 1000 resamplings. Bootstrap values below 50% are not given. The cuttlefish, S. officinalis, was used as the outgroup taxon. The scale bar indicates the number of nucleotide substitutions per site. GenBank accession numbers for the cDNA sequences are as follows: B. undatum, AY138128; C. nemoralis, AY138137; C. chinensis, AY138129; H. reticulata, AY138135; L. littorea, AY138127; M. lineata, AY138131; N. alderi, AY138136; N. freycineti mMDH 9, AY138125; N. freycineti mMDH 10, AY138134; N. lapillus mMDH 9, AF280052, N. lapillus mMDH 10, AF218064; N. lima mMDH 9, AY138133; N. lima mMDH 10, AY138132; P. patula, AY138130; P. vulgata, AY138138; S. officinalis, AY138126.

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Figure 3
figure 3

Neighbor-joining tree on Kimura two-parameter gamma distances using a gamma shape parameter a = 0.43 for 72% of the mature mMDH cDNA from a subset of the taxa presented in Fig. 2. All third-position codons and first-position Leu codons, where Leu is conserved across all sequences, were removed from the analysis. Numbers by interior nodes represent bootstrap values determined from 1000 resamplings. Bootstrap values below 50% are not given. The cuttlefish, S. officinalis, was used as the outgroup taxon. The scale bar indicates the number of nucleotide substitutions per site. GenBank accession numbers are the same as those given in Fig. 2.

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Table 1 shows the results of the test of the heterogeneity of evolutionary rates among different lineages, which examines the null hypothesis that states the evolutionary rates along two branches separated by a node are the same. The result of the χ2 test of rate constancy for all interior nodes under the root is 11.92, which is not statistically significant at 7 degrees of freedom. The values of d S and d N obtained for the full-length ORF variants of mMDH in N. lapillus were 0.79 ± 0.09 and 0.15 ± 0.02, respectively, and the Z-test indicated that there was no evidence of positive selection acting on these variants. The results of the two-cluster test and the Z-test indicate that it is reasonable to employ a molecular clock to estimate a date for the divergence of mMDH 9 and mMDH 10.

Table 1 Two-cluster test of the heterogeneity of evolutionary rates among mMDH lineages
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Divergence Time of mMDH 9 and mMDH 10 of N. lapillus

Figure 4 shows the linearized tree assuming a molecular clock for the mMDH 3′ cDNA end sequences comprising 421 nucleotides after removal of third-position codons and first-position Leu codons. The mMDH 10 sequences from P. patula and N. lapillus, together with the known divergence time of this node, can therefore be used to estimate the divergence time of the lineages leading to mMDH 9 and mMDH 10 from the mMDH variants found in N. lapillus. The height of the ancestral node of the lineages leading to P. patula mMDH 10 and N. lapillus mMDH 10 is 0.0269 ± 0.0064 and the height of the ancestral node for the lineages leading to N. lapillus mMDH 9 and mMDH 10 is 0.0704 ± 0.0092. By dividing 0.0269 by 55 MY, the average rate of nucleotide substitution for mMDH is estimated to be 4.8 × 10−4 per site per year per lineage. The divergence time for mMDH 9 and mMDH 10 under the assumption of a molecular clock is, therefore, approximately 144 MY ago. The height of the node at the divergence of the lineages leading to the extant Muricidae and Buccinidae is 0.0598 ± 0.0102, which indicates this divergence time to be around 122 MY ago. The height of the node at the divergence of N. freycineti and N. lapillus is 0.0047 ± 0.0024, which estimates this divergence to have occurred approximately 9.6 MY ago.

Figure 4
figure 4

Linearized tree constructed assuming a molecular clock for the same mMDH sequences used in Fig. 3. Numbers by nodes represent minimum divergence times estimated assuming a molecular clock. The numbers by nodes in parentheses are the divergence times of the taxa according to the fossil record (Glibert 1959, 1963; Taylor et al. 1980; Kool 1989; Gibbard et al. 1991; Bandel 1993). The cuttlefish, S. officinalis, was used as the outgroup taxon. The scale bar represents the number of nucleotide substitutions per site. The heights of each node together with their standard errors are given in Table 1.

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Discussion

Clinal variation is a useful indicator of ecological and evolutionary processes (Fisher 1930; Endler 1977). Previous studies of molecular variation in mMDH of N. lapillus were undertaken to investigate the genetic basis of intraspecific clinal variation across an environmental transition (Kirby 2000a), because few molecular studies of clinal electrophoretic protein variation existed (Kreitman 1983; Bernardi et al. 1993). Although several protein-coding loci exhibit coincident clinal variations in N. lapillus, mMDH was chosen primarily as the locus for study because molecular variation in mMDH could be determined easily (Kirby 2000b). The extreme variation observed between putative mMDH alleles of N. lapillus (Kirby 2000a) suggested that differentiation in mMDH, and thus between the two haplotypes, was probably more complex than a simple example of coincident intraspecific clinal allelic variations across an environmental gradient (Kirby et al. 1997). Here, using the approach of a molecular clock, the divergence of the mMDH 9 and mMDH 10 variants found in N. lapillus is estimated to have occurred around 144 MY ago.

