An Ancient Transpecific Polymorphism Shows Extreme Divergence in a Multitrait Cline in an Intertidal Snail (Nucella lapillus (L.))

Richard R. Kirby

Plymouth Marine Laboratory, Plymouth, England


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
Clines in intraspecific genetic variation are frequently associated with an environmental transition. Here, divergence among nucleotide sequences of two nuclear loci, cytosolic and mitochondrial malate dehydrogenase (cMDH and mMDH, respectively), is described, in a multitrait cline over a distance of ca. 3 km where shell phenotype, allozyme, mitochondrial DNA haplotype, and centric fusion (Robertsonian translocations) frequencies covary with temperature and humidity and change abruptly in a continuous population of the dog-whelk (Nucella lapillus), a common intertidal snail of the north temperate Atlantic. Protein electrophoresis has already shown two alleles of mMDH varying from fixation of one allele to near fixation of the other, whereas cMDH appears to be monomorphic. The results of this study show a striking disparity in nucleotide sequence divergence among alleles at the two loci, with extreme molecular differentiation in one of them. Four alleles of cMDH were found to have nucleotide and amino acid sequence divergences of 0.4% and 0.3%, respectively. In contrast, the two mMDH cDNA alleles differed by 23% and 20% at the nucleotide and amino acid levels, respectively. Analysis of a 91-bp partial nucleotide sequence of mMDH from Nucella freycineti, the closest relative of N. lapillus, revealed two similar alleles and indicated that the divergence in mMDH in N. lapillus represents an ancient transpecific polymorphism in these Nucella. Together with earlier studies on variation in N. lapillus, it is argued that the polymorphism in mMDH and the clines in N. lapillus represent the presence of two persistent coadapted gene complexes, multitrait coevolving genetic solutions to environmental variation, which may presently enable this snail to exploit a diverse environment successfully.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
Clines in intraspecific genetic variation have been recognized as important and useful indicators of ecological and evolutionary processes since the theoretical work of R. A. Fisher in the 1920s (Fisher 1930Citation ; Endler 1977Citation ). However, despite the potential offered by clines for understanding ecological processes, only a few have been studied at the nucleotide level (Kreitman 1983Citation ; Bernardi, Sordino, and Powers 1993Citation ). This study investigates molecular genetic variation in two loci from a well-characterized cline in a continuous population of Nucella lapillus, where, on the southwestern peninsula of England, between the two headlands of Peartree Point and Prawle Point, coincident step clines occur in shell phenotype, allozyme, genotype, mitochondrial DNA haplotype, and centric fusion (Robertsonian translocations; chromosomal fusions [Robertson 1916Citation ]) frequencies that covary with temperature and humidity over a distance of ca. 3 km (Bantock and Cockayne 1975Citation ; Day 1990Citation ; Kirby, Bayne, and Berry 1994a, 1994bCitation ; Kirby, Berry, and Powers 1997Citation ). This genetic and phenotypic variation in N. lapillus between Peartree Point and Prawle Point agrees with a more widespread mosaic pattern of similar allozyme, chromosomal, and phenotypic variation in N. lapillus that appears to covary with variation in habitat along the English Channel coasts (Staiger 1957Citation ; Hoxmark 1970, 1971Citation ; Bantock and Cockayne 1975Citation ; Day 1990, 1992Citation ).

The possibility that the genetic variation in N. lapillus between Peartree Point and Prawle Point and elsewhere is associated with habitat and reflects secondary contact between two races has previously been suggested (Hoxmark 1970Citation ; Kirby, Berry, and Powers 1997Citation ). The purpose of this study was to further investigate the nuclear component of genetic variation in N. lapillus by examining nucleotide sequence variation in the two protein-coding loci of the malate dehydrogenase (MDH) enzyme system (EC 1.1.1.37). MDHs are enzymes that catalyze the interconversion of malate to oxaloacetate, and animals possess two molecular forms of MDH which function in different cellular compartments, the cytosol (cMDH) and the mitochondria (mMDH). Allozyme electrophoresis of mMDH and cMDH indicates that these two loci are single-copy in N. lapillus, with normal heterodimer formation in mMDH heterozygotes and the absence of null alleles (Day and Bayne 1988Citation ). In this study, genetic variation in cMDH and mMDH was compared in N. lapillus from the cline, and variation in mMDH in N. lapillus was compared with variation in mMDH from its close relative Nucella freycineti, a Pacific intertidal snail which occupies a similar ecological niche (Buyanovsky 1992Citation ; Amano, Vermeij, and Narita 1993Citation ).

MDH loci were chosen because (1) protein electrophoresis has shown two alleles of mMDH in N. lapillus varying clinally from fixation of one allele to near fixation of the other, whereas cMDH appears to be monomorphic (Day 1990Citation ; Kirby, Berry, and Powers 1997Citation ); (2) variation in mMDH in N. lapillus appears to be in complete linkage disequilibrium with variation in EST-3 (EC 3.1.1.1) (Day 1990Citation ; Kirby, Berry, and Powers 1997Citation ), suggesting that mMDH may characterize different genetic compositions (chromosome rearrangements and allozymes) (Day 1992Citation ); (3) mMDH and cMDH comprise a compartmentalized enzyme system with similar catalytic functions and may therefore experience similar selection pressures; (4) other studies of allelic variations in MDHs, covering a diverse range of taxa, have described intraspecific allelic variation that correlates with variation in environmental temperature (Kirby, Berry, and Powers 1977Citation ; Powers and Place 1978Citation ; Powers et al. 1991Citation ; Nielsen, Page, and Crossland 1994Citation ; Harrison, Nielsen, and Page 1996Citation ), suggesting that in some cases variation in MDHs may represent the outcome of natural selection (Nielsen, Page, and Crossland 1994Citation ; Harrison, Nielsen, and Page 1996Citation ).


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
Sample Collection
Molecular variation in mMDH and cMDH in N. lapillus in the region of the cline was determined first from N. lapillus collected from Sharpers Head (referred to as "Site 13" in previous studies [Kirby, Berry, and Powers 1977Citation ]) and Gara Rock, sites where N. lapillus are highly differentiated in the phenotype and mMDH frequencies (mMDH10 = 3.4% and 100%, respectively) characteristic of the cline. Allelic variation in N. lapillus mMDH and cMDH was then further examined in nine individuals of N. lapillus from site 9, a site which is close to the center of the step cline in allele and phenotype frequencies (frequency of mMDH10 at site 9 = 67.5% [Kirby, Berry, and Powers 1997Citation ]).

To investigate the age of the polymorphism in mMDH in Nucella, samples of N. freycineti, the closest relative of N. lapillus, were collected from Onagawa (Miyagi, Japan). Foot muscle tissue was dissected from five individuals, pooled, and placed into 10-ml of RNA-later (Ambion Corp.) and sent at ambient temperature by airmail to the United Kingdom, where the sample was stored at -70°C.

Amplification of N. lapillus cMDH and mMDH cDNAs from Sharpers Head and Gara Rock
A similar amplification and cloning strategy was followed to obtain both cMDH and mMDH cDNA sequences (fig. 1 ) (Kirby 2000Citation ). Briefly, partial nucleotide sequences of N. lapillus cMDH (249 bp) and mMDH (129 bp) were first obtained from two individuals from Sharpers Head and two individuals from Gara Rock by RT-PCR using DNase-I–treated mRNA (extracted from N. lapillus foot muscle tissue) as template and broad taxon degenerate PCR primers (Kirby 2000Citation ). Rapid amplification of cDNA ends (RACE) (Frohman, Dush, and Martin 1988Citation ) techniques using primers designed for these sequences then obtained the 5' and 3' cDNA ends of cMDH and mMDH. The RACE reactions were performed on two cDNA templates obtained from the pooled mRNAs of five individuals collected from either Sharpers Head or Gara Rock. Primer sequences and amplification conditions for obtaining the cMDH 5' and 3' RACE reactions were similar to those in Kirby (2000)Citation . In contrast, due to the differentiation found between mMDH sequences at Sharpers Head and Gara Rock, allele-specific primers (P5 to P8; fig. 1 ) were used to amplify the 5' and 3' cDNA ends from these two sites. Primers were then designed from the 5' and 3' untranslated regions (UTRs) to obtain the full-length cDNAs. Pooled mRNAs were used in the RACE reactions in order to sample alleles from more than one individual, so the primers designed from the 5' and 3' UTRs (used to obtain the full-length sequences) were from unambiguous regions.