The estimate of 144 MY ago for the origin of the mMDH variants derived from a molecular clock may overestimate their persistence however, since positive selection that may have sped evolutionary change soon after their origin may be masked over a long period of time by subsequent purifying selection and neutral substitutions (Zhang et al. 1998). Another source of error arises if the assumption of neutrality of the variable sites used in the analysis is incorrect. Some studies of allelic variations in MDHs (predominantly cytosolic MDH, cMDH) have described intraspecific variations that correlate with variation in environmental temperature (Powers and Place 1978; Powers et al. 1991; Nielsen et al. 1994; Harrison et al. 1996) and which in some cases may reflect positive selection (Nielsen et al. 1994; Harrison et al. 1996). Since mMDH is part of a cooperating enzyme pair with cMDH and plays a key role in energy metabolism, it is reasonable to consider the possibility that some of the variation in mMDH in N. lapillus may confer a fitness advantage in different temperature environments. However, at present there is no evidence to indicate that much of the extensive variation in mMDH, which comprises 233 variable nucleotide sites or 70 variable amino acid sites, is adaptive. The extent of substitution at neutral amino acid sites in mMDH is revealed in the protease cleavage recognition site where the substitution among neutral amino acids is 100%. Three further lines of evidence give indication that much of the remaining variation between mMDH variants of gastropods may be neutral. First, the one-tailed Z-test used to determine whether positive selection is operating on a gene, which compares the relative abundance of synonymous and nonsynonymous substitutions that have occurred between the two gene sequences, showed no support for positive selection; the number of synonymous substitutions per synonymous site was found to be significantly greater than the number of nonsynonymous substitutions per nonsynonymous site. Second, it is useful to compare mMDH and cMDH in N. lapillus. Electrophoretic analysis of cMDH in N. lapillus indicates a single locus and DNA sequence analysis of N. lapillus cMDH has revealed only two variants that differ by just four nucleotides and which result in a single nonsynonymous substitution (Kirby 2000a). Comparison of the rate of molecular evolution of mMDH and cMDH in N. lapillus to other taxa suggests that molecular evolution proceeds at a similar rate in these loci (Table 2), which might be expected for neutral evolution in a cooperating enzyme pair. The difference in nucleotide substitutions between mMDH and cMDH may therefore indicate a long separation of mMDH variants rather than rapid evolutionary divergence. Third, a comparison of the full-length ORF sequences of mMDH 9 and mMDH 10 from N. lapillus reveals that all the amino acid residues considered essential for catalysis and cofactor binding (Goward and Nicholls 1994) are conserved between the mMDH variants and only 4 of the 70 amino acid substitutions occur at sites that otherwise appear conserved amongst other animal mMDHs sequenced thus far (Kirby 2000a). While it is not yet known whether the four amino acids that differ between mMDH variants of N. lapillus but appear conserved in other taxa have physiological consequences, it appears reasonable to conclude that most of nucleotide sites that differ between mMDH variants may be neutral and therefore it is realistic to estimate their age using a molecular clock.

Table 2 Pairwise distances (poisson correction) and their standard errors that relate amino acid sequences of cMDH and mMDH from N. lapillus to other taxa
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An estimate of the probable minimum persistence time for the mMDH variants may be obtained from the fossil record. The divergence of mMDH variants prior to the evolution of the Buccinidae is supported strongly (Fig. 3). According to the fossil record the divergence of the lineages that include the extant Muricidae (P. patula and Nucella [Fig. 4]) and the Buccinidae (B. undatum [Fig. 4]) within the Muricoidea occurred around 112–90 MY ago (Taylor et al. 1980; Bandel 1993; Tracey et al. 1993) and therefore it can be concluded that the evolution of the mMDH variants occurred no later than this time. This estimate of their minimum molecular divergence time is likely to be an underestimate, however, for two principal reasons. First, the first recorded appearance of two distinct taxa in the fossil record is likely to underestimate their actual time of appearance, and second, gene sequences may have begun to diverge prior to the divergence of the taxa themselves, unless of course the mMDH variants represent a gene duplication that occurred at the time of speciation. In this study using molecular data, the divergence time of the mMDH 10 lineages found in the Buccinidae and the Muricidae was estimated to be 127 MY ago, which therefore suggests that mMDH variants may have persisted for at least this long. Unfortunately, the placements of the mMDH sequences of C. chinensis and L. littorea that might help resolve the origin of the mMDH variants further, are only weakly supported in the gene tree (Fig. 3), which agrees with other unsuccessful attempts to resolve the rapid but distant diversification of the modern Caenogastropoda (Ponder and Lindberg 1997; Colgan et al. 2000). The mMDH 9 lineages of N. freycineti and N. lapillus are, however, calculated to have diverged around 10 MY ago. The first recorded appearance of the Atlantic N. lapillus is in the late Pliocene 3.4–1.8 MY ago (Glibert 1959, 1963; Gibbard et al. 1991) after divergence from N. freycineti approximately 4–3 MY ago (Collins et al. 1996), although this is believed to underestimate the divergence of these lineages (Collins et al. 1996). The estimated divergence times of different nodes based upon a molecular clock therefore agree broadly with the fossil record of the taxa providing confidence in the molecular estimates obtained for the time of divergence of mMDH lineages. It therefore seems reasonable to conclude that the mMDH 9 and mMDH 10 lineages reflect an extremely ancient divergence that occurred prior to the radiation of the Muricidae around 100 MY ago (Bandel 1993) and perhaps near the evolution of the Muricoidea, which is believed to have taken place in the Early Cretaceous around 146–132 MY ago (Taylor et al. 1980) during the early evolution of the Neogastropoda.