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Fig. 1.—A, The PCR amplification and cloning strategy used to obtain the full-length open reading frame cDNA sequences of the putative Nucella lapillus mMDH9 and mMDH10 alleles. The upper diagram depicts the full-length mRNA, with the open box representing the coding region and the solid lines representing the 5' and 3' untranslated regions. The two broken lines represent the reverse-transcribed cDNA templates used in the PCR. P1–P12 refer to the oligonucleotide primers used in the reverse transcription and PCR amplification steps, with arrows representing primer orientation. Line a represents the initial partial internal fragment amplified by nested PCR using degenerate primers P1–P4. Line b represents the 5' untranslated (UTR) fragment obtained for both mMDH9 and mMDH10. Lines c and d represent the mMDH10 and mMDH9 3' rapid amplification of cDNA ends (RACE) fragments, respectively (length differences of these products are due to different primer positions). Line e represents the full-length cDNAs amplified using primers P13 and P14, situated in the UTRs. Numbers in brackets are the sizes (bp) of the amplification products. B, The sequences of the PCR primers (5' to 3') used in the amplification reactions

 
The full-length open reading frames (ORFs) of cMDH and mMDH from Sharpers Head and Gara Rock were amplified from the two pooled cDNA templates using a proofreading polymerase. The primers used to amplify full-length sequences of cMDH from both sites were the 5' cMDH UTR primer 5'-TCCGAGTGAAGAAACTTGCCA-3' and the 3' cMDH UTR primer 5'-TCCTCTAGAGCTACTATCAGC-3' (Kirby 2000Citation ). In contrast, allele-specific primers (P13 and P14; fig. 1 ) were used to amplify the full-length mMDH cDNA sequences from Sharpers Head and Gara Rock. These allele-specific primer pairs were also used to try to amplify mMDH cDNAs from the opposing pooled mRNA templates, i.e., mMDH10 allele-specific primers were used on the pooled mRNA from Sharpers Head and mMDH9 allele-specific primers were used on the pooled mRNA from Gara Rock. PCR reactions included 250 ng of each primer, 80 mM total dNTPs, 5 U Pfu DNA polymerase (Stratagene, United Kingdom), 1.5 mM MgCl2, and 50 ng of the pooled cDNA template in a 100-µl reaction volume. The thermal profile involved an initial denaturation of 45 s at 94°C followed by 30 cycles of 45 s at 94°C, 45 s at 56°C, and 3 min at 72°C with a final extension of 10 min at 72°C. Amplification products were cloned, and eight clones of mMDH and cMDH were then manually sequenced on one strand in one orientation (3' to 5') from each site (32 sequences in total).

Analysis of Allelic Variation in N. lapillus cMDH and mMDH from the Center of the Cline
Full-length cMDH and mMDH alleles were obtained from each of nine individuals from the center of the cline by reverse transcription of total RNA template (using primer P11; fig. 1 ) and a two-step nested PCR protocol using primer pairs situated in the 5' and 3' UTRs of the genes. To amplify cMDH cDNAs, a first-step PCR was performed using the cMDH primers given above. A second-step nested PCR was then performed using the 5' cMDH UTR primer 5'-TGCAAACCTCATTTCAGCGGC-3' and the 3' cMDH UTR primer 5'-GATCTACACCACAACAGCCC-3'.

Due to the extensive allelic differentiation found in the mMDH UTRs, it was necessary to conduct two sets of two-step nested PCR reactions on each individual in order to accommodate possible recombinant alleles at this locus. In both sets of amplifications, the first-step reaction simply increased the mMDH cDNA template concentration by using either the mMDH10 5' UTR primer 5'-TTTCACCGGTGAGGTCGTCTG-3' (located 5' of primer P13) or the mMDH9 allele-specific primer P13 with, in each case, the universal 3' primer P11 (fig. 1 ) (amplifications A1 and A2, respectively). In the first set of second-step amplifications, mixed combinations of 5' and 3' mMDH9 and mMDH10 primers were used to include recombinants; i.e., the second-step reaction on template A1 used the nested 5' mMDH10 primer P13 with the 3' mMDH9 primer P14, while the second-step reaction on template A2 used the nested 5' mMDH9 primer (5'-CCGTGCACATCCCAAGATG-3') with the 3' mMDH10 primer P14. In the second set of second-step amplifications, nested 5' and 3' primers were used that were matched to the 5' primers used in the first-step PCRs; i.e., if the first-step 5' primer was specific for mMDH10, the second-step nested 5' and 3' primers were also specific for mMDH10. Amplification conditions were similar to those used to obtain the full-length sequences from Sharpers Head and Gara Rock. Amplification products for cMDH and mMDH were cloned, and a single clone of cMDH from each individual and a single clone from each amplification product of mMDH obtained were sequenced in one orientation.

Analysis of mMDH Variation in N. freycineti
Partial internal 129-bp cDNA sequences of mMDH were amplified from N. freycineti mRNA by RT-PCR (cMDH was not examined from N. freycineti) using the nested degenerate primers P1–P4 (fig. 1 ) and methods similar to those used to amplify internal fragments of mMDH from N. lapillus (Kirby 2000Citation ). Due to the use of broad taxon degenerate PCR primers, the amplification and cloning of N. freycineti mMDH was repeated on three separate occasions to verify the sequence obtained (five clones were sequenced in one orientation on each occasion).

Nucleotide and Amino Acid Sequence Analysis
DNA and amino acid sequence alignments were performed using CLUSTAL W (Thompson, Higgins, and Gibson 1994Citation ). Unrooted phylogenetic trees relating N. lapillus mMDH and cMDH amino acid sequences to other MDHs were constructed using PHYLIP (Felsenstein 1993Citation ) by neighbor-joining on pairwise amino acid distances calculated according to the formula of Kimura (1983)Citation . Support for the branching order of the trees was provided by bootstrap analysis performed with 500 replications.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
Variation in N. lapillus cMDH at Sharpers Head and Gara Rock
The ORF of the putative N. lapillus cMDH is 1,002 nt long and codes for a putative cMDH protein subunit of 334 aa (Kirby 2000Citation ). At the electrophoretic level, N. lapillus cMDH appears to be monomorphic in the region of the cline (Day 1990Citation ). The four nucleotide sequences of the cMDH cDNA internal fragments cloned from the two individuals from each site were all identical, so the same pairs of PCR primers were used to obtain the 5' and 3' cDNA ends and full ORFs from the two pooled mRNA samples from each site. Two alleles were found among the 16 full-length cMDH sequences examined from the mRNA samples from each site. These two alleles differed by four polymorphic nucleotide sites, giving a nucleotide sequence divergence of 0.4%. These four nucleotide substitutions all involved third-position changes (ORF nucleotide positions [nt]: 42 [C/T], 466 [T/C], 612 [G/A], and 636 [G/T]). Three of the substitutions were synonymous, while the fourth (nt 636), a nonsynonymous transversion, changed the 212th amino acid from the basic lysine to the uncharged asparagine. No variation was observed among the eight sequences obtained from the pooled mRNA from Gara Rock, which all possessed the allele "C, T, G, G." Two of the sequences from the pooled mRNA from Sharpers Head exhibited the allele "C, T, G, G," whereas the other six exhibited the allele "T, C, A, T."