In N. lapillus, protein electrophoresis studies and molecular data indicate that mMDH 9 and mMDH 10, together with variation in other biochemical loci that differentiate the two haplotypes of N. lapillus, segregate like alleles presently (Day and Bayne 1988; Day 1990; Kirby 2000a). However, it is impossible to determine from available data whether mMDH 9 and mMDH 10 are true allelic variants that have since followed separate evolutionary pathways, in which case they would represent an ancient allelic trans-specific polymorphism or if they are paralagous loci that diverged after a gene duplication; clines that involve the presence or absence of genes as opposed to allelic variants have been reported previously (Flint et al. 1986). On the present evidence of the extent of molecular evolution, paralagous loci would represent a more parsimonious explanation than maintenance of a trans-specific polymorphism by natural selection. Whether or not the mMDH variants in N. lapillus arose as alleles or paralagous loci, the chromosomal variation in this snail, through the effect that chromosomal fusions can have on linkage groups (White 1978) and recombination (Gregorius and Herzog 1989), could have played a role in the creation of the haplotypes of N. lapillus and the present allelic behavior of mMDH variants. The chromosomal variation could therefore explain how the suite of associated genetic variations are maintained in their present form in N. lapillus (Darlington 1939; Wallace 1955; Lewontin and Kojima 1960).

N. lapillus is a member of the Muricidae within the Muricoidea of the Neogastropoda. The Neogastropoda are thought to have arisen approximately 146–132 MY ago (Taylor et al. 1980) and represent the most derived order of the Caenogastropoda (an ancient group that arose during the Ordovician 510 to 439 MY ago (Bandel 1993)). The Muricoidea are one of the most important major groups of the Neogastropoda and the Muricidae, which includes Nucella, contain by far the greatest majority of members specialized for life in the intertidal and upper shore (Vermeij and Carlson 2000). In their recent analysis, Vermeij and Carslon (2000) envisage the intertidal Muricidae as restricted groups of animals able to adapt to and specialize in the physically rigorous upper shore environment, virtually in retreat from the more biotically demanding habitats lower down the shore or in the subtidal.

The physical stresses such as temperature that influence life in the intertidal habitat have probably changed little qualitatively over geological time, although they may have varied quantitatively, and they are therefore likely to have been important selective agents throughout the evolution of intertidal species. It is not known whether the genetic variations in shell shape and other traits associated with the haplotypes in N. lapillus reflect similarly ancient variations or if their association extends throughout their evolution. Nevertheless, it is interesting to observe that similar shell shapes to those associated with variation in mMDH in N. lapillus presently (Fig. 1) also occur in fossil Nucella from the early Pleistocene (Cambridge and Kitching 1982) and in other extant intertidal Muricidae whose lineages are older than Nucella (Crothers 1983, 1984; Kitching 1986). Similar shell shape variation also occurs in other lineages of the Caenogastropoda such as representatives of the intertidal Littorinimorpha (which some consider may be paraphyletic with the Neogastropoda [Ponder and Lindberg 1997]) in association with the same environmental variables (Newkirk and Doyle 1975; Grahame and Mill 1992) and, in some studied cases, similar biochemical polymorphisms (Newkirk and Doyle 1979).

Although it is difficult to estimate accurately the time of divergence of the mMDH variants found in the Neogastropoda, they have clearly persisted for a long time, at least 100 MY and perhaps as long as 144 MY. It is therefore tempting to speculate whether the divergence of mMDH 9 and mMDH 10 might reflect an ancient genetic event like a chromosomal mutation such as a centric fusion, inversion, or gene duplication that established the conditions for their independent evolution (whether as alleles or paralagous loci) together with genetic variations in other linked traits. If that were the case, the mMDH 9 and mMDH 10 haplotypes of N. lapillus may reflect ancient and persistent examples of genetic variations that have been important in the evolutionary ecology of these taxa for more than 100 million years and which may have since been maintained by a combination of purifying and balancing selection.