Variation in N. lapillus mMDH at Sharpers Head and Gara Rock
Allelic differentiation in mMDH was far greater than that observed in cMDH. The internal partial mMDH cDNA fragments obtained from the two individuals from Sharpers Head and Gara Rock using the nested degenerate primers, while identical within each site, were highly divergent between sites. This differentiation was taken to reflect the mMDH9 and mMDH10 alleles, respectively, and allowed us to design the allele-specific primers used in the RACE reactions and to obtain the full-length cDNAs. The full ORFs of the putative N. lapillus mMDH9 pre-protein obtained from Sharpers Head and the putative MDH10 pre-protein obtained from Gara Rock differed by 23% (233 substitutions in 1,046 nucleotide sites, with 56, 31, and 146 substitutions in the first, second, and third codon positions, respectively) (fig. 2 ). This nucleotide divergence resulted in 70 amino acid substitutions between the mMDH pre-protein alleles (fig. 2 ), giving a 20% amino acid sequence divergence. The mMDH9 and mMDH10 pre-protein subunit alleles also differed in length by one amino acid residue (342 and 341 amino acids, respectively) due to the creation of a termination codon by a single substitution (AAG-TAG) in the penultimate codon of mMDH9. All of the amino acid residues considered essential for catalysis and cofactor binding (Goward and Nicholls 1994Citation ) were found to be conserved in both alleles, and only four amino acid substitutions occurred at sites that otherwise appeared conserved among all other animal mMDHs sequenced thus far (fig. 2 ). No amplification products were obtained when either the mMDH9 allele-specific primers with the pooled mRNA from Gara Rock or the mMDH10 allele-specific primers with the pooled mRNA from Sharpers Head were used, suggesting that mMDH9 and mMDH10 cDNAs, respectively, were absent from these samples.



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Fig. 2.—Aligned cDNA and amino acid sequences for the open reading frame of the putative Nucella lapillus mMDH9 and mMDH10 alleles (GenBank accession numbers AF280052 and AF218064, respectively). The alignment shows the high degree of sequence divergence between the two alleles. Identical bases and residues are indicated by dots. The vertical line indicates the putative site of cleavage of the leader sequence from the mature protein (Kirby 2000Citation ); nucleotides and amino acid residues of the mature protein are numbered with respect to this site. Boxes indicate residues that differ from those appearing conserved among all animal mMDHs sequenced thus far. A full alignment of N. lapillus mMDH10 to other mMDHs has been presented elsewhere (Kirby 2000Citation )

 
Figure 3 shows the phylogenetic relationships of N. lapillus MDHs to other MDHs, together with the extent of the interlocus disparity in sequence divergence within cMDH compared with that found within mMDH in N. lapillus. The pairwise distances (table 1 ) between either the mMDH9 or the mMDH10 allele and other mMDHs are similar to those between the N. lapillus cMDH and cMDHs of other taxa. However, in striking contrast, the intraspecific divergence observed between the N. lapillus mMDH9 and mMDH10 alleles far exceeds that between mMDHs from different genera.



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Fig. 3.—Unrooted neighbor-joining trees relating (A) cMDH and (B) mMDH amino acid sequences. Nucella lapillus cMDH alleles are referred to by the sample site (GenBank accession numbers AF280051 and AF218065 for the Sharpers Head ["T, C, A, T"]) and Gara Rock ["C, T, G, G"] alleles, respectively), whereas the allelic variants of mMDH are referred to by their electrophoretic mobility (Day 1990Citation ). The trees show that while intraspecific genetic variation in the N. lapillus cMDH locus compares favorably with interspecific genetic differentiation among other cMDHs, divergence between N. lapillus mMDH9 and mMDH10 is far greater than that observed between mMDHs of less related taxa. Values by nodes indicate bootstrap values greater than 50%. The scale bar below each tree indicates the length of each branch in terms of the number of amino acid substitutions per site. cMDH and mMDH amino acid sequences were obtained from the SwissProt and GenBank databases

 

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Table 1 Pairwise Distances (Kimura 1983) from cMDH and mMDH Amino Acid Sequences of Nucella lapillus from Sharpers Head and Gara Rock to Other Eukaryotic mMDHs and cMDHs

 
Variation in cMDH and mMDH Among N. lapillus from the Center of the Cline
Three cMDH alleles were observed among the nine individuals examined from the center of the cline, each involving polymorphisms at the same four nucleotide sites that varied at Sharpers Head and Gara Rock. Two individuals possessed the cMDH allele "C, T, G, G" found at Gara Rock, six individuals possessed a new allele, "C, T, A, G," and one individual possessed a new allele, "T, T, G, G." The allele "T, C, A, T," found at Sharpers Head, was not observed among these nine individuals. Since only one cMDH clone was sequenced from each individual, it is not known whether these individuals were homozygous or heterozygous for these alleles.

No mMDH amplification products were obtained from the two-step nested PCRs using mixed pairs of mMDH9 and mMDH10 primers, indicating the rarity of certain mMDH recombinant types in this sample. Similarly, no mMDH amplification products were obtained from three individuals using matched 5' and 3' mMDH9 primers, whereas amplification products were obtained from these animals using matched 5' and 3' mMDH10 primers; this suggests that these individuals were mMDH10 homozygotes. The remaining six individuals proved to be mMDH9,10 heterozygotes, since amplification products were obtained using either pair of matched mMDH 5' and 3' primers. The 6 mMDH9 and 12 mMDH10 alleles sequenced from these nine individuals were identical to the mMDH9 and mMDH10 alleles obtained from Sharpers Head and Gara Rock.

Variation in mMDH in N. freycineti
The total sequence obtained between the nested degenerate mMDH primers P3 and P4 (fig. 1 ) was 91 nt. Figure 4 shows the nucleotide and amino acid sequences of this 91-bp internal fragment of mMDH from N. freycineti and N. lapillus. Two alleles were observed among the 15 clones sequenced from N. freycineti, and these were very similar to mMDH9 and mMDH10 of N. lapillus. The two partial sequences of the N. freycineti mMDH alleles differed by 22 substitutions, compared with 21 nucleotide substitutions between the same regions of the two N. lapillus mMDH alleles. The mMDH9 and mMDH10 alleles of N. freycineti differed from the homologous N. lapillus mMDH9 and mMDH10 alleles by three and one synonymous nucleotide substitutions, respectively.



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Fig. 4.—Partial nucleotide and amino sequences between amplification primers P3 and P4 (fig. 1 ) of the two alleles of Nucella freycineti mMDH (Nfr mMDH9 and Nfr mMDH10) compared with Nucella lapillus mMDH9 and mMDH10 (Nla mMDH9 and Nla mMDH10). Sites at which nucleotides and amino acid residues were identical across all sequences are represented by dots. Amino acid residues appear below the second nucleotide in the codon. Arrows above and below the sequences indicate the positions at which nucleotides differ between the N. freycineti and N. lapillus mMDH9 and the N. freycineti and N. lapillus mMDH10 alleles, respectively. Numbers to the right of the sequence indicate the position of the internal fragment with respect to the full-length mMDH sequences (fig. 2 )

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
The nucleotide and amino acid sequence divergence between the mMDH9 and mMDH10 alleles in N. lapillus was far greater than anticipated based upon the differentiation in mtDNA across the cline (0.03%) (Kirby, Berry, and Powers 1977Citation ) and far greater than that observed among the four cMDH alleles obtained from the same samples. The partial cDNA sequences of the mMDH alleles of N. freycineti indicate that the extreme divergence between the N. lapillus mMDH9 and mMDH10 alleles represents a very ancient transpecific polymorphism in Nucella. The extent of differentiation between mMDH9 and mMDH10 is emphasized by the terminal eight amino acids of the mtDNA leader peptide of mMDH that forms the protease cleavage recognition site (Gavel and von Heijne 1990Citation ) (fig. 2 ). Apart from the conserved amino acids of phenylalanine and serine (residue positions -8 and -7), the only evolutionary constraint on this region is that it is eight amino acids long; the amino acid sequence divergence is 100% (55% nucleotide substitution) in this region.

The similarity in evolutionary distances in the gene trees of cMDH and mMDH between both cMDH and mMDH alleles of N. lapillus and other taxa (fig. 3 and table 1 ) suggests that the rates of evolution are roughly the same in these two loci. Unfortunately, there are too few mMDH sequences available from closely related taxa with which to reasonably compare molecular evolution of mMDH in N. lapillus to estimate a time of divergence of the mMDH9 and mMDH10 alleles. The Muricidae, which include Nucella, are members of the Neogastropoda a group within the Caenogastropoda (Ponder and Lindberg 1997Citation ) and date to the Albian of the mid-late Cretaceous (Taylor, Morris, and Taylor 1980Citation ). The fossil record of the genus Nucella spans an interval of about 25 Myr from the early Miocene to the recent (Cambridge and Kitching 1982Citation ), and according to this fossil record and estimates based on rates of evolution of mtDNA in Nucella (Collins et al. 1996Citation ), N. lapillus and N. freycineti are believed to have diverged somewhere between 4 and 3 MYA. The extent of differentiation between the partial mMDH9 and mMDH10 cDNAs in N. lapillus and N. freycineti, compared with the divergence between the homologous mMDH alleles from these two species, suggests that the divergence of the two evolutionary lineages might even precede Nucella.

Since the polymorphism in mMDH is transpecific, it is most likely that its persistence (and the multitrait cline) reflects balancing selection acting directly on mMDH9 and mMDH10 or on variation in mMDH as part of a wider linkage group in what Dobzhansky (1937Citation , pp. 307–308) envisaged as a coadapted gene complex. In a coadapted gene complex, the gene pool of the population comprises groups of genes that interact in a beneficial way. In this latter context, the chromosomal rearrangement may be an important component of genetic variation in N. lapillus in this region. In contrast to cMDH, for which four alleles were obtained among N. lapillus from Sharpers Head, Gara Rock, and the center of the cline, the only mMDH alleles that were obtained were mMDH9 and mMDH10. The absence of other mMDH alleles, especially in the sample of individuals from the center of the cline, tentatively suggests that recombination may be reduced at mMDH in these snails. Support for reduced recombination at mMDH is also obtained from the absence of "recombinant" alleles as electrophoretic variants (none have been observed in over 1,000 snails examined to date), which, if they occurred, should be seen due to the considerable amino acid divergence between mMDH9 and mMDH10. Reduced recombination at mMDH, together with linkage disequilibrium between mMDH and EST-3 (Day 1990, 1992Citation ) and associations between the mMDH genotype and the shell phenotype (Kirby, Bayne, and Berry 1994aCitation ), are observations that are all consistent with two separately evolving coadapted genetic lineages in N. lapillus. The chromosomal variation in N. lapillus (chromosomal variants are polymorphic within certain populations, and intermediate karyotypes are common in them [Bantock and Cockayne 1975Citation ]) provides a convenient mechanism by which genetic variation could now be maintained and evolve separately. Robertsonian translocations reduce recombination (White 1978Citation , pp. 45–105), causing genetic isolation between alleles (Gregorius and Herzog 1989Citation ). If the different genetic constitutions of N. lapillus on either side of the cline represent two coadapted gene complexes, the Robertsonian polymorphism could provide a mechanism for maintaining them (Darlington 1939Citation , pp. 87–92; Wallace 1955Citation ) conferring an important selective advantage on N. lapillus (Wright 1931Citation ; Lewontin and Kojima 1960Citation ). Unfortunately, there is no information available on the karyotype of N. freycineti to indicate whether similar chromosomal rearrangements might also explain the maintenance of the polymorphism in mMDH in this species.

Whether variation in mMDH (or cMDH) in N. lapillus is itself adaptive or, more simply, covaries with genetic variation in other traits (such as shell shape; Kirby, Bayne, and Berry 1994aCitation ) still needs to be determined. It is not yet known whether the two amino acid residues that differ between mMDH9 and mMDH10 at sites that are conserved among other known animal mMDHs (fig. 2 ) have physiological consequences in N. lapillus. In other organisms, however, differences in fitness among MDH alleles have been found to be associated with environmental temperature (Powers and Place 1978Citation ; Nielsen, Page, and Crossland 1994Citation ; Harrison, Nielsen, and Page 1996Citation ). The shell shape differences on either side of the cline in N. lapillus are related to temperature, which is an important factor in the biology of N. lapillus in this habitat (Kirby, Berry, and Powers 1997Citation ). Since mMDH plays a key role in energy metabolism through the malate-aspartate shuttle and citric acid cycle, it would not be unreasonable to suggest that some of the variation between N. lapillus mMDH alleles may confer a fitness advantage in different temperature environments. If this is so, the ancient nature of the mMDH alleles makes it tempting to speculate on whether the divergence of these alleles might have coincided with the radiation of the Nucella that involved the colonization of new warm and cold water environments (Amano, Vermeij, and Narita 1993Citation ).

Since the discovery of considerable protein polymorphism in natural populations, there has been debate as to how this variation may be maintained (Lewontin 1974Citation , pp. 189–271; Nei 1975Citation , pp. 154–174; Wright 1978Citation , pp. 242–322; Kimura 1983Citation , pp. 253–299). As further polymorphic protein coding loci have been sequenced, it has been shown that allelic variation that may be transpecific can persist for extremely long evolutionary times when maintained by balancing selection (O'hUigin, Sato, and Klein 1997Citation ; Filatov and Charlesworth 1999Citation ; Flajnik et al. 1999Citation ; Su and Nei 1999Citation ). The differentiation between mMDH9 and mMDH10 in N. lapillus and N. freycineti, compared with the differentiation between the homologous mMDH alleles of these two species, suggests a very ancient divergence in mMDH that, if the fossil record and mtDNA of Nucella are reliable (Cambridge and Kitching 1982Citation ; Collins et al. 1996Citation ), may predate Nucella. Transpecific polymorphisms, like that seen in mMDH in Nucella, are macroevolutionary phenomena (Mayr 1963Citation , pp. 586–620) (as opposed to microevolutionary intraspecific polymorphisms), so other macroevolutioary phenomena such as coadaptation (Dobzhansky 1937Citation , pp. 307–308) and epigenetic interactions (Waddington 1957Citation , pp. 188) are likely to be associated with them. The physical stresses associated with life in the intertidal environment (Lewis 1964Citation , pp. 217–221) have probably changed little over geological time, although their intensity may have varied, and they are likely to have been important selective agents. Today, the persistence of variation in mMDH (and the multitrait cline in N. lapillus between Peartree Point and Prawle Point) may reflect selection on two separate genetic lineages that are perhaps coadapted to different environments (adaptive peaks; Wright 1931Citation ) and help N. lapillus exploit its niche successfully. The variations in shell shape associated with variation in mMDH (and other loci) in N. lapillus are inherited (Kirby, Bayne, and Berry 1994aCitation ); however, it is not known whether the ages of these polymorphisms are similar. Similarly, it is also not yet known if the association between shell shape and variation in mMDH in N. lapillus occurs in other Nucella (or related taxa) or if this association is a more recent outcome of chromosomal rearrangement in this particular snail. Nevertheless, it is interesting to observe that heritable variations in shell shape, like those seen in N. lapillus, occur in association with variation in temperature in closely related intertidal molluscs also belonging to the Caenogastropoda and whose lineages are older than Nucella (Vermeij 1973Citation ; Newkirk and Doyle 1975, 1979Citation ; Janson and Sundberg 1983Citation ; Grahame and Mill 1992Citation ). The study of the origin and molecular evolution of variation in mMDH in Nucella and related taxa consequently comprises ongoing research.


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
GenBank database accession numbers for mMDH and cMDH cDNA sequences reported in this study are as follows: Sharpers Head mMDH9, AF280052; Gara Rock mMDH10, AF218064; Sharpers Head cMDH "T, C, A, T" allele, AF280051; Gara Rock cMDH "C, T, G, G" allele, AF218065.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
I wish to thank R. J. Berry and J. Harris for valuable discussion, Prof. J. Quattro for time in his laboratory, Dr. M. Osada for providing samples of N. freycineti, and two anonymous referees and the editors of Molecular Biology and Evolution for useful comments on the manuscript.


    Footnotes
 
Richard Thomas, Reviewing Editor

1 Keywords: cline coadaptation macroevolution malate dehydrogenase Nucella lapillus, transpecific Back

2 Address for correspondence and reprints: Richard R. Kirby, Plymouth Marine Laboratory, Citadel Hill, The Hoe, Plymouth PL1 2PB, United Kingdom. E-mail: r.kirby{at}pml.ac.uk Back


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Accepted for publication August 7, 2000.